Reinhard Kirnbauer

Serologically diagnosed infection with human papillomavirus type 16 and risk for subsequent development of cervical carcinoma: nested case-control study

Abstract Objective: To study human papillomavirus type 16 in the aetiology of cervical carcinoma. Design: Within a cohort of 18814 Finnish women followed for up to 23 years a nested case-control study was conducted based on serological diagnosis of past infection with human papillomavirus type 16. Subjects: 72 women (27 with invasive carcinoma and 45 with in situ carcinoma) and 143 matched controls were identified during the follow up. Main outcome measure: Relative risk of cervical carcinoma in presence of IgG antibodies to human papillomavirus type 16. Results: After adjustment for smoking and for antibodies to various other agents of sexually transmitted disease, such as herpes simplex virus type 2 and Chlamydia trachomatis, the only significant association was with infection with human papillomavirus type 16 (odds ratio 12.5; 95% confidence interval 2.7 to 57, 2P<0.001). Conclusion: This prospective study provides epidemiological evidence that infection with human papillomavirus type 16 confers an excess risk for subsequent development of cervical carcinoma. Key messages Key messages Human papillomavirus type 16 is the main micro-organism linked to the development of cervical cancer Prospective studies of infection with this virus and cervical cancer have not been reported because of ethical and clinical difficulties and because diagnosis of past infections with the virus has not been possible In this nested case-control study in over 18000 Finnish women who donated blood to a serum bank 25 years ago we were able to measure past infection with human papillomavirus type 16 with new serological tools The results show that infection with the virus confers an increased risk of developing cervical cancer

Chimeric papillomavirus-like particles expressing a foreign epitope on capsid surface loops.

Neutralization capsid epitopes are important determinants for antibody-mediated immune protection against papillomavirus (PV) infection and induced disease. Chimeric L1 major capsid proteins of the human PV type 16 (HPV-16) and the bovine PV type 1 (BPV-1) with a foreign peptide incorporated into several capsid surface loops self-assembled into pentamers or virus-like particles (VLP). Binding patterns of neutralizing monoclonal antibodies (MAb) and immunization of mice confirmed (i) that regions around aa 282-286 and 351-355 contribute to neutralization epitopes and identified the latter region as an immunodominant site and (ii) that placing a foreign peptide in the context of an assembled structure markedly enhanced its immunogenicity. Pentamers disassembled from wild-type HPV-16 and BPV-1 VLPs displayed some of the neutralization epitopes that were detected on fully assembled VLPs, but were deficient for binding a subset of neutralizing MAb that inhibit cell attachment.

Cytokine release by peripheral blood mononuclear cells is affected by 8-methoxypsoralen plus UV-A.

Psoralen plus UV‐A (PUVA) is an effective therapy for psoriasis but also for other inflammatory dermatoses. The precise mechanisms of action, however, are not absolutely clear. Therefore, the effect of PUVA on the release of the proinflammatory cytokines interleukin (IL)‐1, IL‐6, IL‐8 and tumor necrosis factor alpha (TNFα) was studied. Peripheral blood mononuclear cells (PBMC) obtained from humans were incubated with 8‐methoxypsoralen (8‐MOP) and exposed to UV‐A (20 kJ/m2). This treatment resulted in a significant reduction of IL‐6 and IL‐8 amounts in the supernatants. In addition, an inhibition of IL‐1β and TNFα production by lipopolysaccharide (LPS)‐stimulated PBMC was observed upon PUVA treatment. Accordingly, northern blot analysis showed decreased levels of mRNA encoding for IL‐1β, IL‐6, IL‐8 and TNFα in PUVA‐treated PBMC. Finally PBMC were obtained from psoriatics undergoing oral photochemotherapy before the beginning and after completion of treatment. The PBMC collected after PUVA spontaneously produced significantly less IL‐6 and IL‐8 in comparison to the respective samples obtained before therapy. A similar suppression of IL‐1β and TNFα by in vivo PUVA was found in LPS‐stimulated PBMC. The present data demonstrate that PUVA both in vitro and in vivo suppresses the production of the proinflammatory cytokines IL‐1β, IL‐6, IL‐8 and TNFα by PBMC. Because these cytokines are important in the mediation of inflammatory reactions, one may speculate that the inhibitory effects could contribute to the antiinflammatory activity of PUVA.

Human papillomavirus type 16 capsids expose multiple type-restricted and type-common antigenic epitopes.

