A Reverse Structure-based Design of HPV E7 Inhibitor.

BACKGROUND: Human papillomavirus (HPV) is a small, non-enveloped double-stranded circular DNA virus. The high-risk types of HPV are claimed to be responsible for over 99% of cervical cancers. One of the essential HPV oncoproteins, E7, is responsible for escaping from G1/S cell cycle arrest in HPV-infected cells by binding to the retinoblastoma protein (pRb) through its LXCXE binding site. OBJECTIVE: To design a peptide inhibitor targeting HPV E7 through an in silico approach. METHODS: In this study, the LXCXE binding domain of pRb is used as a target to design peptide inhibitors using a reverse structure-based approach. The designed amino acid sequence from the B pocket of pRb, named peptide Y, was further investigated in vitro analysis. The cytotoxicity of the peptide was analysed in two cell lines, namely, CaSki, containing an integrated HPV16 genome, and HaCaT, an immortalized keratinocyte cell. Cell cycle analysis was also carried out in both cell lines treated with peptides. RESULTS: In the in silico approach, a 9-amino acids peptide sequence formed 4 conventional hydrogen bonds with LXCXE motif was selected for in vitro assay. Based on the cytotoxicity analysis, the peptide showed low toxicity in both cell lines, where the cell viability remained over 74% when treated with peptide Y. The peptide also caused an accumulation of cells in G0/G1 (+5.4%) and S phase (+10.2%) and a reduction of cells in the G2/M phase (-14.9%) in the CaSki cells with no significant effect on normal cells, indicating it is a potential HPV inhibitor. CONCLUSION: A peptide inhibitor, peptide Y, that was designed from the LXCXE binding motif in pRb can inhibit HPV E7 by causing a cell accumulation effect in G0/G1, and S phases of the cell cycle in the HPV transformed cell lines. These findings could contribute to HPV E7 peptide inhibitor in the future.

Human papillomavirus 68 prevalence and genetic variability based on E6/E7 genes in Sichuan.

HPV68 is a common HR-HPV, its persistent infection is closely related with the occurrence of cervical cancer. In this study, 2939 (27.60%, 2939/10650) positive samples were detected, and 174 (5.92%, 174/2939) were HPV68. 150 HPV68 E6-E7 were successful sequenced, 4 non-synonymous mutations were detected in E6, and E7 were 12. N133S non-synonymous mutations of HPV 68 E6 and C67G, T68 A/M of HPV68 E7 are E6, E7 positive selection sites, they all located in the key domains and major motifs of E6/E7 protein, the above amino-acid substitutions changed the protein structure, disturbed the interaction with other protein or cellular factors and make a difference in epitopes affinity, may affect the pathogenicity and adaptability of HPV68 to the environment. The enrichment of HPV68 data is of great significance for understanding the inherent geographical and biological differences of HPV68 in China.

Characterization of the High-Affinity Fuzzy Complex between the Disordered Domain of the E7 Oncoprotein from High-Risk HPV and the TAZ2 Domain of CBP.

The intrinsically disordered N-terminal region of the E7 protein from high-risk human papillomavirus (HPV) strains is responsible for oncogenic transformation of host cells through its interaction with a number of cellular factors, including the TAZ2 domain of the transcriptional coactivator CREB-binding protein. Using a variety of spectroscopic and biochemical tools, we find that despite its nanomolar affinity, the HPV16 E7 complex with TAZ2 is disordered and highly dynamic. The disordered domain of HPV16 E7 protein does not adopt a single conformation on the surface of TAZ2 but engages promiscuously with its target through multiple interactions involving two conserved motifs, termed CR1 and CR2, that occupy an extensive binding surface on TAZ2. The fuzzy nature of the complex is a reflection of the promiscuous binding repertoire of viral proteins, which must efficiently dysregulate host cell processes by binding to a variety of host factors in the cellular environment.

