Reduced immunogenicity of DNA vaccine plasmids in mixtures.

We measured the ability of nine DNA vaccine plasmids encoding candidate malaria vaccine antigens to induce antibodies and interferon-gamma responses when delivered alone or in a mixture containing all nine plasmids. We further examined the possible immunosuppressive effect of individual plasmids, by assessing a series of mixtures in which each of the nine vaccine plasmids was replaced with a control plasmid. Given alone, each of the vaccine plasmids induced significant antibody titers and, in the four cases for which appropriate assays were available, IFN-gamma responses. Significant suppression or complete abrogation of responses were seen when the plasmids were pooled in a nine-plasmid cocktail and injected in a single site. Removal of single genes from the mixture frequently reduced the observed suppression. Boosting with recombinant poxvirus increased the antibody response in animals primed with either a single gene or the mixture, but, even after boosting, responses were higher in animals primed with single plasmids than in those primed with the nine-plasmid mixture. Boosting did not overcome the suppressive effect of mixing for IFN-gamma responses. Interactions between components in a multiplasmid DNA vaccine may limit the ability to use plasmid pools alone to induce responses against multiple targets simultaneously.

Codon optimization of gene fragments encoding Plasmodium falciparum merzoite proteins enhances DNA vaccine protein expression and immunogenicity in mice.

In contrast to conventional vaccines, DNA and other subunit vaccines exclusively utilize host cell molecules for transcription and translation of proteins. The adenine plus thymine content of Plasmodium falciparum gene sequences (approximately 80%) is much greater than that of Homo sapiens (approximately 59%); consequently, codon usage is markedly different. We hypothesized that modifying codon usage of P. falciparum genes encoded by DNA vaccines from that used by the parasite to those resembling mammalian codon usage would lead to increased P. falciparum protein expression in vitro in mouse cells and increased antibody responses in DNA-vaccinated mice. We synthesized gene fragments encoding the receptor-binding domain of the 175-kDa P. falciparum erythrocyte-binding protein (EBA-175 region II) and the 42-kDa C-terminal processed fragment of the P. falciparum merozoite surface protein 1 (MSP-1(42)) using the most frequently occurring codon in mammals to code for each amino acid, and inserted the synthetic genes in DNA vaccine plasmids. In in vitro transient-expression assays, plasmids containing codon-optimized synthetic gene fragments (pS plasmids) showed greater than fourfold increased protein expression in mouse cells compared to those containing native gene fragments (pN plasmids). In mice immunized with 0.5, 5.0, or 50 microg of the DNA plasmids, the dose of DNA required to induce equivalent antibody titers was 10- to 100-fold lower for pS than for pN plasmids. These data demonstrate that optimizing codon usage in DNA vaccines can improve protein expression and consequently the immunogenicity of gene fragments in DNA vaccines for organisms whose codon usage differs substantially from that of mammals.

Erythrocyte-binding activity of Plasmodium yoelii apical membrane antigen-1 expressed on the surface of transfected COS-7 cells.

Malaria merozoite surface and apical organellar molecules facilitate invasion into the host erythrocyte. The underlying molecular mechanisms of invasion are poorly understood, and there are few data to delineate roles for individual merozoite proteins. Apical membrane antigen-1 (AMA-1) is a conserved apicomplexan protein present in the apical organelle complex and at times on the surface of Plasmodium and Toxoplasma zoites. AMA-1 domains 1/2 are conserved between Plasmodium and Toxoplasma and have similarity to the defined ligand domains of MAEBL, an erythrocyte-binding protein identified from Plasmodium yoelii. We expressed selected portions of the AMA-1 extracellular domain on the surface of COS-7 cells to assay for erythrocyte-binding activity. The P. yoelii AMA-1 domains 1/2 mediated adhesion to mouse and rat erythrocytes, but not to human erythrocytes. Adhesion to rodent erythrocytes was sensitive to trypsin and chymotrypsin, but not to neuraminidase. Other parts of the AMA-1 ectodomain, including the full-length extracellular domain, mediated significantly less erythrocyte adhesion activity than the contiguous domains 1/2. The results support the role of AMA-1 as an adhesion molecule during merozoite invasion of erythrocytes and identify highly conserved domains 1/2 as the principal ligand of the Plasmodium AMA-1 and possibly the Toxoplasma AMA-1. Identification of the AMA-1 ligand domains involved in interaction between the parasite and host cell should help target the development of new therapies to block growth of the blood-stage malaria parasites.

