Monoclonal antibodies to human immune interferon and their use in a sensitive solid-phase ELISA.

Two stable hybridoma cell lines secreting specific antibodies against human gamma interferon (HuIFN-gamma) were established. Both monoclonal antibodies (designated as MD-1 and MD-2) belong to the IgG1/kappa subclass and neutralize the antiviral activity of natural and recombinant DNA derived HuIFN-gamma (nHuIFN-gamma and rHuIFN-gamma respectively), although MD-1 is far more effective than MD-2. MD-1 and MD-2 recognize different epitopes and do not compete with each other in binding to HuIFN-gamma as concluded from competition assays. In a 'Western' blot, both antibodies reacted with the 20 kDa and 25 kDa polypeptides present in nHuIFN-gamma preparations. A sandwich enzyme immunoassay using microtiter plates coated with unlabeled MD-2 was developed. Biotinylated MD-1 was used as the second antibody. Bound MD-1 was detected by an avidin/alkaline phosphatase enzyme reaction. This immunoassay is highly specific and as sensitive as a bioassay. A radioimmunoassay using MD-2 coated on polystyrene balls and 125I-labeled MD-1 as the second antibody showed a sensitivity comparable to that of the enzyme immunoassay.

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.

The purification and characterization of rat gamma interferon by use of two monoclonal antibodies.

Two mouse monoclonal antibodies, designated DB-1 and DB-2, were isolated and used for the purification and characterization of recombinant rat interferon gamma (rRIF-gamma) derived from Chinese hamster ovary (CHO) cells. The two antibodies belong to different classes (DB-1 is an IgG1 and DB-2 an IgA) and display similar epitope specificities as shown in competition binding experiments. Both antibodies, raised against rRIF-gamma, exhibited high affinity for rat and mouse gamma interferon and efficiently neutralized the antiviral activity of both animal interferon species. Affinity chromatography analysis showed that a column with immobilized DB-1 was capable of complete binding of rat and mouse gamma interferon, both natural and recombinant DNA-derived. As visualized by SDS-polyacrylamide gel electrophoresis and Western blot analysis, the purified rRIF-gamma preparation consisted of at least seven molecular forms with Mr values ranging between 14 000 and 25 000, with a relative abundance of a 18 000 Mr protein. Gel permeation chromatography of crude rRIF-gamma gave coincident peaks of rRIF-gamma proteins (all different forms) and interferon activity corresponding to a Mr value of 45 000. The results suggest that the molecular heterogeneity was due to differential glycosylation and was not the consequence of a proteolytic degradation process.

Hepatitis delta virus cDNA sequence from an acutely HBV-infected chimpanzee: sequence conservation in experimental animals.

Hepatitis delta virus (HDV) RNA was isolated from the serum of a chimpanzee acutely infected with hepatitis B virus (HBV) and superinfected with HDV. Interference of HDV with HBV resulted in decreased HBV DNA levels in the serum. This interference did not change the size of the two HBV specific RNAs present in the liver of the chimpanzee. The complete cDNA sequence of the HDV RNA (5th passage) was determined. Comparison of this cDNA sequence with our previously published sequence (4th passage), located in the variable domain of HDV, was highly conserved. The HDV strain used for these infections originated from a human HDV isolate also used for five to seven HDV passages in chronic HBV carrier chimpanzees (subtypes adw and ayw) or woodchucks chronically infected with woodchuck hepatitis virus (WHV). The complete HDV cDNA sequence showed an extreme conservation (up to 99.8% homology) with the previously published animal-derived HDV cDNA sequences irrespective of passage number and animal species. In contrast a markedly lower homology (85-89%) was found when compared with 3 human-derived HDV cDNA sequences. Comparison of our complete cDNA sequence with the human-derived cDNA sequences showed that the nucleotide changes in the human-derived isolates were restricted to specific regions on the genome and to specific basepair substitutions. The hepatitis Delta antigen (HDAg) is highly conserved both in the human- and animal-derived cDNA sequences showing mainly conservative amino acid changes.

Cloning and expression of the chromosomal immune interferon gene of the rat.

The chromosomal immune interferon gene of the rat (IFN-gamma) was identified by screening a recombinant rat lambda phage library with a human IFN-gamma cDNA probe. In contrast to the genes of other rat IFNs, this rat IFN-gamma chromosomal gene contains introns and its structural organization closely resembles that of the human and murine IFN-gamma genes. The rat IFN-gamma gene encodes a signal sequence of 19 amino acids followed by the mature IFN-gamma protein of 137 amino acids. The gene was expressed under control of the simian virus 40 (SV40) early promoter in Chinese hamster ovary (CHO) cells deficient in dihydrofolate reductase (DHFR) after co-transformation with a plasmid containing the mouse DHFR gene. Initial transformants with a DHFR+ phenotype produced IFN-gamma titres ranging from 20 to 1600 units/ml. After stepwise increases in the concentration of methotrexate (MTX) in the growth medium of transformed CHO cells, MTX-resistant clones producing 80 000-100 000 units per ml were isolated. Protein analysis of supernatants of these MTX-resistant cells by polyacrylamide gel electrophoresis revealed a product with an apparent mol. wt. of 18 000 daltons which was not detectable in the growth medium of DHFR+ transformants that did not produce IFN. The product was identified as rat IFN-gamma and constituted approximately 5% of the proteins excreted from these cells.

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.

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.

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.