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This paper presents a novel flow‐through reverse transcription‐polymerase chain reaction (RT‐PCR) microreactor with optimized thermal and fluidic characteristics. It integrates the steps of reverse transcription of the initially applied RNA sample and the amplification of specific DNA fragments of the cDNA formed. The microreactor comprises a heating plate with different temperature zones and an interchangeable fluidic chip with serpentine microchannels. The heating plate provides temperature zones for reverse transcription, hot start activation, denaturation, annealing and extension. The fluidic chip contains a serpentine microchannel with integrated features for segmented sample stream generation and dosing operations, which is guided over the temperature zones according to the thermal protocol of RT‐PCR. Reverse transcription is performed in an integrated microchannel section immediately before the amplification of the cDNA's, generated from the primarily applied RNA sample. One feature of this chip system is the generation of a segmented flow for high‐throughput analysis of RT‐PCR samples. It is shown that RT‐PCR reactions can be performed successfully in the microreactor with continuous and segmented flow regimes. The aim of the experiments was the detection of the HPV 16 DNA genome and of viral oncogene transcripts (E6/E7), respectively. Both markers are of importance in medical diagnostics and will be applied in further studies for the identification of oncogene positive cells in cell populations by means of flow‐through in‐situ RT‐PCR.

Purpose:Molecular genotyping relies on the identification of specific microbial DNA sequences. Accurate genotyping not only requires discrimination between low- and high-risk pathogens for effective diagnosis or disease management but also requires the identity of the specific strain or type of the microbe involved in pathogenesis. The majority of these assays require DNA amplification followed by genome identification either through sequencing or hybridization to specific oligonucleotide probes. We evaluated the use of a DNA microchip assay as a simple and easy-to-use procedure for genotyping. Methods:Various methodological parameters were optimized for single-base mismatch discrimination on a DNA microarray. The fabrication procedures involved substrate chemistry for immobilization. The effect of various buffers and features associated with oligonucleotide sequences were standardized. The assay was evaluated on a low-density genotyping chip containing the sequences of various (Human Papilloma Virus) HPV subtypes. Results:The specific subtype was identified with high specificity by hybridization in miniaturized condition. Conclusions:The DNA microchip provides a rapid and cost-effective genotyping procedure for microbial organisms and can be implemented easily in any laboratory.

Cervical cancer has been shown to be highly associated with human papillomavirus (HPV) infection. The viral oncogenes E6 and E7 are constantly expressed by the tumor cells and are therefore targets for immunotherapy. In the present study we investigated the potential of HPV16 L1E7 chimeric virus-like particles (CVLP) to activate specific cytotoxic T lymphocytes in human blood donors. CVLP were expressed by recombinant baculovirus and purified. Direct incubation of freshly isolated peripheral blood lymphocytes (PBL) with CVLP resulted in induction of proliferation and growth of T cell lines. To enhance antigen presentation we also loaded dendritic cells with CVLP and used them to activate naive T cells. Growing cell lines were mainly CD3 positive (>95%) with a predominant CD4-positive and a minor CD8-positive component. Analysis of Tcell specificity was carried out by an interferon-γ ELISpot assay. Dendritic cells pseudoinfected with CVLP or pulsed with human leukocyte antigen (HLA)-A*0201-restricted peptide E711–20 or with a newly identified HPV16 peptide L1323–331 were used as stimulator cells. T cells responsive to CVLP were found in the cultures with frequencies of 0.5%–0.7%. Frequencies to peptides were around 0.1%. These T cells had cytolytic activity toward autologous B-lymphoblastic cell lines either pseudoinfected with CVLP or pulsed with HLA-A*0201-restricted peptides. They also lysed the HPV16- and HLA-A*0201-positive cervical cancer cell line CaSki, whereas HLA-A*0201-negative SiHa cells were not lysed. We conclude from our data that CVLP show promise for a therapeutic vaccine in patients with HPV16-positive cervical intraepithelial neoplasia lesions or cervical cancer. © 2001 Wiley-Liss, Inc.

The complete nucleotide sequence of the circular double‐stranded DNA of the genital human papillomavirus type 6b (HPV6b) comprising 7902 bp was determined and compared with the DNA sequences of human papillomavirus type 1a (HPV1a) and bovine papillomavirus type 1 (BPV1). All major open reading frames are located on one DNA strand only. Their arrangement reveals that the genomic organization of HPV6b is similar to that of HPV1a and BPV1. The putative early region includes two large open reading frames E1 and E2 with marked amino acid sequence homologies to HPV1a and BPV1 which are flanked by several smaller frames. The internal part of E2 completely overlaps with another open reading frame E4. The putative late region contains two large open reading frames L1 and L2. The L1 amino acid sequences are highly conserved among analyzed papillomavirus types. By sequence comparison, potential promoter, splicing and polyadenylation signals can be localized in HPV6b DNA suggesting possible mechanisms of genital papillomavirus gene expression.