Induction of biologically active antibodies in mice, rabbits, and monkeys by Plasmodium falciparum EBA-175 region II DNA vaccine.

BACKGROUND: Plasmodium falciparum merozoites bind to and invade human erythrocytes via specific erythrocyte receptors. This establishes the erythrocytic stage of the parasite life cycle that causes clinical disease resulting in 2-3 million deaths per year. We tested the hypothesis that a Plasmodium falciparum ligand, EBA-175 region II (RII), which binds its erythrocyte receptor glycophorin A during invasion, can be used as an immunogen to induce antibodies that block the binding of RII to erythrocytes and thereby inhibit parasite invasion of erythrocytes. Accordingly, we immunized mice, rabbits, and monkeys with DNA plasmids that encoded the 616 amino acid RII. MATERIALS AND METHODS: DNA vaccine plasmids that targeted the secretion of recombinant RII protein with and without the universal T-cell helper epitopes P2P30 were used to immunize mice, rabbits, and Aotus monkeys. RII specific antibodies were assessed by IFA, ELISA, blocking of native [35S] labeled EBA-175 binding to human erythrocytes, and growth inhibition assays, all in vitro. RESULTS: The RII DNA plasmids were highly immunogenic as measured by ELISA and IFA. The anti-RII antibodies blocked the binding of native EBA-175 to erythrocytes, and rosetting of erythrocytes on COS-7 cells expressing RII. Most important, murine and rabbit anti-RII antibodies inhibited the invasion of merozoites into erythrocytes. We immunized nonhuman primates and showed that the RII-DNA plasmids were immunogenic and well tolerated in these monkeys. Monkeys were challenged with parasitized erythrocytes; one of three monkeys that received RII DNA plasmid was protected from fulminant disease. After challenge with live parasites, anti-RII antibody titers were boosted in the immunized monkeys. CONCLUSIONS: By proving the hypothesis that anti-RII antibodies can block merozoite invasion of erythrocytes, these studies pave the way for the clinical evaluation of EBA-175 as a receptor-blockade vaccine.

Endostatin binds tropomyosin. A potential modulator of the antitumor activity of endostatin.

The mechanism of action of Endostatin, an endogenous inhibitor of angiogenesis and tumor growth, remains unknown. We utilized phage-display technology to identify polypeptides that mimic the binding domains of proteins with which Endostatin interacts. A conformed peptide (E37) was identified that shares an epitope with human tropomyosin implicating tropomyosin as an Endostatin-binding protein. We show that recombinant human Endostatin binds tropomyosin in vitro and to tropomyosin-associated microfilaments in a variety of endothelial cell types. The most compelling evidence that tropomyosin modulates the activity of Endostatin was demonstrated when E37 blocked greater than 84% of the tumor-growth inhibitory activity of Endostatin in the B16-BL6 metastatic melanoma model. We conclude that the E37 peptide mimics the Endostatin-binding epitope of tropomyosin and blocks the antitumor activity of Endostatin by competing for Endostatin binding. We postulate that the Endostatin interaction with tropomyosin results in disruption of microfilament integrity leading to inhibition of cell motility, induction of apoptosis, and ultimately inhibition of tumor growth.

Sequence diversity and antigenic polymorphism in the Plasmodium yoelii p235 high molecular mass rhoptry proteins and their genes.

