Human challenge trials in vaccine development, Rockville, MD, USA, September 28-30, 2017.

The International Alliance for Biological Standardization organized the second workshop on human challenge trials (HCT) in Rockville, MD, in September 2017. The objective of this meeting was to examine the use of HCT, in response to the continuing human suffering caused by infectious diseases, preventable by the development of new and improved vaccines. For this, the approach of HCT could be valuable, as HCT can provide key safety, tolerability, immunogenicity, and efficacy data, and can be used to study host-pathogen biology. HCT can generate these data with speed, efficiency and minimal expense, albeit not with the same level of robustness as clinical trials. Incorporated wisely into a clinical development plan, HCT can support optimization or down-selection of new vaccine candidates, assuring that only the worthiest candidates progress to field testing. HCT may also provide pivotal efficacy data in support of licensure, particularly when field efficacy studies are not feasible. Many aspects of HCT were discussed by the participants, including new and existing models, standardization and ethics. A consensus was achieved that HCT, if ethically justified and performed with careful attention to safety and informed consent, should be pursued to promote and accelerate vaccine development.

High-risk human papillomavirus types in HIV-infected and HIV-uninfected young women in KwaZulu-Natal, South Africa: implications for vaccination.

BACKGROUND: High-risk human papillomavirus (hr-HPV) infections and low-grade squamous intraepithelial lesions occur frequently in young women. The available vaccines cover up to seven hr-HPV genotypes (HPV16, HPV18, HPV31, HPV33, HPV45, HPV52 and HPV58) and two low-risk HPV types (HPV6 and HPV11). The objective of this study was to describe the hr-HPV genotypes present among HIV-uninfected and HIV-infected young women in rural high schools. METHODS: Cervicovaginal lavages were obtained from sexually active young women recruited from high schools in KwaZulu-Natal (n = 1223). HPV testing was done by the polymerase chain reaction using GP5+/GP6 + primers and enzyme immunoassay. HIV testing was done using rapid test kits. RESULTS: Of the 1223 cervicovaginal lavages, 301 (25%) were positive for hr-HPV. The HPV prevalence was higher in HIV infected (32.20%, 95% CI: 0.27-0.38) than in HIV-uninfected women (22.50%, 95% CI: 0.21-0.26), (p = .001). Similarly, multiple infections were slightly more common in HIV infected (59.32%) than in HIV-uninfected women (53.51%), (p = .37). The nine predominant genotypes in descending order were HPV types 16 (n = 99, 22.10%), 51 (n = 58, 12.91%), 18 (n = 56, 12.50%), 35 (n = 50, 11.10%), 33 (n = 47, 10.82%), 56 (n = 42, 9.31%), 45 (n = 34, 7.60%), 52 (n = 32, 7.14%) and 59 (n = 31, 6.91%). HPV 35, 51, 56 and 59 (40.62%), which are not covered by any vaccine, were among the most prevalent in the schools of KwaZulu-Natal. CONCLUSION: Four of the most predominant high-risk HPV types in this region are not covered by the new nine-valent HPV vaccine.

Expression analysis on archival material: comparison of 5 commercially available RNA isolation kits for FFPE material.

BACKGROUND: Formalin-fixed paraffin-embedded (FFPE) tissue is the most common tissue specimen widely available. Moreover, long clinical follow-up is on hand. Therefore, FFPE material is a precious source of material for identifying predictive and/or prognostic biomarkers in cancer research on the basis of gene expression. However, the main drawback of FFPE tissue is the significant reduction in quantity and quality of the extracted RNA. The aim of this study is the comparison of different commercially available kits for the RNA isolation in FFPE tissue material. METHODS: Five commercially available RNA isolation kits were tested and the concentration, purity, integrity, and raw cycle threshold values were determined. RESULTS: The mean total RNA concentrations were as follows: Qiagen 25957±19417 ng, Ambion 8249±2898 ng, SA Biosciences 8070±3700 ng, and Macherey-Nagel 622±394 ng. The mean A260/A280 ratios were as follows: Qiagen: 1.81, SA Biosciences: 0.66, Ambion: 1.03, and Macherey-Nagel: 1.04. The mean A260/A230 ratios were as follows: Qiagen: 1.88, SA Biosciences: 1.61, Ambion: 1.54, and Macherey-Nagel: 1.88. The RNA extractions from Epicentre could not be measured by the Nanodrop and, therefore, were excluded from further analysis. The mean RNA integrity number (range, 2.09 to 2.47) and the mean raw cycle threshold values (range, 33.43 to 35.37) were more or less the same for all the tested RNA isolation kits. CONCLUSIONS: Altogether, on the basis of the number of adequate isolations, the kit from Qiagen seems to be the most appropriate kit to be used in our further studies that require RNA isolation from FFPE material.

The presence of Y chromosomal deoxyribonucleic acid in the female vaginal swab: possible implications for human papillomavirus testing.