The transcriptional promoter of the candidate E6‐E7 transforming gene region of human papillomavirus (HPV)‐16 (P97) was active in transiently transfected cervical carcinoma cells when linked to the HSV‐1 tk or bacterial cat genes. Sequences 5′ to P97 contain a short enhancer element responding to cellular factor(s) in uninfected human foreskin keratinocytes and in cervical carcinoma cells, but not in human or animal fibroblasts. The E2 trans‐activator products of HPV‐16 or of the related bovine papillomavirus (BPV)‐1 further elevated HPV‐16‐driven transcripts in co‐transfections, and required the presence of E2‐binding ACC(N)6GGT cores in cis. A ‘short E2’ C‐terminal repressor gene product (sE2) of HPV‐16 or the BPV‐1 sE2 repressor not only inhibited viral E2 trans‐activation, but also suppressed enhancer response to keratinocytic factors. Suppression by the sE2 products was abolished by deletion of the E2‐binding cores in cis or by a mutation in the sE2 DNA binding domain. The keratinocyte‐dependent enhancer is likely to contribute to the epithelial cell tropism of HPV‐16, and may direct persistent E6‐E7 gene transcription in response to cellular factors in cervical carcinoma cells in which the viral E2 genes are inactive.

The transcription of human papillomavirus type 18 (HPV 18) is selectively suppressed in non‐tumorigenic HeLa x fibroblast or HeLa x keratinocyte cell hybrids by 5‐azacytidine. In contrast, viral gene expression is not influenced by 5‐azacytidine in both tumorigenic hybrid segregants and in the parental HeLa cells. The suppression mechanism seems to operate at the level of initiation of transcription since nuclear run‐on experiments show the absence of elongated nascent viral RNA, whereas the transcription of cellular reference genes remains unaffected. Down‐regulation of HPV 18 mRNA correlates directly with cessation of cellular growth and can be abolished using the protein synthesis inhibitor cycloheximide. Furthermore human keratinocytes immortalized by HPV 16 but still retaining the non‐tumorigenic phenotype reveal the same inhibitory effect on viral transcription after treatment with 5‐azacytidine. These results support a model of a postulated intracellular control mechanism, directed against papillomavirus transcription, which can be induced by 5‐azacytidine and appears to correlate with the presence of specific chromosomes in non‐tumorigenic cells.

The chromosomal locations of cellular sequences flanking integrated papillomavirus DNA in four cervical carcinoma cell lines and a primary cervical carcinoma have been determined. The two human papillomavirus (HPV) 16 flanking sequences derived from the tumor were localized to chromosome regions 20pter----20q13 and 3p25----3qter, regions that also contain the protooncogenes c-src-1 and c-raf-1, respectively. The HPV 16 integration site in the SiHa cervical carcinoma-derived cell line is in chromosome region 13q14----13q32. The HPV 18 integration site in SW756 cervical carcinoma cells is in chromosome 12 but is not closely linked to the Ki-ras2 gene. Finally, in two cervical carcinoma cell lines, HeLa and C4-I, HPV 18 DNA is integrated in chromosome 8, 5' of the c-myc gene. The HeLa HPV 18 integration site is within 40 kilobases 5' of the c-myc gene, inside the HL60 amplification unit surrounding and including the c-myc gene. Additionally, steady-state levels of c-myc mRNA are elevated in HeLa and C4-I cells relative to other cervical carcinoma cell lines. Thus, in at least some genital tumors, cis-activation of cellular oncogenes by HPV may be involved in malignant transformation of cervical cells.

