Human papillomavirus type 16 viral load is decreased following a therapeutic vaccination.

In the dose-escalation phase of a Phase I clinical trial in which six subjects each were vaccinated with PepCan at the 50, 100, 250, and 500 µg per peptide dose, the 50 µg dose showed the best histological regression rate. Ten additional subjects were vaccinated at this dose in the final dose phase. As with the dose-escalation phase, no dose-limiting toxicities were observed. Overall, the histological regression rates were 50% at the 50 µg dose (7 of 14) and 100 µg dose (3 of 6), and 45 % overall (14 of 31). Of subjects in whom HPV type 16 (HPV 16) was detected at entry, it became undetectable in three subjects after vaccination, and the viral loads significantly decreased in nine subjects in whom HPV 16 infection was detected at entry and exit (p = 0.008). Immune profiling revealed increased T-helper type 1 cells after vaccinations (p = 0.02 and 0.0004 after 2 and 4 vaccinations, respectively). T-helper type 2 cells initially increased after two vaccinations (p = 0.01), but decreased below the baseline level after four vaccinations although not significantly. Pre-vaccination regulatory T cell levels were significantly lower in histological responders compared to non-responders (p = 0.03). Feasibility of testing plasma for multiplex cytokine/chemokine analysis and of performing proteomic analysis of PBMCs was examined for potentially identifying biomarkers in the future. While these analyses are feasible to perform, attention needs to be given to how soon the blood samples would be processed after phlebotomy. As sufficient safety of PepCan has been demonstrated, enrollment for the Phase II clinical trial has been opened.

A novel use of a statewide telecolposcopy network for recruitment of participants in a Phase I clinical trial of a human papillomavirus therapeutic vaccine.

BACKGROUND: Historically, recruitment and retention of young women in intervention-based clinical trials have been challenging. In August 2012, enrollment for a clinical trial testing of an investigational human papillomavirus therapeutic vaccine called PepCan was opened at our institution. This study was an open-label, single-arm, single-institution, dose-escalation Phase I clinical trial. Women with recent Papanicolaou smear results showing high-grade squamous intraepithelial lesions or results that could not rule out high-grade squamous intraepithelial lesion were eligible to enroll. Patients with biopsy-confirmed high-grade squamous intraepithelial lesion were also eligible. Colposcopy was performed at the screening visit, and participants became eligible for vaccination when the diagnosis of high-grade squamous intraepithelial lesion was confirmed with biopsy and other inclusion criteria were met. The aim of this study was to identify strategies and factors effective in recruitment and retention of study participants. METHODS: Potential vaccine candidates were recruited through direct advertisement as well as referrals, including referrals through the Arkansas telecolposcopy network. The network is a federally funded program, administered by physicians and advanced practice nurses. The network telemedically links rural health sites and allows physician-guided colposcopy and biopsies to be conducted by advanced practice nurses. A variety of strategies were employed to assure good retention, including face-to-face contact with the study coordinator at the time of consent and most of study visits; frequent contact using text messaging, phone calls, and e-mails; and creation of a private Facebook page to improve communication among research staff and study participants. A questionnaire, inquiring about motivation for joining the study, occupation, education, household income, number of children, and number of sexual partners, was administered at the screening visit with the intent of identifying factor(s) associated with recruitment and retention. RESULTS: A total of 37 participants were enrolled between September 2012 and March 2014. The largest proportion of participants (46%) was enrolled from the telecolposcopy network. Others were enrolled through outside institutions (43%), in-house referrals (8%), or direct advertisement (3%). Most participants were motivated to join the study to take care of their health issues. Only two participants joined the Facebook private page. Of the 24 participants who qualified for vaccination, only 1 terminated early due to an unanticipated move. CONCLUSION: The availability of a large number of potential participants from the telecolposcopy network increased recruitment to this clinical trial by 85% over other traditional means of recruitment. The telecolposcopy network is not only a means of providing a gynecological service to women who otherwise would forego care but also a novel and valuable resource in recruiting participants for a clinical trial.

Distribution of human papillomavirus (HPV) types and anti-HPV T-cell immune responses among different racial/ethnic groups in Central Arkansas.

The distribution of human papillomavirus (HPV) types and T-cell immune responses were compared among European American, African American, and Hispanic American populations being followed for abnormal Papanicolaou smear results who were attending the same university medical center clinic in Central Arkansas. Statistically significant differences were found for HPV types 55, 58, and 83 among the 37 HPV types tested. However, there were no differences in T-cell immune responses among these racial/ethnic groups. These results are unlikely to have an impact on therapeutic HPV vaccine development since the most prevalent HPV type among all racial/ethnic groups was HPV type 16.

CD4+ T-cell response against human papillomavirus type 16 E6 protein is associated with a favorable clinical trend.

