HK1140229A - Vaccine composition comprising a mixture of a recombinant protein containing a c-terminal p19 fragment of the msp-1 protein from plasmodium falciparum and another recombinant p19 protein from a plasmodium homologous to p. falciparum - Google Patents

Description

The invention relates to novel active ingredients of vaccines derived from the major surface protein of Merozoite forms of a Plasmodium infectious to mammals, including humans, more commonly known as MSP-1.

This protein has been extensively studied, is synthesized in the schizont stage of Plasmodium parasites, notably Plasmodium falciparum, and is expressed as a major constituent of the surface of the mesozoite during both the hepatic and erythrocytic stages of malaria (1, 2, 3, 4). Due to the predominance and conservation of this protein in all known Plasmodium species, it has been suggested that it may be a candidate for the development of malaria vaccines (5, 6).

The same was true for fragments of this protein, particularly natural cleavage products which are observed to form, for example during the parasite invasion of the red blood cells of the infected host. e cleavage products include the C-terminal fragment with a molecular weight of 42 kDa (7.8), which is in turn cleaved again into an N-terminal fragment with a conventional apparent molecular weight of 33 kDa and a C-terminal fragment with a conventional apparent molecular weight of 19 kDa (9) which normally remains attached to the parasite membrane after the modifications to which it is subject, by means of glycosylphosphatidylinositol (GPI) (10, 11) groups.

It is still found in the early ring stage of the intraerythrocyte development cycle (15, 16), hence the observations that this 19 kDa fragment may play an unknown but probably essential role in re-invasive processes, which has led to the assumptions already made in the past that this protein may be a particularly effective target for future vaccines.

It will be understood that the frequent references in the following to p42 and p19 proteins from a certain type of Plasmodium are understood to refer to the corresponding C-terminal cleavage products of the MSP-1 protein of that Plasmodium, or by extension to products containing substantially the same amino acid sequences, obtained by genetic recombination or chemical synthesis using conventional techniques, e.g. Applied System type synthesizer or Merrifield type solid phase synthesis. For the sake of convenience, references to recombinant p42 and 19 p are obtained from 42 and p 19 in relation to techniques involving at least one step of genetic engineering.

Due to the difficulty of obtaining large quantities of P.falciparum parasites and the impossibility of growing P.vivax in vitro, it became clear that the only way to produce an anti-malarial vaccine required the use of techniques that allow the use of recombinant peptides or proteins.

The recombinant proteins for the C-terminal portion of P.falciparum MSP-1 produced and tested in monkeys (12, 40, 41) include: a p19 fused with a glutathione S-transferase produced in E. coli (40), a p42 fused with a glutathione S-transferase produced in E. coli (12), a p19 fused with a tetanus anatoxin polypeptide carrier of T-helper epitopes produced in S. cerevisiae (12), a p42 produced in a baculovirus system (41).

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

A composition containing the p42 fusion protein with a glutathione-S-transferase produced in E.coli in combination with a complete Freund adjuvant did not have a protective effect in the two Aotus monkey types (A.nancymai and A.vociferans) to which it was administered.

Some researchers (Chang et al.) have also reported immunisation trials in rabbits with a recombinant p42 protein produced in a baculovirus system and containing an amino acid sequence in common with P.falciparum (18). The latter authors report that this recombinant p42 behaves in rabbits in a substantially similar manner to the whole recombinant MSP-1 protein (gp195). This p42 protein in combination with a complete Freund adjuvant was tested in a non-human primate susceptible to infection by P.falciparum., A. grisaeus, A. lemurinemembra (40).Although the antibodies produced by the test were similar to the controls, they had a longer latency period. It is however risky to conclude that the antibodies thus induced are protective against the parasites themselves in humans. It should be noted that there are currently no very satisfactory experimental models 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.falciparum, are artificial systems, requiring adaptation of parasite strains and often splenectomy of animals to obtain significant parasites. Consequently, the vaccination results of these models may have only limited predictive value for humans.

In any case, the question arises as to what the actual vaccination rate would be with such recombinant proteins, given the finding - reported below - that the presence of hypervariable regions in the p42 from Plasmodiums of the same species, and more particularly in the corresponding p33s, would in many cases make the immunoprotective efficacy of antibodies induced in persons vaccinated with a p42 from a Plasmodium strain against infection by other strains of the same species random (13).

It can even be assumed that the high N-terminal polymorphism of p42 plays a significant role in immune escape, often seen in this type of parasite.

The purpose of the present invention is to produce recombinant vaccine proteins that escape these difficulties and whose protective effect can be verified in truly significant experimental models, or even directly in humans.

The invention relates in particular to vaccine compositions against a human infectious Plasmodium parasite, containing as active substance a recombinant glycosylated protein or not, the essential constitutive polypeptide sequence of which is: a 19 kilodalton (p19) C-terminal fragment of the surface protein 1 (MSP-1 protein) of the merozoite form of a human infectious Plasmodium parasite, which C-terminal fragment normally remains anchored on the surface of the parasite at the end of its phase of penetration into human red blood cells during an infectious cycle, or a part of this fragment, if it is also capable of inducing an immune response capable of inhibiting a parasitic response in vivo to the corresponding parasite; or a peptide immunologically equivalent to this p19 fragment or the above part of this fragment; and that recombinant protein, which also contains unstable conformational epitopes in a reducing medium and preferably constitutes the majority of epitopes recognised by human antiserums formed against the corresponding Plasmodium.

The presence of these conformational epitopes could play an important role in the protective efficacy of the active substance of vaccines. They are particularly found in active substances which also have the other characteristics defined above when produced in a baculovirus vector system. If necessary, it is mentioned below that the term baculovirus vector system means the whole of the type-vector itself and the cell lines, including insect cells which can be transfected by a baculovirus site modified by a sequence to be transfected into these baculovirus ligands with the resulting expression of preferred sequence transferred. These cells from two baculovirus partners, which have been described in this article, have been used in the same way as the baculovirus system itself. The following are examples of cells which can be transfected by the same system and which have been used in the same location.

In particular, that recombinant protein is recognised by human antiserums formed against the corresponding Plasmodium or against a homologous Plasmodium when it is in the unreduced state or in a non-irreversible reduced state, but not recognised or poorly recognised by those same antiserums when it is irreversibly reduced.

The instability of these conformational epitopes in the reducing medium can be demonstrated by the test described in the examples below, in particular in the presence of β-mercaptoethanol.

From this point of view, the recombinant protein produced by S. Longacre et al. (14) can be used in such formulations. It is recalled that S. Longacre et al. succeeded in producing a recombinant p19 from P. vivax MSP-1 in a baculovirus vector system containing a nucleotide sequence coding for Plasmodium vivax p19, in particular by transfection of insect cell cultures [Spodoptera frugiperda lineage (Sf9) ] with vector baculoviruses containing, under the control of the polyhedrin promoter, a sequence coding for the following peptide fragments defined below, the sequences of which were placed in the order in which the baculovirus was used: a length of not more than 10 mm,which had mutated (in ATT) the methionine codon initiating expression of this protein;a 5'-terminal nucleotide fragment coding for a 32-amino acid peptide corresponding to the N-terminal part of MSP-1, including the MSP-1 signal peptide;either a nucleotide sequence coding for p19 or a sequence coding for p42 of the MSP-1 protein of Plasmodium vivax, these sequences also being, as appropriate, or provided with (anchor forms ),

For p42, the sequences derived from the C-terminal region of MSP-1 therefore extended from the amino acid Asp 1325 to the amino acid Leu 1726 (anchored form) or Ser 1705 (soluble form) and for p19, the sequences extended from the amino acid Ile 1602 to the amino acid Leu 1726 (anchored form) or Ser 1705 (soluble form), it being understood that the complete amino acid sequences of p42 and p19 with the terminal initial amino acids indicated above derive from the P. Belem gene isolate which was sequenced (20).

