ABSTRACT
Cervical cancer caused by Human papillomavirus (HPV) is one of the most common causes of cancer death in women worldwide. Even though the disease can be avoided by immunization, the expensive price of HPV vaccines makes it hard to be accessed by women in middle-low-income countries. Thus, the development of generic HPV vaccines is needed to address inequalities in life-saving access. This study aimed to develop the HPV52 L1 VLP-based recombinant vaccine using Pichia pastoris expression system. The l1 gene was codon-optimized based on P. pastoris codon usage resulting CAI value of 0.804. The gene was inserted into the pD902 plasmid under the regulation of the AOX1 promoter. The linear plasmid was transformed into P. pastoris BG10 genome and screened in YPD medium containing zeocin antibiotic. Colony of transformant that grown on highest zeocin concentration was characterized by genomic PCR and sequencing. The positive clone was selected and expressed using BMGY/BMMY medium induced with various methanol concentrations. The SDS-PAGE and Western blot analyses showed that 55 kDa L1 protein was successfully expressed using an optimum concentration of 1% methanol. The self-assembly of HPV52 L1 protein was also proven using TEM analysis. Moreover, we also analyzed the B-cell epitope of HPV52 L1 protein based on several criteria, including antigenicity, surface accessibility, flexibility, and hydrophilicity. We assumed that epitope 476GLQARPKLKRPASSAPRTSTKKKKV500 could be developed as an epitope-based vaccine with a neutralizing antibody response toward HPV52 infection. Finally, our study provided the alternative for developing low-cost HPV vaccines, either VLP or epitope-based.
Subject(s)
Human Papillomavirus Viruses , Papillomavirus Vaccines , Female , Humans , Methanol/metabolism , Capsid Proteins/genetics , Pichia/genetics , Pichia/metabolism , Papillomavirus Vaccines/genetics , Papillomavirus Vaccines/metabolism , Epitopes/metabolism , Codon/metabolismABSTRACT
Human papillomavirus (HPV) imposes an increased risk of developing cervical, anal and oropharyngeal cancer. In the Western world, HPV infection is currently the major cause of oropharyngeal cancer. The effectiveness of HPV vaccines for oral or oropharyngeal HPV infection is yet to be determined. This study conducted a systematic literature search in Pubmed and Embase. Studies investigating the impact of HPV vaccines on oral or oropharyngeal HPV infection were enrolled. This review reports the relative prevention percentage (RPP), including a risk of bias assessment as well as a quality assessment study. Nine studies were included (48,777 participants): five cross-sectional studies; one randomized community trial study (RCT); one longitudinal cohort study; and two case-control studies. A significant mean RPP of 83.9% (66.6-97.8%) was calculated from the cross-sectional studies, 82.4% in the included RCT and 83% in the longitudinal cohort study. Further, two case-control studies that measured antibody response in participants immunized with HPV vaccines were included. Respectively, 100% and 93.2% of participants developed HPV-16 Immunoglobulin G (IgG) antibodies in oral fluids post-vaccination. Analysis of the studies identified a significant decrease in vaccine-type oral or oropharyngeal HPV infections in study participants immunized with HPV vaccines across study designs and heterogenous populations. Further, a significant percentage of participants developed IgG antibodies in oral fluid post-vaccination.
Subject(s)
Papillomavirus Infections/prevention & control , Papillomavirus Vaccines/pharmacology , Alphapapillomavirus/pathogenicity , Case-Control Studies , Cross-Sectional Studies , Female , Humans , Longitudinal Studies , Male , Mouth/virology , Mouth Neoplasms/prevention & control , Mouth Neoplasms/virology , Oropharyngeal Neoplasms/prevention & control , Oropharyngeal Neoplasms/virology , Oropharynx/virology , Papillomaviridae/immunology , Papillomaviridae/pathogenicity , Papillomavirus Infections/metabolism , Papillomavirus Vaccines/metabolism , Pre-Exposure Prophylaxis/methods , VaccinationABSTRACT
OBJECTIVES: Human papillomavirus infection (HPV) is the most common viral infection which is causes of cervical, penal, vulvar, anal and, oropharyngeal cancer. E7 protein of HPV is a suitable target for induction of T cell responses and controlling HPV-related cancer. The aim of the current study was to designed and evaluated a novel fusion protein containing the different E7 proteins of the HPV 16, 18, 6 and 11, linked to the cell-penetrating peptide HIV-1 Tat 49-57, in order to improve cytotoxic immune responses in in-vitro and in-vivo. RESULTS: In this study whole sequence of HPV16,18,6,11 E7-Tat (47-57) and HPV16,18,6,11 E7 cloned into the vector and expressed in E. coli (BL21). The purified protein was confirmed by SDS page and western blotting and then injected into the C57BL/6 mice. The efficiency of the fusion protein vaccine was assessed by antibody response assay, cytokine assay (IL-4 and IFN-γ), CD + 8 cytotoxicity assay and tumor challenge experiment. Result showed that fusion proteins containing Adjuvant (IFA,CFA) could express higher titer of antibody. Also, we showed that vaccination with E7-Tat and, E7-Tat-ADJ induced high frequencies of E7-specific CD8 + T cells and CD107a expression as well as IFN-γ level and enhanced long-term survival in the therapeutic animal models. CONCLUSION: Our finding suggested that this novel fusion protein vaccine was able to induce therapeutic efficacy and immunogenicity by improving CD8 + T cell in TC-1 tumor bearing mice; so this vaccine may be appreciated for research against HPV and tumor immunotherapies.