We have amplified by the polymerase chain reaction, cloned, and sequenced genomic segments of 118 human papillomavirus type 16 (HPV-16) isolates from 76 cervical biopsy, 14 cervical smear, 3 vulval biopsy, 2 penile biopsy, 2 anal biopsy, and 1 vaginal biopsy sample and two cell lines. The specimens were taken from patients in four countries--Singapore, Brazil, Tanzania, and Germany. The sequence of a 364-bp fragment of the long control region of the virus revealed 38 variants, most of which differed by one or several point mutations. Phylogenetic trees were constructed by distance matrix methods and a transformation series approach. The trees based on the long control region were supported by another set based on the complete E5 protein-coding region. Both sets had two main branches. Nearly all of the variants from Tanzania were assigned to one (African) branch, and all of the German and most of the Singaporean variants were assigned to the other (Eurasian) branch. While some German and Singaporean variants were identical, each group also contained variants that formed unique branches. In contrast to the group-internal homogeneity of the Singaporean, German, and Tanzanian variants, the Brazilian variants were clearly divided between the two branches. Exceptions to this were the seven Singaporean isolates with mutational patterns typical of the Tanzanian isolates. The data suggest that HPV-16 evolved separately for a long period in Africa and Eurasia. Representatives of both branches may have been transferred to Brazil via past colonial immigration. The comparable efficiencies of transfer of the African and the Eurasian variants to the New World suggest pandemic spread of HPV-16 in past centuries. Representatives of the African branch were possibly transferred to the Far East along old Arab and Indonesian sailing routes. Our data also support the view that HPV-16 is a well-defined virus type, since the variants show only a maximal genomic divergence of about 5%. The small amount of divergence in any one geographic location and the lack of marked divergence between the Tanzanian and Brazilian African genome variants two centuries after their likely introduction into the New World suggest a very slow rate of viral evolution. The phylogenetic tree therefore probably represents a minimum of several centuries of evolution, if not an age equal to that of the respective human races.

DNA from one biopsy sample of invasive cancer of the cervix contained sequences hybridizing with human papillomavirus (HPV) type 11 DNA only under nonstringent conditions. This DNA was molecularly cloned in lambda phage. Under stringent conditions of hybridization it cross-hybridized to a minor extent (less than 0.1%) with HPV types 10, 14, and 15 and showed no homology with DNA of other human HPV types. We therefore propose to designate it tentatively as HPV 16. HPV 16 DNA was used as a probe to test additional cancer biopsy samples from cervical, vulval, and penile cancer, as well as benign genital warts (condylomata acuminata) and cervical dysplasias for the presence of homologous sequences. In 61.1% (11/18) of cervical cancer samples from German patients sequences were found hybridizing with HPV 16 DNA under conditions of high stringency. In contrast, only 34.8% (8/23) of cancer biopsy samples from Kenya and Brazil revealed this DNA. Vulval and penile cancer biopsy samples hybridized to 28.6% (2/7) or 25% (1/4), respectively. Only 2 out of 33 condylomata acuminata contained HPV 16 DNA. Both positive tumors harbored in addition HPV 6 or HPV 11 DNA. The data thus indicate that HPV 16 DNA prevails in malignant tumors, rendering an accidental contamination with papillomavirus DNA from adjacent papillomas rather unlikely. The rare presence in benign genital papillomas in addition to common genital papillomaviruses suggests a dependence of HPV 16 replication on helper virus.

The L1 genes of two human papillomavirus type 16 (HPV16) isolates derived from condylomata acuminata were used to express the L1 major capsid protein in insect cells via recombinant baculoviruses. Both L1 major capsid proteins self-assembled into virus-like particles (VLP) with high efficiency and could be purified in preparative amounts on density gradients. The yield of VLP was 3 orders of magnitude higher than what has been obtained previously, using L1 derived from the prototype HPV16. DNA sequence comparison identified a single nonconserved amino acid change to be responsible for the inefficient self-assembly of the prototype L1. VLP were also obtained by expressing L1 of HPV6, HPV11, and cottontail rabbit papillomavirus, indicating that L1 from a variety of papillomaviruses has the intrinsic capacity to self-assemble into VLP. Coexpression of HPV16 L1 plus L2 by using a baculovirus double-expression vector also resulted in efficient self-assembly of VLP, and the average particle yield increased about fourfold in comparison to when L1 only was expressed. Coimmunoprecipitation of L1 and L2 and cosedimentation of the two proteins in a sucrose gradient demonstrated that L2 was incorporated into the particles. The ability to generate preparative amounts of HPV16 L1 and L1-L2 VLP may have implications for the development of a serological assay to detect anti-HPV16 virion immune responses to conformational epitopes and for immunoprophylaxis against HPV16 infection.

Two human papillomavirus type 16 (HPV 16)-immortalized human keratinocyte cell lines (HPK) were shown to have retained the ability for differentiation after subcutaneous injection into nude mice. These properties were maintained even at late passage. HPK cells gave rise to transiently growing cysts which exhibited an epitheliumlike architecture. Moreover, differentiation-specific markers such as cytokeratin 10, involucrin, and filaggrin were shown to be expressed in an ordered succession. RNA-RNA in situ hybridization revealed heterogeneous and low levels of HPV 16 E6-E7 RNA in the basal layer of the cysts. In contrast, in progressively growing tumors induced by HPK cells containing an activated ras oncogene (EJ-ras) or in tumors induced by the cervical carcinoma cell line CaSki, high levels of E6-E7-specific RNA could be detected. Irrespective of the growth potential of these cell lines in nude mice, viral transcription was always more evident in the basal layer and in proliferatively active cells rather than in differentiated cells. This contrasts with viral gene expression in HPV 16 positive low-grade cervical dysplasia, in which abundant viral transcriptional activity was mapped to the upper third of the epithelium. It is suggested that the physical state of the viral DNA, i.e., integrated viral DNA in the cell lines as opposed to extrachromosomal DNA in low-grade cervical dysplasia, may influence viral gene regulation.

