Characterization of a human monoclonal antibody against Shiga toxin 2 expressed in Chinese hamster ovary cells.

Shiga toxin-producing Escherichia coli infections can often lead to the development of hemolytic-uremic syndrome (HUS) in a small percentage of infected humans. Patients with HUS receive only supportive treatment as the benefit of antibiotic therapy remains uncertain. We have previously reported the generation and preclinical evaluation of neutralizing human monoclonal antibodies (HuMAbs) against the Shiga toxins (Stx). In this paper, we describe the expression in Chinese hamster ovary (CHO) cells of 5C12 HuMAb, which is directed against the A subunit of Stx2. The cDNAs of the light and heavy chain immunoglobulin (Ig) variable regions of 5C12 HuMAb were isolated and cloned into an expression vector containing human IgG1 constant regions. The vector was transfected into CHO cells, and transfectants secreting Stx2-specific antibody were screened by an Stx2-specific enzyme-linked immunosorbent assay. The CHO-produced recombinant 5C12 (r5C12) showed similar specificity and binding affinity to Stx2 as the parent hybridoma-produced 5C12. More significantly, the r5C12 displayed the same neutralizing activity as the parent 5C12 in vitro and in vivo. In the mouse toxicity model, both antibodies significantly and equally prolonged survival at a dose of 0.312 microg/mouse. The data showed that since r5C12, produced in CHO cells, was equally effective as the parent 5C12, it is our choice candidate as a potential prophylactic or therapeutic agent against hemolytic-uremic syndrome.

Genetic analysis of a Cryptosporidium parvum human genotype 1 isolate passaged through different host species.

Cryptosporidium parvum TU502, a genotype 1 isolate of human origin, was passaged through three different mammalian hosts, including humans, pigs, and calves. It was confirmed to be genotype 1 by PCR-restriction fragment length polymorphism analysis of the Cryptosporidium oocyst wall protein gene, direct sequencing of PCR fragments of the small subunit rRNA and beta-tubulin genes, and microsatellite analysis. This isolate was shown to be genetically stable when passaged through the three mammalian species, with no evidence of the emergence of new subpopulations as observed by a genotype-specific PCR assay. TU502 oocysts from different sources failed to infect gamma interferon knockout mice, a characteristic of genotype 1 isolates. The genotypic and phenotypic characterization of TU502 is significant since it is the isolate selected to sequence the genome of C. parvum genotype 1 and is currently used in several research projects including human volunteer studies.

Variability among Cryptosporidium parvum genotype 1 and 2 immunodominant surface glycoproteins.

Published genomic differences between Cryptosporidium parvum genotype 1 (human-derived) and genotype 2 (animal and human-derived) isolates suggest that these may belong to two distinct species. This is of significant interest since genotype 1 isolates are associated with sporadic cases of human cryptosporidiosis in 30-40 % of cases in contrast to 60-70 % of cases caused by genotype 2. The lower genetic sequence similarity between genotype 1 and 2 surface glycoproteins (gp40/15) suggests that antigenic differences should also occur, a feature that was investigated in this study. Using immune and convalescent serum samples from gnotobiotic piglets previously inoculated with genotype 1 and 2 isolates, we demonstrated that C. parvum gp15 was immunodominant for both genotype 1 and 2 isolates. Lower genetic sequence similarity between genotype 1 and 2 Cpgp40/15 did correspond to gp15 protein differences as detected by Western blot. Moreover, we confirmed that gp15 contains epitopes that are also immunodominant. Deglycosylation of C. parvum proteins resulted in decreased ability of gp15, gp23 and gp900 to react with homologous polyclonal antibodies, suggesting that these proteins also express carbohydrate epitopes. Taken together, our data suggest that there is a high phenotypic variability between C. parvum genotype 1 and 2 isolates at the level of gp15. We contemplate that gp15 surface glycoprotein plays an important role in the biology of C. parvum as a potent inducer of immune response and a possible virulence factor.

Extensive polymorphism in Cryptosporidium parvum identified by multilocus microsatellite analysis.

Restriction fragment length polymorphism and DNA sequence analysis discern two main types of Cryptosporidium parvum. We present a survey of length polymorphism at several microsatellite loci for type 1 and type 2 isolates. A total of 14 microsatellite loci were identified from C. parvum DNA sequences deposited in public databases. All repeats were mono-, di-, and trinucleotide repeats of A, AT, and AAT, reflecting the high AT content of the C. parvum genome. Several of these loci showed significant length polymorphism, with as many as seven alleles identified for a single locus. Differences between alleles ranged from 1 to 27 bp. Karyotype analysis using probes flanking three microsatellites localized each marker to an individual chromosomal band, suggesting that these markers are single copy. In a sample of 19 isolates for which at least three microsatellites were typed, a majority of isolates displayed a unique multilocus fingerprint. Microsatellite analysis of isolates passaged between different host species identified genotypic changes consistent with changes in parasite populations.