In Silico Design and Immunological Studies of Two Novel Multiepitope DNA-Based Vaccine Candidates Against High-Risk Human Papillomaviruses.

Human papillomaviruses (HPV)-16 and 18 are the most prevalent types associated with cervical cancer. HPV L1 and L2 capsid proteins and E7 oncoprotein play crucial roles in HPV-related diseases. Hence, these proteins were proposed as target antigens for preventive and therapeutic vaccines. In this study, two multiepitope DNA-based HPV vaccine candidates were designed using in silico analysis including the immunogenic and conserved epitopes of HPV16/18 L1, L2 and E7 proteins (the L1-L2-E7 fusion DNA), and of heat shock protein 70 (HSP70) linked to the L1-L2-E7 DNA construct (the HSP70-L1-L2-E7 fusion DNA). Next, the expression of the L1-L2-E7 and HSP70-L1-L2-E7 multiepitope DNA constructs was evaluated in a mammalian cell line. Finally, immunological responses and antitumor effects of the DNA constructs were investigated in C57BL/6 mice. Our data indicated high expression rates of the designed multiepitope L1-L2-E7 DNA (~ 56.16%) and HSP70-L1-L2-E7 DNA (~ 80.45%) constructs in vitro. The linkage of HSP70 epitopes to the L1-L2-E7 DNA construct significantly increased the gene expression. Moreover, the HSP70-L1-L2-E7 DNA construct could significantly increase immune responses toward Th1 response and CTL activity, and induce stronger antitumor effects in mouse model. Thus, the designed HSP70-L1-L2-E7 DNA construct represents promising results for development of HPV DNA vaccine candidates.

Design of a multi-epitope vaccine against cervical cancer using immunoinformatics approaches.

Cervical cancer, caused by human papillomavirus (HPV), is the fourth most common type of cancer among women worldwide. While HPV prophylactic vaccines are available, they have no therapeutic effects and do not clear up existing infections. This study aims to design a therapeutic vaccine against cervical cancer using reverse vaccinology. In this study, the E6 and E7 oncoproteins from HPV16 were chosen as the target antigens for epitope prediction. Cytotoxic T lymphocytes (CTL) and helper T lymphocytes (HTL) epitopes were predicted, and the best epitopes were selected based on antigenicity, allergenicity, and toxicity. The final vaccine construct was composed of the selected epitopes, along with the appropriate adjuvant and linkers. The multi-epitope vaccine was evaluated in terms of physicochemical properties, antigenicity, and allergenicity. The tertiary structure of the vaccine construct was predicted. Furthermore, several analyses were also carried out, including molecular docking, molecular dynamics (MD) simulation, and in silico cloning of the vaccine construct. The results showed that the final proposed vaccine could be considered an effective therapeutic vaccine for HPV; however, in vitro and in vivo experiments are required to validate the efficacy of this vaccine candidate.

Genetic variation of E6 and E7 genes of human papillomavirus 52 from Central China.

Cervical cancer is the fourth most common malignant tumor in women worldwide and is closely related to human papillomavirus (HPV). Women have the highest susceptibility to HPV-52 type in Jingzhou, China. In this study, E6-E7 sequences of 183 HPV-52 positive samples were amplified by a polymerase chain reaction and sequenced. HPV-52 E6-E7 gene variations were analyzed. The phylogenetic tree was constructed using the Kimura 2-parameter method. The secondary structure of the protein was analyzed. The selective pressure to E6-E7 genes was estimated using PAML. In addition, the B cell epitopes of the E6-E7 sequences in HPV-52 were predicted by the ABCpred server. In E6 sequences, 15 single nucleotide variants were observed, including 6 nonsynonymous variants and 9 synonymous variants. In E7 sequences, 19 single nucleotide variants occurred, including 10 nonsynonymous variants and 9 synonymous variants. Six amino acid variants, including 3 nonconservative substitutions, were found in sequences encoding the alpha helix. Eight amino acid variants, including three nonconservative substitutions, occurred in sequences encoding the strand. Through phylogenetic analysis, the E6-E7 sequences were mainly distributed in B lineage. In HPV-52 E6-E7 sequences, no positively selected site was found. The nonconservative substitutions, such as K93R, K93E in E6, T37I, and D38N in E7, affected multiple hypothetical epitopes in the B cell. This study provides information for the investigation of HPV epidemic characters. The discovery of new variants of HPV-52 may lay the basis for the development of the virus diagnosis, further study of cervical cancer, and vaccine design in Central China.

