Sequence of the immunodominant epitope for the surface protein on sporozoites of Plasmodium vivax

Plasmodium vivax is one of the four malaria parasites that cause disease in humans. The structure of the immunodominant repeating peptide of the circumsporozoite (CS) protein of P. vivax was determined. A fragment of P. vivax DNA that encodes this tandemly repeating epitope was isolated by use of an oligonucleotide probe whose sequence is thought to be conserved in CS protein genes. DNA sequence analysis of the P. vivax clone indicates that the CS repeat is nine amino acids in length (Gly-Asp-Arg-Ala-Asp-Gly-Gln-Pro-Ala). The structure of the repeating region was confirmed with synthetic peptides and monoclonal antibodies directed against P. vivax sporozoites. This information should allow synthesis of a vaccine for P. vivax that is similar to the one being tested for P. falciparum.     

Circumsporozoite protein of Plasmodium vivax: gene cloning and characterization of the immunodominant epitope

The gene encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium vivax has been cloned. The deduced sequence of the protein consists of 373 amino acids with a central region of 19 tandem repeats of the nonapeptide Asp-Arg-Ala-Asp/Ala-Gly-Gln-Pro-Ala-Gly. A synthetic 18-amino acid peptide containing two tandem repeats binds to a monoclonal antibody directed to the CS protein of Plasmodium vivax and inhibits the interaction of this antibody with the native protein in sporozoite extracts. The portions of the CS gene that do not contain repeats are closely related to the corresponding regions of the CS genes of two simian malarias, Plasmodium cynomolgi and Plasmodium knowlesi. In contrast, the homology between the CS genes of Plasmodium vivax and Plasmodium falciparum, another malaria parasite of humans, is very limited.     

Expression of Plasmodium falciparum circumsporozoite proteins in Escherichia coli for potential use in a human malaria vaccine

The circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum may be the most promising target for the development of a malaria vaccine. In this study, proteins composed of 16, 32, or 48 tandem copies of a tetrapeptide repeating sequence found in the CS protein were efficiently expressed in the bacterium Escherichia coli. When injected into mice, these recombinant products resulted in the production of high titers of antibodies that reacted with the authentic CS protein on live sporozoites and blocked sporozoite invasion of human hepatoma cells in vitro. These CS protein derivatives are therefore candidates for a human malaria vaccine.     

DNA cloning of Plasmodium falciparum circumsporozoite gene: amino acid sequence of repetitive epitope

A clone of complementary DNA encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum has been isolated by screening an Escherichia coli complementary DNA library with a monoclonal antibody to the CS protein. The DNA sequence of the complementary DNA insert encodes a four-amino acid sequence: proline-asparagine-alanine-asparagine, tandemly repeated 23 times. The CS beta-lactamase fusion protein specifically binds monoclonal antibodies to the CS protein and inhibits the binding of these antibodies to native Plasmodium falciparum CS protein. These findings provide a basis for the development of a vaccine against Plasmodium falciparum malaria.     

Protection against malaria by vaccination with sporozoite surface protein 2 plus CS protein.

The circumsporozoite (CS) protein has been the target for development of malaria sporozoite vaccines for a decade. However, immunization with subunit vaccines based on the CS protein has never given the complete protection found after immunization with irradiated sporozoites. BALB/c mice immunized with irradiated Plasmodium yoelii sporozoites produced antibodies and cytotoxic T cells against a 140-kilodalton protein, sporozoite surface protein 2 (SSP2). Mice immunized with P815 cells that had been transfected with either SSP2 or CS genes were partially protected, and those immunized with a mixture of SSP2 and CS transfectants were completely protected against malaria. These studies emphasize the importance of vaccine delivery systems in achieving protection and define a multi-antigen sporozoite vaccine.     

Crystal structure of the malaria vaccine candidate apical membrane antigen 1

Apical membrane antigen 1 from Plasmodium is a leading malaria vaccine candidate. The protein is essential for host-cell invasion, but its molecular function is unknown. The crystal structure of the three domains comprising the ectoplasmic region of the antigen from P. vivax, solved at 1.8 angstrom resolution, shows that domains I and II belong to the PAN motif, which defines a superfamily of protein folds implicated in receptor binding. We also mapped the epitope of an invasion-inhibitory monoclonal antibody specific for the P. falciparum ortholog and modeled this to the structure. The location of the epitope and current knowledge on structure-function correlations for PAN domains together suggest a receptor-binding role during invasion in which domain II plays a critical part. These results are likely to aid vaccine and drug design.     