The study of viral infectivity and detection of viral capsid antigens of the major cervical cancer-associated human papillomavirus (HPV) type, HPV-16, requires knowledge of which epitopes are exposed in clinical specimens of infected tissue or on intact capsids. To define the antigenic epitopes of HPV-16, antisera to 66 overlapping synthetic peptides corresponding to the HPV-16 capsid proteins L1 and L2 and to seven peptide analogues were tested in immunoperoxidase stainings of consecutive sections from formalin-fixed, paraffin-embedded HPV infected tissue. Antisera against eleven different peptides from L1 and against seven different peptides from L2 recognized the HPV capsid antigen. Most epitopes were only found on the capsid antigen of certain genital HPV types, but four antigenic epitopes in L1 were detectable also in cutaneous wart specimens. All antigenic epitopes in L2 were restricted to genital HPV types and four L2 epitopes were only detectable in HPV-16 or HPV-33 positive specimens. The surface exposure of the antigenic epitopes was investigated by comparing the reactivity of the antipeptide antisera with intact or disrupted virions or capsids of HPV-11, HPV-16 and bovine papillomavirus (BPV). Twenty antipeptide sera from L1 and seven antipeptide sera from L2 were reactive with intact HPV-16 capsids at titres up to 1:146,000. Sixteen of these antisera were also reactive with disrupted HPV-16 capsids. Cross-reactivity with disrupted HPV-11 and BPV was detected for eleven and six antisera, respectively, whereas intact HPV-11 or BPV virions showed only weak cross-reactivity. In conclusion, the HPV-16 L1 and L2 capsid proteins contained multiple antigenic epitopes, most of which were shared with one or several additional HPV types.

Seroreactivity to HPV16 virus-like particles as a marker for cervical cancer risk in high-risk populations

Sexually transmitted genital human papillomavirus (HPV) infection, most often HPV 16, is considered the major etiologic determinant of cervical cancer. However, some studies have found relatively low prevalences of genital tract HPV DNA in some geographical areas, such as Greenland, that have high rates of cervical cancer. We sought to evaluate HPV 16 infection in high‐risk cohorts using a serologic assay that assesses prior exposure as well as current infection and to compare the results with those obtained using a sensitive PCR‐based HPV DNA assay. An ELISA based on HPV 16 virus‐like particles was used to detect IgG serum antibodies in women attending sexually transmitted disease (STD) clinics in Nuuk, Greenland and Copenhagen, Denmark. Using a preassigned cut‐off, 56% of Greenlandic and 41% of Danish women were seropositive (p = 0.02). In Greenlandic women, there was a non‐significant increase in seropositivity with age, and odds ratios for seropositivity were similar for women with more than 5 lifetime sex partners. Seropositivity in the Danish women, however, increased linearly with increases in these 2 factors, which are likely correlates of lifetime exposure to genital HPVs. In contrast, any genital HPV DNA (HPV16 specifically) was detected in 24% and 36% of Greenlandic and Danish women, respectively and was most frequently detected in women below 20. The finding that HPV DNA prevalences, unlike seroprevalences, tended to decrease with increased lifetime risk of infection, provides an explanation for the lack of correlation between HPV DNA prevalences and cervical cancer risk in previous studies of high‐risk populations.

Keratinocyte-Derived Interleukin 3

Interleukin 3 (IL 3) initially was described as a cytokine which is produced by murine T lymphocytes and has multicolony stimulating factor (CSF) activity, activates mast cells and induces the proliferation of hematopoietic stem cell lines. In addition to T cells murine keratinocytes also produce an IL 3-like factor which according to its biological, biochemical and antigenic properties is indistinguishable from murine T cell IL 3. Moreover, by Northern blot analysis murine keratinocytes were found to express mRNA homologous to T cell IL 3 cDNA. Similarly, human keratinocytes have been shown to release an IL 3-like cytokine which also enhances the activity of natural killer cells and stimulates the release of oxygen radicals by granulocytes. However, human IL 3 mRNA could not yet be detected in human epidermal cells or epidermoid carcinoma cell lines. These findings indicate that human keratinocyte IL 3 appears to be distinct from T cell IL 3. Nevertheless, the exact nature of this cytokine remains to be clarified by sequence analysis and gene cloning. Through the production of these cytokines with IL 3-like capacity keratinocytes may participate in the regulation of the activity of different hematopoietic cells and thereby turn on early nonspecific host defense mechanisms against transformed cells and various harmful microbial organisms.

Population dynamics of serologically identified coinfections with human papillomavirus types 11, 16, 18 and 31 in fertile-aged Finnish women

Licensed human papillomavirus (HPV) vaccines are expected to prevent high‐risk (hr) HPV‐infections (most notably types 16 and 18). Whether HPV vaccination will change the distribution of hrHPVs at the population level is open, since competition between HPV types is not well understood. Two stratified random subcohorts (1983–1997 and 1995–2003) of 7,815 and 3,252 women with a minimum of 2 pregnancies (<32 years) were selected from the Finnish Maternity Cohort. Using ELISA based on virus‐like particles (VLP), we determined antibodies to HPV11, 16, 18 and 31 in paired sera of the women and used Poisson regression models to estimate the risk of further infection with other HPV types in those positive for HPV16 or HPV18 at baseline. Baseline HPV16 seropositivity was associated with increased risk of later infections with HPV18 (3.1, 95% CI: 1.7, 5.6). HPV18 seropositivity was associated with increased risk of HPV16 (3.9, 95% CI: 2.5, 6.1). Our observations favor a coinfection rather than superinfection model for the different HPV types and are not suggestive for type‐replacement following HPV vaccination.