Human Papillomavirus E7 Oncoprotein Promotes Proliferation and Migration through the Transcription Factor E2F1 in Cervical Cancer Cells.

BACKGROUND: High-Risk Human Papillomavirus (HR-HPV) persistent infection is the main cause of cervical cancer and its precancerous lesions. A previous study showed that HPV16 and HPV58 infections were the most common infection types in the local region. Some studies also declared that HPV58 E7 variants increased the risk of cervical cancer among Asian populations. OBJECTIVE: This study aimed to determine whether the HPV58 E7 T20I (C632T) variant promotes the malignant behavior of cervical cancer cells and the underlying mechanism of the HR-HPV E7 oncoprotein involved in the development of cervical cancer. METHODS: CCK-8 and clone formation assays were used to detect cell proliferation ability. Transwell assays and cell wound healing assays were used to evaluate cell migration ability. Targeted knockdown of E2F1 expression using specific siRNA, RT-qPCR and Western blot were performed to assess gene expression changes. A chromatin immunoprecipitation assay was used to verify that E2F1 interacted with the TOP2A promoter region. RESULTS: HPV58 E7 and HPV58 E7M oncoproteins increased the proliferation and migration ability of cervical cancer cells. However, the HPV58 E7 T20I variant did not promote malignant behaviors compared with wildtype HPV58 E7. HPV E7 and E7M oncoproteins increased the expression of TOP2A, BIRC5 and E2F1, and knockdown of HPV E7 decreased their expression. Low E2F1 expression reduced the expression of TOP2A and BIRC5 and inhibited the proliferation and migration ability of cervical cancer cells. E2F1 interacted with the TOP2A gene promoter region to promote its transcriptional expression. CONCLUSION: The HPV58 E7 T20I variant did not promote malignant behaviors compared with wild-type HPV58 E7. The HR-HPV E7 oncoprotein enhanced the proliferation and migration of cervical cancer cells, which was considered to be due to the HPV E7 oncoprotein, increasing the expression of BIRC5 and TOP2A by upregulating the transcription factor E2F1.

Employing ATP as a New Adjuvant Promotes the Induction of Robust Antitumor Cellular Immunity by a PLGA Nanoparticle Vaccine.

Tumor vaccines based on synthetic human papillomavirus (HPV) oncoprotein E7 and/or E6 peptides have shown encouraging results in preclinical model studies and human clinical trials. However, the clinical efficacy may be limited by the disadvantages of vulnerability to enzymatic degradation and low immunogenicity of peptides. To further improve the potency of vaccine, we developed a poly(lactide-co-glycolide)-acid (PLGA) nanoparticle, which encapsulated the antigenic peptide HPV16 E744-62, and used adenosine triphosphate (ATP), one of the most important intracellular metabolites and an endogenous extracellular danger signal for the immune system, as a new adjuvant component. The results showed that PLGA nanoparticles increased the in vivo stability, lymph node accumulation, and dendritic cell (DC) uptake of the E7 peptide; in addition, ATP further increased the migration, nanoparticle uptake, and maturation of DCs. Preventive immunization with ATP-adjuvanted nanoparticles completely abolished the growth of TC-1 tumors in mice and produced long-lasting immunity against tumor rechallenge. When tumors were fully established, therapeutic immunization with ATP-adjuvanted nanoparticles still significantly inhibited tumor progression. Mechanistically, ATP-adjuvanted nanoparticles significantly improved the systemic generation of antitumor effector cells, boosted the local functional status of these cells in tumors, and suppressed the generation and tumor infiltration of immunosuppressive Treg cells and myeloid-derived suppressor cells. These findings indicate that ATP is an effective vaccine adjuvant and that nanoparticles adjuvanted with ATP were able to elicit robust antitumor cellular immunity, which may provide a promising therapeutic vaccine candidate for the treatment of clinical malignancies, such as cervical cancer.

