Rapid and precise epitope mapping of monoclonal antibodies against Plasmodium falciparum AMA1 by combined phage display of fragments and random peptides.

We describe an approach for the rapid mapping of epitopes within a malaria antigen using a combination of phage display techniques. Phage display of antigen fragments identifies the location of the epitopes, then random peptide libraries displayed on phage are employed to identify accurately amino acids involved in the epitope. Finally, phage display of mutant fragments confirms the role of each residue in the epitope. This approach was applied to the apical membrane antigen-1 (AMA1), which is a leading candidate for inclusion in a vaccine directed against the asexual blood stages of Plasmodium falciparum. As part of the effort both to understand the function of AMA1 in the parasite life cycle and to define the specificity of protective immune responses, a panel of monoclonal antibodies (MAbs) was generated to obtain binding reagents to the various domains within the molecule. There is a pressing need to determine rapidly the regions recognized by these antibodies and the structural requirements required within AMA1 for high affinity binding of the MAbs. Using phage displaying random AMA1 fragments, it was shown that MAb5G8 recognizes a short linear epitope within the pro-domain of AMA1 whereas the epitope recognized by MAb 1F9 is reduction sensitive and resides within a disulphide-bonded 57 amino acid sub-domain of domain-1. Phage displaying random peptide libraries and mutant AMA1 fragments were employed for fine mapping of the MAb5G8 core epitope to a three-residue sequence in the AMA1 prodomain.

Specificity of the protective antibody response to apical membrane antigen 1.

Apical membrane antigen 1 (AMA1) is considered one of the leading candidates for inclusion in a vaccine against blood stages of Plasmodium falciparum. Although the ama1 gene is relatively conserved compared to those for some other potential vaccine components, numerous point mutations have resulted in amino acid substitutions at many sites in the polypeptide. The polymorphisms in AMA1 have been attributed to the diversifying selection pressure of the protective immune responses. It was therefore of interest to investigate the impact of sequence diversity in P. falciparum AMA1 on the ability of anti-AMA1 antibodies to inhibit the invasion of erythrocytes in vitro by P. falciparum merozoites. For these studies, we used antibodies to recombinant P. falciparum 3D7 AMA1 ectodomain, which was prepared for testing in early clinical trials. Antibodies were raised in rabbits to the antigen formulated in Montanide ISA720, and human antibodies to AMA1 were isolated by affinity purification from the plasma of adults living in regions of Papua New Guinea where malaria is endemic. Both rabbit and human anti-AMA1 antibodies were found to be strongly inhibitory to the invasion of erythrocytes by merozoites from both the homologous and two heterologous lines of P. falciparum. The inhibitory antibodies targeted both conserved and strain-specific epitopes within the ectodomain of AMA1; however, it appears that the majority of these antibodies reacted with strain-specific epitopes in domain I, the N-terminal disulfide-bonded domain, which is the most polymorphic region of AMA1.

CD4+ T cells acting independently of antibody contribute to protective immunity to Plasmodium chabaudi infection after apical membrane antigen 1 immunization.

Apical membrane Ag 1 (AMA1) is a leading malaria vaccine candidate. Homologues of AMA1 can induce protection in mice and monkeys, but the mechanism of immunity is not understood. Mice immunized with a refolded, recombinant, Plasmodium chabaudi AMA1 fragment (AMA1B) can withstand subsequent challenge with P. chabaudi adami. Here we show that CD4+ T cell depletion, but not gammadelta T cell depletion, can cause a significant drop in antiparasite immunity in either immunized normal or immunized B cell KO mice. In normal mice, this loss of immunity is not accompanied by a decline in Ab levels. These observations indicate a role for AMA1-specific Ab-independent T cell-mediated immunity. However, the loss of immunity in normal CD4+ T cell-depleted mice is temporary. Furthermore, immunized B cell KO mice cannot survive infection, demonstrating the absolute importance of B cells, and presumably Ab, in AMA1-induced immunity. CD4+ T cells specific for a cryptic conserved epitope on AMA1 can adoptively transfer protection to athymic (nu/nu) mice, the level of which is enhanced by cotransfer of rabbit anti-AMA1-specific antisera. Recipients of rabbit antisera alone do not survive. Some protected recipients of T cells plus antisera do not develop their own AMA 1-specific Ab response, suggesting that AMA 1-specific CMI alone can protect mice. These data are the first to demonstrate the specificity of any protective CMI response in malaria and have important implications for developing a malaria vaccine.

