Immunization with plasmid DNA encoding hepatitis C virus envelope E2 antigenic domains induces antibodies whose immune reactivity is linked to the injection mode.

Plasmids expressing different domains of the hepatis C virus (HCV) envelope E2 glycoprotein from a genotype 1a isolate were constructed to compare the immunogenic potential of E2 in nucleic acid-based immunizations. One plasmid, pCIE2t, expressed a C-terminally truncated form of E2, while others, pS2.SE2A to pS2.SE2E, encoded the adjacent 60-amino-acid (aa) sequences of E2 (inserts A to E) expressed as a fusion with the hepatitis B virus surface antigen. BALB/c mice were given injections of the plasmids intramuscularly (i.m.) or intraepidermally (i.e.) via a gene gun (biolistic introduction), and induced humoral immune responses were evaluated. The i.e. injections resulted in higher seroconversion rates and antibody titers, up to 100-fold, than did the i.m. injections (P = 0.01 to 0.04). Three restricted immunogenic domains, E2A (aa 384 to 443), E2C (aa 504 to 555), and E2E (aa 609 to 674), that yielded antibody titers ranging from 1:59 to > 1:43,700 could be identified. Subtype 1a- and 1b-derived E2 antigens and synthetic peptides were used in Western blot and enzyme-linked immunosorbent assay analyses, which revealed that the cross-reactivity of the plasmid-induced antibodies was linked both to the type of antigen expressed and to the injection mode. Induced anti-E2 antibodies could immunoprecipitate noncovalent E1E2 complexes believed to exist on the surface of HCV virions. This study allowed us to identify restricted immunogenic domains within E2 and demonstrated that different routes of injection of HCV E2 plasmids can result in quantitatively and qualitatively different humoral immune responses.

Rapid and efficient purification of native histidine-tagged protein expressed by recombinant vaccinia virus.

Vaccinia virus has been used as a vector to express foreign genes for the production of functional and posttranslationally modified proteins. A procedure is described here that allows the rapid native purification of vaccinia-expressed proteins fused to an amino-terminal tag of six histidines. Extracts from cells infected with recombinant vaccinia virus are loaded onto Ni2+.nitrilotriacetic acid (Ni2+.NTA)-agarose and histidine-tagged proteins are selectively eluted with imidazole-containing buffers. In the case of the human serum response factor (SRF), a transcription factor involved in the regulation of the c-fos protooncogene, the vaccinia-expressed histidine-tagged SRF (SRF-6His) could be purified solely by this step to greater than 95% purity. SRF-6His was shown to resemble authentic SRF by functional criteria: it was transported to the nucleus, bound specifically the c-fos serum response element, interacted with the p62TCF protein to form a ternary complex, and stimulated in vitro transcription from the serum response element. Thus, the combination of vaccinia virus expression and affinity purification by Ni2+.NTA chromatography promises to be useful for the production of proteins in a functional and posttranslationally modified form.

Identification of seroreactive regions of the human papillomavirus type 16 protein E4, E6, E7 and L1.

Small fragments of the DNA of human papillomavirus type 16 (HPV-16) were randomly cloned into the bacteriophage fd which expresses the resulting peptides as part of its capsid. Antisera raised against different HPV-16 fusion proteins were used for screening of the phage clones and the reacting peptides were determined by sequencing the inserted HPV-16 DNA fragments of the positive recombinants. Seroreactive regions of the proteins derived from the E4, E6, E7 (two regions) and L1 (three regions) open reading frames could be found by this approach. Of these seven regions, four were defined by at least two overlapping inserts, thus limiting the domains to between 10 and 15 amino acids. In the case of the E4 open reading frame, the same region identified by immunoscreening was also found when synthetic overlapping octapeptides were tested by ELISA with the anti-E4 antiserum. Using an approach to predict 'receptor-like' regions within the respective proteins, five of the seven regions were also identified. From the data on these regions, synthetic peptides were produced and used for the detection of antibodies against HPV-16 proteins in human sera by ELISA.

Production of human monoclonal IgG antibodies reacting with cytomegalovirus (CMV).

