Human papillomavirus-positivity is associated with EREG down-regulation and promoter hypermethylation in head and neck squamous cell carcinoma.

BACKGROUND: Human papillomavirus (HPV) etiology has become evident in head and neck cancers (HNCs) and HPV positivity showed a strong association with its malignant progression. Since aberrant DNA methylation is known to drive carcinogenesis and progression in HNCs, we investigated to determine target gene(s) associated with this modification. METHODS: We characterized epigenetic changes in tumor-related genes (TRGs) that are known to be associated with HNC development and its progression. RESULTS: The expression levels of 42 candidate HNC-associated genes were analyzed. Of these, 7 TGRs (CHFR, RARß, GRB7, EREG, RUNX2, RUNX3, and SMG-1) showed decreased expressions in HPV-positive (+) HNC cells compared with HPV-negative (-) HNC cells. When gene expression levels were compared corresponding to the DNA methylation conditions, GRB7 and EREG showed significant differential expression between HPV+ and HPV- cells, which suggested these genes as primary targets of epigenetic regulation in HPV-induced carcinogenesis. Furthermore, treatment with a demethylation agent, 5-aza-2'-deoxycytidine (5-aza-dc), caused restoration of EREG expression and was associated with hypomethylation of its promoter in HPV+ cells, while no changes was noted in HPV- cells. EREG promoter hypermethylation in HPV+ cells was confirmed using methylation-specific PCR (MS-PCR). CONCLUSION: We conclude that EREG is the target of epigenetic regulation in HPV+ HNCs and its suppressed expression through promoter hypermethylation is associated with the development of HPV-associated HNCs.

Rapid-Release Griffithsin Fibers for Dual Prevention of HSV-2 and HIV-1 Infections.

The biologic griffithsin (GRFT) has recently emerged as a candidate to safely prevent sexually transmitted infections (STIs), including human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus 2 (HSV-2). However, to date, there are few delivery platforms that are available to effectively deliver biologics to the female reproductive tract (FRT). The goal of this work was to evaluate rapid-release polyethylene oxide (PEO), polyvinyl alcohol (PVA), and polyvinylpyrrolidone (PVP) fibers that incorporate GRFT in in vitro (HIV-1 and HSV-2) and in vivo (HSV-2) infection models. GRFT loading was determined via enzyme-linked immunosorbent assay (ELISA), and the bioactivity of GRFT fibers was assessed using in vitro HIV-1 pseudovirus and HSV-2 plaque assays. Afterwards, the efficacy of GRFT fibers was assessed in a murine model of lethal HSV-2 infection. Finally, murine reproductive tracts and vaginal lavage samples were evaluated for histology and cytokine expression, 24 and 72 h after fiber administration, to determine safety. All rapid-release formulations achieved high levels of GRFT incorporation and were completely efficacious against in vitro HIV-1 and HSV-2 infections. Importantly, all rapid-release GRFT fibers provided potent protection in a murine model of HSV-2 infection. Moreover, histology and cytokine levels, evaluated from collected murine reproductive tissues and vaginal lavage samples treated with blank fibers, showed no increased cytokine production or histological aberrations, demonstrating the preliminary safety of rapid-release GRFT fibers in vaginal tissue.

Sustained-release Griffithsin nanoparticle-fiber composites against HIV-1 and HSV-2 infections.

Human immunodeficiency virus (HIV-1) and herpes simplex virus 2 (HSV-2) affect hundreds of millions of people worldwide. The antiviral lectin, Griffithsin (GRFT), has been shown to be both safe and efficacious against HSV-2 and HIV-1 infections in vivo. The goal of this work was to develop a multilayered nanoparticle (NP)-electrospun fiber (EF) composite to provide sustained-release of GRFT, and to examine its safety and efficacy in a murine model of lethal HSV-2 infection. Composites were fabricated from polycaprolactone (PCL) fibers surrounding polyethylene oxide (PEO) fibers that incorporated methoxy poly(ethylene glycol)-b-poly(lactide-co-glycolide) (mPEG-PLGA) GRFT NPs. GRFT loading and release were determined via ELISA, showing that NP-EF composites achieved high GRFT loading, and provided sustained-release of GRFT for up to 90 d. The in vitro efficacy of GRFT NP-EFs was assessed using HIV-1 pseudovirus assays, demonstrating complete in vitro protection against HIV-1 infection. Additionally, sustained-release NP-EFs, administered 24 h prior to infection, prevented against a lethal dose of HSV-2 infection in a murine model. In parallel, histology and cytokine expression from murine reproductive tracts and vaginal lavages collected 24 and 72 h post-administration were similar to untreated mice, suggesting that NP-EF composites may be a promising and safe sustained-delivery platform to prevent HSV-2 infection. Future work will evaluate the ability to provide prolonged protection against multiple virus challenges, and different administration times with respect to infection.