A gene family in Plasmodium yoelii YM encodes p235, a group of high molecular mass erythrocyte-binding rhoptry proteins. Sequence analysis of 6 cDNA clones from the 3' end of expressed p235 genes divided them into two groups corresponding to genes on chromosomes 1, and 5 and 6, respectively. Twelve partial p235 protein sequences, derived from cDNA sequences from the region with greatest protein sequence similarity to Plasmodium vivax RBP2, fell into three groups, together with one chimeric sequence. A comparison of these cDNA sequences with genomic DNA sequences from the same region suggested that only a subset of the gene repertoire is expressed. Three genomic DNA clones, derived from the 5' end of p235 genes designated E1, E2, and E5 and located on chromosome 5/6, were also obtained and aligned with sequences from the known E8 and E3 genes. In the region of overlap there was only approximately 27% protein sequence identity, indicating that the sequences in this p235 N-terminal region are more diverse than at the C-terminal end. This sequence variation in the expressed genes did not result in antigenically different rhoptry proteins as detected with a panel of p235-specific mAbs. Only one schizont out of 500 examined with mAb 25.86 appeared to be an antigenic variant, with all of the developing merozoites in this schizont being mAb 25.86 negative. No other antigenic variants were detected with the other antibodies, and therefore it is likely that these antibodies recognise conserved epitopes.

Protection of Aotus monkeys by Plasmodium falciparum EBA-175 region II DNA prime-protein boost immunization regimen.

Aotus monkeys received 4 doses of Plasmodium falciparum EBA-175 region II vaccine as plasmid DNA (Dv-Dv) or recombinant protein in adjuvant (Pv-Pv) or as 3 doses of DNA and 1 dose of protein (Dv-Pv). After 3 doses, antibody titers were approximately 10(4) in DNA-immunized monkeys and 10(6) in protein-immunized monkeys. A fourth dose did not significantly boost antibody responses in the Dv-Dv only or Pv-Pv only groups, but titers were boosted to approximately 10(6) in monkeys in the Dv-Pv group. Four weeks after the last immunization, the animals were challenged with 10(4) P. falciparum-parasitized erythrocytes. Peak levels of parasitemia were lower in the 16 monkeys that received region II-containing plasmids or proteins than in the 16 controls (geometric mean: 194,178 and 410,110 parasites/microL, respectively; P=.013, Student's t test). Three of 4 monkeys in the Dv-Pv group did not require treatment. These data demonstrate that immunization with EBA-175 region II induces a significant antiparasite effect in vivo.

Plasmodium yoelii: effects of red blood cell modification and antibodies on the binding characteristics of the 235-kDa rhoptry protein.

The 235-kDa rhoptry protein of the rodent malaria parasite Plasmodium yoelii yoelii was shown to bind to the surface of mouse red blood cells in a calcium-independent process, using a erythrocyte-binding assay. This binding is affected by modification of the surface of the red blood cells by enzymatic treatment. Chymotrypsin and trypsin but not neuraminidase treatment of the erythrocytes significantly reduced the binding of the 235-kDa proteins. The binding of an unrelated 135-kDa protein was abolished by treatment with chymotrypsin. Although the 235-kDa proteins bind to both reticulocytes and mature red blood cells, the binding to mature cells was more pronounced. In the presence of hyperimmune infection serum or specific polyclonal antibodies to the 235-kDa protein its binding to erythrocytes was reduced, further demonstrating the specificity of this ligand-receptor interaction.

A recombinant baculovirus-expressed Plasmodium falciparum receptor-binding domain of erythrocyte binding protein EBA-175 biologically mimics native protein.