A human monoclonal antibody (MoAb) reacting with cytomegalovirus (CMV) has been produced using somatic cell hybridization between Epstein-Barr virus (EBV) infected B lymphocytes and a human-mouse heteromyeloma cell line (SHM-D33). The hybrids were selected in HAT medium containing 5 x 10(-7) ouabain. The median level of Ig production was 5 (0.1-20) micrograms/10(6) cells/day. One selected hybridoma (IB-8E9H5) has been maintained in continuous culture for more than 30 months with a stable IgG2, lambda production. Molecular hybridization using EBV-specific probes demonstrate that our hybrids have lost the IR-1 EBV sequence during fusion. Unexpectedly, these blotting experiments revealed the presence of multiple EBNA-1 sequences dispersed among the genomic DNA of the SHM-D33 cell line. Screening for anti-CMV specificity was performed by ELISA and confirmed by immunofluorescence staining. Thus far, three CMV reference strains and 14 local strains are stained by the MoAb as early as 3 h after CMV infection of human fibroblasts, apparently through the recognition of a nuclear viral antigen of 67 kDa. In conclusion, this technique permits (a) the removal of the EBV genome contained in the lymphoblastoid parental cell line and (b) the production of human anti-CMV MoAb with potential applications in the prevention of life threatening CMV infections.

Detection of human papillomaviruses 16-18 in cervical cells by molecular hybridization: relationship with morphonuclear cell image analyses.

Forty-one Feulgen-stained cervical imprint smears were analyzed by means of the SAMBA 200 cell image processor in order to quantitatively score human papillomavirus (HPV) 16-18-induced morphonuclear modifications as assessed by morphometric, densitometric, and textural parameters. Molecular hybridization technology using 16 and 18 type specific genetic probes made it possible to divide our series into three groups: Group 1, containing noninfected smears; Group 2, containing "suspicious", i.e., borderline positive, smears; and Group 3, those related to infected patients. Our results show that nuclei from infected smears are much more hyperchromatic and bigger than those arising from noninfected smears. This quantitative description of HPV 16-18-induced chromatin modifications enabled us to create preliminary data banks which could lead to an objective and reproducible grading of unknown cases. This approach is now being prospectively assessed on a large series of cases because the value of the current study is limited until the data bank is tested against unknown specimens with a broader spectrum of HPV infection.

Structural organization of the bovine thyroglobulin gene and of its 5'-flanking region.

The structural organization of the bovine thyroglobulin gene has been investigated by a combination of Southern genomic blotting and direct analysis of cloned gene fragments isolated from a chromosomal DNA library. The entire locus is spread over more than 200,000 base pairs which makes it one of the largest eukaryotic genes studies to date. The coding information is scattered into at least 42 exons, 34 of which have been precisely identified. A different evolutionary origin of the 5' and 3' regions of the gene is supported by the highly different proportion of exonic material they contain (12% and 3%, respectively) and by the existence of sequence homology between the 3' region of thyroglobulin and acetylcholinesterase. Detailed sequence analysis of the 5' region of the gene and its flanking segment demonstrated that a significant homology exists between bovine and human thyroglobulin sequences, except for the presence within the ruminant promoter region of a 220-base-pair sequence belonging to the bovine monomer repeated family.

Normal and defective expression of the thyroglobulin gene.

Molecular studies of the thyroglobulin (Tg) gene have progressed significantly in recent years. Cloning and sequencing the complete bovine Tg cDNA led to the knowledge of the primary structure of the Tg subunit. This large polypeptidic chain displays a repetitive structure, especially in its amino-terminal half, and bears a striking homology with the acetylcholinesterase molecule of Torpedo californica in its carboxy-terminal portion. The four specific domains known to be involved in the formation of the thyroid hormones have been assigned to both terminal parts of the polypeptide, a location which could play a role in the process leading to hormone release. The very large (greater than 250 kb) Tg gene has been localized on the long arm of chromosome 8 in man, in close linkage with the c-myc oncogene. The study of its structure allowed the characterization of the molecular defect responsible for a congenital flaw in Tg gene expression in a herd of South-African cattle. This work led to the unexpected finding that the Tg pre-mRNA undergoes alternative splicing in normal animals, too. A DNA segment involved in the transcriptional control of Tg gene expression by cAMP has been identified by transfecting primary cultured thyrocytes with recombinant genes.