The association between the CD8+ T-cell responses to human papillomavirus type 16 (HPV-16) E6 protein and a favorable clinical trend has been demonstrated previously. The roles of human papillomavirus (HPV)-specific CD4+ T-cell responses and of regulatory T-cells (Tregs) were examined. Subjects with a recent history of abnormal Papanicolaou smear were eligible, and colposcopy-guided biopsy was performed at enrollment. Interferon-γ enzyme-linked immunospot assay and fluorescent-activated cell sorter analysis to measure the frequencies of Tregs were performed. Subjects with histological diagnoses of cervical intraepithelial neoplasia 1, 2, or 3 were considered to have short-term persistence of cervical abnormality and were called "persistors" (n = 51) while those of normal histology were designated to be "regressors" (n = 33). A significantly higher percentage CD4+ T-cell response was detected in the regressors (15/33 or 45.5%) compared with the persistors (10/51 or 19.6%) (P = .015) for the E6 peptides but not for the E7 peptides. The CD4+ responses to certain E6 regions [E6(16-40), E6(91-115), E6(106-130), and E6(136-158)] were also significantly higher in the regressors. Although there was no difference in the frequencies of Tregs between the two groups, low frequencies of Tregs were significantly associated with positive CD4+ T-cell responses within certain E6 regions [E6(16-40), E6(31-55), E6(76-100), E6(91-115), and E6(106-130)]. The CD4+ and CD8+ T-cell responses to the HPV-16 E6 protein are associated with a favorable clinical trend. The HPV-16 E6 protein should be incorporated in the design of an HPV therapeutic vaccine.

Evaluation of DNA extraction protocols from liquid-based cytology specimens for studying cervical microbiota.

Cervical microbiota (CM) are considered an important factor affecting the progression of cervical intraepithelial neoplasia (CIN) and are implicated in the persistence of human papillomavirus (HPV). Collection of liquid-based cytology (LBC) samples is routine for cervical cancer screening and HPV genotyping and can be used for long-term cytological biobanking. We sought to determine whether it is possible to access microbial DNA from LBC specimens, and compared the performance of four different extraction protocols: (ZymoBIOMICS DNA Miniprep Kit; QIAamp PowerFecal Pro DNA Kit; QIAamp DNA Mini Kit; and IndiSpin Pathogen Kit) and their ability to capture the diversity of CM from LBC specimens. LBC specimens from 20 patients (stored for 716 ± 105 days) with CIN values of 2 or 3 were each aliquoted for each of the four kits. Loss of microbial diversity due to long-term LBC storage could not be assessed due to lack of fresh LBC samples. Comparisons with other types of cervical sampling were not performed. We observed that all DNA extraction kits provided equivalent accessibility to the cervical microbial DNA within stored LBC samples. Approximately 80% microbial genera were shared among all DNA extraction protocols. Potential kit contaminants were observed as well. Variation between individuals was a significantly greater influence on the observed microbial composition than was the method of DNA extraction. We also observed that HPV16 was significantly associated with community types that were not dominated by Lactobacillus iners.

A favorable clinical trend is associated with CD8 T-cell immune responses to the human papillomavirus type 16 e6 antigens in women being studied for abnormal pap smear results.

OBJECTIVE: The goal of this study was to examine the role of CD8 T-cell responses to human papillomavirus type 16 (HPV-16) in a favorable clinical trend in women being studied for abnormal Pap smear results. MATERIALS AND METHODS: Human papillomavirus-deoxyribonucleic acid testing and enzyme-linked immunospot assay using the HPV-16 E6 and E7 antigens were performed. The subjects with subsequent normal histologic diagnoses were considered to be "regressors" (n = 28), whereas those with histologic diagnoses of cervical intraepithelial neoplasia 1, 2, or 3 were considered to have short-term persistence of cervical abnormality and were designated to be "persistors" (n = 37). RESULTS: There was a higher percentage of CD8 T-cell responses to the E6 antigen in the regressors (15/28 or 53.6%) when compared with the persistors (10/37 or 27.0%; p = .04), but there was no recorded response difference for the E7 antigen. Results were the same when the analyses for E6 included only subjects who were high-risk HPV-positive (p = .01). CONCLUSIONS: The CD8 T-cell immune responses to the HPV-16 E6 antigens but not to E7 antigens are associated with a favorable clinical trend regardless of HPV types currently detected.

Cervical Microbiome and Response to a Human Papillomavirus Therapeutic Vaccine for Treating High-Grade Cervical Squamous Intraepithelial Lesion.

