Barbara Kell

High prevalence of human papillomavirus type 16 infection among children

Infection with high‐risk human papillomaviruses (HPV), is the most significant risk factor for cervical cancer and it may be possible to prevent this malignancy by immunisation. Before immunisation programmes can be designed, however, it is necessary to know the age of acquisition and all routes of infection for these viruses. Sexual transmission is well documented and vertical transmission has also been demonstrated, although the frequency of transmission remains controversial. We previously showed that vertical transmission frequently results in persistent infection, and now present data on the prevalence of HPV‐16 DNA (the most prevalent high‐risk HPV type) in healthy children. Buccal samples from 267 healthy children aged 3–11 years were tested for HPV DNA by generic PCR (MY09/MY11), and a HPV‐16 specific nested PCR. Reverse transcriptase (RT)‐PCR was used to determine the prevalence of transcriptionally active HPV‐16 infection in a subset of children. HPV‐16 DNA was detected by nested PCR in 138 of 267 (51.7%) samples, whereas HPV DNA was detected in only 45 (16.8%) specimens by generic PCR, that has a lower analytical sensitivity. There were no significant differences in prevalence according to age or sex. Early region mRNA was detected by RT‐PCR in six (11.3%) of 53 HPV‐16 E5 DNA positive samples. HPV‐16 E5 DNA sequences from 10 children confirmed the identity of the sequences detected and identified 13 HPV‐16 variants. J. Med. Virol. 61:70–75, 2000.

Detection of E5 oncoprotein in human papillomavirus type 16-positive cervical scrapes using antibodies raised to synthetic peptides

Polyclonal antibodies were raised to partial and full-length synthetic peptides of human papillomavirus type 16 (HPV-16) E5. Antisera specificity for HPV-16 E5 was demonstrated by their ability to recognize not only their peptide immunogens but also full-length peptide and a glutathione S-transferase-E5 fusion protein. The most reactive antiserum, PE-6, raised to a full-length peptide, was used in Western blot analysis to identify HPV-16 E5 protein from exfoliated cervical cells. A strong, single band at approximately 20K was detected in two of six HPV-16-positive samples from women with a history of low-grade cervical intraepithelial neoplasia. The apparent M(r) by SDS-PAGE suggests that HPV-16 E5 forms homodimers in vivo, but not through cysteine linkage.

Cervical lesions are associated with human papillomavirus type 16 intratypic variants that have high transcriptional activity and increased usage of common mammalian codons.

Human papillomavirus type 16 (HPV-16) is a major cause of cervical neoplasia, but only a minority of HPV-16 infections result in cancer. Whether particular HPV-16 variants are associated with cervical disease has not yet been clearly established. An investigation of whether cervical neoplasia is associated with infection with HPV-16 intratypic variants was undertaken by using RFLP analyses in a study of 100 HPV-16 DNA-positive women with or without neoplasia. RFLP variant 2 was positively associated [odds ratio (OR)=2.57] and variant 5 was negatively associated with disease (OR=0.2). Variant 1, which resembles the reference isolate of HPV-16, was found at a similar prevalence among those with and without neoplasia. Variants 1 and 2 were also more likely to be associated with detectable viral mRNA than variant 5 (respectively P=0.03 and P=0.00). When HPV-16 E5 ORFs in 50 clones from 36 clinical samples were sequenced, 19 variant HPV-16 E5 DNA sequences were identified. Twelve of these DNA sequences encoded variant E5 amino acid sequences, 10 of which were novel. Whilst the associations between HPV-16 E5 RFLP variants and neoplasia could not be attributed to differences in amino acid sequences, correlation was observed in codon usage. DNA sequences of RFLP variant 2 (associated with greatest OR for neoplasia) had a significantly greater usage of common mammalian codons compared with RFLP pattern 1 variants.