Similar results were obtained by implementing in the same vector systems the nucleotide sequences coding for Plasmodium cynomolgi p42 and p19 P.cynomolgi has a double interest: it is a parasitic species very close to P.vivax that is infectious to macaques. It can also infect humans Natural hosts of P.cynomolgi, rhesus monkeys and toad monkeys, are also available to test the protective effectiveness of P.cynomolgi MSP-1 in natural systems. The rhesus monkey is considered one of the most representative species of immune responses in humans.

In particular, excellent results were obtained in vaccination trials in monkeys with two recombinant polypeptides p42 and, most importantly, P. cynomolgi-derived, soluble p19 soluble, produced in a baculovirus system and purified on an affinity column with monoclonal antibodies that recognize the corresponding regions of the native MSP-1 protein. The following observations were made: the six monkeys immunized with p19 alone (three monkeys) and p19 and p42 together (3 monkeys) all showed almost sterile immunity after test infection. The results in the three monkeys immunized with p42 were significant. Two of them behaved as the previous ones, but no less than three monkeys with P42 were immunocompromised (3 monkeys) but no less than one monkey with P42 was immunocompromised (3 monkeys) but no less than one monkey with Freundemia (3 monkeys) showed significant immunocompromised (3 monkeys) if they were not tested.

A second test infection showing that monkeys given p19 alone were protected for at least six months. A second test of p19 vaccination in combination with alum in this system (P.cynomolgi/toque monkey) demonstrated significant protection for 2 of the 3 monkeys. This is the first time that MSP-1 or another recombinant antigen has demonstrated a protective effect in the presence of alum (42).

The results of particularly effective tests in macaques with recombinant polypeptides produced in a baculovirus system using a recombinant p19 from P.cynomolgi show that recombinant polypeptides containing recombinant p19 from other Plasmodiums respectively must behave in the same way. They are more prominent for malaria in humans than the results of tests with P.vivax or P.falciparum in their artificial hosts .

Recombinant baculovirus proteins derived from a C-terminal portion of MSP-1 (p19) have a very significant protective anti-malarial effect in a natural system, which is the most representative model for assessing the protective effect of MSP-1 in humans.

The protective effect obtained could be even better as the p19 form lacks the hypervariable region of the N-terminal part of p42, the effect of which may be deleterious in natural situations where the vaccinated subject is exposed to significant polymorphism.

The 19 kDa C-terminal fragment with a sequence present in the active substance of the vaccine may be limited to the p19 sequence itself, in the absence of any polypeptide sequence normally upstream of the p19 sequence in the corresponding MSP-1 protein. However, it is clear that the essential constitutive polypeptide sequence of the active substance may still contain a polypeptide sequence on the C-terminal side belonging to the 33 kDa N-terminal fragment (p33) still associated with p19 in the corresponding p42 before natural cleavage of the latter, and whenever the presence of this fragment is not such as to alter the immunological properties of the active substance of the vaccine.As will be seen below, particularly with regard to the description of the examples, the C-terminal sequences of p33 from different strains of the same Plasmodium species (see C-terminal part of the peptide sequences from region III in Figure 4) also show a substantial degree of sequence homology or conservation, for example of at least 80% in different strains of Plasmodiums infectious to humans, so that they are not likely to fundamentally alter the vaccinating properties of the active substance (the sequence of which corresponds to region IV) in Figure 4.In particular, in the case of this figure, the presumed cleavage site between p19 and p33 region III is between the leucine and asparagine residues in a particularly well-conserved region (LNVQTQ).

Normally the C-terminal polypeptide sequence of p33, when present, contains less than 50 amino acid residues, or even less than 35, or even less than 10 amino acid residues.

Conversely, the essential constitutive polypeptide sequence of the active substance of a vaccine may not contain the entire sequence coding for p19, naturally provided that p19 retains the ability to induce protective antibodies against the parasite. In particular, the above fragment part has a molecular weight of 10 to 25 kDa, including 10 to 15 kDa. Preferably, this polypeptide fragment part contains at least one of the two EGF regions (abbreviation of Epidermal Growth Factor ).

It is clear that the professional is able to distinguish between active fragments and those which would cease to be active, in particular experimentally by producing modified vectors containing inserts from p19 of different lengths, respectively isolated from fragments obtained from the p19 coding sequence, by reaction with appropriate restriction enzymes or by exonucleolytic enzymes which would have been kept in contact with the p19 coding fragment for varying periods of time; the ability of the products of expression of these inserts in corresponding eukaryotic cells, insect cells, transformed by the modified vectors, to exert a protective effect, which can then be described, in particular in the experimental conditions which will be further from the p19 coding fragment; in particular, in vitro, the ability of the products of expression of these inserts in corresponding eukaryotic cells, insect cells, transformed by the modified vectors, to exert a protective effect, which can then be described, in particular in the experimental conditions which will be more closely related to the parasite, in particular in vitro, should be tested for examples of parasite inhibition.

The invention also covers any vaccine composition in which the essential constitutive polypeptide sequence of the active substance is a peptide capable of inducing a cellular and/or humoral immune response equivalent to that produced by p19 or the fragment as defined, provided that the addition, deletion or substitution in its sequence of certain amino acids by others would not result in a significant change in the ability of the modified peptide - hereinafter referred to as immunologically equivalent peptide - to also inhibit the above parasitemia.

The p19 fragment can also naturally be associated either on the N-terminal or C-terminal side or via peptide binding to another plasmodial protein fragment with vaccinating potential such as a protein ( Duffy Binding Protein from P. vivax (29) or EBA-175 from P. falciparum (30) and (31) with a specifically cysteine-rich region), provided that its ability to inhibit a parasite normally introduced in vivo by the corresponding parasite is not altered but instead amplified.

The fragment encoding p19 or part of it may also contain, upstream of the N-terminal of p19, a peptide sequence that is even different, for example to a C-terminal fragment of the signal peptide used, such as that of the MSP-1 protein. This sequence preferably contains less than 50 amino acids, for example 10 to 40 amino acids.

These observations also extend to p19 from other Plasmodiums, in particular P.falciparum, the dominant species of parasite responsible for one of the most serious forms of malaria.

However, the techniques mentioned above for the production in a baculovirus system of a recombinant p19 derived from P.vivax or P.cynomolgi are difficult to transpose as they are for the production of a recombinant p19 from P. falciparum with satisfactory yield, if only to obtain appreciable quantities to permit immunoprotection tests.

