epidermal-growth-factor and Malaria--Falciparum

Pfs28 is a Plasmodium falciparum malaria transmission-blocking vaccine candidate that is anchored to the parasite surface through a C-terminal glycosylphosphatidylinositol (GPI) moiety, and plays a role in parasite survival in the mosquito midgut. Pfs28 contains epidermal growth factor (EGF)-like domains and is part of a family of sexual stage malaria proteins that includes the related vaccine antigen Pfs25. The lack of structural definition of Pfs28 and the immune response to this candidate has limited further malaria vaccine development for this antigen. Here, we present the crystal structure of Pfs28, examine its conservation with P. vivax Pvs28, and evaluate the cross-reactivity of Pfs28 to antibodies that recognize Pfs25. Pfs28 is comprised of four EGF-like domains stabilized by ten disulfide bridges with an overall architecture that highly resembles Pfs25. Despite the high sequence and structural similarity between these antigens, no cross reactivity of Pfs28 to anti-Pfs25 monoclonal antibodies could be demonstrated.

Topics: Animals; Antibodies, Protozoan; Antigens, Protozoan; Antigens, Surface; Epidermal Growth Factor; Malaria; Malaria Vaccines; Malaria, Falciparum; Malaria, Vivax; Plasmodium falciparum; Protozoan Proteins

Using bioinformatics analyses of the unfinished malaria genome sequence, we have identified a novel protein of Plasmodium falciparum that contains two epidermal growth factor (EGF)-like domains near the C-terminus of the protein. The sequence contains a single open reading frame of 1572bp with the potential to encode a protein of 524 residues containing hydrophobic regions at the extreme N- and C-termini which appear to represent signal peptide and glycosylphosphatidylinositol (GPI)-attachment sites, respectively. RT-PCR analysis has confirmed that the novel gene is transcribed in asexual stages of P. falciparum. Antibodies to the EGF-like domains of the novel protein are highly specific and do not cross-react with the EGF-like domains of MSP1, MSP4, MSP5 or MSP8 expressed as GST fusion proteins. Antisera to the C-terminal fragments react with two bands of 80 and 36kDa in P. falciparum parasite lysates whereas antisera to the most N-terminal fusion protein only recognises the 80kDa band, suggesting that the novel protein may undergo processing in a similar way to MSP1 and MSP8, but with fewer cleavage events. Immunoblot analysis of stage-specific parasite samples reveals that the protein is present in trophozoites, schizonts and in isolated merozoites. The protein partitions in the detergent-enriched phase after Triton X-114 fractionation and is localised to the surfaces of trophozoites, schizonts and free merozoites in an apical distribution. Based on the accepted nomenclature in the field we now designate this protein MSP10. We have shown that the MSP10 fusion proteins are in a conformation that can be recognised by human immune sera and that there is very limited sequence diversity in an approximately lkb region of MSP10, encompassing the two EGF-like domains. A sequence similar to MSP10 can be identified in the available P. yoelii genomic sequence, offering the possibility of ascertaining whether this novel protein can induce host protective responses in an in vivo model.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Polarity; Epidermal Growth Factor; Female; Humans; Life Cycle Stages; Malaria, Falciparum; Mice; Mice, Inbred BALB C; Molecular Sequence Data; Plasmodium falciparum; Protein Structure, Tertiary; Protozoan Proteins; Rabbits; Sensitivity and Specificity

Antibodies that bind to antigens expressed on the merozoite form of the malaria parasite can inhibit parasite growth by preventing merozoite invasion of red blood cells. Inhibitory antibodies are found in the sera of malaria-immune individuals, however, the specificity of those that are important to this process is not known. In this paper, we have used allelic replacement to construct a Plasmodium falciparum parasite line that expresses the complete COOH-terminal fragment of merozoite surface protein (MSP)-1(19) from the divergent rodent malaria P. chabaudi. By comparing this transfected line with parental parasites that differ only in MSP-1(19), we show that antibodies specific for this domain are a major component of the inhibitory response in P. falciparum-immune humans and P. chabaudi-immune mice. In some individual human sera, MSP-1(19) antibodies dominated the inhibitory activity. The finding that antibodies to a small region of a single protein play a major role in this process has important implications for malaria immunity and is strongly supportive of further understanding and development of MSP-1(19)-based vaccines.

