CA2245727A1 - 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

CA 0224 ~ 727 l998-08-0 ~
WO 97/30159 PCT / FRg7 / 00291 , ~ RECOMBBED PROTEIN ~ CONTAINING A C-TERMINAL FRAGMENT OF MSP-I TDE PLASMODIUM

The invention relates to new active principles of vaccines.
o derived from the major surface protein of merozoite forms of a infectious plasmodium for mammals, especially humans, more generally known as MSP-1.
This protein has already been the subject of numerous studies. She is synthesized in the schizont stage of P ~ asmod ~ um type parasites, including Plasmodium falciparum, and is expressed as one of major constituents of the surface of merozoites as well during the hepatic stage than during the erythrocytic stage of malaria (1, 2, 3, 4). Due to the predominance and conservation in all the known Plasmodium species of this protein it has been suggested that she could represent a candidate for the constitution of vaccines anti-malarial drugs (~, 6).
It was the same for fragments of this protein, particularly natural cleavage products which are observed formation, for example during the parasite invasion of erythrocytes from the infected host. Among these cleavage products, the C-terminal fragment having a molecular weight of 42 kDa (7.8) which is at in turn cleaved again into an N-terminal fragment having a weight conventional molecular apparent of 33 kDa and in a fragment C-terminal with a conventional apparent molecular weight of 19 kDa (9) which normally remains attached to the parasite membrane after CA 0224 ~ 727 1998-08-0 ~
WO 97/30 ~ 59 PCT / E ~ 97/00291 modifications of which he himself is the object, through groups of the - type glycosylphosphatidylinositol (GPI) (10, 11 ~.
We still find it at the early ring stage of the intraerythrocytic development (15, 16), hence the observations which have been made that this 19 kDa fragment could play a role not yet known, but undoubtedly essential in the reinvasive processes. Of the flow from the assumptions already made in the past that this protein could be a particularly effective target for possible vaccines.
It will be understood that the references often made in the following to proteins p42 and p19 from a certain type of Plasmodium are understood to relate to C-terminal cleavage products correspondents of the MSP-1 protein of this Plasmodium, or, by extension, to products containing substantially the same sequences in amino acids, obtained by genetic recombination or by synthesis chemical according to conventional techniques, for example by synthesizer type ~ Applied System> ~ or by synthesis on solid phase type ~ Merrifield ~. For the convenience of language, references to ~ p42 recombinants, ~ and recombinant ~ p19 ~ refer to ~ p42 ~

2 () and ~ p19 ~ obtained by techniques comprising at least one step of genetic engineering.
Given 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 use of techniques allowing the use of peptides or recombinant proteins. However, MSP-1 is very difficult to produce in whole because of its ~ large size of around 200 kDa, a ~ a which led researchers to take an interest in the C-terminal part - whose function, still unknown, is probably the most important.

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WO 97/301 ~ 9 PCT / FR97 / 00291 Under the title, recombinant proteins relating to part C-terminal of MSP-1 of ~ .falciparum, which have been produced and tested in Ie monkey (12, 40, 41), we will mention:
. a p19 fused with a glutathione-S-transferase produced in E coli (~ 4 ~;)), . a p42 fused with a glutathione-S-transferase produced in E. coli;
a p19 fused with a polypeptide derived from a tetanus toxoid and carrying helper T cell epitopes produced in o S cerevisiae (12), . a p42 produced in a baculovirus system (41).

A composition containing the fusion protein p1 g with a glutathione-S-transferase produced in E.coli in association with alum or s liposomes had no protective effect on any of the six Aotus monkeys nancymai vaccinated (40).
A composition containing the p42 fusion protein with a glutathione-S-transferase produced in E.coli in association with a Freund's complete adjuvant did not have a protective effect in two types of Aotus monkeys (A.nancymai and A.vociferans) to which they had been administered. ~ a p19 protein produced in S.cerevisiae a exerted a protective effect in two Aotus monkeys of type A.nancymai (12). However, it did not have a protective effect in two monkeys Aotus type A. vociferans.
2 ~ Some researchers (Chang et al.) Have also reported rabbit immunization assays with protein recombinant p42 produced in a baculovirus system and containing a amino acid sequence in common with P.falciparum (18). So these do recent authors indicate that this recombinant p42 behaves 3t) in rabbits in much the same way as the protein MSP-1 whole recombinant (gp195). This p42 protein in combination with a CA 0224 ~ 727 1998-08-0 ~

Freund's complete adjuvant has been the subject of a vaccination trial in a - - non-human primate susceptible to infection by P.falciparum, Aotus, lemurinus grisemembra (40). Results show that 2 out of 3 animals were completely protected and the third, although showing a s parasitaemia similar to controls, had a latent period more long. It is nevertheless risky to conclude that the protective character in humans, antibodies thus induced against the parasites themselves same. Remember that there are currently no models very satisfactory experimental results in primates for P. vivax and P.falciparum. The Saimiri model, which was developed for P.falciparum and P. vivax, and the Aotus model for P.falcipanrm, are systems artificial, requiring the adaptation of parasite strains and often the animal splenectomy to obtain significant parasitaemias. In Consequently, the vaccination results from these models do not s may have only limited predictive value for humans.
Besides that we can therefore wonder about what would be a real rate vaccination likely to be obtained with such recombinant proteins, the presence in p42 from Plasmodiums of the same species, and more particularly in p33 2 () corresponding, hypervariable regions could make random in many cases the immunoprotective efficacy of the antibodies induced in people vaccinated with p42 from a strain of Plasmodium against infection by other strains of the same species (13).
2 ~ The important polymorphism of the central regions of p42 could even play a significant role in the immune system, often observed for this type of parasites.
But it turns out that p42 from various infectious PJasmodiums for humans have hypervariable regions concentrating : ~ (i mainly in their regions lll, and even more in their regions ll respective: see the publication by Longacre, S. (1 3) in which the CA 0224 ~ 727 1998-08-0 ~

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p.cynomolgy, P.vivax (Belem) and P.vivax (Sal-i) p42 sequences have - ~ been aligned. The sequence ~ consensus ~ of ~ attached Figure 4 a ~ outée to the sequences of p42 of these three parasites testifies to it.
We will refer to the article by Longacre, S. (13) in which are set out the conditions under which the said regions were determined. Note that the attached Figure 4 is none other than reproduction of Figure 1 from Longacre's article. The reader is therefore invited to refer to the legend of this figure. This also appear the relative sizes of the four regions of p42 (region IV
corresponding to the sequence of p19) expressed in percentages vis-à-vis the size of the entire p42 coding sequence.
As also follows from Figure 4 of this asks, the percentages of homology are high between the sequences of regions I of P.cynomolgi and P.vivax: 84% in region 1, 86% in region IV. On the other hand, this percentage of homology decreases sharply in region III (69%) and even more so in region 11 (47%).
A first objective of the invention is therefore to provide active ingredients in p42 vaccines better able to protect I host cont ~ e the immune escape which was discussed above.
2 () It goes without saying that the fragmentation of p42 which has just been envisaged can also be extended to P. falciparum, the parasite mainly responsible for acute forms of malaria in humans, and this all the more so since the locations of the zones separating regions 1, II, III and IV of the constituent sequences of P.cynomolgi and of the two varieties of 2: ~ P.vivax were determined by analogy with corresponding sites, previously identified in P.falciparum, as described by (34) (3 ~) The invention relates more particularly to compositions immunizing against an infectious Plasmodium parasite for

- 3 () humans, containing as active ingredient a recombinant protein WO 97/30159 PCT / FRg7 / 00291 glycosylated or not, whose constitutive polypeptide sequence - - - essential is that ~ either from the p42 from which the region ll have been deleted and, where appropriate, all or part of region III;
~ either of part of this fragment when it is also able to induce an immune response capable of inhibiting parasitaemia in vivo by the corresponding parasite;
. either of a peptide immunologically equivalent to this fragment p42 or to the above part of this fragment, and this recombinant protein further comprising epitopes conformational 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 protein recombinant, glycosylated or not derived from p42 and containing both essential parts of region I and region IV defined above for the constitution of immunogenic compositions, in particular of vaccines.
The above recombinant protein, glycosylated or not, contains the case if necessary also the preserved part of the region III which is situated on the side C-2 () terminal of p33, close to p19, in particular the one between amino acids 255 to 273, or more particularly still between amino acids 255 to 270 (see numbering of the consensus sequence of Figure 4).
In other words, all or a little-preserved part of the region lll can be delated from the N-terminal part of region III.
For the convenience of language, reference will often be made in which following a partially deleted ~ p42 ~ to designate the p42 modified, as defined above.
The presence in this active ingredient of the above-mentioned epitopes ~ 3 (~ conformational could play an important role in efficiency protective that it is likely to confer on the vaccinated host. Is the CA 0224 ~ 727 l998-08-0 ~