Animal propagation and genomic survey of a genotype 1 isolate of Cryptosporidium parvum.

Human cryptosporidiosis is attributed to two major Cryptosporidium parvum genotypes of which type 1 appears to be the predominant. Most laboratory investigations however are performed using genotype 2 isolates, the only type which readily infects laboratory animals. So far type 1 has only been identified in humans and primates. A type 1 isolate, obtained from an individual with HIV and cryptosporidiosis, was successfully adapted to propagate in gnotobiotic piglets. Genotypic characterization of oocyst DNA from this isolate using multiple restriction fragment length polymorphisms, a genotype-specific PCR marker, and direct sequence analysis of two polymorphic loci confirmed that this isolate, designated NEMC1, is indeed type 1. No changes in the genetic profile were identified during multiple passages in piglets. In contrast, the time period between infection and onset of fecal oocyst shedding, an indicator of adaptation, decreased with increasing number of passages. Consistent with other type 1 isolates, NEMC1 failed to infect mice. A preliminary survey of the NEMC1 genome covering approximately 2% of the genome and encompassing 200 kb of unique sequence showed an average similarity of approximately 95% between type 1 and 2 sequences. Twenty-four percent of the NEMC1 sequences were homologous to previously determined genotype 2 C. parvum sequences. To our knowledge, this is the first successful serial propagation of genotype 1 in animals, which should facilitate characterization of the unique features of this human pathogen.

Identification of a full-length cDNA for an endogenous retrovirus of miniature swine.

Endogenous retroviruses of swine are a concern in the use of pig-derived tissues for xenotransplantation into humans. The nucleotide sequence of porcine endogenous retrovirus taken from lymphocytes of miniature swine (PERV-MSL) has been characterized. PERV-MSL is a type C retrovirus of 8,132 bp with the greatest nucleic acid sequence identity to gibbon ape leukemia virus and murine leukemia virus. Constitutive production of PERV-MSL RNA has been detected in normal leukocytes and in multiple organs of swine. The copy numbers of full-length PERV sequences per genome (approximately 8 to 15) vary among swine strains. The open reading frames for gag, pol, and env in PERV-MSL have over 99% amino acid sequence identity to those of Tsukuba-1 retrovirus and are highly homologous to those of endogenous retrovirus of cell line PK15 (PK15-ERV). Most of the differences in the predicted amino acid sequences of PK15-ERV and PERV-MSL are in the SU (cell attachment) region of env. The existence of these PERV clones will enable studies of infection by endogenous retroviruses in xenotransplantation.

Cytotoxic properties of DAB486EGF and DAB389EGF, epidermal growth factor (EGF) receptor-targeted fusion toxins.

Elevated expression of the receptor for epidermal growth factor (EGF) is a characteristic of several malignancies including those of the breast, bladder, prostate, lung, and neuroglia. To therapeutically target the cytotoxic action of diphtheria toxin to EGF receptor-expressing tumor cells, we have constructed a hybrid gene in which the sequences for the binding domain of diphtheria toxin have been replaced by those for human EGF. The resulting fusion toxins, DAB486EGF and DAB389EGF, bind specifically to the EGF receptor and inhibit protein synthesis in a variety of EGF receptor expressing human tumor cell lines with an IC50 as low as 0.1 pM. Comparisons of DAB486EGF and DAB389EGF showed that DAB389EGF was consistently 10- to 100-fold more cytotoxic than DAB486EGF. Like diphtheria toxin, the cytotoxic action of DAB389EGF results from ADP-ribosylation of elongation factor-2 and is sensitive to the action of chloroquine. Studies of the kinetics of cellular intoxication showed that a 15-min exposure of EGF receptor-expressing A431 cells to DAB389EGF results in complete protein synthesis inhibition within 4 h. Furthermore, inhibition of protein synthesis results in elimination of human tumor cell colonies. These findings show that DAB389EGF is a potential therapeutic agent for a wide variety of EGF receptor-expressing solid tumors.

Pharmacokinetics of the recombinant fusion protein DAB486IL-2 in animal models.

