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We studied determinants of efficient encapsidation of circular DNA, incorporating a PV early region DNA sequence (nt 584-1978) previously shown to enhance packaging of DNA within papillomavirus (PV)-like particles (VLPs). Insect coelomic cells (Sf-9) and cultured monkey kidney cells (Cos-1) were transfected with an 8-kb reporter plasmid incorporating the putative BPV packaging sequence and infected with BPV1 L1 and L2 recombinant baculovirus or vaccinia virus. Heavy (1.34 g/ml) and light (1.30 g/ml) VLPs were produced, and each packaged some of the input plasmid. In light VLPs, truncated plasmids, which nevertheless incorporated the PV-derived DNA packaging sequence, were more common than full-length plasmids. Packaging efficiency of the plasmid was estimated at 1 plasmid per 10(4) VLPs in both Cos-1 and Sf-9 cells. In each cell type, expression of the BPV1 early region protein E2 in trans doubled the quantity of heavy but not light VLPs and also increased the packaging efficiency of full-length circular plasmids by threefold in heavy VLPs. The resultant pseudovirions incorporated significant amounts of E2 protein. Pseudovirions, comprising plasmids packaged within heavy VLPs, mediated the delivery of packaged plasmid into Cos-1 cells, whereby "infectivity" was blocked by antisera to BPV1 L1, but not antisera to BPV1 E4. We conclude that (a) packaging of DNA within PV L1+L2 pseudovirions is enhanced by BPV1 E2 acting in trans, (b) E2 may be packaged with the pseudovirion, and (c) E2-mediated enhancement of packaging favors 8-kb plasmid incorporation over incorporation of shorter DNA sequences., (Copyright 2000 Academic Press.)

Squamous differentiation of keratinocytes is associated with decreases in E2F-1 mRNA expression and E2F activity, and these processes are disrupted in squamous cell carcinoma cell lines. We now show that E2F-1 mRNA expression is increased in primary squamous cell carcinomas of the skin relative to normal epidermis. To explore the relationship between E2F-1 and squamous differentiation further, we examined the effect of altering E2F activity in primary human keratinocytes induced to differentiate. Promoter activity for the proliferation-associated genes, cdc2 and keratin 14, are inhibited during squamous differentiation. This inhibition can be inhibited by overexpression of E2F-1 in keratinocytes. Overexpression of E2F-1 also suppressed the expression of differentiation markers (transglutaminase type 1 and keratin 10) in differentiated keratinocytes. Blocking E2F activity by transfecting proliferating keratinocytes with dominant negative E2F-1 constructs inhibited the expression of cdc2 and E2F-1, but did not induce differentiation. Furthermore, expression of the dominant negative construct in epithelial carcinoma cell lines and normal keratinocytes decreased expression from the cdc2 promoter. These data indicate that E2F-1 promotes keratinocyte proliferation-specific marker genes and suppresses squamous differentiation-specific marker genes. Moreover, these data indicate that targeted disruption of E2F-1 activity may have therapeutic potential for the treatment of squamous carcinomas. Oncogene (2000).

The presence of T-helper epitopes within the E7 transforming protein of human papillomavirus type 18 (HPV18) was sought using a series of overlapping synthetic 15-20 mer peptides spanning the entire 105 amino acid sequence of this protein. Two H-2k restricted T-helper epitopes were defined, comprising 44VNHQHLPARRA55 and 81DDLRAFQQLF90 as the minimal T proliferative epitopes. Peptides containing these epitopes were able to provide cognate help to B epitopes from HPV18E7 protein for production of antibody to this protein in vivo in CBA/CaH mice. No H-2b or H-2d restricted epitopes were demonstrable, and in H-2d mice this was associated with poor antibody response to the E7 protein. There is no "promiscuous" T-helper epitope in HPV18 E7 comparable to the 49DRAHYNI55 sequence in HPV16 E7, and restricted T-helper epitope availability may be a determinant of poor immune responses to this protein after natural infection.

Synthetic peptides can be tailor-made to include any B or T epitopes desired from a single or multiple antigens or organisms. However, peptides in general are not very immunogenic and have not proven easy to incorporate into immunogenic vaccines. ISCOMs is an adjuvant system that has the capability not only to enhance the humoral immunogenicity of a protein but has also been shown to induce cell-mediated immune responses in animals. Synthetic peptide ISCOM vaccines are few because of the difficulty in incorporation of these peptides into ISCOMs. We have shown in this study that non-immunogenic peptides could be made immunogenic by polymerisation, and these polymers could be incorporated into ISCOMs to give highly immunogenic vaccines. Synthetic 20mer peptides containing known B and T-helper epitopes from the E7 protein of the cervical cancer associated human papillomavirus type 16 (HPV16 E7) have been used here as model immunogens. We have compared the humoral immunity induced by these peptides as polymers or as copolymers with a lipid binding 20mer peptide (LAP 20), with or without incorporation into ISCOMs. Unpolymerised peptide elicited no measurable antibody. When polymerised peptide was administered with CFA, or in phosphate-buffered saline (PBS) without adjuvant, or incorporated into ISCOMs, antibodies recognising both the immunising peptide and HPV16 E7 protein were produced. For equal quantities of administered peptide (5 micrograms), ISCOMs gave higher titres of antibody than CFA or PBS. Polymerised peptides induced high antigen-specific IgG2a:IgG1 ratios, which increased with multiple immunisations. These data indicate that polymerised peptides could be incorporated into ISCOMs to form efficient immunogens which may elicit a Th1 type response.

