A Novel Human Papillomavirus 16 L1 Pentamer-Loaded Hybrid Particles Vaccine System: Influence of Size on Immune Responses.

Cervical cancer remains the second-most prevalent female malignancy around the world, leading to a great majority of cancer-related mortality that occurs mainly in developing countries. Developing an effective and low-cost vaccine against human papillomavirus (HPV) infection, especially in medically underfunded areas, is urgent. Compared with vaccines based on HPV L1 viruslike particles (VLPs) in the market, recombinant HPV L1 pentamer expressed in Escherichia coli represents a promising and potentially cost-effective vaccine for preventing HPV infection. Hybrid particles comprising a polymer core and lipid shell have shown great potential compared to conventional aluminum salts adjuvant and is urgently needed for HPV L1 pentamer vaccines. It is well-reported that particle sizes are crucial in regulating immune responses. Nevertheless, reports on the relationship between the particulate size and the resultant immune response have been in conflict, and there is no answer to how the size of particles regulates specific immune response for HPV L1 pentamer-based candidate vaccines. Here, we fabricated HPV 16 L1 pentamer-loaded poly(d,l-lactide- co-glycolide) (PLGA)/lecithin hybrid particles with uniform sizes (0.3, 1, and 3 µm) and investigated the particle size effects on antigen release, activation of lymphocytes, dendritic cells (DCs) activation and maturation, follicular helper CD4+ T (TFH) cells differentiation, and release of pro-inflammatory cytokines and chemokines. Compared with the other particle sizes, 1 µm particles induced more powerful antibody protection and yielded more persistent antibody responses, as well as more heightened anamnestic responses upon repeat vaccination. The superior immune responses might be attributed to sustainable antigen release and robust antigen uptake and transport and then further promoted a series of cascade reactions, including enhanced DCs maturation, increased lymphocytes activation, and augmented TFH cells differentiation in draining lymph nodes (DLNs). Here, a powerful and economical platform for HPV vaccine and a comprehensive understanding of particle size effect on immune responses for HPV L1 pentamer-based candidate vaccines are provided.

E7 oncoprotein of human papillomavirus: Structural dynamics and inhibitor screening study.

Human papillomavirus (HPV) has been the primary causative agent of cervical cancer, the most threatening cancer affecting millions of women worldwide. HPV, a small non enveloped DNA virus of high and low risk types contain intrinsically disordered region and it also plays significant role in the development of cervical cancer. HPV E7 contains an ordered Zinc finger motif that binds to pRB and alters its function. It utilizes both disordered N-terminal and structured C-terminal regions for cellular transformation. In this study, we have focused extensively on the evolutionary relationships of E7 among various HPV types and generated a 3D homology model of full length HPV 16 E7, since the structure have not been solved till date. We also analysed the stable conformation and atomic flexibility of modelled E7 through molecular dynamics simulation at 100 ns. To understand the disordered based binding sites of E7 oncoprotein, Molecular recognition features (MoRFs) analysis was carried out on the E7 oncoprotein. The validated model was taken forward for the identification of potential lead compounds and the most prominent compounds were selected for the molecular dynamics simulation of the 100 ns for the stability analysis. Overall, this study highlights the holistic E7 regions including important disordered based binding sites analysed through the MoRFs. The potential inhibitor compound that targets the structured C-terminal region of E7 oncoprotein were subjected for the pharmacological properties analysis and further validated for the binding modes of the compounds with the target structure. This study helps in providing a better intuition to develop a potent anti-HPV agent.

Surface modification of tri-calcium phosphate nanoparticles by DOPE and/or anti-E6 antibody to enhance uptake of antisense of E6 mRNA.

The main aim of this study was to evaluate the uptake of E6 mRNA antisense into cervical cancer cells, induced by human papilloma virus (HPV). In this study, the carrier of the antisense was tri-calcium phosphate nanoparticles (TCP NPs) conjugated with dioleoyl phosphatidyl ethanolamine (DOPE) and/or anti-E6 antibody. At first, TCP NPs were synthesized, coated with carboxy-polyethylene glycol, and then conjugated with anti-E6 antibody and/or DOPE by carbodiimide cross-linker. Then, a single stranded DNA, which was complementary (antisense) of E6 mRNA, was attached to each one. Finally, the uptake of conjugated and unconjugated TCP NPs into HelaS3 cells was separately evaluated by Fourier transform infrared spectroscopy, optical microscopy, and fluorescent microscopy. Also, the cytotoxicity of these carriers was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Overall, 4 types of TCP NPs were used in this study, including 1) TCP NPs conjugated with DOPE (TCP NPs/DOPE), 2) TCP NPs conjugated with DOPE and antibody (TCP NPs/DOPE/Anti-E6 Ab), 3) TCP NPs conjugated with antibody (TCP NPs/Anti-E6 Ab), and 4) TCP NPs which not conjugated with DOPE and antibody (unconjugated TCP NPs). Uptake tests showed that although all types of TCP NPs could transfer antisense of E6 mRNA into HelaS3 cells, TCP NPs/DOPE and TCP NPs/DOPE/Anti-E6 Ab had more uptake than TCP NPs/Anti-E6 Ab and unconjugated TCP NPs. Moreover, MTT assay showed that TCP NPs/DOPE was more toxic than TCP NPs/DOPE/Anti-E6 Ab, TCP NPs/Anti-E6 Ab, and unconjugated TCP NPs. It can be concluded that TCP NPs/DOPE/Anti-E6 Ab is a good choice for oligonucleotide delivery, because of higher uptake and less toxicity, compared with other formulations.

Human papilloma virus 16 E6 protein as a target for curcuminoids, curcumin conjugates and congeners for chemoprevention of oral and cervical cancers.