Immunogenicity of synthetic peptides from circumsporozoite protein of Plasmodium falciparum

In a study of recombinant proteins that might be useful in developing a vaccine against malaria, synthetic peptides from the circumsporozoite (CS) protein of Plasmodium falciparum were found to be immunogenic for mice and rabbits. Antibody to peptides from the repeating region of the CS protein recognized native CS protein and blocked sporozoite invasion of human hepatoma cells in vitro. Antibodies to peptides from regions I and II had no biologic activity, although antibody to region I recognized processed CS protein by Western blot analysis. These data support the feasibility of developing a vaccine against the sporozoite stage of the malaria parasite by using synthetic peptides of the repeating region of the CS protein conjugated to a carrier protein.     

Conserved repetitive epitope recognized by CD4+ clones from a malaria-immunized volunteer

T cell clones obtained from a human volunteer immunized with Plasmodium falciparum sporozoites specifically recognized the native circumsporozoite (CS) antigen expressed on P. falciparum sporozoites, as well as bacteria- and yeast-derived recombinant falciparum CS proteins. The response of these CD4+ CD8- cells was species-specific, since the clones did not proliferate or secrete gamma interferon when challenged with sporozoites or recombinant CS proteins of other human, simian, or rodent malarias. The epitope recognized by the sporozoite-specific human T cell clones mapped to the 5' repeat region of the CS protein and was contained in the NANPNVDPNANP sequence.     

Rationale for development of a synthetic vaccine against Plasmodium falciparum malaria

Protective immunity against malaria can be obtained by vaccination with irradiated sporozoites. The protective antigens known as circumsporozoite (CS) proteins, are polypeptides that cover the surface membrane of the parasite. The CS proteins contain species-specific immunodominant epitopes formed by tandem repeated sequences of amino acids. Here it is shown that the dominant epitope of Plasmodium falciparum is contained in the synthetic dodecapeptide Asn-Ala-Asn-Pro-Asn-Ala-Asn-Pro-Asn-Ala-Pro or (NANP)3. Monoclonal antibodies and most or all polyclonal human antibodies to the sporozoites react with (NANP)3, and polyclonal antibodies raised against the synthetic peptide (NANP)3 react with the surface of the parasite and neutralize its infectivity. Since (NANP)3 repeats are present in CS proteins of P. falciparum from many parts of the world, this epitope is a logical target for vaccine development.     

Limited immunological recognition of critical malaria vaccine candidate antigens

Current vaccine development strategies for malaria depend on widespread immunological responsiveness to candidate antigens such as the zygote surface antigens and the sporozoite coat protein, the circumsporozoite (CS) protein. Since immunological responsiveness is controlled mainly by genes mapping within the major histocompatibility complex (MHC), the humoral immune response to the zygote surface antigens and the cytotoxic T lymphocyte (CTL) response to the CS protein were examined in MHC-disparate congenic mouse strains. Only two of six strains responded to the 230-kilodalton zygote surface antigen and another two strains responded to the 48/45-kilodalton surface antigen. From two mouse strains, expressing between them five different class I MHC molecules, there was recognition of only a single CTL epitope from the CS protein, which was from a polymorphic segment of the molecule. The restricted CTL response to this protein parallels the restricted antibody response to this protein observed in humans and mice. These findings suggest that subunit malaria vaccines now being developed may be ineffective.     