Differences in incidence and co-occurrence of vaccine and nonvaccine human papillomavirus types in Finnish population before human papillomavirus mass vaccination suggest competitive advantage for HPV33

To understand likelihood of type replacement after vaccination against the high‐risk human papillomavirus (HPV) types, we evaluated competition of the seven most common genital HPV types in a population sample of unvaccinated, fertile‐aged Finnish women. First trimester sera from two consecutive pregnancies were retrieved from 3,183 Finnish women (mean age, 23.1 years) of whom 42.3% had antibodies to at least one HPV type (6/11/16/18/31/33/45) at the baseline. Antibody positivity to more than one HPV types by the second pregnancy was common among the baseline HPV seropositives. However, compared to baseline HPV‐seronegative women, significantly increased incidence rate ratios (IRRs), indicating an increased risk to seroconvert for another HPV type, were consistently noted only for HPV33 among baseline HPV16 or HPV18 antibody (ab)‐positive women: HPV16ab only → 16&33ab IRR 2.9 [95% confidence interval (CI) 1.6–5.4] and HPV18ab only → 18&33ab IRR 2.5 (95% CI 1.1–6.0), irrespectively of the presence of antibodies to other HPV types at baseline: HPV16ab → 16&33ab IRR 3.2 (95% CI 2.0–5.2) and HPV18ab → 18&33ab IRR 3.6 (95% CI 2.1–5.9). Our findings suggest a possible competitive advantage for HPV33 over other genital HPV types in the unvaccinated population. HPV33 should be monitored for type replacement after HPV mass vaccination.

Anal Human Papillomavirus Testing with Digene’s Hybrid Capture 2 Using Two Different Sampling Methods

PurposeThe incidence of human papillomavirus detection in the anal canal is rising. Efficient anal screening by cytology is hampered because of poor specificity. Human papillomavirus (HPV) testing is proposed in addition to Papanicolaou (Pap) testing for the detection of cervical neoplasia. The purpose of this study was to determine the usefulness of a human papillomavirus-DNA detection test to detect human papillomavirus-associated disease and to compare two different methods of sample collection.MethodsIn 555 patients, anal samples were obtained by using a cervical brush and a Dacron swab to test for high-risk and low-risk human papillomavirus-DNA. Patients positive for human papillomavirus-DNA underwent anoscopy. Biopsies were taken from visible lesions.ResultsLow-risk human papillomavirus-DNA was found in 325 of 555 patients (58.6 percent) and high-risk human papillomavirus-DNA in 285 of 555 patients (51.4 percent). Positive results confined to one single test method were higher for Dacron swab sampling (2.3 vs. 4.3 percent for low-risk human papillomavirus, P < 0.0001; 3.1 vs. 4.9 percent for high-risk human papillomavirus, P < 0.001). A positive correlation of relative light units was found for both sampling methods in the total cohort (P < 0.0001) as well as for patients who tested human papillomavirus-positive by both sampling techniques (P < 0.0001). Sampling with Dacron swabs yielded higher relative light units values compared with sampling with cervical brush for low-risk human papillomavirus-DNA and high-risk human papillomavirus-DNA.ConclusionsAnal screening for human papillomavirus-DNA by hybrid capture 2 is a useful method for detection of human papillomavirus-associated disease. Sample collection using Dacron swabs identifies more human papillomavirus-positive patients, and yields higher relative light unit values than using the cervical brush. Further studies are needed to determine the exact value of hybrid capture 2 in the screening for (pre)cancerous lesions of the anal canal.

Risk of being seropositive for multiple human papillomavirus types among Finnish and Ugandan women.

Abstract Although infections with multiple human papillomavirus (HPV) types have been reported widely, more information is needed on the occurrence of the different types. We determined the distribution of seroprevalences to multiple HPV types in Finland and Uganda to compare the epidemiology of the different HPV types in the 2 populations. Serum samples were obtained from 2784 Finnish and 1964 Ugandan women (mean ages 22 y and 25 y, respectively) of whom 44% and 57%, respectively, had antibodies to at least 1 of the 7 HPV types (6, 11, 16, 18, 31, 33, 45) tested (p < 0.001). Multiple HPV antibody positivity was common. HPV45-seropositive Finns had a higher risk of having antibodies to other high-risk HPV types: HPV18 (odds ratio (OR) = 10.9), HPV31 (OR 6.1), HPV33 (OR 12.2), than their Ugandan counterparts: HPV18 (OR 3.4), HPV31 (OR 2.2), HPV33 (OR 3.3). Increased estimates for being double antibody-positive were also noted among HPV18- and HPV16-seropositive women, but there were no major differences between HPV16-seropositive Finns and Ugandans. In addition to biological and behavioural factors, iatrogenic and societal factors (screening vs no screening) may also result in the different occurrence of infections with the high-risk HPV types in Finland and Uganda.