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To investigate the efficiency of encapsidation of plasmid by papillomavirus virus-like particles (PV VLPs), and the infectivity of the resultant PV pseudovirions, Cos-1 cells were transfected with an 8-kb plasmid incorporating a green fluorescent protein (GFP) reporter gene (pGSV), and infected with bovine PV (BPV-1) L1/L2 recombinant vaccinia virus to produce BPV1 pseudovirions. Approximately 1 in 1.5 x 10(4) of dense (1.35 g/ml) PV pseudovirions and 0.3 in 10(4) of less-dense (1.29 g/ml) pseudovirions packaged an intact pGSV plasmid. The majority (>75%) of packaged plasmids contained deletions, and the deletions affected all tested genes. After exposure of Cos-1 cells to BPV-1 pseudovirions at an MOI of 40,000:1, 6% of cells expressed GFP, giving a calculated efficiency of delivery of the pGSV plasmid, by pseudovirions which had packaged an intact plasmid, of approximately 5%. Plasmid delivery was not effected by purified pGSV plasmid, was blocked by antiserum against BPV-1, and was not blocked by DNase treatment of pseudovirions, confirming that delivery was mediated by DNA within the pseudovirion. We conclude that a major limitation to the use of PV pseudovirions as a gene delivery system is that intact plasmid DNA is not efficiently selected for packaging by VLPs in cell-based pseudovirions production systems.

Transport of BPV-1 virus from the cell membrane to the nucleus was studied in vitro in CV-1 cells. At reduced temperature (4 degrees C), BPV-1 binding to CV-1 cells was unaffected but there was no transport of virions across the cytosol. Electron microscopy showed BPV-1 virions in association with microtubules in the cytoplasm, a finding confirmed by co-immunoprecipitation of L1 protein and tubulin. Internalization of virus was unimpaired in cells treated with the microtubule-depolymerizing drug nocodazole but virions were retained in cytoplasmic vesicles and not transported to the nucleus. We conclude that a microtubule transport mechanism in CV-1 cells moves intact BPV-1 virions from the cell surface to the nuclear membrane., (Copyright 2001 Academic Press.)

Chimeric papillomavirus (PV) virus-like particles (VLPs) based on the bovine papillomavirus type 1 (BPV-1) L1 protein were constructed by replacing the 23-carboxyl-terminal amino acids of the BPV1 major protein L1 with an artificial "polytope" minigene, containing known CTL epitopes of human PV16 E7 protein, HIV IIIB gp120 P18, Nef, and reverse transcriptase (RT) proteins, and an HPV16 E7 linear B epitope. The CTL epitopes were restricted by three different MHC class I alleles (H-2(b), H-2(d), HLA-A*0201). The chimeric L1 protein assembled into VLPs when expressed in SF-9 cells by recombinant baculovirus. After immunization of mice with polytope VLPs in the absence of adjuvant, serum antibodies were detected which reacted with both polytope VLPs and wild-type BPV1L1 VLPs, in addition to the HPV16E7 linear B cell epitope. CTL precursors specific for the HPV16 E7, HIV P18, and RT CTL epitopes were also detected in the spleen of immunized mice. Polytope VLPs can thus deliver multiple B and T epitopes as immunogens to the MHC class I and class II pathways, extending the utility of VLPs as self-adjuvanting immunogen delivery systems., (Copyright 2000 Academic Press.)

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.)

We have previously described a DNA-packaging assay using bovine papillomavirus type 1 (BPV-1) virus-like particles (VLPs) and have identified a region of the BPV genome that assists in packaging. In this study, we identify a specific BPV sequence involved in DNA packaging by BPV-1 VLPs. In the initial screening of BPV-1 genomic sequences essential for DNA packaging, we observed that a plasmid with deletions between nucleotides (nt) 948 and 2113 failed to be packaged into BPV-1 VLPs. However, plasmids containing nt 948 to 2113 were efficiently packaged, suggesting that this 1.2-kb fragment contains a packaging enhancement sequence (PES). Further mapping of the BPV-1 genome showed that this packaging sequence lies between nt 1506 and 1625. Furthermore, this packaging sequence is also recognized by HPV6b VLPs, suggesting that a common packaging mechanism may be used by the two papillomavirus types. Given the phylogenetic difference between these two viral types, it is likely that other papillomavirus types may also use the same packaging mechanism. Identification of the PES has allowed a minimal viral genome sequence to be used in the packaging assay, improving the usefulness of the assay in studying the process of papillomavirus DNA encapsidation., (Copyright 1999 Academic Press.)

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has attracted widespread interest since it was demonstrated to be fluorescent in vivo when expressed in other organisms. In order to investigate papillomavirus life cycle which hampered by the unavailability of conventional cell culture system, we constructed a chimeric bovine papillomavirus (BPV) type 1 virus-like particles (VLPs) containing GFP. It was found that fluorescent VLPs could be assembled from L2 protein in which GFP is inserted into the N-terminal region of L2 (aa 88). The fluorescent VLPs could also be assembled from a GFP/L2 fusion protein in which part of the L2 sequence had been deleted. In vitro, fluorescent VLPs could bind to CV-1 cells, and this VLP/cell interaction could be analyzed by FACS assay. These results demonstrated that GFP could incorporate into BPV1 VLPs without disruption of the VLP structure. Fluorescent VLPs might be a useful tool for study of papillomavirus virus/cell interaction.