The complete nucleotide sequence of the circular double‐stranded DNA of the genital human papillomavirus type 6b (HPV6b) comprising 7902 bp was determined and compared with the DNA sequences of human papillomavirus type 1a (HPV1a) and bovine papillomavirus type 1 (BPV1). All major open reading frames are located on one DNA strand only. Their arrangement reveals that the genomic organization of HPV6b is similar to that of HPV1a and BPV1. The putative early region includes two large open reading frames E1 and E2 with marked amino acid sequence homologies to HPV1a and BPV1 which are flanked by several smaller frames. The internal part of E2 completely overlaps with another open reading frame E4. The putative late region contains two large open reading frames L1 and L2. The L1 amino acid sequences are highly conserved among analyzed papillomavirus types. By sequence comparison, potential promoter, splicing and polyadenylation signals can be localized in HPV6b DNA suggesting possible mechanisms of genital papillomavirus gene expression.

The transcriptional promoter of the candidate E6‐E7 transforming gene region of human papillomavirus (HPV)‐16 (P97) was active in transiently transfected cervical carcinoma cells when linked to the HSV‐1 tk or bacterial cat genes. Sequences 5′ to P97 contain a short enhancer element responding to cellular factor(s) in uninfected human foreskin keratinocytes and in cervical carcinoma cells, but not in human or animal fibroblasts. The E2 trans‐activator products of HPV‐16 or of the related bovine papillomavirus (BPV)‐1 further elevated HPV‐16‐driven transcripts in co‐transfections, and required the presence of E2‐binding ACC(N)6GGT cores in cis. A ‘short E2’ C‐terminal repressor gene product (sE2) of HPV‐16 or the BPV‐1 sE2 repressor not only inhibited viral E2 trans‐activation, but also suppressed enhancer response to keratinocytic factors. Suppression by the sE2 products was abolished by deletion of the E2‐binding cores in cis or by a mutation in the sE2 DNA binding domain. The keratinocyte‐dependent enhancer is likely to contribute to the epithelial cell tropism of HPV‐16, and may direct persistent E6‐E7 gene transcription in response to cellular factors in cervical carcinoma cells in which the viral E2 genes are inactive.

The transcription of human papillomavirus type 18 (HPV 18) is selectively suppressed in non‐tumorigenic HeLa x fibroblast or HeLa x keratinocyte cell hybrids by 5‐azacytidine. In contrast, viral gene expression is not influenced by 5‐azacytidine in both tumorigenic hybrid segregants and in the parental HeLa cells. The suppression mechanism seems to operate at the level of initiation of transcription since nuclear run‐on experiments show the absence of elongated nascent viral RNA, whereas the transcription of cellular reference genes remains unaffected. Down‐regulation of HPV 18 mRNA correlates directly with cessation of cellular growth and can be abolished using the protein synthesis inhibitor cycloheximide. Furthermore human keratinocytes immortalized by HPV 16 but still retaining the non‐tumorigenic phenotype reveal the same inhibitory effect on viral transcription after treatment with 5‐azacytidine. These results support a model of a postulated intracellular control mechanism, directed against papillomavirus transcription, which can be induced by 5‐azacytidine and appears to correlate with the presence of specific chromosomes in non‐tumorigenic cells.

Human papillomavirus type-16 (HPV-16) transcription in two human keratinocyte cell lines (HPK) immortalized by transfection of viral DNA in vitrowas analyzed by nucleotide sequencing of cDNA clones, and in addition by primer extension analysis and S1 nuclease and exonuclease VII digestion of poly(A)+RNA. A novel mRNA species which probably initiates in the E7 ORF and in which the 5′-part of the E1 ORF (splice donor at position (pos.) 880) is joined to an exon comprising the entire E2 ORF (splice acceptor at pos. 2708) was found in both cell lines. This mRNA has the potential to encode a full-length E2 protein, which is known to function as a repressor of transcription initiated at P97. cDNAs derived from the late region of the viral genome and the use of a late polyadenylation signal at pos. 7320–7325 are described. In agreement with RNA data published by others the major promoter for HPV-16 transcription is located at pos. 97. mRNA species encoding full-length or truncated forms of the E6 protein, and species characterized by an E1iÂE4 splice junction (which provided the E4 open reading frame (ORF) with an ATG triplet) were identified.

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