Malarial pre-erythrocytic stage polypeptide molecules

Details Malarial pre-erythrocytic stage polypeptide molecules Malarial pre-erythrocytic stage polypeptide molecules

1998-02-06

2001-02-20

Chin, Christopher L. (Department: 1641)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S006120, C435S069300, C435S320100, C514S04400A, C514S895000, C530S350000, C530S822000

Reexamination Certificate

active

06191270

ABSTRACT:

BACKGROUND OF THE INVENTION
The parasites responsible for malaria in man display different morphologies in the human host and express different antigens depending on their location in the body. The morphological and antigenic differences of these parasites during their life cycles in man enable different stages of development in the liver and in the blood to be defined: the sporozoite, the infectious form injected by the vector mosquito, transforms rapidly into a schizont in the host's hepatocytes and thereafter infects the erythrocytes. The intrahepatic localization of
P.falciparum
manifests itself in the expression of a group of antigens specific to this stage of development and which are highly immunogenic under the natural conditions of exposure to the disease. This clinically silent phase is at present the only one against which a very strong, sterilizing immunity can be induced experimentally in man, by injecting irradiated sporozoites capable of entering the hepatocyte and of developing therein but without being able to lead on to the blood stage of the disease. Accordingly, the inventors have concentrated the bulk of their efforts on these two pre-erythrocytic stages. However, these stages are also the most intricate ones to study, and hence the least understood, since it is difficult or even impossible to obtain biological material, the only in vitro study model affords a very low yield and the best animal model remains the chimpanzee, the use of which is limited and expensive.
In order to gain access to the antigens of the pre-erythrocytic stages, the inventors used sera of individuals who had resided for 25 years in a region where the disease is endemic but who were on permanent prophylaxis with chloroquine. These individuals were regularly subjected to infected mosquito bites but did not develop any complete blood infection. Their serum hence contained antibodies directed essentially against the pre-erythrocytic stages, which was verified by immunofluorescence (IF) and western blotting on the 3 stages of the parasite.
The use of these sera for screening a library of genomic DNA of the parasitic clone of
P.falciparum,
the library being constructed in expression vectors in a phage lambda gt11 (V. Rosario, Science 212, 1981, pp. 1037-1038; and Thaithong et al., Transactions of Royal Society of Tropical Medicine and Hygiene, 1984, 78:242-245), led to the demonstration of polypeptides of the pre-erythrocytic stage, in particular the SALSA (sporozoite liver stage antigen) polypeptides described in EP A-0,407,230 and LSA-1 (liver stage antigen) described in WO 92/13884. The present invention relates to new polypeptide molecules specific to the pre-erythrocytic stage, and to their use as active principle of antimalarial vaccine or in methods of diagnosis of the disease.
SUMMARY OF THE INVENTION
The invention is the outcome of the demonstration by the inventors of the special properties of a particular antigen referred to as LSA-3 and of its fragments, which are seen to be candidates with a strong potential for producing an antimalarial vaccine, for the following reasons:
a) when a fraction of LSA-3 was used in combination with another antigen of the same stage of development of the parasite, such as LSA-1, to immunize chimpanzees, the animal responding to both molecules or only to LSA-3 displays the feature of not having parasites in the blood, of having a substantial decrease of the parasites in the liver and of manifesting a substantial recruitment of mononuclear cells indicating a response in terms of cellular immunity;
b) in regions where the disease is endemic, a very clear correlation is observed between the protection of individuals against natural infection by sporozoites and their responses in terms of antibodies against LSA-3;
c) in eight human volunteers immunized by injection of irradiated sporozoites, antibodies directed against LSA-3 are found in each of the four individuals resisting sporozoite infection and in none of the other four volunteers who developed a blood infection;
d) antibodies obtained against the peptide DG729 in WO 92/13884, already described, give a cross-reaction with the sporozoite and liver stages of the murine parasite
P.yoelii,
which permits a significant exploitation of the mouse model. In vitro, the human antibodies immunopurified on DG729 are capable, even at very low concentrations, of blocking the entry of
P.yoelii
sporozoites into mouse hepatocytes. In vivo, mice immunized with DG729 are fully or partially protected against infection by
P.yoelii
sporozoites;
e) lastly, some epitopes, in particular in the non-repeat portions of the molecule, stimulate the secretion of interferon-&ggr; by monocytes, this mediator enabling the intrahepatic development of the parasite to be inhibited (S. Mellouk et al., The Jour. Of Immun. 139, 4192-4195, 1987);
f) the sequence of the region of LSA-3 corresponding to a (lipo)peptide NR2 was analysed in 27 samples: 4 laboratory strains (NF54, K1, Palo Alto, T9/96), 3 Madagascan isolates, 3 Burmese isolates, 5 Brazilian isolates, 7 isolates from the Ivory Coast and 5 Thai isolates. No mutation was observed on the 300 base pairs analysed, that is to say 100% conservation in this immunologically important region containing one or more B, Th and CTL epitopes;
g) information about the structure of the antigen, and in particular of a peptide RE, and more especially about the central repeat region from which the peptide RE was designed and which contains one or more major B epitopes, was obtained from the hydrophobic cluster plot of the sequence available in the clone T9/96 (630 amino acids) (Gaboriot et al., (1987): Hydrophobic cluster analysis: an efficient new way to compare and analyse amino acid sequences, FEES Letters, 224: 149-155); this method predicts a very strong propensity for &agr;-helical organization. The repeat region displays remarkable regularity in the spacing of the valine and isoleucine residues, alternating with acid or proline residues. The arrangement of the hydrophobic groups at the surface of this helix is reminiscent of a hydrophobic border gradually shifting from one face of the helix to the other according to a constant general orientation along the molecule, and probably related to a coiled-coil structure or packaging as seen in
FIG. 4
b
which depicts the HCP (hydrophobic cluster plot) of the peptide sequence of the clone DG729;
h) after demonstrating that there was a very wide range of immune responses to the LSA-3 antigen, we analysed the capacity of the responder cells to localize around the parasites in the liver. In mice immunized with the recombinant antigens, intraportal injection of each of the peptides absorbed on 10 &mgr;m polystyrene beads enables an afflux of lymphocytes around the antigen (mimicking the parasite) to be visualized after 48 hours, followed on the 5th day by a substantial recruitment of cells belonging to the macrophage line.
All these properties, some of which will be demonstrated in detail in the experiments described later, show that the LSA-3 antigen displays both good antigenicity and good immunogenicity.
The inventors were able to confirm and define the specificity of the stages of expression of the molecule; in the sporozoites, this expression was confirmed by the surface immunofluorescence of several strains and isolates. In western blot (or immunoblot) analysis, the LSA-3 molecule appears as a protein of molecular weight 200,000 daltons. While the messenger RNAs of sporozoites could not be obtained in sufficient amounts for a northern blot analysis, reverse PCR experiments confirmed the expression of LSA-3 at this stage. In infected hepatocytes, LSA-3 is observed in the parasitophorous vacuole of the parasite by immunofluorescence using antibodies against the repeat and non-repeat regions of the protein, as well as by electron microscopy.
A fragment of LSA-3 designated 729S, as well as three peptides designated NRI and NRII included in the non-repeat portion and 729R included in the repeat portion, have been described in Appli

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