It has been shown previously that recombinant virus-like particles (VLPs) of papillomavirus can induce VLP-specific humoral and cellular immune responses following parenteral administration. To test whether mucosal administration of bovine papillomavirus type 1 (BPV1) VLPs could produce mucosal as well as systemic immune responses to VLPs, 50 micrograms chimeric BPV1 VLPs containing an HPV16 E7 CTL epitope (BPVL1/E7 VLP) was administered intranasally to mice. After two immunisations, L1-specific serum IgG and IgA were observed. L1-specific IgG and IgA were also found in respiratory and vaginal secretions. Both serum and mucosal antibody inhibited papillomavirus VLP-induced agglutination of RBC, indicating that the antibody induced by mucosal immunisation may recognize conformational determinants associated with virus neutralisation. For comparison, VLPs were given intramuscularly, and systemic and mucosal immune responses were generally comparable following systemic or mucosal delivery. However, intranasal administration of VLP induced significantly higher local IgA response in lung, suggesting that mucosally delivered HPV VLP may be more effective for mediating local mucosal immune responses. Intranasal immunisation with HPV6b L1 VLP produced VLP-specific T proliferative responses in splenocytes, and immunisation with BPVL1 VLP containing an HPV16 E7 CTL epitope induced E7-specific CTL responses. We conclude that immunisation with papillomavirus VLPs via mucosal and intramuscular routes, without adjuvant, can elicit specific antibody at mucosal surfaces and also systemic VLP epitope specific T cell responses. These findings suggest that mucosally delivered VLPs may offer an alternative HPV VLP vaccine strategy for inducing protective humoral immunity to anogenital HPV infection, together with cell-mediated immune responses to eliminate any cells which become infected.

Encapsidation of circular DNA by papillomavirus capsid protein was investigated in Cos-1 cells. Plasmids carrying both an SV40 origin of replication (ori) and an E. coli ori were introduced into Cos-1 cells by DNA transfection PV capsid proteins were supplied in trans by recombinant vaccinia viruses. Pseudovirions were purified from infected cells and their packaged DNA was extracted and used to transform E. coli as an indication of packaging efficacy. VLPs assembled from BPV-1 L1 alone packaged little plasmid DNA, whereas VLPs assembled from BPV-1 L1 + L2 packaged plasmid DNA at least 50 times more effectively. BPV-1 L1 + L2 VLPs packaged a plasmid containing BPV-1 sequence 8.2 +/- 3.1 times more effectively than a plasmid without BPV sequences. Using a series of plasmid constructs comprising a core BPV-1 sequence and spacer DNA it was demonstrated that BPV VLPs could accommodate a maximum of about 10.2 kb of plasmid DNA, and that longer closed circular DNA was truncated to produce less dense virions with shorter plasmid sequences. The present study suggests that packaging of genome within PV virions involves interaction of L2 protein with specific DNA sequences, and demonstrates that PV pseudovirions have the potential to be used as DNA delivery vectors for plasmids of up to 10.2 kb.

To evaluate an antigen delivery system in which exogenous antigen can target the major histocompatibility complex (MHC) class I pathway, a single human papillomavirus (HPV) 16 E7 cytotoxic T lymphocyte (CTL) epitope and a single HIV gp160 CTL epitope were separately fused to the C-terminus of bovine papillomavirus 1 (BPV1) L1 sequence to form hybrid BPV1L1 VLPs. Mice immunized with these hybrid VLPs mounted strong CTL responses against the relevant target cells in the absence of any adjuvants. In addition, the CTL responses induced by immunization with BPV1L1/HPV16E7CTL VLPs protected mice against challenge with E7-transformed tumor cells. Furthermore, a high titer-specific antibody response against BPV1L1 VLPs was also induced, and this antiserum could inhibit papillomavirus-induced agglutination of mouse erythrocytes, suggesting that the antibody may recognize conformational determinates relevant to virus neutralization. These data demonstrate that hybrid BPV1L1 VLPs can be used as carriers to target antigenic epitopes to both the MHC class I and class II pathways, providing a promising strategy for the design of vaccines to prevent virus infection, with the potential to elicit therapeutic virus-specific CTL responses.

Bovine papillomavirus type 1 (BPV-1) virions were produced in vitro using vaccinia virus (VV) recombinants expressing the BPV-1 L1 and L2 capsid proteins. Particles morphologically resembling papillomaviruses were observed in the nucleus of cells infected with a VV recombinant for the BPV-1 L1 protein, and greater numbers of similar particles were seen in the nuclei of cells infected with a VV double recombinant for L1 and L2. Virus-like particles (VLPs) assembled in cells infected with the VV double recombinant for BPV-1 L1 and L2, and not those assembled in cells infected with the VV recombinant for BPV-1 L1 alone, were able to package BPV-1 DNA. Transcription of the BPV-1 E1 viral open reading frame was observed after a mouse fibroblast cell line was exposed to VLPs produced using a BPV-1 L1/L2 VV recombinant in a cell line containing episomal BPV-1 DNA. E1 transcription was not observed when the VLPs were pre-incubated with antibodies to the capsid protein of BPV-1. This system should allow an in vitro approach to the definition of the BPV-1 cellular receptor.