We used a capture ELISA to quantify the E7 protein of human papillomavirus type 18 (HPV-18). In HeLa cells, which express low levels of immunoreactive E7 protein (iE7), iE7 had a mean half-life of 13.5 min. In HPV-18 E7 recombinant baculovirus (E7rec BV)-infected Sf21 cells, which express higher levels of E7, the half-life of iE7 was much longer (90 min and > 24 h, with two different E7rec BVs). For two transformed human cervical cell lines expressing HPV-18 E7, exposure of the cells to hydrocortisone resulted in a twofold increase in steady-state levels of the E7 protein: no similar effect was observed with progesterone, oestrogen or testosterone. The half-life of iE7 was unaltered by hydrocortisone or progesterone exposure. An immunoassay which distinguished Ser33-phosphorylated E7 from E7 not phosphorylated at this residue (Ser33dephospho-E7), showed that in HeLa and Sf21 cells the majority of E7 was phosphorylated: the half-life of both species of E7 was similar in HeLa cells, but the half-life of Ser33dephospho-E7 was much shorter (90 min) in Sf21 cells than that of Ser33phospho-E7 (> 24 h). A HeLa-fibroblast fusion cell line with tumorigenic potential (CGL-1) had a similar ratio of dephospho-E7 to total E7 (0.06), as a similar fusion cell line (CGL-4) with no tumorigenic potential (0.03). We conclude that E7 is a labile phosphoprotein, and that the expression and steady-state level of the E7 protein in eukaryotic cells may be influenced by the hormonal environment of the cells.

Proteins derived from the E6, E7 and L1 ORFs of HPV16 and the E7 ORF of HPV18 were produced in insect cells using a baculovirus expression system. HPV ORFs were inserted into baculovirus transfer vectors pAcYM1 or pVL1393/2, and recombinant baculoviruses isolated using a combination of limiting dilution and plaque assay. Using HPV-specific antisera and monoclonal antibodies HPV proteins were identified in lysates of Spodoptera frugiperda (Sf-21) cells infected with HPV-recombinant baculovirus. Immunoreactive HPV16 E7 protein produced in Sf-21 cells had an apparent M(r) of 19 kDa, larger than that predicted from the amino acid sequence, and similar to that of native HPV16 E7 protein in HeLa and CaSki cells. The apparent M(r) of recombinant HPV18-E7, HPV16-L1 and HPV16-E6 proteins was equivalent to the M(r) values predicted from the amino acid sequence. Thermostability studies revealed that the half-life of HPV16-E7 protein in Sf-21 cell lysate was approx. 20 h at 4 degrees C, 2 h at 22 degrees C, and less than 30 min at 37 degrees C. HPV16 L1, HPV16 E7 and HPV18 E7 proteins were predominantly localised in the nucleus of recombinant baculovirus-infected Sf-21 cells, whereas recombinant HPV 16 E6 protein was localised in both the cytoplasm and nucleus of infected insect cells. Northern blot analysis of RNA derived from insect cells infected with vAc16E6E7, a recombinant baculovirus containing both HPV16 E6 and E7 ORF's, revealed the presence of only E6 ORF transcripts, suggesting that the splicing of RNA products derived from the E6 and E7 ORF's, as observed in cervical cancer-derived cell lines, is not performed in insect cells. Baculovirus-derived HPV proteins have similar biological properties to the native proteins and should be suitable for studies on the immunology of HPV.

ELISA capture assays were established for the E7 transforming proteins of HPV16 and HPV18, based on a range of previously characterised polyclonal and monoclonal antibodies. No cross-reactivity was observed in the ELISAs between HPV18 E7 and HPV16 E7. Immunoreactive E7 protein (iE7) was measured in a series of HPV-transformed cell lines, and ranged from 0.6 to 17.7 ng iE7/mg cell protein. iE7 was labile at 22 degrees C (t1/2 = 37 min) but relatively more stable at 4 degrees C (t1/2 = 210 min). HPV16 E7 protein at concentrations from 0.10 to 0.69 ng iE7/mg cell protein was detected in 5 of 13 smears from women with abnormal cervical cytology. Assay of E7 protein may play a role in the detection of HPV-induced cervical lesions with malignant potential.

A panel of murine mAb raised against a MS2 replicase/HPV 18 E7 fusion protein included 23 reactive by ELISA with HPV 18 E7 determinants. A total of 19 of the 23 recognized linear epitopes in the N-terminal region of the E7 molecule, while the other four were deduced by binding inhibition assays to recognize conformational determinants in this region. All tested antibodies precipitated a 14-kDa peptide doublet that corresponded with the predicted size of the E7 protein, from HeLa cells, but not from HPV 16 E7 containing CaSki cells. HPV 18 E7 protein was detected by immunolabeling with electron microscopy in both the nucleus and the cytoplasm of HeLa cells with the greater proportion occurring in the cytoplasm. No antibody reacted specifically by indirect immunofluorescence with HeLa cells. Weak cross-reactivity of some mAb with the E6 MS2-replicase fusion protein of HPV 16 was detected by ELISA, but no protein of the appropriate size was immunoprecipitated from CaSki cells. It is concluded that the B cell epitopes on the HPV 18 E7 transforming protein are located in the N-terminal region of the molecule and that some are weakly cross-reactive with HPV 16 E6 protein. E7 protein is either present in HeLa cells at a concentration too low to be detected by indirect immunofluorescence, or the N-terminal epitopes are masked by protein conformation or interaction with cellular or other viral components.