HPV16 E7 protein associates with the protein kinase p33CDK2 and cyclin A.

E7 is the major transforming protein of human papillomavirus type 16 (HPV16). It has been found to associate with the retinoblastoma protein Rb1. We investigated whether HPV16 E7 protein was associated with other cellular proteins, in particular with those involved in cell cycle control. Immunoprecipitates from CaSki cell extracts with an anti E7 monoclonal antibody contained a histone H1 kinase. Recombinant E7, synthesized in yeast, when mixed with protein extracts from epithelial cells bound histone H1 kinase activity in vitro. The in vivo and the in vitro-formed E7-kinase complex had the same periodicity of activity during the cell cycle, being most active in S and G2/M. Immunoblotting of E7 immunoprecipitates with an antibody raised against the p33CDK2, revealed a 33 kDa protein band not detected by an anti-p34cdc2 antibody, suggesting that the E7-associated kinase activity is due to the p33CDK2. The interaction appears to be via cyclin A, since probing of similar immunoblots showed a 50 kDa band corresponding to cyclin A. The association of E7 with cyclin A appeared to be direct, not involving Rb 1 or other proteins.

HPV16 E7 phosphorylation in fission yeast: characterization and biological effects.

Human papillomavirus type 16 E7 protein (HPV-16 E7), synthesised in Schizosaccharomyces pombe, is both phosphorylated and targeted to the nucleus [Tommasino et al., Gene 93 (1990) 265-270] as is E7 protein synthesized in primate cells. Further analysis of E7 expression in fission yeast indicates that: (i) E7 protein synthesised in S. pombe is phosphorylated only on the Ser residues which are part of a casein kinase II consensus site, as it has been shown to be the cause in human cells, and is tightly associated with the nuclear matrix; (ii) synthesis of wild type, phosphorylated E7 is responsible for a significant increase in S. pombe doubling time; and (iii) E7 phosphorylation is not required for the tight association of the protein with the nuclear matrix, but E7 mutants, in which one (Ser31) or both phosphorylated serines (Ser31 and Ser32) have been substituted, lack any effect on cell-cycle duration.

Synthesis, phosphorylation, and nuclear localization of human papillomavirus E7 protein in Schizosaccharomyces pombe.

The complete E7 protein-encoding open reading frame of human papillomavirus type 16 (HPV-16) was expressed in the fission yeast Schizosaccharomyces pombe, under the control of a cloned yeast promoter. The HPV-16 E7 protein synthesized in S. pombe is a 17-kDa phosphoprotein which is recognized by anti-E7 antibodies (raised in rabbits against E7 fusion protein produced in Escherichia coli). The mobility during sodium dodecyl sulfate-polyacrylamide-gel electrophoresis of native E7 phosphoprotein synthesized in S. pombe is identical to that of the E7 phosphoprotein immunoprecipitated from human CaSki cells. Immunofluorescence staining showed that HPV-16 E7 phosphoprotein is localized in the nuclei of transformed S. pombe. These results indicate that E7 protein synthesized by S. pombe is apparently indistinguishable from HPV-16 E7 protein synthesized in higher eukaryotic cells expressing genes of HPV-16, and also that the phosphorylated, nuclear HPV-16 E7 protein is synthesized in S. pombe in a form compatible with its biological activity.

Typing of human papillomavirus DNAs by restriction endonuclease mapping of the PCR products.

The polymerase chain reaction (PCR) for the diagnosis of human papillomavirus (HPV) infections, and in particular for the study of cervical HPV-associated lesions, is used widely. We identified a novel set of universal primers that are able to amplify a fragment spanning the E1 open reading frame (ORF) from different mucosotropic HPV types. A restriction endonuclease digestion of the amplified products is suggested for accurate typing. In particular, AluI digestion of the amplified fragments yields a distinctive fragment pattern for each 'high-risk' (16, 18, 31 and 33) HPV sequence, thus distinguishing them from 'low-risk' (6b and 11) HPV sequences.

Physicochemical characterisation of the pertussis vaccine.

The characterisation of an acellular pertussis vaccine composed of a genetically modified pertussis toxin, filamentous haemagglutinin and pertactin is described. The three antigens are submitted to a mild treatment with formaldehyde in the presence of lysine before their use in vaccine formulation. Characterisation is performed by amino acid analysis, SDS-PAGE, analytical size exclusion chromatography and, in the case of pertactin, isoelectrofocusing. The effect of some variables on pertactin formaldehyde treatment has been studied by means of isoelectrofocusing and mouse immunogenicity.

MF59 adjuvant enhances the antibody response to recombinant hepatitis B surface antigen vaccine in primates.

The effect of the proprietary adjuvant MF59 on the immunogenicity of a recombinant hepatitis B virus (HBV) vaccine, PreS2+SAg, was investigated in baboons. The magnitude and duration of the antibody response to hepatitis B surface antigen (anti-HBs) induced by the HBV/MF59 vaccine was compared with the same antigen combined with alum and with two licensed alum vaccines. After one immunization, the HBV/MF59 vaccine generated anti-HBs titers >10 mIU/mL in a greater proportion of animals, and mean titers were 26- to 84-fold higher than titers from alum vaccines. After a third immunization, the HBV/MF59 vaccine generated titers 38- to 127-fold higher than alum vaccines. Seven months after the third immunization, HBV/MF59 elicited titers 75- to 472-fold higher than alum vaccines. The dramatic immune response elicited by HBV/MF59 in baboons suggests that MF59 may be a desirable adjuvant for use in improved HBV vaccines for humans.