The invention also provides a method to remedy this difficulty to a large extent. It also becomes possible to obtain much higher yields in P.falciparum p19 - and other Plasmodiums when similar difficulties are encountered - by implementing a synthetic substitute nucleotide sequence for the natural nucleotide sequence coding for Plasmodium falciparum p19 in a vector of expression of a baculovirus system, this synthetic nucleotide sequence coding for the same p19, but characterized by a higher proportion of G and C nucleotides than in the natural nucleotide sequence.

In other words, the invention arises from the discovery that the expression in a baculovirus system of a nucleotide sequence coding for a p19 was apparently related to improved compatibility of the successive codons of the nucleotide sequence to be expressed with the cellular machinery of host cells transformable by baculoviruses, as is observed for the natural nucleotide sequences normally contained in these baculoviruses and expressed in infected host cells; hence the mis-expression, if not the total absence of nucleotide sequence expression of P. baculomuccium hence also an explanation for the much higher possible sequence expression of native p19; hence the observation of a much higher sequence of native p19 and P. baculomuccium, as in the case of P. longa and P. G. G. (14) and their sequence, due to the higher sequence of native p19 and P. longa, is also relevant.

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 that vector and capable of being recognized by cells transfectable by that 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 that promoter and code for the peptide sequence: or a 19 kilodalton (p19) C-terminal peptide fragment of the surface protein 1 (MSP-1 protein) of the merozoite form of a parasite of the Plasmodium type other than Plasmodium vivax and infectious to humans, which C-terminal fragment normally remains anchored on the surface of the parasite after penetration into human red blood cells,during an infectious cycle; either part of this peptide fragment, provided that the product of expression of the second sequence in a baculovirus system is also capable of inducing an immune response capable of inhibiting in vivo parasitemia due to the corresponding parasite; or an immunologically equivalent peptide derived from the above-mentioned C-terminal peptide fragment (p19) or the above-mentioned part of the peptide fragment by addition, deletion or substitution of amino acids, without significantly changing the ability of this immunologically equivalent peptide to induce an immune response of the cellular type and/or similar to that produced by this peptide fragment to the above-mentioned part of p19 or to that fragment,and the nucleotide sequence concerned, where applicable, containing between 40% and 60% G and C nucleotides, preferably at least 50% of the total nucleotides of which it is composed; this sequence may be obtained by constructing a synthetic gene in which the natural codons have been changed by G/C-rich codons without any translational change (peptide sequence maintenance).

In the present case, the nucleotide sequence provided by synthetic DNA may have at least 10% of codons modified compared to the sequence of the natural gene or cDNA while retaining the characteristics of the translated natural sequence, i.e. the maintenance of the amino acid sequence.

However, it is not excluded that this G and C nucleotide content could be further increased, since the resulting changes in the amino acid sequence of the recombinant peptide - or immunologically equivalent peptide - produced would not result in a loss of the immunological or even protective properties of the recombinant proteins formed, particularly in the tests which will be illustrated below.

These observations naturally apply to other Plasmodium infectious to humans, especially since native nucleotide sequences coding for the corresponding p19s would have T and A nucleotide levels that are difficult to match with effective expression in a baculovirus system.

The coding sequence for the signal used may be that normally associated with the native sequence of the Plasmodium concerned, but it may also be derived from another Plasmodium, e.g. P.vivax or P.cynomolgi, or from another organism if it is likely to be recognised as a signal in a baculovirus system.

The sequence encoding p19 or a fragment thereof in the vector under consideration is, if applicable, devoid of the anchorage sequence of the parasite-native protein from which it originated, in which case the expressed protein is generally excreted in the culture medium (soluble form). It is also noteworthy in this respect that under the conditions of the invention, the soluble and anchored forms of recombinant proteins produced, particularly when derived from P.falciparum or P.cynomolgi or P.vivax, tend to form oligomers, this property being originally the immunogenic enhancers of recombinant proteins.

The invention also concerns vectors in which the coding sequence contains the 3' end sequence coding for the hydrophobic C'-terminal end 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' end region may be heterologous to the coding sequence for the soluble part of p19, e.g. corresponding to the 3' end sequence from P.vivax or another organism, since it will code for an anchoring sequence of the entire recombinant protein produced in the cell membrane of the host cell as a CD3 or GIP (3142) in the human cell membrane.

The invention also concerns recombinant proteins, which have conformational epitopes recognized by human serums formed against the corresponding Plasmodium

D In general, the invention also applies to any recombinant protein of the type described above, provided that it has conformational epitopes such as those produced in the baculovirus system, in particular those which are found to be unstable in the reducing medium.

The invention naturally concerns the recombinant proteins, whether in the soluble form or in the form with an anchoring region, in particular the cell hosts used in the baculovirus system.

The invention also covers oligomers produced spontaneously in the baculovirus systems used or produced subsequently, using conventional protein oligomerization techniques. The technique commonly used is glutaraldehyde. However, any conventional bridging system between amine and carboxyl functions, as found in proteins, may also be used. For example, any of the techniques described in European patent application 0602079 may be used.

oligomer means a molecule containing 2 to 50 monomer units, each of these monomer units containing p19 or a fragment thereof, as defined above, capable of forming an aggregate. The invention also covers any conjugate product between a p19 or p19 fragment as defined above, on the one hand, and a carrier molecule - for example a polysine-alanine - which can be used for the production of vaccines, on the other hand, by means of covalent or non-covalent bonds.

The invention also relates to vaccine formulations incorporating these oligomeric or conjugated recombinant proteins, including proteins derived from Plasmodium vivax, these observations also extending to the oligomers of these recombinant proteins.

The invention also includes compounds in which the recombinant proteins mentioned above are combined with an adjuvant, e.g. an alum. Recombinant proteins with the extreme C-terminal region allowing their anchoring to the cell membrane in which they are produced are advantageously used in combination with lipids suitable for liposomes and suitable for vaccine production.

The presence of the anchorage region in the recombinant protein, whether homologous or heterologous to the vaccine part itself, is likely to promote the production of cytophilic antibodies, in particular IgG2a and IgG2b in mice, which may have a particularly high protective activity, so that the active substances of vaccines thus formed could not be combined with adjuvants other than lipids used for the liposomal forms.

Further features of the invention will be shown in the following description of examples of recombinant proteins covered by the invention and the conditions under which they may be produced, without these examples being of a nature to limit the scope of the invention.

The test chemical is a solvent that is used to produce a solution of P. falciparum.

The recombinant PfMSP1p19S construction contains DNA corresponding to the 8 base pairs of the leader sequence and the first 32 amino acids of MSP1 from Plasmodium vivax from Met1 to Asp32 (isolate Belem; Del Portillo et al. 1991 P.N.A.S. 88, 4030.) followed by a GluPhe, due to the EcoR1 site binding the two fragments. This is followed by the synthetic gene, described in Figure 1, encoding Plasmodium falciparum Asn1613 to MSP1p19 from Asn1613 to MS1705 (isolate Ser-Palo Alto Chang et al. 1988. Exp. Uganda Parasitol 67,1).

Similarly and for comparison purposes, a recombinant construction 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 corresponding DNA of the strain P.falciparum (FUP) described by Chang et al , Exp. Parasit. 67,1: 1989.