Human papillomavirus (HPV) infection is associated with the vast majority of cervical cancer cases as well as with other anogenital cancers. PepCan is an investigational HPV therapeutic vaccine for treating cervical high-grade squamous intraepithelial lesions. The present study was performed to test whether the cervical microbiome influences vaccine responses and to explore host factors as determinants of the cervical microbiome composition in women with biopsy-proven high-grade squamous intraepithelial lesions. In a recently completed Phase I clinical trial of PepCan, histological response rate of 45% (14 of 31 patients), a significant increase in circulating T-helper type 1 cells, and a significant decrease in HPV 16 viral load were reported. DNA, extracted from liquid cytology specimens collected before and after vaccinations, were amplified and then hybridized to a G4 PhyloChip assay to characterize the microbiome. We describe trends that certain bacterial taxa in the cervix may be enriched in non-responders in comparison to responders (Padj = .052 for phylum Caldithrix and Padj = .059 for phylum Nitrospirae). There was no difference in bacterial diversity between the 2 groups. A permutational analysis of variance performed for various demographic and immune parameters showed significant clustering with microbiome beta diversity for race, HPV 16 status, peripheral T-helper type 1 cells, and HLA-B40 (P = .001, .014, .037, and .024, respectively). Further analyses showed significant differences at the empirical Operational Taxonomic Unit level for race and HPV 16 status. As these results are from a small Phase I study, further studies are needed to examine the role of cervical microbiome in response to HPV therapeutic vaccines.

Maternal and perinatal outcomes of indicated inductions of labor.

PURPOSE: The purpose of this study was to evaluate the labor characteristics and maternal/perinatal consequences following indicated induction of labor. METHODS: This retrospective study examined all of the indicated inductions over 24 months and at two institutions evaluated mode of delivery (vaginal versus cesarean) as well as a comprehensive list of labor characteristics and maternal/perinatal outcomes. RESULTS: There were 1577 indicated inductions with 1097/1577 (69.6%) delivering vaginally. Women with a cesarean delivery had lower parity (p < (0).0001) and Bishop's score (p < (0).0001), and higher body mass indices (p = 0.022). Indication for induction due to preeclampsia increased the risk of a cesarean delivery (p = 0.008). Chorioamnionitis, post-partum complications and NICU admissions were greater in the women delivered by cesarean section. CONCLUSIONS: Indicated inductions which result in cesarean delivery were more likely in women with higher body mass indexes, lower parity, and Bishops scores, as well as preeclampsia.

A phase I dose-escalation clinical trial of a peptide-based human papillomavirus therapeutic vaccine with Candida skin test reagent as a novel vaccine adjuvant for treating women with biopsy-proven cervical intraepithelial neoplasia 2/3.

PURPOSE: Non-surgical treatments for cervical intraepithelial neoplasia 2/3 (CIN2/3) are needed as surgical treatments have been shown to double preterm delivery rate. The goal of this study was to demonstrate safety of a human papillomavirus (HPV) therapeutic vaccine called PepCan, which consists of four current good-manufacturing production-grade peptides covering the HPV type 16 E6 protein and Candida skin test reagent as a novel adjuvant. PATIENTS AND METHODS: The study was a single-arm, single-institution, dose-escalation phase I clinical trial, and the patients (n = 24) were women with biopsy-proven CIN2/3. Four injections were administered intradermally every 3 weeks in limbs. Loop electrical excision procedure (LEEP) was performed 12 weeks after the last injection for treatment and histological analysis. Six subjects each were enrolled (50, 100, 250, and 500 µg per peptide). RESULTS: The most common adverse events (AEs) were injection site reactions, and none of the patients experienced dose-limiting toxicities. The best histological response was seen at the 50 µg dose level with a regression rate of 83% (n = 6), and the overall rate was 52% (n = 23). Vaccine-induced immune responses to E6 were detected in 65% of recipients (significantly in 43%). Systemic T-helper type 1 (Th1) cells were significantly increased after four vaccinations (P = 0.02). CONCLUSION: This study demonstrated that PepCan is safe. A significantly increased systemic level of Th1 cells suggests that Candida, which induces interleukin-12 (IL-12) in vitro, may have a Th1 promoting effect. A phase II clinical trial to assess the full effect of this vaccine is warranted.

A Human Papillomavirus Type 16 E6 52-62 CD4 T-Cell Epitope Restricted by the HLA-DR11 Molecule Described in an Epitope Hotspot.