Evolutionary trends of neurofilament proteins in fish.

1. Neurofilament complement was studied in an early chordate (Ciona intestinalis) and six fish species by immunoblot with antisera specific for each of the three mammalian NF subunits. 2. The anti-NF-H and anti-NF-M antisera were characterized as strictly specific for phosphorylated epitopes located in the carboxyterminal domain. 3. The NF-L subunit is absent in primitive chordates and appears first in fish; it can be identified on the basis of its apparent mol. wt, its reactivity with the anti-IFA antibody and with polyclonal antibodies raised to the NF-L subunit of mammals. 4. Primitive chordate neurofilaments are constituted by a single polypeptide of ca 160,000 mol. wt exhibiting only M-type phosphorylation-dependent epitopes. 5. Primitive fish (Acipenser transmontanus, Salmo gairdneri, Scorpaena porcus, Serranus scriba) possess only a single high mol. wt NF subunit reacting with both anti-NF-H and anti-NF-M antiserum while more recent species (Mugil saliens, Perca fluviatilis) possess two high mol. wt NF subunits which are immunologically distinct as to their phosphorylation structures. 6. The existence in some fish species of two high mol. wt NF polypeptides suggests that the process of gene duplication and diversification supposed to have given rise to the two high mol. wt NF subunits of mammals and birds has occurred repeatedly in vertebrate evolution, and may be regarded as a case of convergent evolution.

Phosphorylated epitopes of neurofilaments have been conserved during chordate evolution.

We have characterized some rabbit polyclonal responses as strictly specific for phosphorylated epitopes located in the carboxyterminal (tail) domain of the H or the M subunits of mammalian neurofilaments. These antibodies have been used to confirm the occurrence in lizard neurofilaments of a single heavy subunit cross-reacting with both H and M from mammals. A heavy subunit with similar cross-reactivity has been detected in neurofilaments preparations from fishes, whereas more primitive Chordata possess a HMW polypeptide cross-reacting with only the M subunit. We could also demonstrate in frog spinal cord two distinct heavy subunits cross-reacting with either the M or the H subunit from mammals, a fact which suggests a convergent evolution for phosphorylated epitopes of neurofilaments.

Heterogeneous localization of epitopes along axonemes of mammalian cilia.

The specificity of four monoclonal antibodies, raised against mammalian ciliary axonemes, was determined by both immunofluorescence and immunoblot experiments. Three antibodies reacted with epitopes which are differentially located along axonemal length. Among these, antibody 3.12 recognized an epitope common to different dynein heavy chains, reacted only with tracheal cilia and specifically stained the proximal portion of the ciliary axoneme.

Immunization of mice by oral colonization with live recombinant commensal streptococci.

To test the use of recombinant streptococci as live vaccine vectors, colonization/immunization experiments were performed with Streptococcus gordonii expressing heterologous cell-surface antigens. Three isogenic strains of S. gordonii were used: a wild-type, a recombinant expressing the M6 protein of Streptococcus pyogenes, and a recombinant expressing the E7 protein of human papillomavirus type 16 as a fusion with the M6 protein. A single dose of live bacteria was used to inoculate outbred mice, and it was found that: (i) mice were stably colonized by a single intranasal/oral inoculum of S. gordonii; (ii) recombinant strains were equally effective as wild-type in colonizing mice; (iii) two months after the inoculum, oral/pharyngeal swabs of 83.3% of animals were still positive for isolation of S. gordonii; (iv) recombinant S. gordonii isolated from colonized mice were always positive for expression of the heterologous antigens; (v) live bacteria induced a systemic immune response, since sera of mice colonized with recombinant S. gordonii contained IgG specific for the heterologous cell-surface antigens; (vi) this immune response depended upon the effective colonization by live bacteria, since killed bacteria did not induce such a response.

Delivery and expression of a heterologous antigen on the surface of streptococci.

We have developed a system in which a foreign antigen replaces nearly all of the surface-exposed region of the fibrillar M protein from Streptococcus pyogenes and is fused to the C-terminal attachment motif of the M molecule. The fusion protein is thus expressed on the surface of Streptococcus gordonii, a commensal organism of the oral cavity. The antigen chosen to be expressed within the context of the M6 molecule was the E7 protein (98 amino acids) of human papillomavirus type 16. Stable recombinant streptococci were obtained by integrating genetic constructs into the chromosome, exploiting in vivo homologous recombination. The M6-E7 fusion protein expressed on the S. gordonii surface was shown to be immunogenic in mice. This is the first step in the construction of recombinant live vaccines in which nonpathogenic streptococci as well as other gram-positive bacteria may be used as vectors to deliver heterologous antigens to the immune system.