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

It is noted that 57 codons of the 93 codons of the native coding sequence for P.falciparum p19 were modified (for the third nucleotide in 55 of them and the first and third nucleotides in the remaining two codons). New codons were added at the 5' end to introduce the peptide signal under the conditions indicated above and to introduce an EcoRI site for cloning, on the one hand, and two codons not present in P.falciparum p19 were added to obtain termination signals for expression. The individualised letters respectively placed above the successive acids correspond to the respective amino acid codons. The two sequences have the same stop signs in relation to the same nucleotide sequences. (*) The two stop signs in the two sequences are underlined in relation to the same amino acid sequences.

The test chemical is a chemical that is used to test the pH of the soil.

The PfMSP1p19A construction has characteristics of the previous one except that the synthetic sequence (Figure 1 B) codes for the MSP1p19 of Plasmodium falciparum (Uganda-Palo alto isolate) from Asn1613 to Ile1726 followed by two stop codons of TAA. This construction results in a recombinant protein that is anchored in the plasma membrane of cells infected by a glycosyl phosphatidyl inositol (GPI) type structure.

Figure 1C is representative of the sequence of the recombinant PfMSP1P19S protein before the signal sequence is cut.

Figure 1D represents the sequence of the recombinant protein PfMSP1p19S after cutting the signal sequence. The amino acids highlighted in Figures 1C and 1D come from the EcoR1 site used to join the nucleotide sequences derived from the N-terminal portion of P.vivax MSP1 (with signal sequence) and P.falciparum MSP1p19.

Figure 2 - Immunoaffinity purified soluble recombinant antigen PfMSP1p19 was analyzed by immunoblot after SDS-PAGE in the presence (reduced) or absence (unreduced) of B-mercaptoethanol. The samples are loaded onto gel after heating at 95°C in the presence of 2% SDS In these conditions only covalent-type bonds (disulfide bridges) can resist disintegration. The left blot was detected with a monoclonal antibody that reacts with a linear epitope of natural p19. The right blot was detected with a mixture of 13 antiseptic-compliant subjects with malaria immunity due to Plasmodium falciferum. These results show that the majority of human polymorphs are recombinant with the polymorphs of the human genome.

Figure 2B Human antiserum immunoblot analysis of MSP-1 p19 purified from P. vivax and P. cynomolgi recombinant under non-reduced (NR) conditions, reduced only in the loading medium (R) and irreversibly reduced (IR): This work is based on the idea that the baculovirus expression system reproduces correctly and in a large proportion the conformational epitopes present in vivo on the C-terminal portion of MSP-1. The best way to measure this property (and may be the only possible way in the absence of purified native proteins corresponding to p19) is to study the reactivity of recombinant proteins with the erum of individuals exposed to malaria,Err1:Expecting ',' delimiter: line 1 column 108 (char 107)Immunoblot was detected with a mixture of 25 human antiserums from subjects with acquired immunity to malaria due to Plasmodium vivax. V and C refer to MSP-1 derived proteins from P. vivax and P. cynomolgi respectively. Remarkably, irreversibly reduced recombinant proteins show no reactivity with human antiserum while non-irreversibly reduced or unreduced proteins show good reactivity. (Unreduced Pv MSP-1 p19 is somewhat weak because in its glycosylated state it does not bind very well to nitrocellulose paper.) These results show that recognition of the MSP-119 molecules by human bacterovirus antiserum is largely in theIf the system is not completely dependent on the reduction-sensitive conformational epitopes that are reproduced in this system.

The samples were then analysed by immunoblot in the presence (reduced) or absence (non-reduced) of B-mertoethaneol. Fractions 5 to 12 of isoelectrofocusing, as well as two total extracts of merzoites made in the presence (Tex) or absence (T) of polymer detergent were analysed. The immunoblot was therefore isolated with antibodies specific to the polymers p1919 and p1919; the results of these experiments suggest that the presence of this molecule in vivo may be due to a high degree of activity in the digestion of the polymers p19 (P19) in the body.

Figure 3B: Differential contribution of p42 and p19 antigens to the human anti-MSP-1 response of P. vivax. The recognition of P. vivax antibodies MSP-1 p42 and p19 by antiserum of individuals with acquired immunity to P. vivax was compared by ELISA inhibition technique as follows: a mixture of 25 human antiserums from subjects with acquired immunity to malaria from P. vivax was diluted at 1:5000 and incubated for 4 hours at room temperature either alone or in the presence of a P. vivax purified 1 mM p42 or p19 recombinant solution. This mixture was transferred to a microtitre vessel previously coated for 18 hours at 4°C with 500 ml of P. vivax purified or absorbed p19 ng,After washing with PBS containing 0 1 % Tween 20, a goat IgG mouse antibody combined with peroxidase was added and the mixture was incubated for 1 hour at 37°C and enzymatic activity was revealed by reading the optical density at 492 nm The inhibition percentage was calculated based on 100% values of antiserum reactivity with microtent-coated antigen in the absence of a competitor antigen The statistical data were calculated using the Statview program. Each bar represents the average inhibition percentage of a pair of competitive/absorbed antigen based on 4 to 12 lines and the vertical determinations correspond to a 95% confidence interval.The asterisks (*) indicate antigens produced in the presence of tunicamycin, thus without N-glycosylation. Important parameters of these measures are antiserum dilution at 1:5000 which is in the sensitive region of ELISA curves and competing antigen concentrations at 1 mM which include competition by low affinity epitopes. Thus, the data reflect the maximum similarity between the two antigens compared. The results show that most if not all of the epitopes of p42 known by the human antigen are present on p19 as in the presence of the latter, the antigen activity of the human antigen is inhibited against p42 as much as it is by the same p42 antigen.However, about 20% of the p19 epitopes recognized by human antiserum are not or are not accessible on p42 since the human antiserum's reactivity to p19 is much less inhibited by p42 than by p19 itself. Such p19 specific epitopes can be formed or revealed only after p42 is cut into p19 and p33. These results are not affected by glycosylation, showing that the effect is due to a difference between the peptide components of p19 and p42 and not a difference in glycosylation.These results highlight the fact that p19 has a distinct immunological identity from p42.

The test chemical is a chemical that is used to test the pH of the test medium.

The DNA used for the above construction was obtained from a clone of the strain of Plasmodium cynomolgi ceylonesis (22-23), which was maintained by successive transitions in its natural host (Macaca sinica) and cyclic transmissions via mosquitoes (27).

Blood parasites were obtained from infected monkeys at the mature schizophrenic stage when parasitemia 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 fragment of 1200 base pairs was then produced using the PCR reaction using the oligonucleotides highlighted in Figure 4 and derived from P.vivax. The 5' oligonucleotide comprised an EcoRI restriction site and the 3' oligonucleotide two synthetic stop TAA codons followed by a BglII restriction site. This fragment was introduced by ligation and via these EcoRI and BglII sites into the plasmid pVLSV200. already containing the signal sequence of the MSP-1 protein from P.vivax (19).