Immunisation with recombinant AMA-1 protects mice against infection with Plasmodium chabaudi.

The Plasmodium merozoite surface antigen apical membrane antigen-1 (AMA-1) has previously been shown to provide partial protection to Saimiri and rhesus monkeys immunised with recombinant Plasmodium fragile or parasite-derived Plasmodium knowlesi AMA-1, respectively. In the study reported here we have used the Plasmodium chabaudi/mouse model system to extend our pre-clinical assessment of an AMA-1 vaccine. We describe here the expression of the full-length Plasmodium chabaudi adami AMA-1 and the P. chabaudi adami AMA-1 ectodomain using both baculovirus and Escherichia coli. The ectodomain expressed in E. coli, which contained an N-terminal hexa-his tag, was purified by Ni-chelate chromatography and refolded in vitro in the presence of oxidised and reduced glutathione to generate intramolecular disulphide bonds. In a series of vaccine trials, in both inbred and outbred mice, highly significant protection was obtained by immunising with the refolded AMA-1 ectodomain. Protection was shown to correlate with antibody response and was dependent on intact disulphide bonds. Passive transfer of antibodies raised in rabbits against the refolded AMA-1 ectodomain was also protective. In view of this demonstration that E. coli expression of a soluble P. chabaudi AMA-1 domain can generate a vaccine that is effective in mice, we are pursuing a similar approach to generating a vaccine against P. falciparum for testing in human volunteers.

Isolation from phage display libraries of single chain variable fragment antibodies that recognize conformational epitopes in the malaria vaccine candidate, apical membrane antigen-1.

Phage display of single chain variable fragment (scFv) antibodies is a powerful tool for the selection of important and useful antibody specificities. We have constructed such a library from mice protected from malaria challenge by immunization with recombinant Plasmodium chabaudi DS apical membrane antigen (AMA-1). Panning on refolded AMA-1 enriched a population of scFvs which specifically bound the antigen. The single chain antibodies recognize conformational epitopes on AMA-1 from the P. chabaudi DS strain but not on AMA-1 of the 556KA strain of P. chabaudi. A subset of the antibody fragments recognized AMA-1 from the human malaria parasite Plasmodium falciparum. Nucleotide sequencing revealed that at least four unique scFv genes were selected by the panning procedure. These scFv antibodies are valuable reagents for probing the structure and function of AMA-1 and will be used to test the feasibility of using recombinant antibodies in a passive immunization therapy against malaria.

A cryptic T cell epitope on the apical membrane antigen 1 of Plasmodium chabaudi adami can prime for an anamnestic antibody response: implications for malaria vaccine design.

We have investigated the proliferative and Th cell responses to the Plasmodium chabaudi adami DS homologue of the Plasmodium falciparum apical membrane Ag 1 (AMA-1), a leading malaria vaccine candidate. Immunodominant T cell epitopes were defined following immunization of BALB/c mice with Escherichia coli-expressed, refolded P. c. adami DS AMA-1 recombinant protein and testing cells from the draining lymph nodes for responses against a series of overlapping peptides spanning P. c. adami AMA-1. A limited number of major T cell sites were identified in both conserved and variable regions of the protein. Several cryptic epitopes that evoked T cell responses following immunization with peptides, but not after protein immunization, were also identified. Adoptive transfer of a T cell line specific for a conserved cryptic epitope (corresponding to residues 31-50) provided help for an anti-AMA-1 protein-specific Ab response following in vivo challenge with P. c. adami parasitized RBC, such that AMA-1-specific Abs appeared more rapidly in recipient mice than in controls. Furthermore, T cells specific for cryptic epitopes afforded partial protection against P. c. adami infection in nude mice. The identification of conserved cryptic Th cell epitopes has important implications for malaria vaccine design.