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particularly found in the active ingredients presenting by - - elsewhere the other characteristics defined above, when they have been produced in a baculovirus vector system. If it is needed, it is mentioned below only by the expression ~ vector system baculovirus ~ we mean the set that constitutes the type vector baculovirus itself and cell lines, especially cells of insects transfectable by a baculovirus modified by a sequence to transfer to these cell lines resulting in the expression of this sequence trans ~ erée. Favorite examples of these two partners o 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 that cells infected with this baculovirus can be used instead and place of the one who was chosen.
The unstable nature in a reducing environment of these epitopes conformational can be highlighted, in particular by the test described later in the examples, especially in the presence of , ~ -mercaptoethanol.
From this point of view, recombinant proteins derived from p42 recombinant produced by S. Longacre et al. (14) can be set works in such compositions. It is recalled that S. Longacre et al.
succeeded in producing a recombinant p19 from MSP-1 of P.vivax in a baculovirus vector system containing a sequence nucleotide encoding Plasmodium vivax p19, in particular by 2: ~ transfection of insect cell cultures [Spodoptera line frugiperda (Sf9)] with vector baculoviruses containing, under the control of the polyhedrin promoter, a sequence coding for the fragments ,. peptides defined below, the sequences of which were placed in the following order in the baculovirus vector used:

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WO 97/30159 PCT / FR97 / 00 ~ 91 5'-terminal fragment of 35 base pairs of the signal sequence of the polyhedrine, from which the methionine codon had been mutated (into ATT) initiating the expression of this protein;
~ a 5'-nucleotide terminal fragment coding for a peptide of 32 amino acids corresponding to the N-terminal part of MSP-1, y including the signal peptide of MSP-1;
either a nucleotide sequence coding for p19, or a Plasmodium MSP-1 p42 coding sequence vivax, these sequences also being, as appropriate, either provided (forms ~ anchored ~), or devoid (soluble forms) of the regions of the 3 ′ end of these nucleotide sequences whose products of expression C-terminal extremes are deemed 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-terminal region of MSP-1 therefore extended from amino acid Asp 1325 to acid amino acid Leu 1726 (anchored form) or amino acid Ser 1705 (form soluble) and for p19, the sequences extended from the amino acid lle 1602 with the amino acid Leu 1726 (anchored form) or with the amino acid Ser 1705 2 () (soluble form), it being understood that the amino acid sequences complete with p42 and p19 whose initial and terminal amino acids have have been indicated in the foregoing arise from the gene for the Belem isolate of P.vivax which has been sequenced (20).
Similar results have been obtained by implementing 2 :) in the same vector systems of nucleotide sequences coding for 1 ~ 3 p19 and p42 of Plasmodium cynomolgj. P.cynomo / gi presents 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. he can also infect humans. We also have access to natural hosts 3 () by P.cynomolgi, rhesus monkeys and toque monkeys, to test CA 0224 ~ 727 1998-08-0 ~

') The protective efficacy of P.cynomolgi's MSP-1 in systems - - natural. The rhesus monkey is considered one of the most representative of immune reactions in humans.
In particular, excellent results have been obtained in 5 vaccination carried out in the toque monkey with two polypeptides recombinants: soluble p42 and p1 9, derived from P.cynomolgi, respectively produced in a baculovirus system and purified on affinity column with monoclonal antibodies recognizing ies corresponding regions of the native MSP-1 protein. The observations following were made: the six monkeys immunized with the single p19 (three monkeys) and the 19th and p42 together (3 monkeys) all testified to a almost sterile immunity after challenge infection. The obtained results 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 witnesses immunized with PBS buffer in the presence of Freund's adjuvant (3 monkeys) or not immunized (3 monkeys), it was nonetheless patent.
The results of particularly effective trials carried out in macaque with recombinant polypeptides produced in a system 2 () baculovirus implementing a p42, in combination with a p19 recombinant of P.cynomolgi, establish that polypeptides recombinants containing respectively recombinant p42 from other Plasmodiums must behave in the same way. These trials are ~ more telling ~ for malaria in humans than results of tests carried out with P.vivax or P. ~ alciparum in their ~ hosts artificial ~.
Recombinant baculovirus proteins, derived from part C-terminal of MSP-1 (p42 ~, and more particularly the p42 partially deleted, have a very significant antimalarial protective effect in a - 30 natural system, which constitutes the model for evaluating the protective effect of MSP-1 most representative for humans.

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The protective effect obtained could be all the better as p42 - partially deleted is devoid of the hypervariable region of the part N-terminal of p42, the effect of which may be harmful in situations in which the vaccinated subject is confronted with a important polymorphism But the deletion of region ll and all or part of region lll normally leads to better results. It is clear that the man of the profession would have no difficulty in making fusion proteins between regions I and IV of the corresponding p42, or even between a o region I and a region IV respectively from two p42 from two different varieties of P / asmodium. It goes without saying that these fusion proteins may also contain linking elements still corresponding to parts of region ll, preferably of the region III, preferably chosen from among the best preserved. As s of example, mention will be made of the C-terminal polypeptide sequence of the p33, when present, includes less than 50 acid residues amino or even less than 35, or even less than 10 acid residues amines.
However, the polypeptide sequence of the p42 protein 2 () partially deleted may not contain the entire sequence coding for p1g (or from region IV ~, naturally provided that the latter retains the ability to induce protective antibodies against the parasite. In particular the above ~ fragment part has a weight molecular from 10 to 25 kDa, in particular from 10 to 15 kDa. Preferably, 2: ~ this part of the polypeptide fragment contains at least one of the two EGF regions (abbreviation of the English expression ~ Epidermal Growth Factor ~).
Naturally the same observations apply to region I of the recombinant protein according to the invention.
3 () It is clear that the skilled person can make a difference between active fragments and those that would cease to be active, especially CA 0224 ~ 727 1998-08-0 ~
WO 97/30159 PCT / E ~ R97 / 00291 .

experimentally by producing modified vectors containing, by - - example, inserts comprising parts of p42 and in particular of p42 deleted, of different lengths, respectively produced from the coding sequence for p42, if appropriate, partially deleted, by reaction with appropriate restriction enzymes, or exonucleolytic enzymes that would have been kept in contact with the fragment coding for initial p42, if necessary, partially deleted for varying times; the ability of the expression products of these inserts in corresponding eukaryotic cells, including insect cells, transformed by the corresponding modified vectors, to exert a protective effect, which can then be tested, in particular in the experimental conditions which will be described later, with regard to the examples. In particular, the expression products of these inserts should be able to inhibit a parasitaemia induced tn vivo by the parasite whole correspondent.
Likewise, the invention includes all immunogenic compositions, even in which the constitutive polypeptide sequence essential of the active ingredient would consist of a peptide capable of inducing an equivalent cell and / or humoral immune response ~ e 2 () to that produced by the partially deleted protein as defined above above, since the addition, deletion or substitution in its sequence of certain amino acids by others would not center a significant modification of the capacity of the peptide thus modified below called ~ immunologically equivalent peptide ~ - also to inhibit 2 ~ above parasitaemia.
Partially deleted p42 can of course also be combined whether on the N-terminal side or on the C-Terminal side or through of a peptide bond, to another fragment of plasmodial protein having a vaccinating potential like for example a protein (~ Duffy - 3 () Binding Protein ~ by P. vivax (29) or EBA-175 by P. falciparum (30 ~ and ~ 31) whose region is specifically rich in cysteine), provided that -CA 0224 ~ 727 1998-08-0 ~