The sequences of P.cynomolgi and corresponding P.vivax sequences were aligned. The black arrows indicate the presumed primary and secondary cleavage sites. They were determined by analogy with the known sites in P.falciparum (27, 28). Vertical lines and horizontal arrows locate the boundaries of the four regions that were studied. Region 4 corresponds to the sequence coding for P.cynomolgi p19. Glycosylation sites are framed and the preserved cystins are underlined.

The recombinant construction PcMSP1p19S contains DNA corresponding to the 8 base pairs of the sequence leader and the first 32 amino acids of MSP1 from Plasmodium vivax from Met1 to Asp32 (isolated Belem; Del Portillo et al. 1991 P.N.A.S. 88, 4030.) followed by a GluPhe, due to the EcoR1 site binding the two fragments. All this is followed by the sequence coding for MSP1p19 from Plasmodium cynomolgi (Ceylon strain) from Lys276 to Ser380.

Purification of the recombinant PfMSP1p19 protein by immuno-affinity chromatography with a monoclonal antibody specifically recognising Plasmodium falciparum p19.

The chromatography resin was prepared by binding 70 mg of a monoclonal antibody (obtained from a G17.12 hybridome registered at the CNCM (Paris, France) on 14 February 1997 under No. I-1846; this G17.12 hybridome was constructed from myeloma X63 Ag8 653 producing IgG 2a/k recognizing P.falciparum p19) to 3g of CNBr-Sepharose 4B and then activated (Pharmacia) by standard methods detailed in the instructions provided by Pharmacia. The culture surfaces containing PfMSP119p were incubated in ultrafine batch with chromatography resin for 16 hours at 4°C. The wash was performed once with 0.1% dihydrogen phosphate and once with 0.7% dihydrogen phosphate; all the samples were washed with 0.7 to 0.5% sodium phosphate and 0.7 to 0.7 to 0.7 to 0.7 to 0.0 MPSP1 (PBSP2P) and were purified with a pH of 0.0 to 0.0 mL, with a concentration of sodium phosphate and a pH of 0.0 to 0.7 to 0.0 mL, with a concentration of 0.0 to 0.0 to 0.0 mL of sodium phosphate, and a pH of 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.0 to 0.

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

A vaccination trial was conducted in male Saimiri sciureus boliviensis, aged 2-3 years, who had not undergone splenectomy. Three monkeys were injected 3 times intramuscularly at 3 weeks intervals with a mixture of approximately 50-100 μg each of recombinant soluble PvMSP1p42 and p19 (19), purified by immuno-affinity. The complete and incomplete Freund adjuvant was used as follows: 1st injection: 1:1 FCA/ FIA; 2nd injection: 1:4 FCA/ FIA; 3rd injection: FIA. These adjuvant formulations were then mixed 1:1 with the PBS antigen. The five monkeys received the glutathione-transferase (Glutamase) antigen (Glutamase) produced in the same test. The test was carried out on the same day with a protective dose of 2.5 mg/ kg in vitro in all animals infected. The test was conducted on the same day with a protective dose of 2.5 mg/ kg in vitro.

The curves in Figure 5 represent the change in parasitemia measured in number of parasitic haematites per microlitre of blood (on the logarithmic ordering scale) as a function of time after infection (in days).

Examination of the figure shows a very sharp reduction in parasitemia as a result of vaccination

Test for vaccination with recombinant MSP1 from Plasmodium cynomolgi (p42 and p19) in macaque macaque, Macaca sinica.

Fifteen captive monkeys were used as follows: (1) 3 animals injected with 100 μg soluble PcMSP1p42; 3 animals injected with 35 μg (first injection) or 50 μg (second and third injections) soluble PcMSP1p19; (3) 3 animals injected with a mixture of PcMSP1p42 and p19; (4) 3 animals injected with the adjuvant plus PBS, (5) 3 animals not injected. Freund' s complete and incomplete adjuvant was used as described above- The injections were given intramuscularly at 4 week intervals The test infection was done by injecting 2,105 negative Plasmodium cynomegaly haematites 4 weeks after the last injection The parasites were evaluated for parasitism in all animals with parasitism expressed in the parasitic field of the test organism, with 400 parasitism parasites being evaluated as parasitic in the test organism.

Figures 6A-6G illustrate the results obtained, each showing the parasitemia (expressed as a percentage of parasitic haematia on the axis of the logarithmic scale) observed in the test animals as a function of the time after infection (in days on the axis of the abscesses).

The results are: in Figure 6A; unvaccinated control animals; Figure 6B refers to animals that had received saline containing additionally Freund's adjuvant; Figure 6C is an overlay of Figures 6A and 6B, in order to show the relative results resulting from the administration of Freund's adjuvant to animals (the variations are obviously not significant); Figure 6D provides results obtained after vaccination with p42; Figure 6E refers to animals vaccinated with p19 alone; and finally, Figure 6F refers to animals vaccinated with a mixture of p19 and p42.

Although p42 does provide some protection, as shown in Figures 6E and 6F, the protection provided by the recombinant p19 of the invention is considerably improved.

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

The figures used to compile the graphs (6A-6F) are given in Figure 6G.

P.cynomolgi/tooth monkey vaccination trial; second test infection in monkeys vaccinated with p19 alone and controls (Figures 8)

Six months later, without further immunization, the 3 monkeys receiving MSP-1 p19 alone with FCA/FIA (Figure 6E) and the 3 monkeys receiving a saline solution containing Freund's adjuvant (Figure 6B) as well as 2 new unvaccinated naïve monkeys developed a new test infection by injection of 1,106 haematites infected with Plasmodium cynomolgi. Protection was assessed by determining daily parasiticity in all animals by examining the smears with giemsa. The parasites were classified as negative only after counting 400 smear fields. The parasites are expressed as a percentage of parasitic haematites (the figures used to compile the 8A-C graphs are specified in Figure 8D).The six immunized animals that had received a test infection six months earlier had no detectable parasitemia except for 1 animal in each group that had a parasitemia of 0.008% for 1 day (Figures 8A and 8B). Both naïve controls show classic parasitemia with a maximum of 0.8% and for 21 days (Figure 8C). Thus, the 3 animals vaccinated with MSP-1 p19 were as protected six months later as the 3 controls that had a complete classic infection after the first test infection, despite either no or very low parasitemia after the first test infection. These results suggest that the duration of p19 protection is at least six months.

Test of vaccination with p19 in combination with alum in the P.cynomolgi/toque monkey system (Figures 9)

Previous positive protective results have been obtained using Freund's complete adjuvant (FCA) or incomplete adjuvant (FIA). However, the only adjuvant currently accepted in humans is alum. For this reason, we conducted a vaccination trial with P. cynomolgi MSP-1 p19 in the monkey tooth in the presence of alum as adjuvant. Six captive monkeys were used as follows: (1) 3 animals injected with 4 doses of 50 mg MSP-1 p19 recombinant P. cynomolgi with 20 mg alum (2) 3 animals injected with 4 doses of body water and 10 mg alum. The injections were given intramuscularly 4 times in 4 weeks.The test infection was made by injecting 2,105 P. cynomolgi-infected monkeys 4 weeks after the last injection. Protection was assessed by determining daily parasitemia in all animals by examining smears with giemsa. Parasitemia was classified as negative only after counting 400 smear fields. Parasitemia is expressed as a percentage of parasitic hematemia. The results of this experiment are as follows: 2 of 3 monkeys immunized with recombinant p19 had approximately 30 times less total parasitemia during the duration of infection (Figures 9A and 9B) than the 3 immunocontrol monkeys immunized with water and aluminum (Figure 9D) after the test infection.The third monkey immunized with p19 (Figure 9C) was not very different from the controls. For the Plasmodium cynomolgi p19 vaccination trial in the monkey tooth, Macaca sinica, described in Figure 9, the figures used to compile the graphs (9A-9D) are specified (Figure 9E). Although these results are somewhat less dramatic than previous ones (Figures 6, 8), this is the first time that significant protection has been observed for recombinant MSP-1 in alum.