The disulfide bond structure of Plasmodium apical membrane antigen-1.

Apical membrane antigen-1 (AMA-1) of Plasmodium falciparum is one of the leading asexual blood stage antigens being considered for inclusion in a malaria vaccine. The ability of this molecule to induce a protective immune response has been shown to be dependent upon a conformation stabilized by disulfide bonds. In this study we have utilized the reversed-phase high performance liquid chromatography of dithiothreitol-reduced and nonreduced tryptic digests of Plasmodium chabaudi AMA-1 secreted from baculovirus-infected insect cells, in conjunction with N-terminal sequencing and electrospray-ionization mass spectrometry, to identify and assign disulfide-linked peptides. All 16 cysteine residues that are conserved in all known sequences of AMA-1 are incorporated into intramolecular disulfide bonds. Six of the eight bonds have been assigned unequivocally, whereas the two unassigned disulfide bonds connect two Cys-Xaa-Cys sequences separated by 14 residues. The eight disulfide bonds fall into three nonoverlapping groups that define three possible subdomains within the AMA-1 ectodomain. Although the pattern of disulfide bonds within subdomain III has not been fully elucidated, one of only two possible linkage patterns closely resembles the cystine knot motif found in growth factors. Sites of amino acid substitutions in AMA-1 that are well separated in the primary sequence are clustered by the disulfide bonds in subdomains II and III. These findings are consistent with the conclusion that these amino acid substitutions are defining conformational disulfide bond-dependent epitopes that are recognized by protective immune responses.

Protective immune responses to apical membrane antigen 1 of Plasmodium chabaudi involve recognition of strain-specific epitopes.

Apical membrane antigen 1 (AMA-1), an asexual blood-stage antigen of Plasmodium falciparum, is an important candidate for testing as a component of a malaria vaccine. This study investigates the nature of diversity in the Plasmodium chabaudi adami homolog of AMA-1 and the impact of that diversity on the efficacy of the recombinant antigen as a vaccine against challenge with a heterologous strain of P. chabaudi. The nucleotide sequence of the AMA-1 gene from strain DS differs from the published 556KA sequence at 79 sites. The large number of mutations, the nonrandom distribution of both synonymous and nonsynonymous mutations, and the nature of both the codon changes and the resulting amino acid substitutions suggest that positive selection operates on the AMA-1 gene in regions coding for antigenic sites. Protective immune responses induced by AMA-1 were strain specific. Immunization of mice with the refolded ectodomain of DS AMA-1 provided partial protection against challenge with virulent DS (homologous) parasites but failed to protect against challenge with avirulent 556KA (heterologous) parasites. Passive immunization of mice with rabbit antibodies raised against the same antigen had little effect on heterologous challenge but provided significant protection against the homologous DS parasites.

Protective immunity induced in squirrel monkeys with recombinant apical membrane antigen-1 of Plasmodium fragile.

Saimiri sciureus boliviensis monkeys were immunized with the Plasmodium fragile form of the merozoite apical membrane antigen-1 produced using the baculovirus expression system and combined with Montanide ISA 720 adjuvant. Following three immunizations, monkeys were challenged with 10,000 P. fragile trophozoite parasites. Antibody titers determined by fluorescence microscopy indicated an enhanced response following the second immunization. Four of five control animals had parasite counts > 5% 18-26 days following challenge. Four of five immunized monkeys had reduced levels of maximum parasitemia or delays in accumulated parasite counts, suggestive of protection. Rechallenge of the animals with P. falciparum resulted in three of four adjuvant control animals developing patent parasitemia whereas none of five immunized animals were infected, suggesting some level of heterologous protection.