Isolation of the gene for a glycophorin-binding protein implicated in erythrocyte invasion by a malaria parasite

Plasmodium falciparum, the most lethal of the malarial parasites that infect humans, undergoes three cycles of development in its vertebrate host and elicits stage-specific immune responses. This stage specificity of the immune response has made it difficult to isolate antigens that would be useful in developing a vaccine against malaria. A complementary DNA clone for a glycophorin-binding protein of Plasmodium falciparum merozoites has been isolated and characterized. The protein interacts with glycophorin, the erythrocyte receptor, during invasion of the host cell by the parasite. Antigenic determinants of this protein expressed in Escherichia coli have been used to produce antibodies to a glycophorin-binding protein. The antibodies show schizont-specific immunofluorescence and react with the merozoite protein. The primary sequence of these determinants reveals a 150-nucleotide tandem-repeating sequence coding for a 50-amino-acid repeat. The characterization of the Plasmodium falciparum glycophorin-binding protein represents one approach toward designing serologic agents to block the parasite's development in the vertebrate host.     

Oral Salmonella typhimurium vaccine expressing circumsporozoite protein protects against malaria

Immunization with radiation-attenuated malaria sporozoites induces potent cellular immune responses, but the target antigens are unknown and have not previously been elicited by subunit vaccines prepared from the circumsporozoite (CS) protein. A method is described here for inducing protective cell-mediated immunity to sporozoites by immunization with attenuated Salmonella typhimurium transformed with the Plasmodium berghei CS gene. These transformants constitutively express CS antigens and, when used to immunize mice orally, colonize the liver, induce antigen-specific cell-mediated immunity, and protect mice against sporozoite challenge in the absence of antisporozoite antibodies. These data indicate that the CS protein contains T cell epitopes capable of inducing protective cell-mediated immunity, and emphasize the importance of proper antigen presentation in generating this response. Analogous, orally administered vaccines against human malaria might be feasible.     

Cell-adhesive motif in region II of malarial circumsporozoite protein

The segment of the malarial circumsporozoite (CS) protein designated Region II is highly conserved among different malarial species. A similar sequence is also present in several other proteins, including thrombospondin, properdin, and a blood-stage antigen of Plasmodium falciparum. By means of peptides synthesized from sequences of the Plasmodium vivax CS protein in the vicinity of Region II, it was found that two overlapping 18- to 20-amino acid peptides promoted the adhesion of a variety of human hematopoietic cell lines. The amino acid sequence valine-threonine-cysteineglycine (VTCG), contained within this common motif, was shown to be the critical sequence for the observed cell-adhesive properties.     

Human monoclonal antibodies to Pf 155, a major antigen of malaria parasite Plasmodium falciparum

Pf 155, a protein of the human malaria parasite Plasmodium falciparum, is strongly immunogenic in humans and is believed to be a prime candidate for the preparation of a vaccine. Human monoclonal antibodies to Pf 155 were obtained by cloning B cells that had been prepared from an immune donor and transformed with Epstein-Barr virus. When examined by indirect immunofluorescence, these antibodies stained the surface of infected erythrocytes, free merozoites, segmented schizonts, and gametocytes. They bound to a major polypeptide with a relative molecular weight of 155K and to two minor ones (135K and 120K), all having high affinity for human glycophorin. The antibodies strongly inhibited merozoite reinvasion in vitro, suggesting that they might be appropriate reagents for therapeutic administration in vivo.     

Plasmodium vivax transmitted from man to monkey to man

Blood forms of human vivax malaria infected splenectomized night monkeys (Aotus trivirgatus). Anopheles albimanus mosquitoes transmitted the in fection from a monkey to two human volunteers; parasites and symptoms ap peared 11 days later. Blood forms of vivax malaria from each of the two humans infected other night monkeys.     

Naturally acquired antibodies to sporozoites do not prevent malaria: vaccine development implications

The first human vaccines against the malaria parasite have been designed to elicit antibodies to the circumsporozoite protein of Plasmodium falciparum. However, it is not known whether any level of naturally acquired antibodies to the circumsporozoite protein can predict resistance to Plasmodium falciparum malaria. In this study, 83 adults in a malaria-endemic region of Kenya were tested for circumsporozoite antibodies and then treated for malaria. They were monitored for the development of new malaria infections for 98 days. Antibody levels, as determined by four assays in vitro, were indistinguishable between the 60 individuals who did and the 23 who did not develop parasitemia during follow-up, and there was no apparent relation between day of onset of parasitemia and level of antibodies to circumsporozoite protein. Unless immunization with sporozoite vaccines induces antibodies that are quantitatively or qualitatively superior to the circumsporozoite antibodies in these adults, it is unlikely that such antibodies will prevent infection in areas with as intense malaria transmission as western Kenya.     