Curcumin (diferuloyl methane) and its naturally occurring analogs viz. demethoxy, bisdemethoxy and cyclocurcumin, present in rhizomes of curcuma species turmeric, have been shown to inhibit the proliferation of a wide variety of tumor cells. Target nuclear protein HPV 16 E6 (PDB ID: 2fk4) is the major protein actively participating in oral and cervical cancers. In silico studies indicate that curcumin and its natural analogs have effective binding with different active sites on HPV 16 E6 protein, ideal target for restoring the tumor suppressor function of p53 and thus allowing the apoptosis of infected cells. The main limitation in the use of curcuminoids as therapeutic agents is their low bioavailability. Since piperine is known to enhance curcumin bioavailability to more than two thousand times by inhibiting its efflux, a conjugate of curcumin-piperic acid was also used. Although curcumin has been found to have strongest binding with this target and the two curcuminoids, demethoxy and bisdemethoxy curcumin have lower but comparable affinity, chlorogenic acid amongst the naturally occurring analogs has been found to have best binding affinity amongst all the analogs. Although curcumin-piperoyl conjugate does not show very encouraging results, it is likely to have potential activity in vitro and in vivo. These results throw light on the SAR of curcuminoids, leading to future designing of potent, non-toxic drugs for oral and cervical cancers.

Treatment of established lesions caused by high-risk human papilloma virus using a synthetic vaccine.

Here, we review a novel vaccine modality, characterized by the administration of long (23 to 45 amino acids) synthetic peptides in incomplete Freund's adjuvant (mineral oil based, Montanide ISA-51), delivered subcutaneously. Such vaccines were first demonstrated to be much more potent in preclinical T-cell response induction and tumor therapy experiments than were short major histocompatibility complex class I-binding peptides. Nevertheless, a recent study has shown the clinical efficacy of an anchor-modified short gp100 peptide in melanoma patients. We now review the evidence and mode of action of a long peptide vaccine consisting of 13 overlapping peptides, together covering the entire length of the 2 oncogenic proteins E6 and E7 of high-risk human papilloma virus type 16 (HPV16), causing complete regression of all lesions and eradicating virus in 9 of 20 women with high-grade vulvar intraepithelial neoplasia. The nature and strength of the vaccine-induced T-cell response correlated significantly with the clinical response. This vaccine promises to be of use not only in patients with premalignant lesions caused by high-risk HPV16 but also in malignant tumors caused by this virus, including HPV16-positive cervical cancer, vulvar cancer, anal cancer, and head and neck cancer.

Reactive oxygen species play a central role in the activity of cationic liposome based cancer vaccine.

Recently, we developed a simple and potent therapeutic liposome cancer vaccine consisting of a peptide antigen and a cationic lipid. The molecular mechanism of the adjuvanticity of cationic liposome was studied and described in the current report. First, cationic DOTAP liposome, but not the neutral liposome DOPC, was shown to generate reactive oxygen species (ROS) in mouse bone marrow-derived dendritic cells (BMDC). ROS generation by DOTAP was required for ERK and p38 activation and downstream chemokine/cytokine induction. Furthermore, ROS were shown to be involved in the expression of the co-stimulatory molecules CD86/CD80 induced by DOTAP. However, as the DOTAP concentration increased from 50 to 800 microM, the apoptotic marker Annexin V and ROS double positive cells increased, suggesting that high dose of DOTAP-generated ROS causes cell apoptosis. In vivo, optimal amount of ROS in the draining lymph nodes (DLN) and anti-tumor (HPV positive TC-1 tumor) activity induced by E7 peptide (antigen derived from E7 oncoprotein of human papillomavirus (HPV) type 16) formulated in 100 nmol DOTAP were attenuated by incorporating DOPC in the formulation, suggesting that ROS are essential for the vaccine induced anti-tumor activity. Moreover, 600 nmol DOTAP/E7 generated huge amount of ROS in the DLN and showed no activity of tumor regression. Interestingly, 600 nmol DOTAP/E7-induced ROS were tuned down to the same level induced by 100 nmol DOTAP/E7 by adding DOPC in the formulation and this formulation showed tumor regression activity. In conclusion, DOTAP is an active DC stimulator resulting in the activation of ERK and p38 and induction of chemokines, cytokines and co-stimulatory molecules mediated by appropriate amount of ROS. Our data elucidated an important mechanism of adjuvant activity of cationic liposome and could facilitate rational design of synthetic lipid based adjuvants and vaccine formulation.

Podophyllotoxin directly binds a hinge domain in E2 of HPV and inhibits an E2/E7 interaction in vitro.

Podophyllotoxin (PT), a strong cytotoxic agent from berberidaceae, has been known to inhibit tubulin polymerization. Although PT has been used for developing anticancer drugs as one of seed compounds, clinical treatment by itself has been unsuccessful because of the side effects, except one example in the treatments of warts. In this study, we screened peptides binding to PT with T7 phage display clonings in order to obtain more information about molecular mechanism of the action. A selected phage clone has a specific amino acid sequence to be SVPSRRRPDGRTHRSSRG. A homology search by protein database BLAST showed that this sequence had a similarity to a hinge domain (HD) of E2 protein in human papillomavirus (HPV) type 1a which is known to cause plantar warts. Surface plasmon resonance (SPR) analysis showed that PT bound to a recombinant HPV 1a E2 protein giving a K(D)=24.1microM which has compared with those of other domains of E2 protein. Also we demonstrated whether PT inhibited HD interaction or not. E7 protein of HPV has been known to be an oncoprotein and was reported to interact with HD of E2 protein. We demonstrated that an E2/E7 interaction was inhibited by the addition of PT in this report. And we showed the bindings of PT to other types of HPV. Our results suggest that PT is potential as a tool for clarifying the molecular mechanism of HPV.