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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 partially deleted p42 or part thereof of it, may also contain, upstream of its N- end terminal, an even different peptide sequence, for example a C-terminal fragment of the signal peptide of the MSP-1 protein. This sequence preferably comprises less than 50 amino acids, per example of 10 to 35 amino acids.
These observations extend similarly to p42 o partially deleted from other Plasmodium, in particular P.falciparum, the dominant species of parasites, responsible for one of the most severe forms of malaria.
But the techniques recalled above for production in a baculovirus system of a recombinant p42 from P. vivax or s P. cynomolgi are difficult to transpose as such to the production of a recombinant P. falciparum p42 with a satisfactory yield, if only to obtain appreciable quantities authorizing the carrying out immunoprotection tests.
The inventior also provides a process that largely remedies 2 () party to this difficulty. It also becomes possible to obtain returns much more important in p42 of P falciparum - and others plasmodiums when encountering similar difficulties - by putting implementing a synthetic nucleotide sequence of substitution for the natural nucleotide sequence coding for Plasmodium p42 2 :) falciparum in an expression vector of a baculovirus system, this synthetic nucleotide sequence encoding the same p42, but being characterized by a higher proportion of nucleotides G e ~ C than in the natural nucleotide sequence.
It is clear to those skilled in the art that this result can be obtained by - 3 () taking advantage both of its ability to synthesize DNAs by synthesis nucleotide and choose from the possibilities offered by the code CA 0224 ~ 727 1998-08-0 ~
WO 97/301! I9 PCT / FR97 / 00291 genetic, to substitute, whenever the genetic code authorizes it, - - synthetic codons with higher C and / or G contents than the natural codons which, within the native sequences coding for the p42 corresponding natives, 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 more high.
In other words, the invention stems from the discovery that expression in a baculovirus system of a nucleotide sequence o coding for a p42, partially deleted or not, was apparently linked improved compatibility of successive codons in the sequence nucleotide to be expressed with ~ cell machinery ~ cells hosts transformable by bacuioviruses, as observed for natural nucleotide sequences normally contained s in these baculoviruses and expressed in infected host cells; from where bad expression, if not sometimes the total absence of expression of a native nucleotide sequence of P. faJciparum; hence also a possible explanation for the more effective expression observed of p42 of P.vivax in a baculovirus system by Longacre et al. (19) and, as the inventors have also observed, from the sequence of P.cynomoJgi from corresponding native p42 nucleotide sequences, due to their relative contents in nucleotides G and C much higher than those of the native nucleotide sequences coding for the p42s of P.falclparum.
2 ~ The invention therefore also relates, more generally, to a vector modified recombinant baculovirus type containing under the control of a promoter contained in this vector and likely to be recognized by , cells transfectable by this vector, a first sequence nucleotide coding for a signal peptide exploitable by a system - '3 () baculovirus, 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 sequence - - constitutive, that:
~ either of a peptide fragment encoding p42 or a p42 of which have been deleted region II and, where appropriate, all or part of region III;
~ either of part of this peptide fragment as soon as the product expression of the second sequence in a baculovirus system is also able to inhibit parasitaemia normally induced in vivo by the corresponding parasite;
. either an immunologically equivalent peptide derived from the above C-terminal peptide fragment (p19) or the above portion of fragment peptide by addition, deletion or substitution of amino acids not causing a significant change in the capacity of this immunologically equivalent peptide to induce a response cell-type and / or humoral immunology similar to that s produced by this peptide fragment p19 or the aforementioned part of this fragment, and said nucleotide sequence having the case of a content of nucleotides G and C between 40 and 60%, preferably at least 50% of the totality of the nucleotides of which it is made up. This sequence 2 (t can be obtained by construction of a synthetic gene in which the natural codons have been changed to G / C rich codons without their translation be modified (maintenance of the peptide sequence).
In this case, said nucleotide sequence, provided by a DNA
synthetic, may have at least 10% of codons modified compared to to the sequence of the natural gene or cDNA while retaining the characteristics of the translated nature ~ e sequence, that is to say the maintenance of the amino acid sequence.
However, it is not excluded that this content of nucleotides G and C
could be further increased, since the modifications which 3 (~ would result in the amino acid sequence of the peptide recombinant -or immunologically equivalent peptide-product CA 02245727 l998-08-05 would not result in loss of immunological properties, or even - - protective, recombinant proteins formed, in particular in essays which will be illustrated later.
These observations naturally apply to other Plasmodium infectious to humans, particularly when sequences native nucleotides encoding the corresponding p42s, if applicable partially deleted, would have T and A nucleotide contents hardly compatible with an effective expression in a system baculovirus.
The coding sequence for the signal used may be that normally associated with the native sequence of the Plasmodium concerned.
But it can also come from another Plasmodium, for example P.vivax or P. cynomolgi or another organism if it is likely to be recognized as a signal in a baculovirus system.
The sequence coding for p42 or a part thereof within the vector considered is, if necessary, devoid of the sequence anchoring of the native protein to the parasite from which it originates, case in which the expressed protein is generally excreted in the medium of culture (soluble form ~.
2 () The invention relates equally to the vectors in which the coding sequence contains the terminal 3 'terminal sequence coding for the hydrophobic C'-terminal sequence of p19 and which is normally involved in inducing the anchoring of the native protein to the cell membrane of the host in which it is expressed. This 3'-terminal end region can moreover be heterologous with respect to the sequence coding for the rest of the corresponding p42, for example match the 3'-terminal sequence from P.vivax or another organization as soon as it codes for an anchoring sequence of all of the recombinant protein produced at the host membrane - - ~ () cell of the bacu ~ ovirus system used. As an example of such anchor sequences we cite the GPI of the CD59 antigen expressible in CA 0224 ~ 727 1998-08-0 ~

insect cells of the spodoptera frugiperda type (32) or the GPI of a - - - human protein CD14 (33).
The invention naturally also relates to proteins recombinant, these proteins comprising conformational epitopes s recognized by human sera formed against Plasmodium corresponding.
In general, the invention also relates to any protein recombinant of the type indicated above, since it contains conformational epitopes as produced in the baculovirus system, especially those who happen to be unstable in a reducing environment.
The invention naturally relates to said proteins.
recombinant, whether in the so-called soluble form or in the form provided with an anchoring region, in particular to the cellular hosts set up works 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 vaccine production, on the other hand, by via cGvalent links or not. The essays 2 () vaccinating using them are also part of the invention.
The invention also relates to vaccine compositions using using these recombinant or conjugated proteins, including moreover proteins from Plasmodium vivax.
Also included in the invention are the compositions in 2: ~ which the aforementioned recombinant proteins are associated with an adjuvant, for example an alum Recombinant proteins comprising the extreme C-terminal region allowing 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 there having been instead of being limited to it, it is possible to have recourse to the lipids described for this purpose by CA 0224 ~ 727 1998-08-0 ~

example in the corporate book ~ Liposomes technological aspects, biological and pharmacological ~ by J. Delattre et al., INSERM edition, 1,993.
The presence of the anchoring region in the recombinant protein, whether it is a homologous or heterologous anchoring region vis-à-vis of the vaccinating part proper, is likely to favor the production of cytophilic antibodies, in particular of the IgG2a and IgG2b type in the mice which could have a particularly high protective activity, to the point that we could dispense with combining the active ingredients of vaccines thus constituted with adjuvants other than the lipids used for the constitution of liposomal forms. This would be an advantage important, since liposomes can be lyophilized in conditions allowing their storage and transport, without cold chains are therefore essential.
Other characteristics of the invention will become apparent during of the following description of examples of recombinant proteins using in play recombinant proteins whose active sequences, or contain those of p42, or are limited to that of the p19 proteins corresponding. Without these examples being strictly speaking 2 () still directly covered by the claims which follow, they do not contribute no less to establishing the operational character of the invention which is the subject of the present application.

Description of the construction P ~ MSP1 p, gS (soluble) (p19 solub ~ e 2 ~ from P.falciparum) The recombinant construction PfMSP1 p ~ gS contains DNA
corresponding to the 8 base pairs of the leader sequence and the 32 first amino acids of MSP1 from Plasmodium vivax from Met1 to Asp32 (Belem isolate; Del Portillo et al. 1991. PN AS 88, 4030.) followed by a 3u GluPhe, due to the EcoR1 site linking the two fragments. The whole is followed by the synthetic gene, described in Figure 1, coding for Plasmodium CA 0224 ~ 727 l998-08-0 ~

1 ~

falciparum MSP1p1g from Asn1613 to Ser, 705 (isolate Uganda-Palo Alto; Chang and al. 1988. Exp. Parasitol. 67.1). Construction is completed by two TAA stop codons. This construction gives rise to a protein recombinant that is secreted into the cell culture supernatant infected.
In the same way and for purposes of comparison, we produced a recombinant construct under conditions similar to those used for the production of p19 above, but working with a coding sequence consisting of a direct copy of DNA
correspondent of the strain P.falcipar ~ lm (FUP) described by Chang et al., Exp. Parasit. 67.1; 1989. The copy of this natural gene ~ extending from asparagine 1613 to serine 1705) was formed by PCR from the gene native.