The kinetics of the in vitro cytotoxicity of DAB486IL-2, a genetically engineered fusion protein containing a portion of diphtheria toxin and human interleukin-2, were examined in the C91/PL cell line, which constitutively expresses IL-2 receptors. Maximal inhibition of protein synthesis was observed by 4-6 h after DAB486IL-2 addition at a concentration of 300 ng/ml. The tissue distribution, urinary excretion, and plasma pharmacokinetics of DAB486IL-2 in the rat and its plasma pharmacokinetics in the monkey were also examined. In rats the primary site of distribution of [35S]-DAB486IL-2 outside the vasculature appears to be the liver, followed by the kidney, spleen, and lung. Persistence of radioactive material in the liver and urinary excretion of metabolic degradation products suggest that labeled protein is metabolized by hepatic tissue. Following i.v. bolus administration of DAB486IL-2, the initial serum half-life for both the rat and the monkey was approximately 5 min. The overall clearance rate of drug for the two species differed, with DAB486IL-2 being cleared from circulation 2-3 times more rapidly in the monkey. Presence of high levels of neutralizing antibodies to diphtheria toxin in the rat significantly influenced the clearance of bioactive DAB486IL-2. However, the question as to whether the presence of in vitro biological activity for the molecule is masked by the presence of antibodies cannot be clearly answered.

Nucleotide sequence and analysis of the plant-inducible locus pinF from Agrobacterium tumefaciens.

Several loci on the tumor-inducing plasmid from Agrobacterium tumefaciens were transcriptionally activated in the presence of wounded plant tissue or extracts. The inducible virulence loci were required for efficient tumor formation. In contrast, the plant-inducible locus pinF was not observed to be absolutely essential for virulence. Mutants in pinF showed an attenuated virulence on a variety of dicotyledonous hosts, and this attenuation became more pronounced with decreasing numbers of bacterial cells in the inoculum. The DNA sequence of a 5.5-kilobase region which included the pinF locus from the octopine-type tumor-inducing plasmid A6 was determined. Four open reading frames consistent with the observed transcription of pinF were observed. Two of the open reading frames, pinF1 and pinF2, coded for polypeptides with relative molecular weights of 47,519 (pinF1) and 46,740 (pinF2). A comparison of the amino acid sequences of pinF1 and pinF2 indicated that they were similar to each other and to known polypeptide sequences for cytochrome P-450 enzymes.

Design, synthesis and expression of a human interleukin-2 gene incorporating the codon usage bias found in highly expressed Escherichia coli genes.

A synthetic gene encoding human interleukin-2 (IL-2) was designed such that the codon usage bias resembled that found in highly expressed Escherichia coli genes. The percentage of preferred codons was increased from 43% in the native cDNA sequence to 85% in the synthetic sequence. The cDNA and synthetic IL-2 genes were placed under the control of the trc promoter and expressed in E. coli JM101. While Northern blot analysis of IL-2 mRNA from each genetic construct demonstrated equivalent message half-lives, immunoblot and bioactivity analyses showed the synthetic gene to direct the synthesis of up to 16 times more IL-2 than the native cDNA sequence.

Characterization of the virB operon from an Agrobacterium tumefaciens Ti plasmid.

The virulence genes of the Agrobacterium tumefaciens Ti plasmid are grouped into six transcription units and direct the transfer of T-DNA into plant cells. We report here the nucleotide sequence of the largest vir operon, virB, from the Ti plasmid pTiA6NC. This operon contains 11 open reading frames, 7 of which show evidence of translational coupling. trpE::virB gene fusions were used to confirm the reading frames of genes virB2, 4, 5, 6, 7, 8, 10, and 11. In addition, the native gene products of virB6 and virB9 were identified using maxicell and in vitro transcription-translation techniques, and the VirB9 protein was found to be proteolytically processed. The codon usage of the predicted virB genes is very similar to the other pTiA6 vir genes and is much less biased than Escherichia coli. Since many of the virB gene products have secretion signals common to exported bacterial proteins, it is likely that they will be membrane-associated. We propose that the VirB proteins are involved in the formation of a transmembrane structure which mediates the passage of the transferred T-DNA molecule through the bacterial and plant cell membranes.

Cytokinin production by Agrobacterium and Pseudomonas spp.

