Title:  Malaria vaccine

United States Patent:  6,579,524

Issued:  June 17, 2003

Inventors:  Corradin; Giampietro (Lausanne, SE); Rogerro; Mario (Epalinges, CH)

Assignee:  RMF Dictagene S.A. (Prilly, CH)

Appl. No.:  506691

Filed:  February 18, 2000

The present invention relates to a vaccine against malaria comprising a polypeptide having the amino acid sequence of the C-terminal part of the circumsporozoite protein of a Plasmodium species, in which polypeptide one or more pairs of cysteine residues are oxidized, and optionally a suitable carrier and/or adjuvant and/or biodegradable microcapsules for use in humans.

Description of the Invention

The present invention relates to a vaccine against malaria. The invention further relates to polypeptides that are capable of eliciting an immunological and protective response against malaria in a subject and the use thereof in prophylaxis.

Malaria is a parasitic disease transmitted during the blood meal of infected mosquitoes which inoculate sporozoites into the mammalian host. Within minutes, sporozoites invade hepatocytes and develop into merozoites intracellularly by asexual schizogony. The merozoites then invade red blood cells, producing the various symptoms associated with the disease. The life-cycle is completed when gametocytes are ingested during the blood meal of the mosquito vectors.

Protective immunity against malaria can be obtained by immunizing mice and humans with irradiation-attenuated sporozoites. This immunity is the result of the effect of neutralizing antibodies recognizing free sporozoites in the blood stream and of CD4⁺ and CD8⁺ T cells which prevent the development of the parasite hepatic forms. Experiments performed in B cell deficient mice have demonstrated that, despite the absence of anti-sporozoite antibodies, protection is induced by irradiated sporozoite immunization. This suggests that T cells specific for proteins present in the intracellular hepatic stage play a predominant role in protection. Therefore, one of the aims in malaria vaccine research is to mimic the protective immune response induced by injection of irradiated sporozoites.

In the research that led to the present invention it was found that a polypeptide of 69 amino acids (PbCS 242-310) encompassing the C-terminal region of the circumsporozoite protein of Plasmodium berghei, which was generated using solid-phase peptide synthesis elicited in BALB/c mice (M-2d) after two subcutaneous injections, in the presence of Incomplete Freund's Adjuvant (IFA) at the base of the tail, (i) high titers of anti-peptide antibodies which also recognize the native P.berghei CS protein, (ii) cytolytic T cells specific for the Major Histocompatibility Complex (MHC) antigen Kd restricted peptide PbCS 245-253, and (iii) partial CD8⁺ -dependent protection against sporozoite-induced malaria. The same frequencies of peptide PbCS 245-253 specific cytotoxic T lymphocytes (CTL) were found by IFN-.gamma. ELISPOT in the draining lymph nodes of animals immunized with the short optimal CTL peptide 245-253 or with the polypeptide 242-310, indicating that the longer polypeptide can be processed and presented in vivo in the context of MHC class I as efficiently as short CTL peptides. Interestingly, even higher levels of CTL activity and protection were observed when the four cysteine residues present in the C-terminal peptide were fully oxidized. Theme findings underline the potential importance of the chemical nature of the C-terminal fragment on the activation of the immune system and concomitant protection. And more generally, as multiple facets of the immune system are stimulated by long synthetic polypeptides, these may provide a valuable alternative to vaccination with recombinant protein fragments or short peptides.

Based on this finding the present invention provides vaccines against malaria, in particular for use in humans, comprising a polypeptide having the amino acid sequence of the C-terminal part of the circumsporozoite protein of a Plasmodium species, which polypeptide at least comprises the four terminal cysteines from which at least one pair is oxidized, the vaccine optionally further comprising a suitable carrier and/or adjuvant and/or biodegradable microspheres. Biodegradable microcapsules are spheres of about 1 to 10 .mu.m and very suitable carriers and/or adjuvants for the vaccine of the invention.

Specifically, the vaccine of the invention is based on the circumsporozoite protein of Plasmodium falciparum, more specifically of Plasmodium falciparum strain NF54.

Preferably the polypeptide in the vaccine consists of at least 42 consecutive amino acids derived from the C-terminal part of the circumsporozoite protein. More in particular, the invention relates to vaccines in which the polypeptide comprises at least the amino acids 342 to 383 of Plasmodium falciparum, even more in particular the amino acids 342 to 383 of the Plasmodium falciparum NF-54 strain.

Preferably, all four cysteines present in the polypeptide derived from the C-terminal part of the circumsporozoite protein of Plasmodium falciparum are oxidized.

It was found that the vaccine of the invention is in particular useful when the adjuvant is Montanide.TM.. Montanide.TM. ISA Adjuvants (Seppic, Paris, France; ISA=Incomplete Seppic Adjuvant) are a group of oil/surfactant based adjuvants in which different surfactants are combined with either a non-metabolizable mineral oil, a metabolizable oil, or a mixture of the two. They are prepared for use as an emulsion with aqueous Antigen solution. The various Montanide ISA group of adjuvants are used as water-in-oil emulsions, oil-in-water emulsions, or water-in-oil-in-water emulsions. The different adjuvants accommodate different aqueous phase/oil phase ratios, because of the variety of surfactant and oil combinations. The performance of these adjuvants is said to be similar to Incomplete Freund's Adjuvant (IRA) for antibody production; however the inflammatory response is usually less.

The invention further relates to polypeptides having the amino acid sequence of the C-terminal part of the circumsporozoite protein of a Plasmodium species, which polypeptides comprise at least 42 consecutive C-terminal amino acids of which one or more cysteine pairs are oxidized.

In a particular embodiment of the invention the polypeptide comprises the amino acids 342 to 383 of Plasmodium falciparum NF-54 strain.

It is preferred that all cysteine residues present in the polypeptide are oxidized.

Furthermore, the invention relates to such polypeptide for use in a vaccine against malaria and to the use of such a polypeptide for the preparation of a vaccine against malaria.

Claim 1 of 11 Claims

What is claimed is:

1. A composition which elicits a neutralizing antibody response and/or a CD8+ specific response against malarial parasites upon in vivo administration to a human subject comprising a polypeptide and a carrier, wherein said polypeptide has an amino acid sequence which:

(a) is at least 42 residues in length and no more than 69 residues in length;

(b) correspond to a sequence in the C-terminal 69 amino acids of the circumsporozoite protein of a Plasmodium species; and

(c) has at least 4 cysteine residues, at least one of which is oxidized.

 

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