Figure 10: Vaccination trial with a recombinant p19 of Plasmodium falciparum in squirrel monkeys

Twenty Saïmiri sciureus guyanensis monkeys (squirrel monkeys) aged approximately 3 years and bred in captivity were used as follows: (1) 4 animals injected with 50 mg of Pf MSP-1 p19 soluble in the presence of Freund adjuvant as follows: 1st injection: 1:1 FCA/ FIA; 2nd injection: 1:4 FCA/ FIA; 3rd injection: FIA These adjuvant compositions were then mixed with the antigen in PBS 1:1. (2) 2 controls received the animal adjuvant as described for (1) PBS only; (3) 4 injected with 50 mg of Pf MSP-1 p19 soluble in the presence of 10 alligators (Aluva-Gel-S, Serva-Gel (4)); 2 controls received 10 mg of PBS-1 mRNA in animals only; (5) 4 controls were mixed with phosphatidylcholine 300 mg in PBS-1 mRNA and 300 mg of PBS-1 mRNA in PBS-1 mRNA and 300 mg of PBS-1 mRNA in 300 mg of PBS-1 mRNA.This solution was dialysed against PBS with adsorbent Bio-Beads SM-2 (Bio-Rad) and the resulting liposomes were centrifuged and resuspended in PBS The first injection was made with fresh liposomes kept at 4°C and the second and third injections were made with liposomes that had 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 biology water Three injections were made intramuscularly 4 weeks apart The test infection was injected in 1.Protection was assessed by determining daily parasitemia in all animals by examining smears with giemsa. Parasitemia is expressed as a percentage of parasitic haematia. The results of this vaccination trial are presented in Figures 10, A-G.

The groups immunized with Freund's adjuvant or liposome p19 demonstrated parasitemia similar to control groups after a test infection (an animal (number 29) vaccinated with Freund's adjuvant p19 died a few days after the test infection due to reasons independent of vaccination (heart attack)).Irregularities in antigen administration in these 2 groups (poor Freund's emulsion, frozen liposomes) do not allow a complete assessment of the significance of these results. In MS group 2 the total parasitemia during the duration of the test was about 4 times greater than the control animals, I-1 was about 3 times greater and similar to the control animals. This was likely to be difficult to interpret in the experiment. This was due to the fact that the antigen was not used in a certain way, which was not immediately associated with the parasitic effect, which was not immediately demonstrated in the experiment.

Test for vaccination with a recombinant Plasmodium falciparum p19 in squirrel monkeys (same test as in Figure 10)

Captive-bred monkeys were injected with 1 ml of inoculum intramuscularly 2 times at 4 week intervals as follows: (1) 4 animals injected with 50 μg of soluble PfMSP1p19 in the presence of Freund' s adjuvant as follows: first injection 1:1 FCA/ FIA; second injection 1:4 FCA/ FIA; and then mixed 1:1 with the antigen in PBS; (2) 4 animals injected with 50 μg of soluble PfMSP1p19 in the presence of 10 mg of animals; (3) 4 animals injected with approximately 50 μg of PfMSP1p19 anchored GPI reconstituted in liposomes 1 1 in molar cholesterol and phosphatidylcholine.

Red blood cells from a squirrel monkey with 30% parasitemia due to P. falciparum (mostly mature forms) were washed in PBS and the collet was diluted 8 times in the presence of 2% SDS and 2% dithiothreitol and heated to 95° before being loaded on a 7.5% polyacrylamide gel (separation gel) and 4% polyacrylamide gel (stacking gel) (top of gel) After transfer to nitrocellulose the immunoblot analysis (immunoprint) was performed with twenty antisera as follows: (1) twenty pool of twenty monkeys vaccinated with PfMSP119p soluble in adjuvant pool of Freundsin (2) twenty pool of twenty monkeys dilute in adjuvant pool of twenty monkeys with PfMSP119p soluble; (3) twenty pool of twenty monkeys diluted in adjuvant pool of twenty monkeys infected with PMSP119p; (5) twenty pool of twenty monkeys diluted in adjuvant pool of twenty monkeys soluble in adjuvant pool of twenty monkeys; (5) twenty pool of twenty monkeys treated with PMSP119p soluble in the pool of twenty monkeys; (5) twenty-five pool of twenty monkeys treated with PMSP1 (non-infected) and (5) all patients treated with PMSP1P1 infected with PMSP1 were treated with PMSP1 after exposure to five years; (6) twenty-five pool of twenty monkeys were treated with PMSP1 and (5) were treated with PMSP1 (non-infected with PMS1 and (6) PMS1 after exposure to five years).

The results show that the 3 antiserum pools of monkeys vaccinated with PfMSP1p19 react significantly and specifically with complexes of very high molecular weight (found diffusely 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 MSP1p19 in vivo with epitopes that are reproduced in the recombinant PfMSP1p19 molecules synthesized in the baculovirus system, particularly those in oligomer form.

Figure 7 also illustrates these results, which relate to the immunofingerprints produced on gel. The first three columns of the gel illustrate the in vivo response of monkeys to p19 injections [(1) with Freund's adjuvant, (2) with alum, (3) as a liposome] and in particular the existence of high molecular weight complexes supporting the hypothesis of the in vivo aggregation of p19 as an oligomer, specific to the maturation stage (when p42 is cut into p19 and p33).

This vaccination trial also includes a third injection identical to the previous ones.

There are two control animals for each group, namely: 2 control animals injected with PBS and Freund' s adjuvant; 2 control animals injected with PBS and alun; 2 control animals injected with protein-free liposomes; and two control animals injected with PBS without adjuvant.

Figure 7B: The data for this figure are derived from the P. falciparum/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 test infection and the SHI antiserum was diluted 1:250. The results show that the serum of the 4 monkeys vaccinated with p19 was high and specifically responded to very high molecular weight complexes while the monkeys in the other groups vaccinated with p19 and Freund adjuvant protective or protective monkeys showed a lower response to these complexes than the monkeys in the other groups. These monkeys were not treated with p19 and did not respond to the protective activity of other groups, despite having a high and specific response to p19 and a partial controlled effect.

The invention naturally concerns other applications, for example those described below in connection with some of the examples, which are not of a limiting nature.

Therapeutic

The recombinant PfMSP1p19 molecule can be used to produce specific antibodies that may be used by passive transfer for the purpose of appropriate therapy for severe P. falciparum malaria with a risk of mortality.