Immunity to commensal papillomaviruses protects against skin cancer.

Immunosuppression increases the risk of cancers that are associated with viral infection1. In particular, the risk of squamous cell carcinoma of the skin-which has been associated with beta human papillomavirus (ß-HPV) infection-is increased by more than 100-fold in immunosuppressed patients2-4. Previous studies have not established a causative role for HPVs in driving the development of skin cancer. Here we show that T cell immunity against commensal papillomaviruses suppresses skin cancer in immunocompetent hosts, and the loss of this immunity-rather than the oncogenic effect of HPVs-causes the markedly increased risk of skin cancer in immunosuppressed patients. To investigate the effects of papillomavirus on carcinogen-driven skin cancer, we colonized several strains of immunocompetent mice with mouse papillomavirus type 1 (MmuPV1)5. Mice with natural immunity against MmuPV1 after colonization and acquired immunity through the transfer of T cells from immune mice or by MmuPV1 vaccination were protected against skin carcinogenesis induced by chemicals or by ultraviolet radiation in a manner dependent on CD8+ T cells. RNA and DNA in situ hybridization probes for 25 commensal ß-HPVs revealed a significant reduction in viral activity and load in human skin cancer compared with the adjacent healthy skin, suggesting a strong immune selection against virus-positive malignant cells. Consistently, E7 peptides from ß-HPVs activated CD8+ T cells from unaffected human skin. Our findings reveal a beneficial role for commensal viruses and establish a foundation for immune-based approaches that could block the development of skin cancer by boosting immunity against the commensal HPVs present in all of our skin.

Alterations in Serum Zinc and Polyunsaturated Fatty Acid Concentrations in Treatment-Naive HIV-Diagnosed Alcohol-Dependent Subjects with Liver Injury.

Heavy alcohol drinking causes alterations in the metabolism of fatty acids and zinc that participate in inflammation and liver injury. HIV infection has been reported to cause dysregulated polyunsaturated fatty acid (PUFA) and zinc metabolism. In this pilot study, we examined the role of dysregulated PUFA metabolism and zinc deficiency in the liver injury occurring in heavy drinkers with early-stage HIV diagnosis. Fourteen heavy drinking alcohol-dependent (AD) patients [seven with treatment-naive HIV diagnosis (AD+HIV) and seven without HIV infection (AD)] participated in this study. Liver injury, serum zinc, PUFAs, viral load, CD4+ count, and drinking measures using lifetime drinking history (LTDH), and timeline follow-back past 90 days (TLFB90) were evaluated. Liver injury was also assessed in seven age- and gender-matched socially drinking HIV treatment-naive patients who served as disease controls. HIV viral load by itself did not show any correlation with liver injury. Liver enzymes were significantly elevated in both AD+HIV and AD patients, and AD+HIV patients had significantly higher alanine aminotransferase (ALT) levels than did AD patients, even with lower drinking. Serum zinc was significantly lower in AD+HIV patients. Only AD+HIV patients showed a significant elevation in linoleic acid (LA) and alpha-linoleic acid (ALA) levels. Serum zinc and ALT, LA and ALT, and ALA and ALT were significantly associated only in AD+HIV patients. The association between LA and ALT showed a higher effect than did the ALA and ALT association in the AD+HIV patients. Interestingly, AD+HIV subjects (who drank less), nevertheless, showed more liver injury compared with AD patients, who reported heavier drinking. We speculate that the underlying proinflammatory response resulting from zinc deficiency and an elevation in serum LA likely contributed to liver injury in AD+HIV patients, even with a comparatively lower degree of heavy drinking.

Cutaneous Metastasis.

Although rare, cutaneous metastases portend a poor prognosis and are often an indicator of widespread disease. Breast cancer and melanoma are the most common types of cancer that are associated with spread to and within the skin; however, other malignancies, such as lung, colon, head and neck, and hematologic, have been described with a degree of relative frequency. A variety of clinical appearances and syndromes of cutaneous metastases are presented and described in this article. Possible treatment options, including skin-directed therapies and immunotherapies, are also discussed.

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.

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.

Leukotriene B4-receptor-1 mediated host response shapes gut microbiota and controls colon tumor progression.