EBA-175 of Plasmodium falciparum is a merozoite ligand that binds its receptor glycophorin A on erythrocytes during invasion. The ligand-receptor interaction is dependent on sialic acids as well as the protein backbone of glycophorin A. Region II (RII) of EBA-175 has been defined as the receptor-binding domain. RII is divided into regions F1 and F2, which contain duplicated cysteine motifs. We expressed RII in a baculovirus and show that RII binds erythrocytes with a specificity identical to that of the native protein. We found that, consistent with the binding of erythrocytes to COS cells expressing F2, recombinant baculovirus-expressed F2 bound erythrocytes. About 20% of all baculovirus-expressed RII is N-glycosylated, unlike native P. falciparum proteins that remain essentially unglycosylated. However, glycosylation of recombinant RII did not affect its immunogenicity. Antibodies raised against both glycosylated and unglycosylated baculovirus-expressed RII recognized P. falciparum schizonts in immunofluorescence assays and also gave similar enzyme-linked immunosorbent assay titers. Furthermore, these antibodies have similar abilities to block native EBA-175 binding to erythrocytes. These results allow the development of RII as a vaccine candidate for preclinical assessment.

Immunization with parasite-derived apical membrane antigen 1 or passive immunization with a specific monoclonal antibody protects BALB/c mice against lethal Plasmodium yoelii yoelii YM blood-stage infection.

We have purified apical merozoite antigen 1 (AMA-1) from extracts of red blood cells infected with the rodent malaria parasite Plasmodium yoelii yoelii YM. When used to immunize mice, the protein induced a strong protective response against a challenge with the parasite. Monoclonal antibodies specific for P. yoelii yoelii AMA-1 were prepared, and one was very effective against the parasite on passive immunization. A second protein that appears to be located in the apical rhoptry organelles and associated with AMA-1 was identified.

Antibodies against the Plasmodium falciparum receptor binding domain of EBA-175 block invasion pathways that do not involve sialic acids.

The 175-kDa Plasmodium falciparum erythrocyte binding protein (EBA-175) binds to its receptor, sialic acids on glycophorin A. The binding region within EBA-175 is a cysteine-rich region identified as region II. Antibodies against region II block the binding of native EBA-175 to erythrocytes. We identified a P. falciparum strain, FVO, that could not invade erythrocytes devoid of sialic acids due to prior neuraminidase treatment, and in addition, we used a strain, 3D7, that could invade such sialic acid-depleted erythrocytes. We used these two strains to study the capacity of anti-region II antibodies to inhibit FVO and 3D7 parasite development in vitro. Analysis of growth-inhibitory effects of purified FVO anti-region II immunoglobulin G (IgG) with the FVO and 3D7 strains resulted in similar levels of growth inhibition. FVO and 3D7 strains were inhibited between 28 and 56% compared to control IgG. There appeared to be no intracellular growth retardation or killing of either isolate, suggesting that invasion was indeed inhibited. Incubation of recombinant region II with anti-region II IgG reversed the growth inhibition. These results suggest that antibodies against region II can also interfere with merozoite invasion pathways that do not involve sialic acids. The fact that EBA-175 has such a universal and yet susceptible role in erythrocyte invasion clearly supports its inclusion in a multivalent malaria vaccine.

Precise timing of expression of a Plasmodium falciparum-derived transgene in Plasmodium berghei is a critical determinant of subsequent subcellular localization.

The development of transfection technology for malaria parasites holds significant promise for a more detailed characterization of molecules targeted by vaccines or drugs. One asexual blood stage vaccine candidate, apical membrane antigen-1 (AMA-1) of merozoite rhoptries has been shown to be the target of inhibitory, protective antibodies in both in vitro and in vivo studies. We have investigated heterologous (trans-species) expression of the human malaria Plasmodium falciparum AMA-1 (PF83/AMA-1) in the rodent parasite Plasmodium berghei. Transfected P. berghei expressed correctly folded and processed PF83/AMA-1 under control of both pb66/ama-1 and dhfr-ts promoters. Timing of expression was highly promoter-dependent and was critical for subsequent subcellular localization. Under control of pb66/ama-1, PF83/AMA-1 expression and localization in P. berghei was limited to the rhoptries of mature schizonts, similar to that observed for PF83/AMA-1 in P. falciparum. In contrast the dhfr-ts promoter permitted PF83/AMA-1 expression throughout schizogony as well as in gametocytes and gametes. Localization was aberrant and included direct expression at the merozoite and gamete surface. Processing from the full-length 83-kDa protein to a 66-kDa protein was observed not only in schizonts but also in gametocytes, indicating that processing could be mediated outside of rhoptries by a common protease. Trans-species expressed PF83/AMA-1 was highly immunogenic in mice, resulting in a response against a functionally critical domain of the molecule.