HUMAN PAPILLOMAVIRUS TYPE 16 IN INFANTS : USE OF DNA SEQUENCE ANALYSES TO DETERMINE THE SOURCE OF INFECTION

Perinatal transmission of human papillomavirus type 16 (HPV-16) and persistence of virus DNA in infants until 6 months of age has been described. To confirm the origin of infant infections as maternal, we determined the nucleotide sequence of the upstream regulatory region (URR; bp 7540 to 157) of HPV-16 in samples from 13 HPV-16 DNA-positive mothers and their infants at 6 weeks and 2 years of age. Identical HPV-16 variant URR sequences were found in two mother/infant samples and similar variants were found in three sets. Four mothers with samples which contained prototypic HPV-16 sequences delivered infants who also had the prototypic sequence. Four mothers with variant URRs delivered infants who harboured either prototypic or different URR variants. Thus, concordant variants or prototypic sequences were detected in nine of 13 mother/infant samples, indicating that up to 69.2% of HPV-16-positive infants acquire virus from their mothers.

Analytic sensitivities of hybrid-capture, consensus and type-specific polymerase chain reactions for the detection of human papillomavirus type 16 DNA

Human papillomavirus type 16 (HPV‐16) DNA is detected commonly in cervical carcinomas; in this study, we have determined the analytical sensitivities of Hybrid Capture, HPV‐consensus PCR, and three HPV‐16‐specific polymerase chain reactions (PCRs) for the detection of HPV‐16 DNA. Samples investigated included a cervical cancer cell line, cervical scrapes from 20 patients attending colposcopy clinics, and buccal swabs from eight immunosuppressed children. HPV‐16 E7 and E5‐nested PCRs [Cavuslu et al. (1996): Journal of Virological Methods, in press] produced positive signals from samples containing fewer than ten HPV‐16 genomes per reaction. HPV‐consensus PCR [Manos et al. (1989): Cancer Cells 7:209–214] and HPV‐16 PCR using primers of van den Brule et al. [(1990): Journal of Clinical Microbiology 25:2739–2743] were of intermediate sensitivity (i.e., produced positive signals from samples containing 250 and 2,500 HPV‐16 genoms/reaction, respectively) and Hybrid Capture could detect just 50,000 HPV‐16 genomes/reaction. Highest rates of positivity for cervical samples were detected with HPV‐16 E7 or E5‐nested PCRs [50% (10 of 20 samples) and 60% (12 of 20 samples) positive, respectively], intermediate rates with HPV‐consensus PCR and PCRs using the primers of van den Brule et al. [both 35% (7 of 20 samples)], and lowest rates of positivity [25% (5 of 20 samples)] with Hybrid Capture. None of eight buccal swab samples from immunosuppressed children were positive by Hybrid Capture, yet three (37.5%) were positive by HPV‐16 E5‐nested PCR. These data indicate that HPV‐16 type‐specific PCRs should be used for the investigation of specimens that may contain low amounts of HPV‐16 DNA.

Proliferative T-cell responses to human papillomavirus type 16 E5 are decreased amongst women with high-grade neoplasia

Proliferative responses to human papillomavirus type 16 (HPV-16) E5 peptides were determined for short-term cell lines derived from peripheral blood mononuclear cells of 75 women. Cell lines from 16 of the 75 women proliferated in response to stimulation with pooled E5 peptides; this was most common for patients with low-grade squamous cervical intraepithelial lesions (LSIL; 6 of 15 patients, 40%) and less frequent for asymptomatic women with no cervical lesions (4 of 20, 20%), those with high-grade squamous intraepithelial lesions(HSIL; 5 of 33, 15%) and others with cervical cancer (1 of 7, 14%, P = 0.027). Amongst these patients, proliferative responses were exclusive to those that were positive for HPV-16 DNA (12 of 41, 29%; c.f. none of 13 HPV-16 DNA-negative subjects exhibited a proliferative response; P= 0023) and were again most prevalent amongst HPV-16 DNA-positive LSIL (6 of 14, 43%), as compared with HPV-16 DNA-positive HSIL (5 of 23, 22%) or HPV-16 DNA-positive cervical cancer patients (1 of 4, 25%, P > 0.05). In contrast, for asymptomatic women, responsiveness was statistically independent of HPV-16 DNA status, i.e. responsiveness in HPV-16 DNA-positive and DNA-negative subjects was observed in 3 of 15 (20%) and 1 of 5 (20%) cases, respectively (P > 0.05). There were no associations between detection of HPV-16 mRNA and proliferative responses (P> 0.05). These data suggest that HPV-16 E5-specific T-helper activity is depressed amongst women with HSIL lesions.