Topics: Adult; Amino Acid Sequence; Animals; Antibodies, Protozoan; Antibody Specificity; Cell Division; Cell Line; Epidermal Growth Factor; Humans; Malaria, Falciparum; Merozoite Surface Protein 1; Mice; Molecular Sequence Data; Parasitic Sensitivity Tests; Peptide Fragments; Plasmodium chabaudi; Plasmodium falciparum; Protein Structure, Tertiary; Recombinant Fusion Proteins; Sequence Alignment; Transfection

Each of the four epidermal growth factor (EGF)-like domains of the Plasmodium falciparum sexual-stage antigen Pfs25 has been individually expressed as a yeast-secreted recombinant protein (yEGF1 through yEGF4). All four are recognized by the immune sera of animals and humans vaccinated with TBV25H (the corresponding yeast-secreted full-length recombinant form of Pfs25), with antibody titers to yEGF1 and yEGF2 weakly correlating with the ability of the sera to block the transmission of parasites to the mosquito host. All four proteins are poorly immunogenic in mice vaccinated with aluminum hydroxide-absorbed formulations. However, all four successfully primed the mice to mount an effective secondary antibody response after a single boost with TBV25H. Sera from mice vaccinated with yEGF2-TBV25H completely block the development of oocysts in mosquito midguts in membrane-feeding assays. Further, of the four proteins, only the depletion of antibodies to yEGF2 from the sera of rabbits vaccinated with TBV25H consistently abolished the ability of those sera to block oocyst development. Thus, antibodies to the second EGF-like domain of Pfs25 appear to mediate a very potent blocking activity, even at low titers. Vaccination strategies that target antibody response towards this domain may improve the efficacy of future transmission-blocking vaccines.

Topics: Animals; Antibodies, Protozoan; Culicidae; Epidermal Growth Factor; Humans; Malaria Vaccines; Malaria, Falciparum; Mice; Plasmodium falciparum; Protein Structure, Tertiary; Protozoan Proteins; Rabbits; Recombinant Proteins; Vaccination; Vaccines, Synthetic

A major protein found on the surface of the invasive stage of the malaria parasite Plasmodium falciparum, merozoite surface protein-1 (MSP1), has been proposed as a vaccine candidate. Antibodies which recognise a single fragment of this molecule (MSP1(19)), composed of 2 regions related to epidermal growth factor (EGF), also inhibit parasite growth in vitro. It is shown by direct expression of the individual EGF-like domains in Escherichia coli, that the first domain is the target of growth-inhibitory antibodies. A single amino acid difference influences the binding of some antibodies to this domain.

Topics: Amino Acid Sequence; Animals; Antibodies, Monoclonal; Antibodies, Protozoan; Antigens, Protozoan; Antigens, Surface; Base Sequence; DNA, Protozoan; Epidermal Growth Factor; Epitopes; Escherichia coli; Gene Expression; Malaria Vaccines; Malaria, Falciparum; Merozoite Surface Protein 1; Molecular Sequence Data; Plasmodium falciparum; Protein Precursors; Protozoan Proteins; Recombinant Fusion Proteins

Pfs25 is a sexual stage antigen of Plasmodium falciparum that is expressed on the surface of zygote and ookinete forms of the parasite. Monoclonal antibodies directed against native Pfs25 can block completely the development of P. falciparum oocysts in the midgut of the mosquito vector. Thus, this 25-kD protein is a potential vaccine candidate for eliciting transmission-blocking immunity in inhabitants of malaria endemic regions. We have synthesized, by secretion from yeast, a polypeptide analogue of Pfs25 that reacts with conformation-dependent monoclonal antibodies, and elicits transmission-blocking antibodies when used to immunize mice and monkeys in conjunction with a muramyl tripeptide adjuvant. Our results suggest the further evaluation of recombinant DNA-derived Pfs25 in transmission-blocking vaccination studies in humans.

Topics: Animals; Antibodies, Monoclonal; Antibodies, Protozoan; Antigens, Surface; Epidermal Growth Factor; Haplorhini; Immunization; Malaria, Falciparum; Mice; Mice, Inbred BALB C; Plasmodium falciparum; Protein Conformation; Protozoan Proteins; Vaccines, Synthetic