Molecular characterisation of Plasmodium reichenowi apical membrane antigen-1 (AMA-1), comparison with P. falciparum AMA-1, and antibody-mediated inhibition of red cell invasion.

Apical membrane antigen 1 is a candidate vaccine component for malaria. It is encoded by a single copy gene and has been characterised in a number of malaria species as either an 83-kDa de novo product (Plasmodium falciparum; Pf AMA-1) or a 66-kDa product (all other species). All members of the AMA-1 family are expressed during merozoite formation in maturing schizonts and are initially routed to the rhoptries. Processed forms may subsequently be associated with the merozoite surface. Because of the unique occurrence of the 83-kDa form in P. falciparum we were interested to determine whether the phylogenetically closely related chimpanzee malaria Plasmodium reichenowi shared characteristics with Pf AMA-1. Here we show that the molecular structure, the localisation and processing are similar to that of Pf AMA-1 and that in vitro growth inhibitory mAbs reactive with Pf AMA-1 also inhibit P. reichenowi growth in an in vitro assay. Polymorphism in the 83-kDa AMA-1 family was analysed through comparison of Pr ama-1 with Pf ama-1 alleles, which showed the most significant evidence for selection maintaining polymorphism in Domains I-III of AMA-1 in P. falciparum. The most substantial divergence between Pr AMA-1 and Pf AMA-1 sequences was in the N-terminal region unique to the 83-kDa form of AMA-1. It was confirmed that the specific Pr ama-1-type allele was not present among P. falciparum parasites in an African population, and an allele coding for lysine at amino acid 187 was uniquely associated with field isolates in this population.

High-level expression of Plasmodium vivax apical membrane antigen 1 (AMA-1) in Pichia pastoris: strong immunogenicity in Macaca mulatta immunized with P. vivax AMA-1 and adjuvant SBAS2.

The apical membrane antigen 1 (AMA-1) family is a promising family of malaria blood-stage vaccine candidates that have induced protection in rodent and nonhuman primate models of malaria. Correct conformation of the protein appears to be essential for the induction of parasite-inhibitory responses, and these responses appear to be primarily antibody mediated. Here we describe for the first time high-level secreted expression (over 50 mg/liter) of the Plasmodium vivax AMA-1 (PV66/AMA-1) ectodomain by using the methylotrophic yeast Pichia pastoris. To prevent nonnative glycosylation, a conservatively mutagenized PV66/AMA-1 gene (PV66Deltaglyc) lacking N-glycosylation sites was also developed. Expression of the PV66Deltaglyc ectodomain yielded similar levels of a homogeneous product that was nonglycosylated and was readily purified by ion-exchange and gel filtration chromatographies. Recombinant PV66Deltaglyc43-487 was reactive with conformation-dependent monoclonal antibodies. With the SBAS2 adjuvant, Pichia-expressed PV66Deltaglyc43-487 was highly immunogenic in five rhesus monkeys, inducing immunoglobulin G enzyme-linked immunosorbent assay titers in excess of 1:200,000. This group of monkeys had a weak trend showing lower cumulative parasite loads following a Plasmodium cynomolgi infection than in the control group.

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.

Isolation and characterization of monoclonal antibodies directed to rat interferon-gamma.

Eight stable hybridoma cell lines producing monoclonal antibodies reactive with rat interferon-gamma have been generated. The antibodies produced belong to three different immunoglobulin isotypes: IgG1 (4 monoclonal antibodies, designated DB-1, DB-10, DB-12 and DB-13), IgG2a (3 monoclonal antibodies, DB-9, DB-14 and DB-16) and IgA (1 monoclonal antibody, DB-2). The antibodies were characterized in terms of epitope specificity and reactivity with rat, mouse and human interferon-gamma. Three antibodies (DB-1, -2 and -14) show high antiviral neutralizing activity against natural and recombinant DNA derived rat interferon-gamma, whereas the others have low (DB-10, -12, -13 and -16) or no (DB-9) detectable activity. Two antibodies (DB-1 and -2) effectively bind and neutralize mouse interferon-gamma, one antibody (DB-14) exhibits some cross-reactivity and the others show no reactivity towards the mouse lymphokine. None of the antibodies reacts with human interferon-gamma. All antibodies recognize immunoblotted recombinant rat interferon-gamma, although substantial variation in the immunoreactivity existed. Competition binding experiments reveal that the antibodies are directed to three spatially distinct sites on the rat lymphokine. Two non-competing monoclonal antibodies were selected and used for the development of a specific enzyme-linked immunosorbent assay for the detection of rat interferon-gamma in biological fluids.

Sera from irradiated rats contain antibodies to a ubiquitous tumour-associated antigen.

Female rats of the inbred strains BN/BiRij and WAG/Rij were irradiated with 30 kV X-rays, or 15 MeV or 0.5 MeV fast neutrons. Sera were collected several months after irradiation and found to be negative for antibodies reacting with the murine mammary tumour virus as tested by a solid-phase radioimmunoassay and an immunofluorescence absorption test. We found, however, that in an immunofluorescence assay several sera from irradiated rats reacted with a cytoplasmic antigen in rat mammary, ureter and skin carcinoma cell lines as well as a mouse mammary tumour and a transformed BALB/3T3 mouse fibroblast line. No reaction was found with normal fibroblast cell lines of rat or murine origin. Endpoint titres of the sera on tumour cells ranged from 1:20 to 1:160. Of 48 sera from unirradiated rats 18 also stained tumour cells, but usually at the low dilution of 1:10. Irradiation seems to enhance antibody activity to a ubiquitous tumour-associated antigen.