Phylogenetic analysis of Alphapapillomavirus based on L1, E6 and E7 regions suggests that carcinogenicity and tissue tropism have appeared multiple times during viral evolution.

Members of the Alphapapillomavirus genus are causative agents for cervix cancer and benign lesions in humans. These viruses are classified according to sequence similarities in their L1 region. Yet, viral carcinogenicity has been associated with variations in the proteins encoded by the E6 and E7 genes. In order to relate evolutionary history with origin of carcinogenicity, we performed phylogenetic reconstructions using both nucleotide and predicted amino acid sequences of the L1, E6 and E7 genes. Whilst phylogenetic analysis of L1 reconstructed genus evolutionary history, phylogenies based on E6 and E7 proteins support the idea that mutations at amino acids S/Tx [V/L] (E6) and LxCxE (E7) might be responsible for carcinogenic potential. These findings indicate that virulence within Alphapapillomavirus have appeared multiple times during evolution. Our results reveal that oncogenic potential is not a monophyletic clade-specific adaptation but might be the result of positive selection on random mutations occurring on proteins involved in host infection during viral diversification.

MiR-875 and miR-3144 switch the human papillomavirus 16 E6/E6* mRNA ratio through the EGFR pathway and a direct targeting effect.

By employing bioinformatics scanning approaches and luciferase reporter, our previous study showed that two less common human miRNAs, miR-875 and miR-3144, target a conserved site in the genomes of most high-risk human papillomaviruses (HR-HPVs). In this study, we found that the site targeted by miR-875 and miR-3144 overlapped with the 5' alternative splice site of E6E7 transcripts in HPV16. Using HPV16+ SiHa cells, we showed that high levels of miR-875 and miR-3144 reduced the abundance of unspliced E6, while they promoted three E6* spliced transcripts and decreased the expression levels of E6/E7 oncoproteins and epidermal growth factor receptor (EGFR). A potential miR-875 target site was predicted in EGFR. Meanwhile, depletion of EGFR resulted in a failure to promote E6* but maintained the suppression of unspliced E6 driven by miR-875 and miR-3144. The data suggest that these two miRNAs switch the E6/E6* ratio through both the EGFR pathway and direct targeting. Here, we demonstrate for the first time that human miRNAs regulate the HPV splice isoforms. Furthermore, miRNA-875 and miRNA-3144 are only found in vertebrates and Homo sapiens, and the binding site in EGFR is highly conserved in Boreoeutheria. Our findings highlight the tumour-suppressing effect of miRNAs that possibly appeared in the late stage of biological evolution.

Nanoparticle Conjugation of Human Papillomavirus 16 E7-long Peptides Enhances Therapeutic Vaccine Efficacy against Solid Tumors in Mice.