The sequences of both the gene are shown in Figure 1A
synthetic (Bac19 ~ and ~ native gene ~ (PF19).
Note that ~; 7 codons out of the 93 codons in the sequence native coder for p19 from P.fa / ciparum have been modified (for this which is the third nucleotide in 55 of them and the first and 2 () third nucleotides in the two remaining codons ~. Codons new ones were added at the 5 'end to introduce the signal peptide under the conditions which have been indicated above and to introduce a EcoRI site for cloning, on the one hand, and similarly two were added stop codons not present in p19 of P.falciparum in order to obtain 2 :) termination signals of the expression. Individual letters placed respectively above the successive codons correspond to successive respective amino acids. The asterisks (~) refer to to stop codons. The vertical lines underline the nucleotides which are the same in both sequences.
~ 1) -CA 0224 ~ 727 1998-08-0 ~

Construction description PfMSP1p1 ~ A (anchored GPI) (p19 anchored from P. ~ alciparum ~
~ construction PfMSP1p19A has characteristics of the previous except that the synthetic sequence (Figure 1B) codes for the MSP1p19 of s Plasmodium falciparum (isolate Uganda-Palo alto) from Asn1613 to tle1726 followed by two stop codons from TAA. This construction gives rise to a recombinant protein that is anchored in the plasma membrane of cells infected with a structure of the glycosyl phosphatidyl inositol type (GPI).
I () Figure 1 ~ is representative of the protein sequence recombinant PfMSP1p19S before cutting the signal sequence.

Figure 1 D is representative of the protein sequence recombinant PfMSP1pl9S after cutting the signal sequence.

The amino acids underlined in Figures 1 C and 1 D come from of the EcoR1 site used to join the nucleotide sequences derived of the N-terminal part of MSP1 of P.vivax (with signal sequence) and of MSP1 pl9 of P.falciparum.
Figure 2 - The recombinant soluble PfMSP1 pl9 antigen purified by immunoaffinity was analyzed by immunoblotting after SDS-PAGE in presence (reduced) or absence (not reduced) of B-mercaptoethanol. The samples are loaded on gel after heating to 9 ~ C in the presence of 2: ~ 2% SDS. Under these conditions only bonds of the covalent type (disulfide bridges) can resist disaggregation. The blot on the left has been revealed with a monoclonal antibody that reacts with a linear epitope of natural p19 The blot on the right has been revealed with a mixture of 13 human antisera from subjects with acquired immunity to 3 () malaria due to Plasmodium falciparum. These results show that the recombinant baculovirus molecule reproduces epitopes well CA 0224 ~ 727 1998-08-0 ~

~ () conformational in the form of polymer which are recognized in majority by human antiserum.

Figure 3 - The soluble PvMSP1p42 recombinant antigen (Longacre and al. 1994, op.cit.) Was incubated for ~ hours at 37 ~ in the presence of protein fractions derived from the merozoites of P falciparum and separated by isoelectric focusing. Subsequently the samples were analyzed by immunoblotting in the presence (reduced) or absence (not reduced) of B-mercaptoethanol. The fractions ~ to 12 of isoelectrofocusing, as well as two total extracts of merozoites made in the presence (Tex) or absence (T) detergent was analyzed. The immunoblot was revealed with monoclonal antibodies specific for MSP1p42 and p19 of P.vivax. The re ~ sul ~ ats suggest that there is proteolytic activity in merozoites of P.falciparum which can be extracted in detergent. Digestion of p42 in certain fractions seems to cause polymerization of the products digestion (p1 9); 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 2 (3 of about 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 PcMSP1p19S (soluble) (soluble p19 of 2 :) P.cynomolgi ~
The DNA used for the above construction was obtained from a clone of the Plasmodium cynomolgi ceylonesis strain (22-23). This strain was maintained by successive passages in its host natural (Macaca sinica) and cyclic transmissions through 3 () of mosquitoes (27).

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~ 1 Blood parasites have been obtained from infected monkeys at the mature schizont stage when the parasitaemias have reached a level of 5%. They were then purified according to the methods described in ~ 25). DNA has then extracted as described in (26).
A 1200 base pair fragment was then produced by having use of the PCR reaction using the oligonucleotides underlined in Figure 4 and from P.vivax. The 5 'oligonucleotide included an EcoRI restriction site and the 3 'oligonucleotide two codons TAA stop synthetics followed by a Bglll restriction site. This fragment has was introduced by ligation and via these EcoRI and Bglll sites in the plasmid pVLSV200. already containing the signal sequence of the protein MSP-1 from P.vivax (19). The new plasmid (pVLSV200C42) has been used for DNA sequence analysis.
P.cynomolgi sequences and corresponding sequences of P.vivax were brought into alignment. The black arrows indicate the presumed primary and secondary cleavage sites. They were determined by analogy with the sites known in P.falciparum (27, 28). Lines vertical and horizontal arrows locate the boundaries of the four regions that have been studied. Region 4 corresponds to the coding sequence 2 () for p19 of P.cynomolgi. Glycosylation sites are framed and conserved cysteines are underlined. In the lower part of the Figure 4 shows the percentages of identity between the two isolates of P.vivax and P. cynomolgi.
Recombinant construct PcMSP1p19S contains DNA
2 :) corresponding to the 8 base pairs of the sequence ~ leader ~ and the 32 first amino acids of VISP1 from Plasmodium vivax from Met1 to Asp32 (Belem isolate; Del Portillo et al. 1991. PNAS 88, 4030.) followed by a GluPhe, due to the EcoR1 site linking the two fragments. The whole is followed by the coding sequence for the MSP1p19 of Plasmodium cynomolgi : ~ () (Ceylon strain) from Lys276 to Ser380. Construction is completed by two TAA stop codons. This construction gives rise to a protein recombinant that is secreted into the cell culture supernatant infected.

Puri ~ ication of the recombinant pro ~ e PfMSP1 p ~ 9 by immunoaffinity chromatography with a monoclonal antibody specifically recognizing the p19 of Plasmodium falciparum.
The chromatography resin was prepared by binding 70 mg of a monoclonal antibody (obtained from a G17.12 hybridoma deposited at the CNCM (Paris, France) on February 14, 1g97 under the number 1-1846; this hybridoma G17.12 was constructed from X63 Ag8 653 myeloma producing IgG 2a / k recognizing p19 of P.falciparum) at 39 of CNBr-Sepharose 4B activated (Pharmacia) by standard methods detailed in the instructions supplied by Pharmacia. Culture supernatants containing the soluble PfMSP1p19 were incubated in batch with the resin of chromatography for 16 hours at 4 ~ C. The column was washed a times with 20 volumes of 0.05% NP40, 0.5M NaCI, PBS; once with 5 volumes of PBS and once with 2 volumes of 10 mM phosphate sodium, 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 then 2 () concentrated by ultrafiltration and dialyzed against PBS. For the purification of the anchored PfMSP1p19, all washing and elution solutions contained in addition 0.1% 3- (Dimethyl-dodecylammonio) -propane sulfonate (Fluka).

2 ~ Vaccination test for recombinant MSP1 of P / asmod; um vfvax (p42 and p19) in the squirrel monkey Saimiri sc; ureus.
This vaccination trial was carried out on Saimiri sciureus male boliviensis 2 to 3 years old, not splenectomized. Three monkeys were injected 3 times intramuscularly 3 weeks apart with a 3 () mixture of approximately 50 to 100, ug each, of PvMSP1 p42 and soluble p19 recombinant (19), purifies by immunoaffinity. Freund's adjuvant and incomplete was used as follows: 1st injection: 1: 1 FCA / FIA; 2nd - injection 1: 4 FC ~ VFIA; 3rd injection: FIA. These adjuvant compositions were subsequently mixed 1: 1 with the PBS antigen. The five monkeys controls received the glutathione-S-transferase (GST) antigen produced in E. coli according to the same protocol. Test infection was carried out by injecting 2.1 o6 infected red cells with a suitable strain of Plasmodium vivax (Belem) 2.5 weeks after the last injection. The protection was assessed by determining daily parasitaemias in all animals by examining colored smears with giemsa.
The curves in Figure 5 are representative of the variation in the parasitaemia measured in number of parasitized red cells per microliter of blood (on the y-axis on a logarithmic scale) as a function of time after infection (in days). Curve A corresponds to s mean values observed in the three vaccinated monkeys, curve B;
the mean values in five control monkeys, From the examination of the figure, a very strong reduction in the parasitaemia under the effect of vaccination.

