Sujita Khanal

Scalable Production of HPV16 L1 Protein and VLPs from Tobacco Leaves.

Cervical cancer is the most common malignancy among women particularly in developing countries, with human papillomavirus (HPV) 16 causing 50% of invasive cervical cancers. A plant-based HPV vaccine is an alternative to the currently available virus-like particle (VLP) vaccines, and would be much less expensive. We optimized methods to express HPV16 L1 protein and purify VLPs from tobacco (Nicotiana benthamiana) leaves transfected with the magnICON deconstructed viral vector expression system. L1 proteins were extracted from agro-infiltrated leaves using a series of pH and salt mediated buffers. Expression levels of L1 proteins and VLPs were verified by immunoblot and ELISA, which confirmed the presence of sequential and conformational epitopes, respectively. Among three constructs tested (16L1d22, TPL1d22, and TPL1F), TPL1F, containing a full-length L1 and chloroplast transit peptide, was best. Extraction of HPV16 L1 from leaf tissue was most efficient (> 2.5% of total soluble protein) with a low-salt phosphate buffer. VLPs were purified using both cesium chloride (CsCl) density gradient and size exclusion chromatography. Electron microscopy studies confirmed the presence of assembled forms of HPV16 L1 VLPs. Collectively; our results indicated that chloroplast-targeted transient expression in tobacco plants is promising for the production of a cheap, efficacious HPV16 L1 VLP vaccine. Studies are underway to develop plant VLPs for the production of a cervical cancer vaccine.

Human papillomavirus detection in histologic samples of multifocal epithelial hyperplasia: a novel demographic presentation

OBJECTIVE Human papillomavirus (HPV) typing of oral lesions microscopically consistent with multifocal epithelial hyperplasia (MEH) was performed to identify potential novel clinical presentations. STUDY DESIGN MEH (N = 22 lesions, 17 patients) and squamous papilloma control samples (N = 9 lesions, 9 patients) were compared by using polymerase chain reaction-based HPV genotyping. Students t tests were used to compare continuous characteristics. RESULTS Of the study cases, 86.4% of MEH and only 11% of controls were positive for HPV (P = .0002). In MEH lesions, 45.5% contained HPV32, 36.4% HPV6, and 4.5% HPV40. MEH lesions were mostly multifocal (50%) and occurred in HIV-negative patients (81.3%). They predominated on the labial/buccal mucosa (63.3%), and there were significant differences between groups by anatomic site (P < .0001). HPV32, but not HPV6, was detected in known HIV-positive patients. CONCLUSIONS A novel clinical presentation of MEH associated with HPV32 in HIV-negative, middle-aged to older adults is reported here. One case with HPV40 is the first to be reported. Future detection protocols should include HPV32, as it may be currently overlooked.

Human papillomavirus E7 serology and association with p16 immunohistochemistry in squamous cell carcinoma of the head and neck

BACKGROUND Human papillomavirus (HPV)-positive oropharyngeal cancer is associated with improved survival and treatment response as compared to HPV-negative cancers. P16 overexpression is widely accepted as a surrogate marker for HPV positivity. METHODS A total of 92 serum samples from 75 head and neck squamous cell carcinoma (HNSCC) patients were examined for HPV16 and 18 E7 antibodies by ELISA. Available tissue was tested for HPV-DNA by PCR, and p16 immunohistochemistry was obtained from a deidentified database. RESULTS Of 75 HNSCC patients, 25 were HPV E7 seropositive. Seropositivity was strongly associated with cancers of the oropharynx, and correlated with positive p16 immunohistochemistry (IHC) and HPV-DNA. Post-treatment serum was available in a limited subset of patients, revealing a decrease in antibody titers following response to treatment. CONCLUSIONS HPV E7 seropositivity correlated with positive tumor HPV-DNA and p16 expression, and was strongly associated with cancers of the oropharynx. E7 serology warrants further study as a potential biomarker in HPV-positive HNSCC.

High-dose versus weekly cisplatin definitive chemoradiotherapy for HPV-related oropharyngeal squamous cell carcinoma of the head and neck

OBJECTIVES To compare the outcomes and toxicity of high-dose cisplatin (HDC) versus weekly cisplatin (WC) definitive chemoradiotherapy (CRT) for patients with human papillomavirus (HPV) related oropharyngeal squamous cell carcinoma (SCCOPx). METHODS All patients with p16 positive SCCOPx treated with definitive CRT with cisplatin between 2010 and 2014 at a single institution were retrospectively reviewed. CTCAE v 4.03 toxicity criteria were used. The Kaplan-Meier method was used to estimate event-free survival (EFS) and the overall survival (OS). RESULTS Of the 55 patients included, 22 were patients treated with HDC at dose of 100mg/m2 on days 1 and 22; and the remaining 33 patients were treated with WC at 40mg/m2. Both cohorts received a median total dose of cisplatin of 200mg/m2. At median follow-up of 31months, there was one local failure and no distant failures in the HDC cohort. In the WC group, there were 6 total failures (2 local, 4 distant). Estimated 2-year EFS was better in HDC cohort as compared to WC (96% vs. 75%; p=0.04). There was no significant difference in 2-year OS (95% vs. 94%; p=0.40). Weight loss, gastric tube dependence at six months, acute renal injury and grade 3 or 4 hematological toxicity were all similar between both groups. CONCLUSIONS HPV-related SCCOPx treated with definitive CRT with either HDC or WC had similar toxicity profile. HDC had better EFS when compared with WC and this seems to be driven by increased distant failure rates, although the OS was similar.