Inflammation and infection are key promoters of colon cancer but the molecular interplay between these events is largely unknown. Mice deficient in leukotriene B4 receptor1 (BLT1) are protected in inflammatory disease models of arthritis, asthma and atherosclerosis. In this study, we show that BLT1-/- mice when bred onto a spontaneous tumor (ApcMin/+) model displayed an increase in the rate of intestinal tumor development and mortality. A paradoxical increase in inflammation in the tumors from the BLT1-/-ApcMin/+ mice is coincidental with defective host response to infection. Germ-free BLT1-/-ApcMin/+ mice are free from colon tumors that reappeared upon fecal transplantation. Analysis of microbiota showed defective host response in BLT1-/- ApcMin/+ mice reshapes the gut microbiota to promote colon tumor development. The BLT1-/-MyD88-/- double deficient mice are susceptible to lethal neonatal infections. Broad-spectrum antibiotic treatment eliminated neonatal lethality in BLT1-/-MyD88-/- mice and the BLT1-/-MyD88-/-ApcMin+ mice are protected from colon tumor development. These results identify a novel interplay between the Toll-like receptor mediated microbial sensing mechanisms and BLT1-mediated host response in the control of colon tumor development.

T cell-mediated antitumor immune response eliminates skin tumors induced by mouse papillomavirus, MmuPV1.

Previous studies of naturally occurring mouse papillomavirus (PV) MmuPV1-induced tumors in B6.Cg-Foxn1nu/nu mice suggest that T cell deficiency is necessary and sufficient for the development of such tumors. To confirm this, MmuPV1-induced tumors were transplanted from T cell-deficient mice into immunocompetent congenic mice. Consequently, the tumors regressed and eventually disappeared. The elimination of MmuPV1-infected skin/tumors in immunocompetent mice was consistent with the induction of antitumor T cell immunity. This was confirmed by adoptive cell experiments using hyperimmune splenocytes collected from graft-recipient mice. In the present study, such splenocytes were injected into T cell-deficient mice infected with MmuPV1, and they eliminated both early-stage and fully formed tumors. We clearly show that anti-tumor T cell immunity activated during tumor regression in immunocompetent mice effectively eliminates tumors developing in T cell-deficient congenic mice. The results corroborate the notion that PV-induced tumors are strongly linked to the immune status of the host, and that PV antigens are major anti-tumor antigens. Successful anti-PV T cell responses should, therefore, lead to effective anti-tumor immune therapy in human PV-infected patients.

Histologic variation in high grade oral epithelial dysplasia when associated with high-risk human papillomavirus.

OBJECTIVES: Reported cytologic alterations associated with high-risk human papillomavirus (HR-HPV) in oral epithelial dysplasia (HPV-OED) need further characterization. STUDY DESIGN: Archival cases of high-grade oral epithelial dysplasia (hgOED) (N = 38) were assigned a cytologic score (CS) based on the average number of mitotic, karyorrhectic, and apoptotic cells per high-power field. Three groups were then generated on the basis of increasing CS: Focal (group 1, N = 14), Intermediate (group 2, N = 12), and Diffuse (group 3, N = 12). Polymerase chain reaction-based HPV genotyping and p16 immunohistochemistry were performed. RESULTS: HR-HPV was found significantly more in group 3 (83.3%) compared with groups 1 and 2 (group 1&2; 42.9% and 41.7%, respectively; P = .047). HPV16 predominated in HR-HPV-positive cases (90.5%). By location, the tongue or the floor of mouth was associated with all groups (P = .04). Increasing CS was associated with a slightly younger age (P = .04) and increased expression of p16 (P = .005). CS and p16 expression were not sensitive but were highly specific predictors for HR-HPV presence. Based on limited follow-up information, HPV-OED does not differ in clinical aggressiveness compared with conventional OED. CONCLUSIONS: Increased CS in hgOED is strongly associated with HR-HPV (mostly HPV16) and p16 expression. CS and p16 expression are specific predictors of HR-HPV presence. Further molecular study and long-term follow-up of HPV-OED are needed.

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.

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.

An inquiry into the causes and effects of the variolae (or Cow-pox. 1798).

Few papers have had a greater impact on the health of the human species than the simple, yet elegant, observations and clinical trials of Edward Jenner with what was at the time called the Cow Pox. In fact, this was a naturally attenuated rodent (probably rat) pox that could infect horses and, through farriers and farm hands, dairy cattle. While commonly called the Cow Pox at the time, Jenner's transmission studies between humans used infectious materials from horses. His methods provided protection from the serious effects of smallpox infections. In 1977, smallpox was considered to be eradicated, although people continue to be infected by pox viruses from other mammalian species. We consider this to be our 'favorite historical paper' because it emphasizes careful clinical observation followed by relatively simple clinical testing can have a profound influence on human health, even when almost nothing is known about the underlying molecular mechanisms. Continued follow-up with strict attention to detail resulted in a crude but effective way to deal with an epidemic, methods still used today for containing infectious diseases.

Suramin protects from cisplatin-induced acute kidney injury.