2 () Plasmodium recombinant MSP1 vaccination trial cynomolgi (p42 and p19 ~ in the toque monkey, Macaca sinica.
Fifteen captured monkeys were used as follows: (1) 3 animals injected with 100 µg soluble PcMSP1p42; 3 animals injected with 35, ug (1st injection) or 50 ug (2nd and 3rd injections) PcMSP1p, 9 soluble; ~ 3) 3 animals injected with a mixture of PcMSP1p42 and p, g; (4) 3 animals injections with the adjuvant plus PBS; ~ 5) 3 animals not injected. The adjuvant Freund's complete and incomplete was used according to the protocol described above above, the injections were done intramuscularly at 4 weeks interval. The challenge infection was made by injecting 2.105 red cells infected with Plasmodillm cynomolgi 4 weeks after the last CA 0224 ~ 727 1998-08-0 ~
WO 97/301 ~ 9 PCTIEl ~ 97/00291 ~ 4 injection. Protection was assessed by determining parasitaemias daily in all animals by examining parasitaemias with giemsa. Parasitemas were classified as negative only after counting 400 smear fields. Parasitaemias are s expressed as a percentage of parasitized red blood cells.
Figures 6A-6G are illustrative of the results obtained. ~ years each of them appears parasitaemias (expressed in percentages of parasitized red blood cells on the vertical axis logarithmic ~ observed in test animals according to time after infection (in days on the x-axis).
The results concern:
. in Figure 6A; unvaccinated control animals;
~ Ia 6B relates to animals which had received a solution saline additionally containing Freund's adjuvant;
~ Ia 6C is a superposition of Figures 6A and 6B, for the purpose of show 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 a vaccination 2 () with p42;
FIG. 6E relates to animals vaccinated with only p19;
~ Finally, ia Figure 6F relates to animals vaccinated with a mixture of p19 and p42.
The p42 certainly induces a certain level of protection. But as in 2: ~ shown in Figures 6E and 6F, the protection conferred by p19 recombinant according to the invention is considerably improved.
We can hypothesize that improved protection results from a secondary cleavage of p42 which is accompanied by unveiling of free cysteine which subsequently forms disulfide bridges 3 () intermoleculars giving rise to p19 multimers CA 0224 ~ 727 1998-08-0 ~

~ 5 characteristics of this form in the recombinant proteins of the three species tested.
..
The figures used to draw the graphs (6A-6F) are specified in Figure 6G.

P.cynomolgilsinge toque vaccination trial; second infection test on monkeys vaccinated with p19 alone and controls (Figures 8) Six months later, without further immunization, the 3 monkeys having received 1 () MSP-1 p19 alone with FCA / FIA (Figure 6E) and the 3 monkeys that received a saline solution containing Freund's adjuvant (Figure 6B) as well as 2 new naive unvaccinated monkeys have been re-infected 1.16 red cells infected with Plasmodium cynomolgi. Protection was assessed by determining parasitaemias daily in all animals by examining smears with giemsa. Parasitaemias have been classified as negative only after counting 400 smear fields. Parasitaemias are expressed as a percentage of parasitic red blood cells (the figures used for elaborate the graphs 8A-C are specified in Figure 8D). The six 2 () immune animals which had suffered a challenge infection six months previously had no detectable parasitaemia except for 1 animal in each group that had 0.008% parasitemia for 1 day (Figures 8A and 8B). The two naive controls show a classical parasitaemia with a maximum of 0.8% and for 21 days 2 :) (Figure 8C). So the 3 animals vaccinated with MSP-1 p19 were also protected six months later than the 3 controls which presented a classic full infection after the first challenge infection, despite an absence or a very weak parasitaemia after the first infection - - proof. These results suggest that the duration of protection of p19 is : 3U at least six months.

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2 ~

Vaccination trial with p19 in combination with alum in the P.cynomolgifsinge toque system (Figures 9) The previous positive protection results were obtained in using Freund's complete (FCA) or incomplete (FIA) adjuvant.
However, the only adjuvant currently accepted in humans is alum.
For this reason, we did a vaccination trial with MSP-1 p19 of P. cynomolgi in the toque monkey in the presence of alum as adjuvant. Six captured monkeys were used as follows: (1) 3 animals injected with 4 doses of 50 mg recombinant MSP-1 p19 of P. cynomolgi with 20 mg of alum (2) 3 animals injected 4 times with water physiological and 10 mg of alum. The injections were done by intramuscularly 4 weeks apart. Trial infection was done by injecting 2.105 red cells infected with P. cynomolgi 4 weeks later the last injection. Protection was assessed by determining the daily parasitaemia in all animals by examining smears with the giemsa. Parasitaemias have been classified as negative only after counting 400 smear fields. Parasitaemias are expressed as a percentage of parasitized red cells. The results of 2 () this experiment are the following: 2 of the 3 monkeys immunized with p19 recombinant in alum had about 30 times less total parasitemia for the duration of the infection (Figures 9A and 9B) that the 3 monkeys immune controls with physiological water and alum (Figure 9D) after challenge infection. The third monkey immunized with p19 (Figure 2: ~ 9C) was not much different from the controls. For the vaccination trial of Plasmodi ~ / m cynomolgi p19 in the toque monkey, Macaca sinica, described in Figure 9, the figures used to develop the graphs (9A-9D) are specified (Figure 9E). ~ although these results are slightly less spectacular than the previous ones (Figures 6, 8); it's the first time WO 97/30159 PCT / FR97 / OOZ9l ~ 7 ~ significant protection is observed for recombinant MSP-1 in alum.
-Figure 10: Vaccination test with a recombinant p19 of Plasmodium falciparum in the squirrel monkey.
Twenty Saimiri sciureus guyanensis monkeys (squirrel monkey) about 3 years captive students were used as follows: (~) 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; 2nd injection: 1: 4 FCA / FIA; 3rd injection: FIA. These adjuvant compositions then been mixed with the antigen in PBS 1: 1; (2) 2 control animals received Freund's adjuvant as described for (1) with only PBS; (3) 4 animals injected with 50 mg of Pf MSP-1 p19 soluble in presence of 10 mg of alum (Alu-Gel-S, Serva); (4) 2 control animals i received 10 mg of alum with only PBS; (5) 4 animals injected with approximately 50-100 rng Pf MSP-1 p19 anchored GPI reconstituted in liposomes as follows: 300 mmol of cholesterol and 300 mmol of phosphatidyl choline were vacuum dried and resuspended in 330 mM
N-octylglucoside in PBS with 1.4 mg of Pf MSP-1 p1 9, GPI. This 2 () 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 1st injection was made with fresh liposomes maintained at 4 ~ C and the 2nd and 3rd injections were made with liposomes that have been frozen for preservation; (6) 2 animals injected with control liposomes made in the same way absence of the p19 antigen, GPI, as described for (5); (7) 2 animals injected with physiological water. Three injections were made by intramuscularly 4 weeks apart. Trial infection was made by injecting 1,106 red cells infected with Plasmodium falciparum.

CA 0224 ~ 727 1998-08-0 ~

-~ X

Protection was assessed by determining daily parasitaemias in all animals by examining smears with giemsa. The parasitaemias are expressed as a percentage of parasitized red cells. The results of this vaccination trial are set out in Fi ~ ures 10, A-G.
Groups immunized with p ~ 9 by Freund's adjuvant or liposome have demonstrated parasitaemias similar to the groups controls after a challenge infection (one animal (number 29) vaccinated with p19 in adjuvant of Freund died a few days after infection 1 ~ 3 trial for reasons unrelated to vaccination (crisis irregularities in the administration of the antigen in these 2 groups (bad Freund emulsion, frozen liposomes) do not not fully assess the significance of these results. In the alum group 2 animals showed parasitaemias 13 total for the duration of infection approximately 4 times smaller than the controls, I animal approximately 3 times smaller and 1 animal was similar to controls. This experience is a bit difficult to interpret due to variability in controls, possibly due to strain of parasite used for challenge infection that would not have been enough adapted to the Saimiri non-spenectomized model developed only recently in Cayenne. However, the real, albeit imperfect, effect with alum is encouraging since our antigens seem to be the only ones MSP-1 recombinant versions of P. falciparum which have so far been demonstrated some effectiveness in association with alum.