The production of cytokinins by plant-associated bacteria was examined by radioimmunoassay. Strains producing trans-zeatin were identified in the genera Agrobacterium and Pseudomonas. Agrobacterium tumefaciens strains containing nopaline tumor-inducing plasmids, A. tumefaciens Lippia isolates, and Agrobacterium rhizogenes strains produced trans-zeatin in culture at 0.5 to 44 micrograms/liter. Pseudomonas solanacearum and Pseudomonas syringae pv. savastanoi produced trans-zeatin at levels of up to 1 mg/liter. In vitro cytokinin biosynthetic activity was measured for representative strains and was found to correlate with trans-zeatin production. The genetic locus for trans-zeatin secretion (tzs) was cloned from four strains: A. tumefaciens T37, A. rhizogenes A4, P. solanacearum K60, and P. syringae pv. savastanoi 1006. Southern blot analysis showed substantial homology of the Agrobacterium tzs genes to each other but not to the two Pseudomonas genes.

Cloning and nucleotide sequence of the tzs gene from Agrobacterium tumefaciens strain T37.

The trans-zeatin secretion locus (tzs), from the nopaline Ti plasmid of Agrobacterium tumefaciens strain T37, was cloned and the nucleotide sequence determined. This gene is located in the virulence region of pTiT37. The tzs gene is responsible for the secretion of trans-zeatin into bacterial culture medium and in addition has the cytokinin biosynthetic activity, dimethylallylpyrophosphate:AMP dimethylallyltransferase. Sequence analysis showed an open reading frame of 729 nucleotides, capable of encoding a protein of 27,545 daltons. A single new labelled protein of 27,200 daltons was detected in Escherichia coli maxicells expressing the cloned tzs gene. Significant sequence homology was observed between the tzs and the published tmr sequence from pTiT37.

Cloning and sequencing of the granulin gene from the Trichoplusia ni granulosis virus.

A restriction fragment containing the granulin gene from the Trichoplusia ni granulosis virus was located in a blot of viral genomic DNA using a cloned polyhedrin gene as a probe. This fragment was cloned, mapped, subcloned, and the sequence of the coding region and 5' and 3' flanking regions was determined. Although granulin is very similar in size to nuclear polyhedrosis virus polyhedrins, the N-terminal region of granulin demonstrated a high degree of variability with the first 60 amino acids only 28% homologous to the Autographa californica nuclear polyhedrosis virus (AcMNPV) polyhedrin sequence. Between amino acid 60 and the carboxyterminus at amino acid 248, the sequence was very similar (64%) to polyhedrin sequences. Overall the nucleotide and amino acid sequences were 58 and 53%, respectively, related to those of AcMNPV. No introns were evident in the gene.

T-DNA of Agrobacterium tumefaciens encodes an enzyme of cytokinin biosynthesis.

Phytohormone overproduction in crown gall tumors is due to the expression of several T-DNA genes. The data strongly suggest that the tmr gene (transcript 4) is responsible for cytokinin overproduction by encoding dimethylallyl-pyrophosphate:AMP dimethylallyltransferase (DMA transferase), an enzyme directly involved in cytokinin biosynthesis. Cell-free extracts of Escherichia coli strains containing the tmr gene from pTiA6NC had DMA transferase activity. No activity was present in the control strain containing only the plasmid vector. The cytokinins synthesized were isopentenyladenine, isopentenyladenosine, and isopentenyladenosine 5'-monophosphate. DMA transferase activity was also detected in cloned crown gall tumors incited by Agrobacterium tumefaciens wild-type A6NC and a tms mutant. Enzymatic activity in cell-free extracts of E. coli and tumors could be abolished by transposon insertion within the tmr gene.

Cytokinin/auxin balance in crown gall tumors is regulated by specific loci in the T-DNA.

Insertion of the transposon Tn5 into the T-region of the octopine Ti plasmid of Agrobacterium tumefaciens gives rise to crown gall tumors having altered morphology. Three loci within the T-DNA that control tumor morphology have been detected [Garfinkel, D. J., Simpson, R. B., Ream, L. W., White, F. F., Gordon, M. P. & Nester, E. W. (1981) Cell 27, 143-153]. They influence tumor size (tml), production of roots (tmr), or production of shoots (tms). Cytokinin and auxin levels in such mutant tumors were examined by HPLC/radioimmunoassay and HPLC/fluorescence assay, respectively. Free indoleacetic acid levels (in pmol/g) were: uninfected tobacco stem tissues, 128; wild-type A348 tumors, 295; tml mutant tumors, 307; tmr mutant tumors, 129; and tms mutant tumors, 70. Average trans-ribosylzeatin levels were correspondingly: 0.97, 48, 40, 0.54, and 1,400 pmol/g. trans-Ribosylzeatin/indoleacetic acid ratios were as high as 24 in shoot-producing tumors and as low as 0.003 in root-producing tumors. The evidence strongly suggests that tumor phytohormone levels are determined by genes in the T-DNA.