Plasmodium falciparum: two antigens of similar size are located in different compartments of the rhoptry.

Two previously described antigens, AMA-1 and QF3, which are located in the rhoptries of Plasmodium falciparum merozoites have polypeptides of similar relative molecular masses. On immunoblots, antibodies to both antigens recognized polypeptides of relative molecular mass 80,000 and 62,000 in all isolates tested. Two-dimensional electrophoresis showed that the isoelectric points of the two antigens were different. QF3 being more basic than AMA-1. AMA-1 was soluble in Triton X-114 whereas QF3 partitioned into the aqueous phase after temperature-dependent phase separation. In immunoelectron microscopic studies. QF3 was found in the body of the rhoptry whereas AMA-1 was consistently found in the neck of the rhoptry. Both antigens gave a punctate double-dot pattern in mature schizonts and merozoites when visualized by fluorescence microscopy, but AMA-1 antibodies also appeared to label the merozoite surface. QF3 was also detected in ring-infected erythrocytes whereas AMA-1 was not. Synthesis of both antigens was first observed in mature trophozoites and immature schizonts. Pulse-chase experiments showed that the Mr 80,000 polypeptide of the AMA-1 gene was subject to immediate processing to the Mr 62,000 product. This cleavage pattern was not stage specific. The Mr 80,000 polypeptide of QF3 was derived from a short-lived Mr 84,000 precursor polypeptide. Processing of the Mr 80,000 polypeptide to an Mr 62,000 polypeptide was restricted to the period of merozoite maturation and reinvasion. Hence AMA-1 and QF3 are different antigens with polypeptides of similar size but located in different compartments of the merozoite rhoptries.

S-antigen localization in the erythrocytic stages of Plasmodium falciparum.

Localization of the S-antigen of Plasmodium falciparum isolate FCQ27/PNG, from Papua New Guinea, was studied by post-embedding immunoelectron microscopy using affinity-purified rabbit antibodies raised against the repeat region of the antigen. Labelling was found in the parasitophorous vacuole (PV) space of early to late schizonts and in PV-related vesicles within the erythrocyte cytoplasm of schizont-infected cells. Other subcellular structures within the erythrocyte cytoplasm were not labelled. After breakdown of the PV membrane, label was observed around the merozoites, consistent with mixing of the PV contents and erythrocyte cytoplasm. The antigen was not found in uninfected cells, ring stages, trophozoites or associated with free merozoites. Antibodies to FCQ27/PNG S-antigen did not react with other isolates tested, whereas rabbit antibodies to the Palo Alto/Wellcome S-antigen repeat region reacted with isolates FCR3 and ItG2F6 but not with FCQ27/PNG.

Integral membrane protein located in the apical complex of Plasmodium falciparum.

We describe the cloning of a novel antigen of Plasmodium falciparum which contains a hydrophobic domain typical of an integral membrane protein. This antigen is designated apical membrane antigen 1 because it appears to be located in the apical complex. Apical membrane antigen 1 appears to be transported to the merozoite surface near the time of schizont rupture.

Variation in p126, a parasitophorous vacuole antigen of Plasmodium falciparum.

We describe a cDNA clone expressing part of p126, a parasitophorous vacuole antigen of Plasmodium falciparum that is processed to smaller fragments about the time of schizont rupture. The amino acid sequence includes determinants that are antigenic during the course of infection. The sequence contains a string of serine residues but no other repetitive elements. Comparison of the sequence to a previously published sequence demonstrates conserved and variable sequence elements. The genomic context of the single copy gene is conserved in six different isolates. p126 is shown to be identical to Pf140, an antigen previously demonstrated to partially protect Saimiri monkeys against P. falciparum infection.

Patterns of antigen expression in asexual blood stages and gametocytes of Plasmodium falciparum.