Diagnosis

The recombinant PvMSP1p42 and PvMSP1p19 and PfMSP1p19 molecules derived from baculoviruses can and have been used to produce specific monoclonal antibodies in mice These antibodies, in combination with polyclonal anti-MSP1p19 antiserums from another species such as rabbit or goat, may form the basis of a semi-quantitative diagnostic test for malaria and be 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 is to trap and quantify any part of the p19 molecule in the blood.

In this context, the advantages of MSP1p19 are as follows: (i) it is both extremely well conserved within the same species and sufficiently divergent between different species to allow for the easy production of species specific reagents No cross-reaction has been observed between the antibodies derived from PfMSP1 p19 and PvMSP1p19; (ii) the function of MSP1p19, although not known with precision, appears to be sufficiently important for this molecule not to vary significantly or be deleted without lethal effect for the parasite; (iii) it is a major antigen found on all mesozoans and therefore must be detectable, in principle, at low parasiticity and in proportion to the parasiticity; (iii) the antibodies derived from MSP1p19 appear to be more suitable for use against MSP119 and therefore the antibodies appear to have a more recombinant structure.

The micro-organisms identified below were registered in accordance with Regulation 6.1 of the Budapest Treaty on 1 February 1996 under the following numbers: - What?

Références d'identification	Numéros d'enregistrement
PvMSP1p19A	I - 1659
PvMSP1p19S	I - 1660
PfMSP1p19A	I - 1661
PfMSP1p19S	I - 1662
PcMSP1p19S	I - 1663

The invention also relates to the use of these antibodies, preferably pre-fixed to a solid support (e.g. for affinity chromatography), for the purification of p19-type peptides originally contained in a mixture.

The purification process then involves the contact of this mixture with the antibody, the dissociation of the antigen-antibody complex and the recovery of the purified p19-type peptide.

The invention also relates to vaccine compositions, including mixtures of proteins or fragments, including mixtures of the following types:

P19 of P. falciparum and p19 of P. vivax. p19 of P.falciparum and p42 of P.falciparum, where applicable, without its most hypervariable regions, p19 of P.vivax and p42 of P.vivax, where applicable, without its most hypervariable regions, p19 of P.falciparum and p42 of P.falciparum, where applicable, without its most hypervariable regions, and p19 of P.vivax and p42 of P. vivax, where applicable, without its most hypervariable regions.

In this case, the most hypervariable regions are defined as region II or region II and region III in part or in its entirety, the part of region III preferably deleted being that which is juxtaposed to region II (the preserved part on the C-terminal side of p33, close to p19).

The invention is not limited to the production of human vaccines. It is equally applicable to the production of veterinary vaccine formulations using the corresponding proteins or antigens derived from mammalian infectious parasites and produced under the same conditions. In fact, infections of the same type, babesiosis, are known to occur in cattle, canines and equines, one of the antigens of the Babesia species has a strong conformational homology (in particular the two domains EFG-like and cysteine rich) 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 p19 used in these mixtures can also give rise to all the modifications discussed above when considered in isolation.

The Commission shall be assisted by the European Parliament.

(1) Holder, J A et al. (1982) Biosynthesis and processing of a Plasmodium falciparum schizont antigen recognized by immune serum and a monoclonal 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 schizonts . J. Immunol. 134:1951-1946 (((4) Sley, B. et al. (1987) Exhibited by: Plasmodium vivax Exocytophthroxyl antibodies against monoclonal schizonts .(5) Holder, A. A. (1988) Processing of the precursor to the major merozoite surface antigen: structure and role in immunity . Prog. Allergy 41: 72-97.(6) Cooper, J.A. (1993) Merozoite surface antigen-1 of Plasmodium Parasitol. Today 9: 50-54.(7) Holder, A.A., et al. (1987) Processing of the precursor to the major merozoite surface antigen of Plasmodium falciparum Parasitology 94: 199-208.(8) Lyon, J.A. et al. (1986) Epitope map and shedding scheme for the MS-dalton glycoprotein of Plasmodium falciparum Merozoites derived from cloned non-cloned fragments of the Proc.The following is a list of the most commonly used methods of determining the concentration of a substance in a food:The following table shows the results of the analysis of the data collected from the analysis of the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from the data collected from data collected from the data collected from the data collected from the data collected from data collectedThe Plasmodium cynomolgi merozoite surface protein 1 C-terminal sequence and its homologies with other Plasmodium species . Mol. Biochem. Parasitol. 74 :105-111.(20) of Portillo, H.A., et al. (1990) Primary structure of the merozoite surface antigen 1 of Plasmodium vivax reveals sequences conserved between different Plasmodium species . Nat. Proc.l. Acad. Sci. USA 88: 4030-4034.(21) Gibson, H.L., et al. (1992) Structure and expression of the gene for P200vike, a major blood-stage surface antigen of Plasmodium vivax . Mol. Biochem.Plasmodium cynomolgi ceylonensis sub sp. nov. and Plasmodium fragile sp. nov. from monkeys in Ceylon . Ceylon Journal of Medical Science 14 : 1-9.(23) Cochrane, A.H., et al (1986) Further studies on the antigenic diversity of the circumsporozoite 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 enhancement and blocking of infectivity to mosquitos during infections in the natural host, Macaca sinica Exp. Parasitol. 71, 305-313.The N-terminal amino acid sequences of the Plasmodium falciparum (FCBI) merozoite surface antigen of 42 and 36 kilodalton, both derived from the 185-195 kilodalton 34 precursor domains Mol. Biochem. Parasitol. 147-154.28) Blackman M.J., and al. (1991) Proteolytic processing of the Plasmodium falciparum surface protein fragment-1 produces two epidermal-bound growth factors containing The following is a list of the most commonly used and used antibiotics in the treatment of malaria: A family of erythrocyte binding proteins of malaria parasites Proc. Natl. Acad Sci, 89:7085-7089 (30) Sim B.K.L (1995) EBA-175 An erythrocyte-binding ligand of Plasmodium falciparum Parasitology Today, vol.II, no. 6:213-217.(31) Sim B K L (1994) Receptor and ligand domains for invasion of erythrocytes by Plasmodium falciparum Science, 264:1941-1944.(32) Davies, A. (1993), Biochem J. 295 (Pt 889-8963) Expression of the glycosyl-binding protein of Plasmodium falciparum Production of the glycosyl-binding protein by C.S.H.A.C.L. and C.S.H.A.L. Inhibited by C.S.H.A.L. and C.S.H.A.L.L.L. Inhibited by C.S.H.A.L.L.L. using a cell-linking agent Inhibited by C.S.H.L.H.L.L.L.A.L. and C.S.H.L.H.A.L.L.L.L.L.L. Inhibited by C.S.H.L.L.L.L.L.L.L.The following is a list of the species of the genus, which are not included in the list of species of the genus:The following is a list of the most commonly used antibiotics in the United States: Vaccines against Babesiosis using Soluble Parasite Antigens , Parasitology Today, vol.11, n°12.(40) P.A. Burghaus et al. (1996) Immunization of Aotus nancymai with Recombinant C Terminus of Plasmodium falciparum Merozoite Surface Protein 1 in Liposomes and Alun Adjuvant Does Not Induce Protection against a Challenge Infection , Infection and Immunity, 64:3614-3619.(41) S.P. Chang, et al. (1996) A Recombinant Baculovirus 42-Kilodalton C-Terminal Fragment of Plasmodium falciparum Merozoite Surface Protein 1 Protects against Infection by Monkeys, Monkeys and Alun Infection and Infection, 64:3614-3619.(31) The need for immunity and protection of the disease in the development of Malaria, as stated by Miller, (33-247) The Blood and Blood.The Commission has not yet published a report on the implementation of the new rules.