Defective splicing of thyroglobulin gene transcripts in the congenital goitre of the Afrikander cattle.

The structure of thyroglobulin mRNA was analyzed in an inbred herd of Afrikander cattle with hereditary goitre. Northern transfer of RNA from affected animals revealed both a shorter (approximately 7100 bases) and a normal-sized (approximately 8200 bases) thyroglobulin mRNA when hybridized to bovine thyroglobulin cDNA clones. S1 nuclease mapping experiments established that 1100 bases are deleted in the 5' region of the smaller mRNA. Electron microscopy of RNA from animals with goitre hybridized to a bovine genomic DNA clone showed that the region deleted corresponds to exon 9 of the thyroglobulin gene. Southern blot analysis of the exon 9 region revealed differences between affected and control animals with the enzymes PstI and TaqI. Although they could reflect a linkage disequilibrium between the mutation and restriction fragment length polymorphism, it is noteworthy that these differences map in the region of the exon 9/intron 9 junction. Our results show that a genetic lesion in the thyroglobulin gene causes aberrant splicing of the pre-mRNA, and suggest that the responsible mutation is at the exon 9/intron 9 junction.

Presence of hormonogenic and repetitive domains in the first 930 amino acids of bovine thyroglobulin as deduced from the cDNA sequence.

The sequence of the first 2831 nucleotides of bovine thyroglobulin mRNA has been determined from the analysis of a cDNA clone. Following a 41-nucleotide 5' untranslated sequence, a single open-reading frame encoding 930 amino acids was observed. This corresponds to the aminoterminal third of thyroglobulin, preceded by a putative signal peptide of 19 amino acids. The protein sequence was found to be essentially made of the sevenfold repetition of a 60-amino-acid-long building unit, interrupted at fixed positions by unrelated segments of variable length. The presence of an internal homology within the repetitive unit itself suggests that the 5' region of the thyroglobulin gene has evolved from the initial duplication of a relatively short sequence, followed by the serial duplication of the resulting unit. The tyrosine residue at position five has been assigned an important hormonogenic function [Mercken, L., Simons, M.-J. and Vassart, G. (1982) FEBS Lett. 149, 285-287]. This residue is flanked by sequence elements related to the repeated unit, suggesting that the hormonogenic domain evolved also from the basic ancestor sequence.

[Structure and expression of thyroglobulin gene]. / Structure et expression du gène de la thyroglobuline.

Thyroglobulin is composed of two 300000 dalton polypeptide chains, translated from an 8000 base mRNA. Preparation of a full length cDNA and its cloning in E. coli have lead to the demonstration that the polypeptides of thyroglobulin protomers were identical. Used as molecular probes, the cloned cDNA allowed the isolation of a fragment of thyroglobulin gene. Electron microscopic studies have demonstrated that this gene contains more than 90% intronic material separating small size exons (less than 200 bp). Sequencing of bovine thyroglobulin structural gene is in progress. Preliminary results show evidence for the existence of repetitive segments. Availability of cloned DNA complementary to bovine and human thyroglobulin mRNA allows the study of genetic defects of thyroglobulin gene expression in the human and in various animal models.

Molecular cloning of bovine thyroglobulin complementary DNA. Characterization of 2500-base-pair and 1900-base-pair fragments.

Double-stranded thyroglobulin complementary DNA (cDNA) was synthesized from purified 33-S bovine thyroglobulin mRNA. This synthetic structural gene has previously been shown to contain three sites for the restriction endonuclease HindIII, yielding two internal fragments of 1900 and 2500 base pairs respectively. Recombinant molecules were prepared by ligating the HindIII-restricted cDNA to the plasmid pBR322 which had been linearized by the same enzyme. When Escherichia coli was transformed with this mixture, it yielded two kinds of colonies each harboring recombinant plasmids containing one of the two cDNA fragments. Both recombinant molecules hybridized specifically to translatable thyroglobulin mRNA. Sequence homology between the two cloned DNAs could not be detected by cross-hybridization experiments; this argues against the existence of internal structural repetition in thyroglobulin subunits. Together, the two cloned DNA fragments represent 55% of the 8000-base-pair double-stranded thyroglobulin DNA.