The stage specificity and localization of 12 Plasmodium falciparum antigens were determined by immunofluorescence using acetone-fixed parasites reacted with monospecific antibodies against cloned antigens. Antibodies were prepared by immunization of rabbits with recombinant proteins or by affinity purification of human plasma against cloned antigen adsorbents. Most of the antigens occurred predominantly in mature asexual parasites, two were abundant in ring stages and three were absent in rings. Four of the 12 antigens were detected in asexual stages but not in gametocytes. Grouping of antigens by localization within blood stages was difficult because of the complexity of fluorescence patterns observed. With some antibodies, fluorescence was apparently distributed evenly over the parasites, but in other cases label was concentrated within discrete compartments or organelles. Extraparasitic intraerythrocytic fluorescence was also observed.

A second antigenic heat shock protein of Plasmodium falciparum.

We describe here an antigen of Plasmodium falciparum, defined by a cDNA clone designated Ag361. The antigen is a soluble cytoplasmic 70-kD polypeptide present in all isolates analyzed and in all stages of asexual development in the blood. The antigen is a natural immunogen, although it lacks repeating epitopes of many P. falciparum antigens. Ag361 shares extensive sequence homology with the hsp70 proteins of Xenopus laevis, Drosophila melanogaster, Escherichia coli, and man, as well as a previously isolated P. falciparum hsp70 protein. The genome of P. falciparum contains at least five hsp70-like genes, located on at lest four different chromosomes.

Structure of the FIRA gene of Plasmodium falciparum.

The falciparum interspersed repeat antigen (FIRA) plays a dominant role in the human antibody response to the malaria parasite Plasmodium falciparum. We have therefore determined the complete sequence of a genomic clone encoding FIRA. The FIRA gene contains a single intervening sequence, located immediately 3' to the putative hydrophobic core of a signal sequence in the short (100 amino acids) exon 1. The second exon largely encodes blocks of 13 hexapeptide repeats based loosely on the consensus sequence Pro-Val-Thr-Thr-Gln-Glu. The first block encoded 39 hexapeptides followed by about nine blocks of 13 hexapeptides interspersed between a conserved region of 81 amino acids, which is itself repeated along the molecule. Although deletions of repeats in this and four other independent clones make the exact number of blocks uncertain, this structure is supported by genomic blotting studies. As 31 variants of the repeat have been identified so far, we suggest that this extreme repeat variability must have important implications for the host immune response.

A cDNA clone expressing a rhoptry protein of Plasmodium falciparum.

Antibodies from immune humans were used to select a cDNA clone expressing an asexual blood stage antigen of Plasmodium falciparum. The expressed fused polypeptide was used as an affinity reagent to purify human antibodies specific for the corresponding parasite antigen. Western blotting and immunoelectronmicroscopy demonstrated that the antigen was a 105 kDa protein located in the rhoptries of merozoites. The cDNA encodes the carboxy terminus of the rhoptry antigen, a sequence rich both in charged and hydroxy amino acids.

The complete sequence of the gene for the knob-associated histidine-rich protein from Plasmodium falciparum.

'Knobs' at the surface of erythrocytes infected with mature stages of Plasmodium falciparum are believed to be important in adherence of these cells to capillary walls. They contain at least one parasite protein, designated the knob-associated histidine-rich protein (KAHRP). We present here the sequences of a cDNA and chromosomal clone that predict the complete sequence of KAHRP. The gene contains a single intervening sequence, located at the 3' boundary of the hydrophobic core of a putative signal sequence. Exon two encodes a short region that is rich in histidine as well as two separate regions of repetitive sequence, the 5' repeats (five copies related to SKKHKDNEDAESVK) and the 3' repeats (seven copies related to SKGATKEAST). These repeat blocks were both shown to bear epitopes recognized by the human immune system during natural infection by expressing them separately in Escherichia coli, and reacting human antibodies affinity-purified on lysates of the resulting clones with the corresponding synthetic oligopeptides. The 3' end of the molecule, presumably the repetitive region, is a site of size variation in KAHRP from different isolates.