BACKGROUND: Vaginal self-sampling for human papillomavirus (HPV) detection is the focus of recent research. However, it has been shown previously that male DNA can be detected in vaginal swabs. The aim of this study was to investigate whether a female vaginal swab may reflect a male active HPV infection. METHODS: Eleven women volunteered to take vaginal samples. The first sample was taken within hours after unprotected intercourse, the others each following morning for five consecutive days. On these samples, a Y-chromosomal locus, as a surrogate marker for HPV, was amplified by PCR. To investigate the prevalence of male DNA in self-obtained vaginal swabs, 282 swabs from 16 women enrolled in an ongoing HPV follow-up study were tested. RESULTS: All baseline samples from the 11 women were positive for male DNA. In the follow-up samples, positivity ranged from day 1 till day 5, with a sharp drop from day 2 (91%) to day 3 (36%). Of 282 swabs, 23 (8.2%) were positive for male DNA. However, 10 of these 23 swabs were provided by one single woman. DISCUSSION: Since HPV can be deposited by the male in the vagina, either through semen, or through exfoliated epithelial cells, our results are of some importance when testing for HPV in vaginal swabs. It is conceivable that women with acquired immunity to HPV, but with an HPV positive partner, are found to be HPV positive in the swab. However, larger studies on more diverse populations are warranted.

Everything you always wanted to know about HPV (but could not ask your doctor).

OBJECTIVE: To investigate specific information needs of people who search for information about the human papillomavirus (HPV) on the Internet. METHODS: We performed a qualitative analysis of the e-mail questions asked by the visitors of a website with evidence-based information about HPV. The website, hosted by Antwerp University, provided basic information on epidemiology and natural history of HPV in women and men, diagnostic and treatment options, screening, and vaccination. If visitors did not find an answer to their questions, they could mail their question to an e-mail address associated with the website. RESULTS: We received 713 questions posed by 527 e-mail correspondents. The following themes emerged as most important: transmission of HPV, the HPV vaccine, the natural history of the virus, the vicious circle (re-infection between partners), HPV detection in men and women, treatment of men and women, incubation time, pregnancy/fertility, genital warts (in)fidelity, and symptoms of HPV infection. CONCLUSION: Both men and women are seeking health information on HPV on the Internet, often after being counseled by a health care provider. PRACTICE IMPLICATIONS: Practitioners should be prepared for questions on the themes that concern people most. Practitioners may play a role in guidance towards reliable sources of information.

General practitioners' perception of risk factors for cervical cancer development: consequences for patient education.

OBJECTIVE: The introduction of human papillomavirus (HPV) detection in cervical screening will make it necessary to provide appropriate information to the general public. Only 3% of Flemish women could name HPV as the viral agent involved in cervical cancer development. The aim of this study was to investigate whether general practitioners (GPs) have appropriate knowledge of the relationship between HPV and cervical cancer to be able to inform women. METHODS: A questionnaire was developed to measure perception of 20 risk factors for cervical cancer development, on a scale of 1 (unimportant) to 5 (very important). Respondents were also asked to give an estimate of the chances of survival for women, diagnosed with cervical cancer detected by screening. RESULTS: Sixty GPs and 28 trainees filled in the questionnaire. The five most important risk factors in the perception of the respondents were, in order of decreasing importance, viral infection, number of sex partners, sexual behaviour of the partner, unsafe sex, and early start of sexual activity. Fifty-six percent of the GPs expected the chance of survival to be between 80 and 100%, compared to only 31% of the trainees. CONCLUSION: Most GPs are well aware of sexual habits as risk factors for cervical cancer development, including the role of HPV as the viral agent in the etiology. However, they seem to underestimate the role of smoking and are unable to identify the correct chance of survival for women in whom cervical cancer is detected within the frame of the cervical smear program. PRACTICE IMPLICATIONS: Attention should be given to education of medical students and practitioners, in order to allow them to supply patients with sufficient background information to make an informed choice on participating in cervical cancer screening.

Combined Pap and HPV testing in primary screening for cervical abnormalities: should HPV detection be delayed until age 35?

In 2003, the United States Food and Drug Administration has approved the Hybrid Capture 2 assay for use with a Pap test to adjunctively screen women of 30 years and older for the presence of high-risk human papillomavirus (HR-HPV) infection. Although the predictive power of a negative test is strong, the number of false-positives may still be high. We investigated HPV prevalence in relation to age in a group of 2293 women, aged between 20 and 50, with normal cytology. Overall HR-HPV prevalence was 6.9% (95%CI=5.9-8.0%). Regression analysis using 5-year intervals showed that the HR-HPV prevalence did not significantly decline up to age 34, whereas it declined significantly after age 35. This would suggest that postponing HPV detection in primary screening from age 30 to 35 would result in a decrease of almost 50% of the number of women with normal cytology and a transient HPV infection. However, larger scale studies are required to confirm this finding.

Unraveling the mechanism of radiosensitization by gemcitabine: the role of TP53.