Treatment of patients bearing human papillomavirus (HPV)-related cancers with synthetic long-peptide (SLP) therapeutic vaccines has shown promising results in clinical trials against premalignant lesions, whereas responses against later stage carcinomas have remained elusive. We show that conjugation of a well-documented HPV-E7 SLP to ultra-small polymeric nanoparticles (NP) enhances the antitumor efficacy of therapeutic vaccination in different mouse models of HPV+ cancers. Immunization of TC-1 tumor-bearing mice with a single dose of NP-conjugated E7LP (NP-E7LP) generated a larger pool of E7-specific CD8+ T cells with increased effector functions than unconjugated free E7LP. At the tumor site, NP-E7LP prompted a robust infiltration of CD8+ T cells that was not accompanied by concomitant accumulation of regulatory T cells (Tregs), resulting in a higher CD8+ T-cell to Treg ratio. Consequently, the amplified immune response elicited by the NP-E7LP formulation led to increased regression of large, well-established tumors, resulting in a significant percentage of complete responses that were not achievable by immunizing with the non-NP-conjugated long-peptide. The partial responses were characterized by distinct phases of regression, stable disease, and relapse to progressive growth, establishing a platform to investigate adaptive resistance mechanisms. The efficacy of NP-E7LP could be further improved by therapeutic activation of the costimulatory receptor 4-1BB. This NP-E7LP formulation illustrates a "solid-phase" antigen delivery strategy that is more effective than a conventional free-peptide ("liquid") vaccine, further highlighting the potential of using such formulations for therapeutic vaccination against solid tumors. Cancer Immunol Res; 6(11); 1301-13. ©2018 AACR.

Characterization of a new genotype of Betapapillomavirus HPV 17 through L1, E7, E7 and LCR sequences.

Human papillomavirus (HPV) exhibits epithelial and mucosal tropism. HPV type 17 belongs to the Betapapillomavirus genus and molecular cloning experiments have identified two subtypes (17a and 17b) isolated from epidermodysplasia verruciformis (EV). HPV subtypes are characterized by dissimilarities from 2 to 10% at the nucleotide level from their referenced HPV. The aim of this study was to characterize the L1, E6, E7 and LCR sequences from an isolate, which was recovered from the oral mucosa of an asymptomatic 63 year-old woman. The whole late gene 1 (L1) was amplified using several sets of primers. The complete early genes 6 and 7 (E6, E7) and the long control region (LCR) were amplified using specific primers. Potential binding sites for transcriptional factors within the LCR were also investigated. Within these sets, the DNA sequence was altered at 91 positions (68 in L1, 13 in E6, 8 in E7, and 2 in LCR sequences). L1 analysis showed high dissimilarity compared with the HPV 17 prototype, reaching 4% of nucleotide substitutions and leading to a probable third 17 subtype. The E6 oncoprotein presented the highest modification among the sequences studied, with four amino acid changes in comparison with the prototype isolate. One amino acid was modified at a position 62 (S-T), a zinc-binding domain (CxxC(C)29 CxxC). Our findings provide data on genetic variations seen in this genotype, reaching to dichotomic branching and pointing to an evolutionary process.

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.

Structural Insights in Multifunctional Papillomavirus Oncoproteins.

Since their discovery in the mid-eighties, the main papillomavirus oncoproteins E6 and E7 have been recalcitrant to high-resolution structure analysis. However, in the last decade a wealth of three-dimensional information has been gained on both proteins whether free or complexed to host target proteins. Here, we first summarize the diverse activities of these small multifunctional oncoproteins. Next, we review the available structural data and the new insights they provide about the evolution of E6 and E7, their multiple interactions and their functional variability across human papillomavirus (HPV) species.

Supercharged green fluorescent protein delivers HPV16E7 DNA and protein into mammalian cells in vitro and in vivo.

Macromolecules including DNA and proteins serve as important human therapeutics but are limited by their general inability to cross cell membranes. Supercharged proteins have been known as potent tools for delivery of macromolecules into mammalian cells. Thus, the use of these delivery systems is important to reduce the human papillomavirus (HPV)-associated malignancies through improvement of vaccine modalities. In this study, we used a supercharged green fluorescent protein (+36 GFP) for delivery of the full-length HPV16 E7 DNA and protein into mammalian cells and evaluated immune responses, and protective/therapeutic effects of different formulations in C57BL/6 tumor mice model. Our results showed that the complexes of E7 DNA/+36 GFP and also E7 protein/+36 GFP form stable nanoparticles through non-covalent binding with an average size of ∼ 200-300 nm. The efficient delivery of E7 DNA or protein by +36 GFP was detected in HEK-293T cell line for 4 h and 24 h post-transfection. Mice immunization with E7 protein/+36 GFP nanoparticles elicited a higher Th1 cellular immune response with the predominant IgG2a and IFN-γ levels than those induced by E7 protein, E7 DNA, E7 DNA/+36 GFP and control groups (p < .05). Moreover, the E7 DNA/+36 GFP and E7 protein/+36 GFP nanoparticles similarly protected mice against TC-1 tumor challenge (∼67%) as compared to E7 DNA and E7 protein (∼33%), respectively. These data suggest that +36 GFP may provide a promising platform to improve protein and DNA delivery in vitro and in vivo.