Vaccination test with a recombinant Plasmodium p19 ~ Alciparum in the squirrel monkey (same test as Figure 10).
Monkeys reared in captivity were injected with 1 ml of inoculum intramuscularly 2 ~ 4 weeks apart as follows: (1) 4 3 (3 animals injected with 50 μg of soluble PfMSP1p19 in the presence CA 0224 ~ 727 l998-08-0 ~
WO 97/3 ~ 159 PCT / FRg7 / 00291 Freund's adjuvant as follows: 1st injection 1: 1 FCA / FIA; 2nd injection 1: 4 FCAIFIA; and subsequently mixed 1: 1 with the antigen in PBS; ~ 2) 4 animals injected with ~ 0 ~ ug of PfMSP1 p19 soluble in presence of 10 mg of alum; (3) 4 animals injected with approximately 50, ug of PfMSP1p19 anchored- GPI reconstituted into 1: 1 compound liposomes molarity of cholesterol and phosphatidyl choline The animals were bled 17 days after the second injection.
Red blood cells from a squirrel monkey with 30%
parasitaemia due to P. falciparum (mostly mature forms) have been washed in PBS and the pellet was diluted 8 times in the presence of 2% SDS and 2% dithiothreitol and heated to 95 ~ before being loaded onto a gel 7.5% polyacrylamide (separation gel) and 4% (stacking gel) (high gel ~. After transferring the immunoblot analysis to nitrocellulose (immunoblot) was done with antisera as follows: (1) pool :. antisera of 4 monkeys vaccinated with soluble PfMSP1p19 by adjuvant of Freund diluted to the twentieth; (2 ~ antisera pool of 4 monkeys vaccinated with PfMSP1 p1 9 soluble by adding alum diluted to the twentieth; (3) pool antisera of 4 monkeys vaccinated with PfMSP1 p19 anchor in liposomes diluted to the twentieth; (4 ~ the monoclonal antibody, which reacts with an epitope 2 () linear from PfMSP1p19, at ~ 0 ~ ug / ml; (5) pool of SHI90 antisera from about twenty monkeys repeatedly infected with P. falciparum and become resistant to any subsequent infection of P. falciparum, diluted five-fold hundredth; (6) antisera pool of naive monkeys (never exposed to P falciparum) diluted to the twentieth.
2 :) The results show that the 3 pools of antisera of monkeys vaccinated with PfMSP1p19 react significantly and specifically with very high molecular weight complexes (so diffuse in stacking gel) and present in parasite extracts ~ - containing more ripe forms. These results confirm ~) The hypothesis of the presence of a specific aggregation of MSP1p19 in 3 () vivo with epitopes that are reproduced in molecules PfMSP1p1 ~ recombinants synthesized in the baculovirus system, especially those in the form of an oligomer.

s Figure 7 also illustrates these results. It relates to immunoblots produced on gel. The first three columns of the gel illustrate the in vivo response of monkeys to injections of p19 [(1) with Freund's adjuvant, (2) with alum, (3) in the form of liposome3 and in particular the existence of high molecular weight complexes comforting The hypothesis of in vivo aggregation of p19 in the form of an oligomer, specific to the maturation stage (when p42 is cut into p19 and p33).
This vaccination trial also includes a third identical injection to the previous ones. Injection with Freund's adjuvant includes only FIA.

Figure 7B: The data for this figure is derived from the test falciparum / squirrel monkey vaccination procedure (Figure 10 below ~.
figures correspond to the individual monkeys noted in Figure 10. The techniques and methods for this figure are the same as for the 2 () Figure 7 except that the individual antiserum of each monkey noted has been tested after three injections on the day of challenge infection and the SHI antiserum was been 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 other groups vaccinated with p19 and Freund's adjuvant or liposomes show only weak 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, despite the fact that a monkey in the alum group was not protected from controls and another was not was only partially.

The invention naturally relates to other applications, for example - example those set out below in relation to some of the examples, which are in no way limiting.

Therapeutic The recombinant molecules according to the invention can be used to produce specific antibodies that can be used by passive transfer for the purpose of therapy adapted to malaria l ~ t severe due to P. falc; parum with risk of mortality.
Diagnosis The recombinant molecules PvMSP1p42 and PvMSP1p19 and according to the invention, baculovirus derivatives can and have been used to produce murine specific monoclonal antibodies. These antibodies, in s combination with polyclonal anti p42 antisera from another species such as rabbit or goat, can be the basis of a semi-quantitative diagnosis for malaria and able to distinguish between malaria due to P. falciparum, which can be fatal, and malaria due to P. vivax, which is generally not fatal. The principle of this test would be to 2 () trap and quantify any MSP1 molecule containing the p42 part in the blood.
In this context, the advantages of recombinant p42, and particularly partially deleted recombinant p42, are the following:
(i) they are both well preserved within the same species and sufficiently divergent between different species to allow easily produce species-specific reagents in conditions whereby derived antibodies can be produced different Plasmodiums, especially against P.falciparum and P.vivax which 3 () do not give rise to cross reactions;

CA 0224 ~ 727 1998-08-0 ~
WO 97/301 ~; 9 PCT / FR97 / 00291 3 ~

(ii) since the recombinant p42 molecules derived from baculoviruses seem to reproduce more the native structure of corresponding native proteins, the antibodies produced against these proteins would be well suited for diagnostic use.
The microorganisms identified below were deposited as follows rule 6.1. of the Budapest Treaty on February 1, 1996, under the following numbers:

Identification references Registration numbers PvMS P 1 p19A I - 1659 PvMSP1 p19S I - 1660 PfMSP1 p19A I - 1661 PfMSP1 p19S I - 1662 PcMSP1p19S I - 1663 1 ~
The invention also relates to the use of these antibodies, so preferably fixed beforehand on a solid support (for example for affinity chromatography), for the purification of p19 type peptides initially contained in a mixture.
2 () The purification then involves bringing this mixing with the antibody, dissociation of the antigen-antibody complex and recovering the purified p19 peptide.
When it is said in the following claims that p42 is deleted from some or less well preserved parts of the region 2: ~ III, these are preferably regions having at least 10 amino acids and whose degree of conservation in P.vivax, P.cynomolgi and P.falciparum is less than 70% (less than seven identical amino acids out of 10, when aligned.
The polyclonal and monoclonal antibodies of the present invention 3 () presented as recognizing p42, are preferably those which more specifically recognize regions other than region IV, except CA 022457i7 1998-08-OS

The exclusion of region IV itself Preferably they recognize the region I of p42.
The invention also relates to secretory hybridomas of specific antibodies selectively recognizing the p42 of a protein s MSP-1 of the merozoite form of an infectious Plasmodium parasite for humans other than Plasmodium vivax and does not recognize Plasmodium vivax.
In particular, these hybridomas secrete antibodies which do not not recognize p42 of Plasmodium vivax or which recognize o specifically p42 of Plasmodium falciparum.
The invention also relates to a hybridoma characterized in that that it produces a monoclonal antibody that specifically recognizes p42 P. vivax and P. cynomolgi. An F10-3 hybridoma was constructed from of myeloma X63 Ag8 653 producing IgG 2b / k recognizing p42 of P vivax.

The invention also relates to vaccine compositions, also comprising mixtures of proteins or fragments, in particular mixtures of the type:
2 () - 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 the compositions which precede p42 is the case lacking its most hypervariable regions.
For example, its region which corresponds to that of the p19 fragment normally included in p42, is itself partially deleted, this CA 0224 ~ 727 1998-08-0 ~

3 ~

region comprising at least one of the two EGF regions normally contained in this p19.
Or it is devoid of the region ll, 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 equally to the production of vaccine compositions veterinarian using the corresponding proteins or antigens derived from infectious parasites for mammals and produced in same conditions. It is indeed known that infections of the same type, Babesiosis, also occurs in cattle, canines and horses.
one of the Babesia species' antigens has strong homology conformational (notably the two domains ~ EFG-like ~ and wealth in cysteine) 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 p42 used in these mixtures may also give rise to all the modifications question in the above, when taken into consideration 2 () in isolation.

CA 0224 ~ 727 1998-08-0 ~

REFERENCES:
~ 1) Holder, JA et al. (1982) ~ Biosynthesis and processing of a Plasmodium falciparum schizont antigen recognized by immune serum and a monocional antibody ~. J. Exp. Med. 156: 1528-1538.
(2) Howard, R. et al. (1984) ~ Localization of the major Plasmodium falciparum glycoprotein on the surface of mature intracellular trophozoites and schizonts ~. Mol. Biochem. Parasitol. 11: 349-362.
(3) Pirson ~ P. et al. (1985) ~ Characterization with monoclonal antibodies of a surface antigen of Plasmodium falciparum merozoïtes ~. J. Immunol. 134 1946-1951.