Polymerase chain reaction protocols for the detection of DNA from mucosal human papillomavirus types -6, -11, -16, -18, -31 and -33

Individual types of human papillomaviruses (HPV) which infect mucosal surfaces have been implicated as the causative agents for carcinomas of the cervix, anus, penis, larynx and the buccal cavity, occasional periungal carcinomas, as well as benign anogenital warts. The identification of particular HPV types is thus important for: identifying patients with premalignant lesions who are at risk of progression to malignancy; epidemiological studies; studies of the natural history of these viruses; and even medico-legal cases of suspected sexual abuse of children. In this protocol we describe PCR assays for: the identification of DNA from the mucosal HPVs types -6, -11, -16, -18, -31 and -33; a consensus HPV PCR for detecting DNA from 20 characterised mucosal HPVs, as well as more than 25 novel HPVs; and, for a control PCR for beta-globin.

Detection of human papillomavirus type-16 DNA utilising microtitre-plate based amplification reactions and a solid-phase enzyme-immunoassay detection system

The development of a nested polymerase chain reaction (PCR) assay to detect low concentrations of human papillomavirus type-16 (HPV-16) DNA for epidemiological studies is described. The PCR utilises primers located in the E5 open reading frame, has an analytical sensitivity of 4 HPV-16 genomes and does not produce amplicons from other common genital HPVs (types-6, -11, -18, -31 and 33). This assay was carried out in 96-well plates utilising internal primers labelled with dinitrophenol (DNP) and biotin so that amplicons can be captured onto streptavidincoated plates and detected using an alkaline phosphatase-labelled monoclonal antibody to DNP. The assay was effective for detecting HPV-16 DNA in plasmids, cell-lines and, both freshly collected or archival (formalin-fixed/paraffin embedded) clinical specimens. This system is therefore suitable for epidemiological studies to identify individuals infected with HPV-16 DNA in episomal form who may be at increased risk of developing anogenital carcinomas.

Detection of human papillomavirus type 16 in microtitre plate based immuno-enzymatic assays: use to determine E5 gene expression in cervical carcinomas

BACKGROUND E5-based nested polymerase chain reaction (PCR) assays and a PCR-enzyme immunoassay (EIA) to detect human papillomavirus type 16 (HPV-16) DNA have been developed. These assays were designed to detect small amounts of HPV-16 DNA for epidemiological studies of subclinical infection. OBJECTIVES The E5 gene of HPV-16 may be lost in some cell lines derived from cervical carcinomas. The aim of this study was to determine if, and how frequently, E5 gene loss occurs in biopsy samples from patients with cervical lesions. STUDY DESIGN Sixteen HPV-16 (E7) DNA positive and five HPV-16 DNA negative cervical lesions (nineteen cervical carcinomas, two cervical intraepithelial neoplasias) were investigated by E5 nested PCR and EIA. RESULTS Overall, 15 of the 16 (93.75%) HPV-16 E7 positive samples were positive for HPV-16 E5 DNA: 14 of 16 (87.5%) were positive by E5 PCR and 15 of 16 (93.75%) were positive by E5 PCR, nested PCR and by PCR-EIA. One of 14 HPV-16 (E7) DNA positive cervical carcinomas was negative for E5 DNA in all three assays. CONCLUSION Loss of the HPV-16 E5 open reading frame (ORF) is a rare event in HPV-16 positive cervical carcinomas and was detected in just one of 14 (7.1%) cases.

Detection of Class-Specific Antibodies to Baculovirus-Derived Human Papillomavirus Type 16 (HPV-16) Capsid Proteins

Seroepidemiological. studies on the prevalence of HPV16 infections are not possible as HPVs cannot be grown in conventional. cell cultures and premalignant and malignant cervical. lesions contain few intact HPV-16 particles and only low levels of native viral. proteins. Whilst HPV-31b has been propagated in vitro 1 and HPV-16 virions have been produced in nude mice2, virus yields are low. Peptides and prokaryotic fusion proteins have therefore been used as antigens for serological. studies3,4. As such constructs probably represent poor imitations of native viral. proteins5, we have expressed the L1 and L2 proteins of HPV-16 in insect cells (via recombinant Autographa californica nuclear polyhedrosis virus: AcMNPV) for serological. studies.