Fungal mannosylation enhances human papillomavirus 16 E7 therapeutic immunity against TC-1 tumors.

Cervical cancer, resulting from infection with human papillomavirus (HPV)16, remains the fourth most common cancer in women worldwide. Recently, three prophylactic HPV vaccines targeting high-risk HPVs (particularly HPV16 and HPV18) have been implemented to protect younger women. However, individuals with pre-existing infections have no benefit from prophylactic vaccines. Thus, there is an urgent need to develop therapeutic vaccines. HPV16 E7 has been widely utilized as a target for immune therapy of HPV16-associated lesions or cancers, reflecting the sustained existence of this virus in cancerous cells. We developed mannosylated HPV16 E7 (mE7) expressed from Pichia pastoris as a therapeutic vaccine against HPV16-associated cancer. Unmannosylated E7 (E7) was also generated from Pichia pastoris as a control. Mannosylation enhanced the uptake of mE7 by mannose receptors of bone marrow-derived dendritic cells (BMDCs), while the uptake of E7 was unaffected. mE7-uptake BMDCs in vitro induced more IFN-γ secretion by splenocytes of immunized mice than E7. Vaccination of C57BL/6 mice with mE7 combined with adjuvant monophosphoryl lipid A (MPL) elicited stronger Th1 (type 1 T helper cell) responses and E7-specific T cell responses than E7. The mE7 vaccine induced the increased production of IFN-γ, IL-2 and TNF-α, elicited more E7-specific IFN-γ-secreting CD8+ T cells in spleen and peripheral blood mononuclear cells (PMBCs) and promoted stronger E7-specific cytotoxic CD8+ T cell responses compared with E7. Furthermore, TC-1 tumor challenged mice were used to confirm the antitumor activity of the vaccines. As a result, mE7 generated complete antitumor activity against TC-1 tumors, while E7 only provided partial antitumor activity. Taken together, mE7 can be a promising immunotherapy for treating cervical cancer.

Antigenic Peptide Prediction From E6 and E7 Oncoproteins of HPV Types 16 and 18 for Therapeutic Vaccine Design Using Immunoinformatics and MD Simulation Analysis.

Human papillomavirus (HPV) induced cervical cancer is the second most common cause of death, after breast cancer, in females. Three prophylactic vaccines by Merck Sharp & Dohme (MSD) and GlaxoSmithKline (GSK) have been confirmed to prevent high-risk HPV strains but these vaccines have been shown to be effective only in girls who have not been exposed to HPV previously. The constitutively expressed HPV oncoproteins E6 and E7 are usually used as target antigens for HPV therapeutic vaccines. These early (E) proteins are involved, for example, in maintaining the malignant phenotype of the cells. In this study, we predicted antigenic peptides of HPV types 16 and 18, encoded by E6 and E7 genes, using an immunoinformatics approach. To further evaluate the immunogenic potential of the predicted peptides, we studied their ability to bind to class I major histocompatibility complex (MHC-I) molecules in a computational docking study that was supported by molecular dynamics (MD) simulations and estimation of the free energies of binding of the peptides at the MHC-I binding cleft. Some of the predicted peptides exhibited comparable binding free energies and/or pattern of binding to experimentally verified MHC-I-binding epitopes that we used as references in MD simulations. Such peptides with good predicted affinity may serve as candidate epitopes for the development of therapeutic HPV peptide vaccines.