(4) Aley, SB et al. (1987) ~ Plasmodium vivax: Exoerythrocytic schizonts recognized by monoclonal antibodies against blood-stage schizonts ~. Exp.
Parasitol. 64: 188-194.
I ~ (5) Holder, AA (1988) ~ The precursor to major merozoïte surface antigen St! Llf-tU! E and! O! E in immunity ~. Prog. A !! ergy 41: 72-97.
~ 6) Cooper, JA (1993) ~ Merozoïte surface antigen-1 of Plasmodium ~.
Parasitol. Today 9: 50-54.
(7) Holder, AA, et al. (1987) ~ Processing of the precursor to the major 2 (~ merozoite antigens of Plasmodium falciparum ~ Parasitology 94: 199-208.
(8) Lyon, JA et al. (1986) ~ Epitope map and processing scheme for the 195 000-dalton surface glycoprotein of Plasmodium falciparum merozoïtes deduced from cloned overlapping segments of the gene ~. Proc. Natl. Acad.
Sci. USA 83: 2989-2993.
2 ~ (9) Blackman, MJ et al. (1992) ~ Secondary processing of the Plasmodium falciparum merozoïte surface protein-1 (MSP1) by calcium-dependent membrane-bound serine protease: shedding of MSP133 as a noncovalently associated complex with other fragments of the MSOP1 ~. Mol. Biochem.
Parasitol. 50: 307-316.
~ 10) Haldar, K., et al. ~ 1985) ~ Acylation of a Plasmodium falciparum merozoïte surface antigen ~, ia sn-1,2-diacyl glycerol. )). J. Biol. Chem. 260: 4969-4974.

Wo 97/30159 PCT / FR97 / 00291 (11) 8raun Breton, C. et al. (1990) <(Glycolipid anchorage of Pfasmodium falciparum surface antigens ~. Res. Immunol. 141: 743-755.
(12) Kumar, S. et al. (1995) ~ Immunogenicity and in vivo Efficacy of Recombinant Plasmodium falciparum Merozolte surface protein-1 in Aotus Monkeys ~. Molecular Medicine, Vol. 1, 3: 325-332.
(1 ~ '.) Longacre, S. et al. (1994) ~ Plasmodium vivax merozolte surface protein 1C-terminal recombinant proteins in baculovirus ~. Mol. Biochem. Parasitol.
64: 191-205.
(15) McBride, JS et al. (1987) ~ Fragments of the polymorphic Mr 185,000 glycoprotein from the surface of isolated Plasmodium falciparum merozoites form an antigenic complex ~. Me. Biochem. Parasitol. 23: 71-84.
(16) Blackman1 MJ et al. (1990) ~ A single fragment of a malaria merozoïtesurface protein remains on the parasite during red cell invasion and is the the ~ target of invasion-inhibiting antibodies ~. J. Exp. Med. 172: 379-382.
(17) Kaslow, DC et al. (1994) ~ Expression and antigenicity of Plasmodium falciparum major merozoïte surface protein (MSP119) variants secreted from Saccharomyces cerevisiae ~. Mol. biochem. Parasitol. 63; 283-289.
(18) Chang, SP, et al. (1992) ~ A carboxyl-terminal fragment of Plasmodium , 0 falciparum gp195 expressed by a recombinant baculovirus induces antibodies that completely inhibit parasite growth ~. J. Immunol. 149: 548-555.
(19) Longacre, S. (19g5) ~ The P ~ asmodium cynomolgi merozoite surface protein 1 C-terminal sequence and its homologies with other Plasmodium 23 species ~. Mol. Biochem. Parasitol. 74: 105-111.
(20) Del Portillo, HA, et al. (1990) ~ Primary structure of the merozolte surface antigen 1 of Plasmodium vivax reveals sequences conserved between different Plasmodium species ~. Proc. Natl. Acad. Sci. USA 88: 4030-4034.
(21) Gibson, HL, et al. (1992) ~ Structure and expression of the gene for Pv200, a major blood-sta ~ e surface antigen of P / asmodium vivax ~. Mol.
biochem. Parasitol. 50: 325-334.

CA 02245727 I sss - os - 05 wo 97/30159 PCT ~ R97100291 (22) Dissanaike, AS, et al. (1965) ~ Two new malaria parasites, Plasmodium cynomolgi ceylonensis sub sp. nov. and Plasmodium fragile sp. Nov from monkeys in Ceylon ~. Ceylon Journal of A / ledical Science 14: 1-9.
(23) Cochrane, AH, et al. (1986) ~ Further studies on the antigenic diversity of the circumsporozolte proteins of the Plasmodium cynomolgi complex. ~.
Am. J. Trop. Med. Hyg. 35: 479-487.
(24) Naotunne, T. de S., et al. (1990) ~ Plasmodium cynomolgi: serum-mediated biocking and enhancement of infectivity to mosquitos during infections in the natural host, Macaca sinica ~. Exp Parasitol. 71, 305-313.
(25) Ihalamulla, RL et al. (1987) ~ Plasmodium vivax: isolation of mature asexual stages and gametocytes from infected human blood by colloidal silica (Percoll) centrifugation gradient)>. Trans. R. Soc. Too much. Med. Hyg. 81:
25-28.
(26) Kimura, E., et al. (1990) ~ Genetic diversity in the major merozoite surface antigen of Plasmodium falciparum: high prevalence of a third polymorphic form detected in strains derived in strains derived from malaria patients ~.
Gene 91: 57-62.
(27) Heidrich, H.-G., et al. (1989) ~ The N-terminal amino acid sequences of thePlasmodium Falciparum (FCBI) merozoïte surface antigen of 42 and 36 2 () kilodalton, both derived from the 185-195 kilodalton precursor)). Mol.
Biochem. Parasitol. 34: 147-154.
(28) Blackman, MJ ~ et al. (1991) ~ Proteolytic processing of the Plasmodium falciparum merozo ~ te surface protein-1 produces a membrane-bound fragment containing two epidermal growth factor-like domains ~. Mol.
Biochem. Parasitol. 49: 29-34.
(29) Adams, JM et al. (1992) ~ A family of erythrocyte binding proteins of malaria parasites ~. Proc. Natl. Acad. Sci, 89: 7085-7089.
~ 30) Sim BKL (1995) ~ EBA-175: An erythrocyte-binding ligand of Plasmodium falciparum ~. Parasitology Today, vol.ll, n ~ 6: 213-217.
3 () (31) Sim BKL (1994) ~ Receptor and ligand domains for invasion of erythrocytes by Plasmodium falciparum ~. Science, 264: 1941-1944.

CA 0224s727 I sss - bone - bone wo 97/30159 PCT / ER97 / 00291 3x (32) Davies, A. et al. (1g93), Biochem J. 29 ~ (Pt3): 88g-896. ~ Expression of the glycosylphosphatidylinositol-linked complement-inhibiting protein CD59 antigen in insect cells using a baculovirus vector ~.
(33) Haziot A. et al. (1994) J. Immunol 152: 5868. ~ Soluble recombinant CD14 Inhibits LPS-lndùced Tumor Necrosis Factor & Production by Cells in Whole Blood ~.
(34) Chang, SP et al., (1988) ~ Plasmodium falciparum: gene structure and hydropathy profile of the major merozoite surface antigen (gp195) of the Uganda-Palo Alto isolate. Exp. Parasitol. 67 1-11.
o ~ 35) Holder, AS et al. (1985) ~ Primary structure of the precursor to the three major surface antigens of Plasmodium falciparum merozoites, Nature 317:
270-273.
(36) G. Bourdoiseau et al. (May 19g5) ~ Bovine babesiosis ~, Le point veterinarian, vol. 27, n ~ 168.
IS (37) P. Bourdeau et al. (May 1995) ~ Canine babesiosis a Babesia canis ~, Le veterinary point, vol. 27, n ~ 168.
(38) C. Soulé (May 1995) ~ Equine babesiosis ~, Le point veterinaire, vol.27, n ~ 168.
(39) TPM Schetters et al. (1995) <~ Vaccines against Babesiosis using Soluble2 () Parasite Antigens ~, Parasitology Today, vol.11, n ~ 12.
~ 40) PA Burghaus et al. (1996) ~ Immunization of Aotus nancymai with Recombinant C Terminus of Plasmodium fafciparum Merozoite Surface Protein 1 in Liposomes and Alun Adjuvant Does Not Induce Protection against a Challenge Infection ~, Infection and Immunity, 64: 3614-3619.
2s (41) SP Chang, et al. (1996) ~ A Recombinant Baculovirus 42-Kilodalton C-Terminal Fragment of Plasmodium falciparum Merozoite Surface Protein 1 Protects Aotus Monkeys against Malaria ~, Infection and Immunity, 64: 253-~ 42) LH Miller et al. (1997) ~ The Need for Assays Predictive of Protection in. (~ Development of Malaria Bloodstage Vaccines ~, Parasitology Today, vol.13, n ~ 2: 46-47.