Analysis of the processing of Plasmodium falciparum rhoptry-associated protein 1 and localization of Pr86 to schizont rhoptries and p67 to free merozoites.

The processing and localization of Plasmodium falciparum rhoptry-associated protein 1 (RAP-1) products were examined using polyclonal and monoclonal antibodies raised to a recombinant protein containing residues 1-294 of RAP-1. Immunoblot and epitope mapping results with antibodies that selectively bound epitopes in the RAP-1 products Pr86, p82, and p67 showed that p82 and p67 are formed from Pr86 by progressive removal of epitopes from the amino-terminus of the RAP-1 coding sequence. The capacity of Pr86 to form complexes was revealed after size fractionation of parasite proteins radiolabeled in the presence of brefeldin A to prevent processing of Pr86. Fractions containing complexed Pr86 also contained the RAP-2 product p39 and the RAP-3 product p37, suggesting that Pr86, p39 and p37 may form complexes similar to complexes previously reported for p82 and p67 with p39 or p37. Immunofluorescence localization and immunoblot studies revealed that Pr86 is present in the rhoptries, but only transiently, and that it is not detected in segmenting schizonts or extracellular merozoites. p67 and p82, on the other hand, were shown to be major RAP-1 components in purified merozoites. Neither p67 nor p82 were relocalized from the intracellular rhoptries to the merozoite surface under conditions that promoted relocalization of the rhoptry protein PF83/apical membrane antigen 1. These results suggest that processing of Pr86 begins after Pr86 complexes are transported to the forming rhoptries and that two site-selective processing reactions occur in the rhoptries, a rapid cleavage of Pr86 to p82 and a delayed cleavage of p82 to p67. Since p67 is missing from ring-stage parasites (Howard et al., Am J Trop Med Hyg, 1984;33:1055 59), the present results indicate there is a narrow time during which p67 may play a role in merozoite invasion of erythrocytes.

Immunization of Aotus monkeys with recombinant Plasmodium falciparum hybrid proteins does not reproducibly result in protection from malaria infection.

Plasmodium falciparum antigens SERP, HRPII, MSAI, and 41-3 have shown promise as vaccine components. This study aimed at reproducing and extending previous results using three hybrid molecules. Antibody responses were reproduced in Aotus monkeys, but solid protection from a P. falciparum blood-stage challenge that showed an unintendedly enhanced pathogenicity was not observed.

High prevalence of natural antibodies against Plasmodium falciparum 83-kilodalton apical membrane antigen (PF83/AMA-1) as detected by capture-enzyme-linked immunosorbent assay using full-length baculovirus recombinant PF83/AMA-1.