Hidden Structural Codes in Protein Intrinsic Disorder.

Intrinsic disorder is a major structural category in biology, accounting for more than 30% of coding regions across the domains of life, yet consists of conformational ensembles in equilibrium, a major challenge in protein chemistry. Anciently evolved papillomavirus genomes constitute an unparalleled case for sequence to structure-function correlation in cases in which there are no folded structures. E7, the major transforming oncoprotein of human papillomaviruses, is a paradigmatic example among the intrinsically disordered proteins. Analysis of a large number of sequences of the same viral protein allowed for the identification of a handful of residues with absolute conservation, scattered along the sequence of its N-terminal intrinsically disordered domain, which intriguingly are mostly leucine residues. Mutation of these led to a pronounced increase in both α-helix and ß-sheet structural content, reflected by drastic effects on equilibrium propensities and oligomerization kinetics, and uncovers the existence of local structural elements that oppose canonical folding. These folding relays suggest the existence of yet undefined hidden structural codes behind intrinsic disorder in this model protein. Thus, evolution pinpoints conformational hot spots that could have not been identified by direct experimental methods for analyzing or perturbing the equilibrium of an intrinsically disordered protein ensemble.

Blocking IL-10 signalling at the time of immunization does not increase unwanted side effects in mice.

BACKGROUND: Cancer therapeutic vaccine induced cytotoxic T cell (CTL) responses are pivotal for the killing of tumour cells. Blocking interleukin 10 (IL-10) signalling at the time of immunization increases vaccine induced CTL responses and improves prevention of tumour growth in animal models compared to immunization without an IL-10 signalling blockade. Therefore, this immunization strategy may have potential to curtail cancer in a clinical setting. However, IL-10 deficiency leads to autoimmune disease in the gut. Blocking IL-10 at the time of immunization may result in unwanted side effects, especially immune-pathological diseases in the intestine. METHODS: We investigated whether blocking IL-10 at the time of immunization results in intestinal inflammation responses in a mouse TC-1 tumour model and in a NOD autoimmune disease prone mouse model. RESULTS: We now show that blocking IL-10 at the time of immunization increases IL-10 production by CD4+ T cells in the spleen and draining lymph nodes, and does not result in blood cell infiltration to the intestines leading to intestinal pathological changes. Moreover, immunization with papillomavirus like particles combined with simultaneously blocking IL-10 signalling does not increase the incidence of autoimmune disease in Non-obese diabetic (NOD) mice. CONCLUSIONS: Our results indicate that immunization with an IL-10 inhibitor may facilitate the generation of safe, effective therapeutic vaccines against chronic viral infection and cancer.

Efficient Eradication of Established Tumors in Mice with Cationic Liposome-Based Synthetic Long-Peptide Vaccines.

Therapeutic vaccination with synthetic long peptides (SLP) can be clinically effective against HPV-induced premalignant lesions; however, their efficiency in established malignant lesions leaves room for improvement. Here, we report the high therapeutic potency of cationic liposomes loaded with well-defined tumor-specific SLPs and a TLR3 ligand as adjuvant. The cationic particles, with an average size of 160 nm, could strongly activate functional, antigen-specific CD8+ and CD4+ T cells and induced in vivo cytotoxicity against target cells after intradermal vaccination. At a low dose (1 nmol) of SLP, our liposomal formulations significantly controlled tumor outgrowth in two independent models (melanoma and HPV-induced tumors) and even cured 75%-100% of mice of their large established tumors. Cured mice were fully protected from a second challenge with an otherwise lethal dose of tumor cells, indicating the potential of liposomal SLP in the formulation of powerful vaccines for cancer immunotherapy. Cancer Immunol Res; 5(3); 222-33. ©2017 AACR.