Claims (39)

1. Recombinant protein whose polypeptide sequence essential constituent is that:
~ or a C-terminal fragment of 42 kilodaltons (p42) of protein 1 of surface (MSP-1 protein) of the merozoite form of a parasite of the type Plasmodium and infectious for humans, and from which the Region II and, where applicable, part or parts of Region III, in especially the less well-preserved parts;
~ or part of this fragment when it is also able to inhibit parasitaemia normally induced in vivo by the parasite corresponding;
~ or a peptide capable of inducing an immunological response of the type cellular and/or humoral equivalent to that produced by this fragment p42 or the aforesaid part of this fragment, and this recombinant protein comprising, where appropriate, epitopes unstable conformations in a reducing medium and constituting the majority epitopes recognized by human antisera raised against the corresponding Plasmodium. 2, Recombinant protein according to claim 1, characterized in that its region which corresponds to that of the p19 fragment 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 p19 fragment 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 from region I and region IV of p42 and that it is devoid of region II. 5. Recombinant protein according to claim 4, characterized in that its polypeptide sequence is that of p42 from which was deleted region II. 6. Recombinant protein according to claim 5, of which a also deleted the N-terminal region of region III or all of region III. 7. Recombinant protein according to any of claims 1 to 6, characterized in that it also comprises a glycosylphosphatidylinositol (GPI) group of the type that allows an anchor of the p19 fragment whose sequence is normally understood in that of p42 to the cellular host, in particular a eukaryotic cell, preferentially an insect cell infectable by a baculovirus, in which said recombinant protein is expressed. 8. Recombinant protein according to any of claims 1 to 7, characterized in that it lacks the part Extreme hydrophobic C-terminal which is involved 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 cell eukaryote, 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 of claims 1 to 9, characterized in that it contains the sequence aforesaid partially deleted p42 of the MSP-1 protein of Plasmodium falciparum or said part of the corresponding fragment. 11. Recombinant protein according to any of claims 1 to 9, characterized in that it contains the sequence aforesaid partially deleted p42 of the MSP-1 protein of Plasmodium cynomolgi or said part of the corresponding fragment. 12. Recombinant protein according to any of claims 1 to 9, characterized in that it contains the sequence aforesaid partially deleted p42 of the MSP-1 protein of Plasmodium vivax or said part of the corresponding fragment. 13. Recombinant protein according to any of claims 1 to 12, characterized in that it is combined with a carrier molecule usable for the production of vaccines. 14. Vaccinating composition against a parasite of the type Plasmodium infectious for humans, containing as active ingredient 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 type Plasmodium infective to humans other than Plasmodium vivax and not does not recognize Plasmodium vivax. 16. Specific antibody according to claim 15, characterized in what it does not recognize Plasmodium vivax. 17. Specific antibody according to claim 15, characterized in that it specifically recognizes p42 of P.falciparum. 18. Specific antibody according to claim 15, characterized in what it specifically recognizes is p42 of P.vivax. 19. Differential diagnostic procedure for distinguishing between a parasitic infection due to P vivax, on the one hand, and an infection parasitic due to another plasmodium, on the other hand, characterized by the bringing into contact a biological sample infected with a plasmodium with an antibody according to claim 18, on the one hand, and with a antibody according to claim 16 or 17, on the other hand, and the detection depending on the case, 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 to be 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 of which at least a part codes for the peptide sequence:
~ either a C-terminal peptide fragment of 42 kilodaltons (p42) of the surface protein 1 (MSP-1 protein) of the merozoite form of a parasite of the Plasmodium type, infectious for humans, and of which, the case where appropriate, region II has been deleted and, where appropriate, one or more parts of Region III, especially the less well-developed parts preserved thereof;
~ or part of this peptide fragment when the product expression of the second sequence in a baculovirus system is also capable of inhibiting a parasitaemia normally induced in vivo by the corresponding parasite;
~ or a peptide capable of inducing an immunological response of the type cellular and/or humoral equivalent to that produced by this fragment peptide p42 or to the aforesaid part of this fragment, and said nucleotide sequence further having a G and C content between 40 and 60%, preferably at least 50% of all of the nucleotides of which it is made. 21. Modified vector according to claim 20, characterized in that that the aforesaid second polypeptide sequence is consistent with that defined in any one of claims 2 to 9. 22. Modified vector according to claim 20, characterized in that that the second nucleotide sequence is a synthetic sequence. 23. Modified vector according to any one of the 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 the claims 20 to 23, characterized in that the second nucleotide sequence is devoid at its 3' terminal end of the end sequence C-terminal hydrophobic which is involved 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 an insect cell infectable by a baculovirus. 25. Modified vector according to any one of the claims 20 to 24, characterized in that it consists of a modified baculovirus. 26. Organism, especially insect cell of the Sf9 type, transfectable and transfected with the modified vector according to any of claims 20 to 24. 27. Synthetic DNA containing a first sequence nucleotide of which at least a part codes for the peptide sequence:
~ either a C-terminal peptide fragment of 42 kilodaltons (p42) of the surface protein 1 (MSP-1 protein) of the merozoite form Plasmodium falciparum, infectious for humans and of which, where appropriate, have been deleted region II and, where appropriate also, one or more parts of region III, in particular the less well preserved parts of these;
~ or part of this peptide fragment when the product of expression of this DNA in a baculovirus system is capable of inhibiting parasitaemia normally induced in vivo by the parasite corresponding;
~ or a peptide capable of inducing an immunological response of the type cellular and/or humoral equivalent to that produced by this fragment peptide p42 or to the aforesaid part of this fragment, and said nucleotide sequence further having a content of G nucleotides and C between 40 and 60%, preferably at least 50% of the total of nucleotides of which the above synthetic DNA is made. 28. Synthetic DNA according to claim 27, characterized in that that its first nucleotide sequence is lacking at its 3' end terminal of the coding sequence for the C-terminal end region hydrophobic normally involved in the induction of the anchorage of the p19 protein whose sequence is included in that of p42, at the cell membrane of the host in which it is expressed, in particular in an insect cell infectable by a baculovirus. 29. Synthetic DNA according to claim 27 or claim 28, characterized in that the first sequence nucleotide is preceded by a signal nucleotide sequence encoding for a signal peptide normally associated with an MSP-1 protein of Plasmodium, homologous or heterologous with respect to the sequence main. 30. Synthetic DNA according to claim 29, characterized in that that the signal sequence is from P.vivax. 31. Synthetic DNA according to any one of the claims 27 to 30, characterized in that the aforesaid first nucleotide sequence includes a 3'-terminal sequence encoding a polypeptide region anchoring to the cell membrane, said anchoring region attaching to the recombinant protein expressed on the surface of the host membrane cell transformed with a vector containing said synthetic DNA, said 3' sequence being homologous to that of the nucleotide sequence main, or heterologous, in particular that derived from P.vivax. 32. Synthetic DNA according to claim 31, characterized in that that the 3'-terminal sequence is from P.vivax. 33. Synthetic DNA according to any one of the claims 27 to 31, characterized in that it lacks said sequence 3'-terminal. 34. Hybridoma secreting monoclonal antibodies having the specificities of the antibodies according to any one of claims 15 to 18. 35. Method for separating a specificity p42 peptide given from a mixture of peptides, characterized by the setting contact of this mixture of peptides with a corresponding antibody, according to any one of claims 15 to 18, preferably previously fixed on an insoluble support, by the subsequent dissociation of the antigen-antibody compound formed and by the recovery of the peptide p42 purified. 36. Use of the protein according to any of claims 1 to 12 for the preparation of an immunogenic composition capable of inducing an immune response against infection with Plasmodium. 37. Vaccine composition comprising as active principles a mixture of a protein conforming to any of claims 1 to 13 and either of the corresponding p19 or of another recombinant protein, p42 or p19 type, from a parasite homologous to that from which the aforesaid protein is derived. 38. Vaccine composition according to claim 37 characterized in that its mixture of active principles is chosen from the mixtures next:
- 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, where appropriate, devoid of its strongest regions hypervariable. 39. Hybridoma according to claim 34, characterized in that it has was filed with the CNCM under number 1-1846, on February 14, 1997.