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
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
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
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
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
(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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