Cisplatin, a commonly used cancer chemotherapeutic, has a dose-limiting side effect of nephrotoxicity. Approximately 30% of patients administered cisplatin suffer from kidney injury, and there are limited treatment options for the treatment of cisplatin-induced kidney injury. Suramin, which is Federal Drug Administration-approved for the treatment of trypanosomiasis, improves kidney function after various forms of kidney injury in rodent models. We hypothesized that suramin would attenuate cisplatin-induced kidney injury. Suramin treatment before cisplatin administration reduced cisplatin-induced decreases in kidney function and injury. Furthermore, suramin attenuated cisplatin-induced expression of inflammatory cytokines and chemokines, endoplasmic reticulum stress, and apoptosis in the kidney cortex. Treatment of mice with suramin 24 h after cisplatin also improved kidney function, suggesting that the mechanism of protection is not by inhibition of tubular cisplatin uptake or its metabolism to nephrotoxic species. If suramin is to be used in the context of cancer, then it cannot prevent cisplatin-induced cytotoxicity of cancer cells. Suramin did not alter the dose-response curve of cisplatin in lung adenocarcinoma cells in vitro. In addition, suramin pretreatment of mice harboring lung adenocarcinomas did not alter the initial cytotoxic effects of cisplatin (DNA damage and apoptosis) on tumor cells. These results provide evidence that suramin has potential as a renoprotective agent for the treatment/prevention of cisplatin-induced acute kidney injury and justify future long-term preclinical studies using cotreatment of suramin and cisplatin in mouse models of cancer.

Phase 2 Randomized Controlled Trial of Radiation Therapy Plus Concurrent Interferon-Alpha and Retinoic Acid Versus Cisplatin for Stage III Cervical Carcinoma.

PURPOSE: Because a combination of retinoic acid, interferon-alpha, and radiation therapy demonstrated synergistic action and effectiveness to treat advanced cervical cancers in earlier studies, we designed this randomized phase 2 open-label trial to assess efficacy and safety of interferon alpha-2b (IFN) and 13-cis-retinoic acid (RA) administered concomitantly with radiation therapy (IFN-RA-radiation) to treat stage III cervical cancer. METHODS AND MATERIALS: Stage III cervical cancer patients were randomized to study and control groups in a 1:1 ratio. All patients were treated with radiation therapy; study arm patients received IFN (3 × 10(6) IU subcutaneously) 3 times a week for 4 weeks and daily RA (40 mg orally) for 30 days starting on day 1 of radiation, whereas control arm patients received weekly cisplatinum (40 mg/m(2)) for 5 weeks during radiation. Patients were followed for 3 years. The primary endpoint was overall survival at 3 years. RESULTS: Patients in the study (n=104) and control (n=105) groups were comparable for clinicopathological characteristics, radiation therapy-related variables and treatment response. Proportions of disease-free patients in the study and control groups were 38.5% and 44.8%, respectively, after median follow-up of 29.2 months. Hazard ratios were 0.67 (95% confidence interval [CI]: 0.44-1.01) and 0.69 (95% CI: 0.44-1.06) for overall and disease-fee survival, respectively, comparing the study group to control, and demonstrated an inferior outcome with RA-IFN-radiation, although differences were statistically nonsignificant. Kaplan-Meier curves of disease-free and overall survival probabilities also showed inferior survival in the study group compared to those in the control. Acute toxicities of chemoradiation were significantly higher with 2 acute toxicity-related deaths. CONCLUSIONS: Treatment with RA-IFN-radiation did not demonstrate survival advantage over chemoradiation despite being less toxic. The trends predicted an inferior outcome with the RA-IFN combination.

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. Student's 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.

Molecular characterization of novel mucosotropic papillomaviruses from a Florida manatee (Trichechus manatus latirostris).

We isolated two new manatee papillomavirus (PV) types, TmPV3 and TmPV4, from a Florida manatee (Trichechus manatus latirostris). Two PV types were previously isolated from this species. TmPV1 is widely dispersed amongst manatees and a close-to-root PV; not much is known about TmPV2. The genomes of TmPV3 and TmPV4 were 7622 and 7771 bp in size, respectively. Both PVs had a genomic organization characteristic of all PVs, with one non-coding region and seven ORFs, including the E7 ORF that is absent in other cetacean PVs. Although these PVs were isolated from separate genital lesions of the same manatee, an enlarged E2/E4 ORF was found only in the TmPV4 genome. The full genome and L1 sequence similarities between TmPV3 and TmPV4 were 63.2 and 70.3 %, respectively. These genomes shared only 49.1 and 50.2 % similarity with TmPV1. The pairwise alignment of L1 nucleotide sequences indicated that the two new PVs nested in a monophyletic group of the genus Rhopapillomavirus, together with the cutaneotropic TmPV1 and TmPV2.

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.