John Cason

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.

Adjuvanted influenza-H1N1 vaccination reveals lymphoid signatures of age-dependent early responses and of clinical adverse events

Adjuvanted vaccines afford invaluable protection against disease, and the molecular and cellular changes they induce offer direct insight into human immunobiology. Here we show that within 24 h of receiving adjuvanted swine flu vaccine, healthy individuals made expansive, complex molecular and cellular responses that included overt lymphoid as well as myeloid contributions. Unexpectedly, this early response was subtly but significantly different in people older than ∼35 years. Wide-ranging adverse clinical events can seriously confound vaccine adoption, but whether there are immunological correlates of these is unknown. Here we identify a molecular signature of adverse events that was commonly associated with an existing B cell phenotype. Thus immunophenotypic variation among healthy humans may be manifest in complex pathophysiological responses.

High risk genital papillomavirus infections are spread vertically

It is well recognised that high‐risk human papillomaviruses (HPVs) are spread by sexual activity, but the possibility of non‐sexual transmission remains controversial. We present evidence for vertical transmission from at least 30% HPV positive mothers to their infants, resulting in persistent infection in children. That the mother is the source of infant infection has been confirmed by DNA sequencing. We also discuss the evidence for oral HPV‐16 infection in children. In our own studies, HPV‐16 DNA was detected in buccal cells from 48% children, aged 3–11 and transcriptionally active infection was confirmed in some children. Other studies have reported prevalences of 19%‐27% among children less than 11 years of age. Studies that have failed to detect high–risk HPVs in children have used techniques which were insufficiently sensitive to detect the low levels of virus present. Serological studies also suggest that ≤45% prepubertal children have acquired HPV‐16. Thus, convincing evidence is now available for vertical transmission of high risk HPVs, which probably results in widespread infection among children. The consequences of such infections remain to be elucidated. Copyright

Identification of immunogenic regions of the major coat protein of human papillomavirus type 16 that contain type-restricted epitopes.

We have identified regions of the major capsid protein, L1, of the human papillomavirus (HPV) type 16 (HPV-16 L1), that are recognized by five monoclonal antibodies (MAbs) raised to a bacterial fusion protein containing residues 172 to 375 of HPV-16 L1. All five MAbs recognized HPV-16-infected tissue sections by immunohistochemistry, but not sections infected with HPV-1a (cutaneous warts), HPV-6b or -11 (genital warts). MAbs 3D1, 5A4 and 1D6 also recognized HPV-2-infected sections (cutaneous warts); MAb 8C4 recognized only sections containing HPV-16. Four MAbs (8C4, 3D1, 1D6 and 5A4) recognized a synthetic peptide corresponding to residues 269 to 284 of HPV-16 L1; within this region a minimum antibody binding site was identified, a tripeptide 276 to 278. However the complete epitope appears to extend beyond these residues and beyond HPV-16 L1 (269 to 284). The fifth MAb, 1C6, recognized bacterial fusion proteins containing HPV-6b L1, -16 L1 or -18 L1 using immunoblots, yet appeared HPV-16-specific when tested on infected tissue sections. This MAb recognized five amino acids within a different region of HPV-16 L1 (residues 299 to 313).

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.

The influence of zinc status and malnutrition on immunological function in Crohn's disease

Cellular immunity is likely to be important in the pathogenesis of Crohns disease; whether it is abnormal is not clear. The heterogeneity of patients with Crohns disease probably underlies the disparity of reports, but attempts to determine which clinical features influence cellular immunity have been largely unsuccessful. This is probably caused by the omission of nutritional status as a potential factor, even though zinc deficiency has frequently been linked with abnormal immunity. Therefore, a detailed study of nutritional and tissue zinc status, nonspecific cellular immunity, and a measure of phagocytic function was performed in 32 patients with Crohns disease and in a control group of 18 normal subjects and 12 patients with anorexia nervosa. Fourteen patients with Crohns disease, all patients with anorexia nervosa, but none of the normal controls were malnourished. Peripheral blood lymphocyte population levels were normal in patients with Crohns disease and in normal controls, but there was a small decrease in the levels of patients with anorexia nervosa. In vivo delayed hypersensitivity skin test responses were profoundly depressed in patients with anorexia nervosa and decreased in patients with Crohns disease who were malnourished or receiving systemic glucocorticoids. In vitro lymphocyte transformation was reduced in malnourished patients with Crohns disease, but there were only minor changes in patients with anorexia nervosa. There were alterations of in vitro immunoregulation in Crohns disease, but they were not responsible for the abnormal lymphocyte transformation responses in malnourished patients. In vitro phagocytic function was reduced in patients with active Crohns disease. These findings suggest that depressed in vivo and in vitro cellular immunity in malnourished patients with Crohns disease is caused by a qualitative lymphocyte defect and that depressed in vivo but normal in vitro cellular immunity in anorexia nervosa is caused by a quantitative defect. Thus, malnutrition in Crohns disease resembles kwashiorkor; in anorexia nervosa, it resembles marasmus. Tissue zinc status was mostly normal in Crohns disease and anorexia nervosa, and zinc deficiency was not responsible for depressed nonspecific cellular immunity in either condition.

Towards vaccines against human papillomavirus type-16 genital infections

Human papillomavirus type-16 (HPV-16) is strongly associated with cervical carcinoma and cervical intraepithelial neoplasia. It may soon be possible to develop prophylactic vaccines designed to induce neutralizing antibodies to HPV-16 virions in genital secretions and therapeutic vaccines to induce cytotoxic T-cell responses against HPV-16 early proteins in cervical intraepithelial neoplasia and cervical cancers. Although significant advances have been achieved, problems remain before such vaccines can be used routinely.