Genetic selection of a Plasmodium-refractory strain of the malaria vector Anopheles gambiae

The anopheline mosquito is the target in most malaria control programs, primarily through the use of residual insecticides. A mosquito was studied that is refractory to most species of malaria through a genetically controlled mechanism. A strain of Anopheles gambiae, which was selected for complete refractoriness to the simian malaria parasite Plasmodium cynomolgi, also has varying degrees of refractoriness to most other malaria species examined, including the human parasites P. falciparum, P. ovale, and P. vivax for which this mosquito is the principal African vector. Furthermore, the refractoriness extends to other subhuman primate malarias, to rodent malaria, and to avian malaria. Refractoriness is manifested by encapsulation of the malaria ookinete after it completes its passage through the mosquito midgut, approximately 16 to 24 hours after ingestion of an infective blood meal. Fully encapsulated ookinetes show no abnormalities in parasite organelles, suggesting that refractoriness is due to an enhanced ability of the host to recognize the living parasite rather than to a passive encapsulation of a dead or dying parasite. Production of fully refractory and fully susceptible mosquito strains was achieved through a short series of selective breeding steps. This result indicates a relatively simple genetic basis for refractoriness. In addition to the value these strains may serve in general studies of insect immune mechanisms, this finding encourages consideration of genetic manipulation of natural vector populations as a malaria control strategy.     

Induction of Plasmodium falciparum transmission-blocking antibodies by recombinant vaccinia virus

Many candidate antigens of malaria vaccines have limited immunological recognition. One exception is Pfs25, a cysteine-rich, 25-kilodalton sexual stage surface protein of Plasmodium falciparum. Pfs25 is a target of monoclonal antibodies that block transmission of malaria from vertebrate host to mosquito vector. The surface of mammalian cells infected with a recombinant vaccinia virus that expressed Pfs25 specifically bound transmission-blocking monoclonal antibodies. Furthermore, major histocompatibility complex-disparate congenic mouse strains immunized with recombinant Pfs25 elicited transmission-blocking antibodies, demonstrating that the capacity to develop transmission-blocking antibodies is not genetically restricted in mice. Live recombinant viruses may provide an inexpensive, easily administered alternative to subunit vaccines prepared from purified recombinant proteins to block transmission of malaria in developing countries.     

Natural malaria infection in Anopheles gambiae is regulated by a single genomic control region

We surveyed an Anopheles gambiae population in a West African malaria transmission zone for naturally occurring genetic loci that control mosquito infection with the human malaria parasite, Plasmodium falciparum. The strongest Plasmodium resistance loci cluster in a small region of chromosome 2L and each locus explains at least 89% of parasite-free mosquitoes in independent pedigrees. Together, the clustered loci form a genomic Plasmodium-resistance island that explains most of the genetic variation for malaria parasite infection of mosquitoes in nature. Among the candidate genes in this chromosome region, RNA interference knockdown assays confirm a role in Plasmodium resistance for Anopheles Plasmodium-responsive leucine-rich repeat 1 (APL1), encoding a leucine-rich repeat protein that is similar to molecules involved in natural pathogen resistance mechanisms in plants and mammals.     

Construction of synthetic immunogen: use of new T-helper epitope on malaria circumsporozoite protein

The circumsporozoite (CS) protein of Plasmodium falciparum is the focus of intense efforts to develop an antisporozoite malaria vaccine. Localization of sites for T-cell recognition on this molecule is critical for vaccine design. By using an algorithm designed to predict T-cell sites and a large panel of H-2 congenic mice, a major nonrepetitive T-cell site was located. When a synthetic peptide corresponding to this site was covalently linked to the major B-cell site on the molecule, an immunogen capable of eliciting a high-titer antibody response was formed. This peptide sequence could prime helper T cells for a secondary response to the intact CS protein. The new helper T-cell site is located outside the repetitive region of the CS protein and appears to be the immunodominant T site on the molecule. This approach should be useful in the rational design and construction of vaccines.