The invention also relates to specific antibody-secreting hybridomas selectively recognising p19 of a MSP-1 protein from the merozoite form of a Plasmodium parasite other than Plasmodium vivax and not recognising Plasmodium vivax.

In particular, these hybridomas secrete monoclonal antibodies that do not recognize Plasmodium vivax p19 and specifically recognize Plasmodium falciparum p19.

The invention also concerns a hybridome characterised by the production of a specific antibody that specifically recognises P vivax p19 and P cynomolgi p19; a hybridome F10-3 was constructed from myeloma X63 Ag8 653 producing IgG 2b/k recognising Plasmodium vivax glycoprotein p42.

Claims (15)

1. Composition of vaccine containing as active ingredients a mixture of: - What?

   (a) a recombinant protein including: - What?

   - a 19 kilodalton (p19) C-terminal fragment of the surface protein 1 (MSP-1) of the merozoite form of a Plasmodium falciparum, this C-terminal fragment normally remaining anchored on the surface of the parasite after its penetration into human red blood cells during an infectious cycle, or

   - a part of that p19 fragment which is capable of inducing an immune response capable of inhibiting parasitemia due to a Plasmodium falciparum parasite, and which contains at least one of the two EGF regions contained in that p19 fragment, the protein

   - having unstable conformational epitopes in a reducing medium and constituting the majority of epitopes recognised by human anti-serum formed against Plasmodium falciparum, and

   - recognised by human anti-serums formed against Plasmodium falciparum when in the unreduced state or in a non-irreversible reduced state, but not recognised or poorly recognised by these same anti-serums when irreversibly reduced, and

   (b) another recombinant p19 protein from a Plasmodium parasite homologous to P. falciparum.
2. Vaccination composition according to claim 1, characterised by the p19 fragment or part of a p19 fragment included in the recombinant protein in question in accordance with (a) is: - What?

   (i) likely to be expressed by a recombinant baculovirus vector containing, under the control of a promoter contained in that vector and likely to be recognized by cells transfectable by that vector, DNA containing a synthetic nucleotide sequence encoding that p19 fragment or part of a p19 fragment, the synthetic nucleotide sequence in question, in addition to containing between 40 and 60% of G and C, preferably at least 50% of the total nucleotides of which it is composed, the said DNA, which also contains a nucleotide sequence encoding a signal peptide, upstream of the 5'-terminal end of the said synthetic nucleotide sequence, the signal peptide being capable of being recognised as a signal in a baculovirus system, or where such p19 fragment or part of a p19 fragment is:

   (ii) likely to be produced by an insect cell transfected by a baculovirus vector as defined in (i).
3. Vaccine composition as claimed 2, characterised by the absence of the synthetic nucleotide sequence at its 3'-terminal of the sequence coding for the hydrophobic C-terminal end region normally involved in inducing the anchoring of the p19 fragment to the cell membrane of the host in which it is expressed, including in a baculovirus-infectious insect cell.
4. Composition of vaccine according to claim 1 or 2, characterised by the protein in question also having a glycosylphosphatidylinositol (GPI) group of the type that allows the p19 fragment to be anchored to the cell host, in particular a eukaryotic cell, preferably an insect cell infectious by a baculovirus, in which the recombinant protein is expressed.
5. A vaccine composition according to any of claims 2 to 4 characterised by the synthetic nucleotide sequence being preceded by a signal nucleotide sequence coding for a signal peptide normally associated with a Plasmodium MSP-1 protein, homologous or heterologous to the synthetic nucleotide sequence.
6. Vaccine composition according to claim 5, characterised by the signal sequence being derived from P. vivax.
7. Composition of vaccine according to any of claims 1 to 6, characterised by the signal sequence being derived from P. falciparum.
8. A vaccine composition according to any of claims 2 to 7 characterised by the fact that the DNA includes in addition, upstream of the synthetic nucleotide sequence, a nucleotide sequence encoding a polypeptide sequence containing less than 50 residues, including less than 35 residues or even less than 10 residues of the C-terminal portion of the 33 kilodalton (p33) fragment of MSP-1.
9. A composition of a vaccine according to any of claims 1 to 8 characterised by the fact that the said p19 fragment or part of a p19 fragment which is included in the recombinant protein according to (a) has a molecular weight which is between 10 and 25 kDa, including 10 and 15 kDa.
10. Composition of vaccine according to any of the claims 2, 3, 6, characterised by: - What?

    the synthetic nucleotide sequence is coded for a 19 kilodalton (p19) C-terminal fragment of surface protein 1 (MSP-1) of the merozoite form of a Plasmodium falciparum from Asn1613 to Ser1705,

    and upstream of the 5'-terminal end of this synthetic nucleotide sequence, the DNA includes: - What?

    - the 8 base pairs of the leading sequence of Plasmodium vivax surface protein 1, and

    - a sequence encoding the thirty-two amino acids of Plasmodium vivax surface protein 1 from Met1 to Asp32, And downstream from the 3'-terminal end of this synthetic nucleotide sequence, the DNA includes two stop TAA codons.
11. Composition of vaccine according to any of the claims 2, 4, 6, characterised by: - What?

    the synthetic nucleotide sequence is coded for a 19 kilodalton (p19) C-terminal fragment of surface protein 1 (MSP-1) of the merozoite form of a Plasmodium falciparum from Asn1613 to Ile1726,

    and upstream of the 5'-terminal end of this synthetic nucleotide sequence, the DNA includes:

    - the 8 base pairs of the leading sequence of Plasmodium vivax surface protein 1, and

    - a sequence encoding the thirty-two amino acids of Plasmodium vivax surface protein 1 from Met1 to Asp32, And downstream from the 3'-terminal end of this synthetic nucleotide sequence, the DNA includes two stop TAA codons.
12. Composition of vaccine according to any of claims 1 to 11, characterised by the fact that the said baculovirus vector in (i) is the virus registered at the C.N.C.M. under number I-1661, or the virus registered at the C.N.C.M. under number I-1662.
13. Composition of a vaccine according to any of claims 1 to 12, characterised by the protein referred to in (a) being conjugated to a carrier molecule usable for the production of vaccines.
14. Composition of vaccine according to any of claims 1 to 13, characterised by the recombinant protein referred to in (b) being Plasmodium vivax p19.
15. Composition of vaccine according to any of claims 1 to 14, characterised by the fact that in addition to the recombinant protein referred to in (a), it includes: - What?

    - P19 of P. vivax,

    - P42 of P. falciparum, where appropriate deleted from its most hypervariable regions, and

    - P. vivax p42, where appropriate deleted from its most hypervariable regions.