Targeting synthetic Human Papillomavirus (HPV) L2 disulfide-induced N-terminus conformational epitopes for pan-HPV vaccine development

BACKGROUND Current vaccines against Human Papillomavirus (HPV) are highly effective and based on recombinant virus-like particles (VLPs) of the major capsid protein L1. Since these vaccines are HPV type-specific and expensive for global implementation, an alternative, broader-spectrum immunogen would be the N-terminus of the minor capsid protein L2 that induces low titered broadly cross-neutralizing antibodies. Here we analyzed the reactivity of different synthetic L2 peptides containing N-terminus amino acids 17-36 in order to test their antigenicity. METHODS Different synthetic peptides were designed to target the 17-36 amino acid sequences, present in highly antigenic amino-terminus of L2 protein. Six different peptides including Cys22-Cys28 disulfide bonded cyclized L2 peptide were examined for their antigenicity against mouse monoclonal antibody RG-1 and rabbit polyclonal antisera to HPV L2 by enzyme-linked immunosorbent assay (ELISA). RESULTS Here we report that the cyclized form of synthetic L2 peptide, which is formed through Cys22-Cys28 disulfide bridges, has the highest reactivity to antibodies than other synthetic L2 peptides. CONCLUSION A cyclized L2 peptide has potential to be an excellent candidate to formulate a low-cost, broadly protective pan-oncogenic HPV vaccine.

Identification of G-quadruplex forming sequences in three manatee papillomaviruses

The Florida manatee (Trichechus manatus latirotris) is a threatened aquatic mammal in United States coastal waters. Over the past decade, the appearance of papillomavirus-induced lesions and viral papillomatosis in manatees has been a concern for those involved in the management and rehabilitation of this species. To date, three manatee papillomaviruses (TmPVs) have been identified in Florida manatees, one forming cutaneous lesions (TmPV1) and two forming genital lesions (TmPV3 and TmPV4). We identified DNA sequences with the potential to form G-quadruplex structures (G4) across the three genomes. G4 were located on both DNA strands and across coding and non-coding regions on all TmPVs, offering multiple targets for viral control. Although G4 have been identified in several viral genomes, including human PVs, most research has focused on canonical structures comprised of three G-tetrads. In contrast, the vast majority of sequences we identified would allow the formation of non-canonical structures with only two G-tetrads. Our biophysical analysis confirmed the formation of G4 with parallel topology in three such sequences from the E2 region. Two of the structures appear comprised of multiple stacked two G-tetrad structures, perhaps serving to increase structural stability. Computational analysis demonstrated enrichment of G4 sequences on all TmPVs on the reverse strand in the E2/E4 region and on both strands in the L2 region. Several G4 sequences occurred at similar regional locations on all PVs, most notably on the reverse strand in the E2 region. In other cases, G4 were identified at similar regional locations only on PVs forming genital lesions. On all TmPVs, G4 sequences were located in the non-coding region near putative E2 binding sites. Together, these findings suggest that G4 are possible regulatory elements in TmPVs.

Viral DNA integration and methylation of human papillomavirus type 16 in high-grade oral epithelial dysplasia and head and neck squamous cell carcinoma

This study evaluated the integration and methlyation of human papillomavirus type 16 (HPV16) in head and neck squamous cell carcinoma (HNSCC) and its oral precursor, high-grade oral epithelial dysplasia (hgOED). Archival samples of HPV16-positive hgOED (N = 19) and HNSCC (N = 15) were evaluated, along with three HNSCC (UMSCC-1, -47 and -104) and two cervical cancer (SiHa and CaSki) cell lines. HgOED cases were stratified into three groups with increasing degrees of cytologic changes (mitosis, karyorrhexis and apoptosis). The viral load was higher and the E2/E6 ratio lower (indicating a greater tendency toward viral integration) in group 3 than in groups 1 or 2 (p = 0.002, 0.03). Methylation was not observed in hgOED cases and occurred variably in only three HNSCC cases (26.67%, 60.0% and 93.3%). In HNSCC cell lines, lower E7 expression correlated with higher levels of methylation. HgOED with increased cytologic change, now termed HPV-associated oral epithelial dysplasia (HPV-OED), exhibited an increased viral load and a tendency toward DNA integration, suggesting a potentially increased risk for malignant transformation. More detailed characterization and clinical follow-up of HPV-OED patients is needed to determine whether HPV-OED is a true precursor to HPV-associated HNSCC and to clarify the involvement of HPV in HNSCC carcinogenesis.