Gemcitabine has excellent radiosensitizing properties, as shown in both preclinical and clinical studies. Radiosensitization correlated with the early S-phase block of gemcitabine. In the present study, we investigated the role of TP53 in the radiosensitizing effect of gemcitabine. Isogenic A549 cells differing in TP53 status were treated with gemcitabine during the 24 h prior to irradiation. Cell survival was determined 7 days after irradiation by the sulforhodamine B test. In addition, cell cycle perturbation was determined by flow cytometry and TP53 expression by Western blot analysis. Gemcitabine caused a concentration-dependent radiosensitizing effect in all cell lines. Transformed A549 cells were less sensitive to the cytotoxic effect of gemcitabine. The cell cycle arrest early in the S phase was dependent on the drug dose but was comparable in the different cell lines and was not related to functional TP53. Using isogenic cell lines, we have shown that neither TP53 status nor the transfection procedure influenced the radiosensitizing effect of gemcitabine. Since both the radiosensitizing effect at equitoxic concentrations and the cell cycle effect of gemcitabine were independent of TP53 expression, it is likely that TP53 protein does not play a crucial role in the radiosensitizing mechanism of gemcitabine.

Human papillomavirus in a rural community in Zimbabwe: the impact of HIV co-infection on HPV genotype distribution.

Cervical cancer is a leading cause of cancer-related deaths in developing countries, and the human papillomavirus (HPV) is linked etiologically to cervical cancer. Hence, a vaccine which prevents HPV-associated cervical cancer would have the most impact in developing countries, including the African continent. The type-specific immune response towards HPV virus-like particles, in combination with geographical variation in the prevalence of HPV, necessitates the presence of multiple HPV type antigens in a single vaccine cocktail in order to provide relevant protection. We aimed to investigate whether co-infection with HIV, which is highly prevalent in Africa, plays a role in HPV genotype distribution. After informed consent, HPV detection by GP5+/6+ PCR and HIV detection by serology was carried out on 236 women from the rural north-western part of Zimbabwe. The prevalence of HPV was higher in HIV positive women (54%) than in HIV negative women (27%). Certain HPV types (HPV types 11, 39, 43, 51, and 59, P-values ranging from 0.017 to 0.067) occurred more frequently in HIV positive women. Only high-risk HPV, and not HIV, was associated significantly with cervical intraepithelial neoplasia in multiple regression analysis. In conclusion, a high prevalence of HPV was found in a rural community, where regular Papanicolaou (Pap) smears would be a logistic and economic impossibility, but where free vaccination programmes against other infections are already established. The results suggest that HIV co-infection may have an impact on HPV genotype distribution.

Can cervical cancer screening be stopped at 50? The prevalence of HPV in elderly women.

Although the relation between cervical cancer and the human papillomavirus (HPV) has been established beyond doubt, the introduction of HPV detection in cervical cancer screening is halted, primarily by the high rate of false positivity in relation to morbidity, since the majority of women infected with HPV will not develop lesions. To counteract overconsumption of cervical cancer screening in elderly women, we wanted to test the hypothesis that women of 50 years or older who are HPV-negative and have a cytologically normal smear might be encouraged to refrain from further screening. As a first step, the prevalence of high-risk HPV in a population of 1,936 women of 50 years and older was investigated. After an initial decline, a slightly higher prevalence can be seen with increasing age. There is a decrease in the prevalence of multiple infections with age, paralleled by an increase in single infections, especially of HPV type 16 in the eldest-age group. However, neither the decrease in multiple infections nor the increase in single infections is statistically significant. The data obtained in this study show that, even in the presence of a slight increase in the HPV prevalence in elderly women, approximately 94% of the elderly women can be withdrawn from the cervical cancer screening. However, a follow-up study will be necessary to determine the frequency of (re)infection as well as the course of an HPV infection in elderly women.

Comparison of the sulforhodamine B assay and the clonogenic assay for in vitro chemoradiation studies.

PURPOSE: Since there is a growing interest in preclinical research on interactions between radiation and cytotoxic agents, this study focused on the development of an alternative to the very laborious clonogenic assay (CA). METHODS: The colorimetric sulforhodamine B (SRB) assay was compared to the clonogenic assay for radiosensitivity testing in two lung cancer cell lines (A549, H292), one colon cancer cell line (HT-29) and one breast cancer cell line (MCF-7). In addition, the combination of the radiosensitizing agent gemcitabine and radiation was investigated with both assays. RESULTS: The dose-response curves obtained with the SRB assay and the CA were very similar up to 6 Gy. The radiosensitivity parameters (SF(2), alpha, beta, MID and ID(50)) obtained from the SRB assay and the CA were not significantly different between H292, A549 and MCF-7 cells. The radiation dose-response curves for A549 and H292 cells pretreated with 4 n M gemcitabine for 24 h clearly showed a radiosensitizing effect with both assays. The dose-enhancement factors obtained with the SRB assay and the CA were 1.80 and 1.76, respectively, for A549 cells, and 1.52 and 1.41 for H292 cells. CONCLUSIONS: The SRB assay was shown to be as useful as the more traditional CA for research on chemotherapy/radiotherapy interactions in cell lines with moderate radiosensitivity. This assay will be used for more extensive in vitro research on radiosensitizing compounds in these cell lines.