Cell-mediated immune responses to the human papillomavirus type 16 (HPV 16) E6 protein have been shown to be important in viral clearance and in regression of cervical lesions. Here, detailed analyses of a novel HPV 16 E6 CD4 T-cell epitope from a subject with cervical intraepithelial neoplasia 1 are described. This subject had demonstrated HPV 16 CD4 T-cell responses to multiple regions within the E6 protein. Isolation and cloning of CD4 T-cells were performed by magnetic selection of interferon-γ secreting cells and limiting dilution. A single HPV 16-specific T-cell clone isolated was shown to have a specificity to HPV 16 E6 52-62 restricted by the HLA-DR11 molecule. Homologous sequences (≥70% amino acid homology) were identified for HPV types 31, 33, 45, 58, 73, but cross-recognition was demonstrated only for HPV 45. Based on work performed by our group and others, it is known that this short peptide contains multiple CD4 and CD8 T-cell HPV epitopes and would be an ideal region to incorporate into a design of vaccines and immunotherapies against HPV-associated malignancies.

Use of interferon-γ enzyme-linked immunospot assay to characterize novel T-cell epitopes of human papillomavirus.

A protocol has been developed to overcome the difficulties of isolating and characterizing rare T cells specific for pathogens, such as human papillomavirus (HPV), that cause localized infections. The steps involved are identifying region(s) of HPV proteins that contain T-cell epitope(s) from a subject, selecting for the peptide-specific T cells based on interferon-γ (IFN-γ) secretion, and growing and characterizing the T-cell clones (Fig. 1). Subject 1 was a patient who was recently diagnosed with a high-grade squamous intraepithelial lesion by biopsy and underwent loop electrical excision procedure for treatment on the day the T cells were collected(1). A region within the human papillomavirus type 16 (HPV 16) E6 and E7 proteins which contained a T-cell epitope was identified using an IFN- g enzyme-linked immunospot (ELISPOT) assay performed with overlapping synthetic peptides (Fig. 2). The data from this assay were used not only to identify a region containing a T-cell epitope, but also to estimate the number of epitope specific T cells and to isolate them on the basis of IFN- γ secretion using commercially available magnetic beads (CD8 T-cell isolation kit, Miltenyi Biotec, Auburn CA). The selected IFN-γ secreting T cells were diluted and grown singly in the presence of an irradiated feeder cell mixture in order to support the growth of a single T-cell per well. These T-cell clones were screened using an IFN- γ ELISPOT assay in the presence of peptides covering the identified region and autologous Epstein-Barr virus transformed B-lymphoblastoid cells (LCLs, obtained how described by Walls and Crawford)(2) in order to minimize the number of T-cell clone cells needed. Instead of using 1 x 10(5) cells per well typically used in ELISPOT assays(1,3), 1,000 T-cell clone cells in the presence of 1 x 10(5) autologous LCLs were used, dramatically reducing the number of T-cell clone cells needed. The autologous LCLs served not only to present peptide antigens to the T-cell clone cells, but also to keep a high cell density in the wells allowing the epitope-specific T-cell clone cells to secrete IFN-γ. This assures successful performance of IFN-γ ELISPOT assay. Similarly, IFN- γ ELISPOT assays were utilized to characterize the minimal and optimal amino acid sequence of the CD8 T-cell epitope (HPV 16 E6 52-61 FAFRDLCIVY) and its HLA class I restriction element (B58). The IFN- γ ELISPOT assay was also performed using autologous LCLs infected with vaccinia virus expressing HPV 16 E6 or E7 protein. The result demonstrated that the E6 T-cell epitope was endogenously processed. The cross-recognition of homologous T-cell epitope of other high-risk HPV types was shown. This method can also be used to describe CD4 T-cell epitopes(4).

Low-grade fibromyxoid sarcoma presenting clinically as a primary ovarian neoplasm: a case report.

Low-grade fibromyxoid sarcoma is an uncommon, deceptively bland mesenchymal neoplasm that typically occurs in the deep soft tissues of the proximal extremities of young to middle-aged patients. Intra-abdominal low-grade fibromyxoid sarcomas are distinctly rare. We describe the first reported example of this sarcoma involving the ovary. The 42-year-old patient presented with progressing abdominal pain and urinary frequency. Computed tomographic imaging of the abdomen and pelvis showed a 14-cm left-sided pelvic mass. The patient underwent surgical resection and, intraoperatively, a left ovarian mass was identified that extended to both the left hypogastric artery and the left ureter. The resected specimen consisted of a 17-cm tan, fleshy mass containing grossly recognizable nodules of gelatinous, myxoid tissue and a small rim of normal ovary. Microscopic examination of the neoplasm revealed a cytologically bland spindle cell proliferation set in a collagenized stroma that abruptly transitioned to nodules of a myxoid stroma with a well-formed capillary vasculature, features characteristic of low-grade fibromyxoid sarcoma. On the basis of the diagnosis, the patient was clinically staged as a retroperitoneal sarcoma with secondary ovarian involvement. The patient has stable residual disease 11 months postoperatively. This case adds to the literature of intra-abdominal low-grade fibromyxoid sarcoma and expands the list of malignant mesenchymal neoplasms that may involve the ovary.