The 83-kilodalton (kD) apical membrane antigen of Plasmodium falciparum (PF83/AMA-1) is a potential asexual blood stage vaccine component. This antigen has been expressed as a full-length, nonfusion, recombinant baculovirus protein (PF83-7G8-1) using the authentic predicted signal peptide for appropriate postsynthetic routing. When purified by a novel high-performance, ion exchange chromatography (HPIEC) method, PF83-7G8-1 induced polyclonal antibodies in rats that immunoprecipitated both 83- and 66-kD forms of PF83/AMA-1 from 35S-methionine metabolically labeled parasite extracts. Using HPIEC-purified PF83-7G8-1 in combination with a rat monoclonal antibody against the highly conserved carboxy-terminal (CT) region of PF83/AMA-1, we developed a CT-capture-enzyme-linked immunosorbent assay to measure naturally acquired responses against the entire PF83/AMA-1 molecule. Analysis of populations from villages in Guinea-Bissau and in an area of high malarial transmission in Senegal demonstrated a very high prevalence (94-100%) of naturally acquired serum IgG responses to PF83/AMA-1. Analysis of these natural responses showed that PF83/AMA-1 may be a well-recognized asexual parasite antigen. A statistically significant age-related change in antibody levels to PF83/AMA-1 was observed in Guinea-Bissau. No such correlation was observed in the Senegalese population, although an age-related antibody response was seen for total parasite antigen. No significant correlation was observed between PF83/AMA-1 responses and the parameters of parasite load and malaria-related fever.

Differential localization of full-length and processed forms of PF83/AMA-1 an apical membrane antigen of Plasmodium falciparum merozoites.

A well conserved 83-kDa apical membrane antigen of Plasmodium falciparum, PF83/AMA-1, is the analogue of PK66/AMA-1, a 66-kDa P. knowlesi protective merozoite protein. PK66/AMA-1 is expressed in late-stage schizonts; is localized within the merozoite apex; and is processed to a 44/42-kDa doublet at, or around, the time of schizont rupture. The processed forms can associate with the merozoite surface. We were interested to further analyze the timing of synthesis and processing, and subcellular localization of PF83/AMA-1, a malaria vaccine candidate, using monoclonal antibodies (mAbs) developed against PF83/AMA-1. Using [35S]methionine metabolically labeled asexual blood stage parasites, in combination with indirect single and dual immunofluorescence, we have determined that, in similar fashion to PK66/AMA-1, protein expression of PF83/AMA-1 is restricted to late-stage schizonts with greater than 8 nuclei. PF83/AMA-1 is post-synthetically processed rapidly by cleavage of an N-terminal peptide to a 66-kDa molecule. Both the 83- and the 66-kDa molecules are initially localized at the merozoite apex. In P. falciparum (7G8 strain and CVD-1 clone) the full-length 83-kDa molecule remains apically restricted following merozoite release. However, the processed 66-kDa form can become circumferentially associated with the merozoite surface at or around the time of schizont rupture and merozoite release. After merozoite invasion a processed form of PF83/AMA-1 is present in early ring stage parasites. Comparative analysis of a rhoptry associated protein RAP-1, shows a co-ordinated and compartmentalized release of rhoptry components.

Ion-exchange-immunoaffinity purification of a recombinant baculovirus Plasmodium falciparum apical membrane antigen, PF83/AMA-1.

A two-step purification regime has been developed for a quantitatively minor, putatively transmembrane, M(r) 83,000, apical membrane blood stage vaccine candidate antigen of Plasmodium falciparum (PF83/AMA-1), that has been expressed as a full-length baculovirus recombinant protein, PF83-7G8-1. The first step utilizes a new approach to high-performance ion-exchange chromatography (HPIEC) in which elution conditions are not only defined by charge, but also by hydrophobicity. HPIEC fractionation involves successive sodium chloride gradient anion-exchange elutions (A and B), where a change in the non-ionic detergent polyoxyethylenealkylether C10E5 concentration between elutions A and B (from 0.01% to 0.1% (w/v) respectively), results in a fraction that comprises from 2% to 9% PF83-7G8-1. Subsequent column immunoaffinity purification of this fraction on Q-Sepharose CL 4B-28G2dc1 mAb yields a PF83-7G8-1 preparation that is 56% pure. Rat mAb 28G2dc1 recognizes a C-terminal region that is conserved and cross reactive within the AMA-1 family, thus permitting recombinant and native full-length AMA-1 molecules from other species to be purified for molecular analysis. Immunological and molecular characterisation of the vaccine-related characteristics of purified PF83/AMA-1 are now underway.