Subject(s)
Alphapapillomavirus/metabolism , HIV-1/genetics , Lung Neoplasms/virology , Papillomavirus E7 Proteins/metabolism , Papillomavirus Infections/prevention & control , Papillomavirus Vaccines/administration & dosage , Peptide Fragments/genetics , tat Gene Products, Human Immunodeficiency Virus/genetics , Alphapapillomavirus/genetics , Alphapapillomavirus/immunology , Animals , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/administration & dosage , Cancer Vaccines/immunology , Cancer Vaccines/metabolism , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/growth & development , Female , HIV-1/metabolism , Human papillomavirus 11/genetics , Human papillomavirus 11/metabolism , Human papillomavirus 16/genetics , Human papillomavirus 16/metabolism , Human papillomavirus 18/genetics , Human papillomavirus 18/metabolism , Human papillomavirus 6/genetics , Human papillomavirus 6/metabolism , Humans , Lung Neoplasms/prevention & control , Mice , Mice, Inbred C57BL , Papillomavirus E7 Proteins/genetics , Papillomavirus Vaccines/immunology , Papillomavirus Vaccines/metabolism , Recombinant Fusion Proteins/administration & dosage , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Vaccination , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology , Vaccines, Synthetic/metabolismABSTRACT
No licensed therapeutic human papillomavirus (HPV) vaccine is currently available, so it remains a high priority to develop a therapeutic HPV vaccine or prophylactic/therapeutic HPV vaccine for cervical cancer. In this current study, we designed an HPV vaccine including CpG oligodeoxynucleotides 1826 as an adjuvant and HPV16 E7 43-77 peptide as antigen, which contains a CD8 T cell epitope (E7 49-57), and two CD4 T cell epitopes (E7 43-77 and E7 50-62). The prophylactic and therapeutic effect on cervical cancer induced by a single administration of vaccine, were comprehensively evaluated by examining the tumor size and the percentage of tumor-free/bearing mice. The cellular immunity and modulation of immunosuppressive cells induced by the vaccine were evaluated by examining intracellular cytokine staining (ICS) of splenocytes and FCM, respectively. Antigen-specific cytotoxic T-lymphocyte (CTL) responses were investigated using in vivo cytolytic assay. The results showed that the single administration of vaccine elicited significant prophylactic as well as therapeutic effect on cervical cancer. The increased cellular immunity mediated by CD4â¯+â¯IFN-γâ¯+â¯T cells and CD8â¯+â¯IFN-γâ¯+â¯T cells, and the decreased numbers of immunosuppressive cells including regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) were induced by the vaccine. Antigen-specific CTL response was also induced by vaccination. These findings suggested that significant anti-tumor effect of the vaccine may result from the induction of increased cellular immunity and decreased immunosuppressive cells.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Cancer Vaccines/immunology , Epitopes, T-Lymphocyte/metabolism , Human papillomavirus 16/physiology , Myeloid-Derived Suppressor Cells/immunology , Papillomavirus E7 Proteins/metabolism , Papillomavirus Infections/immunology , Papillomavirus Vaccines/immunology , Peptides/metabolism , T-Lymphocytes, Regulatory/immunology , Uterine Cervical Neoplasms/immunology , Adjuvants, Immunologic , Animals , Cells, Cultured , Cytotoxicity, Immunologic , Female , Humans , Interferon-gamma/metabolism , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Oligodeoxyribonucleotides/immunology , Papillomavirus Vaccines/metabolism , Vaccination , Vaccines, SubunitABSTRACT
Here, we describe a process for expression, purification, and characterization of truncated human papillomavirus type-6 (HPV-6) L1 virus-like particles (VLPs). The scalable cultivation process in a WAVE Bioreactor at the 10-L scale was optimized to express HPV-6 L1 VLPs using the baculovirus insect expression system. A hollow fiber membrane system was used for the integrated operation, including concentration, diafiltration, extraction, and clarification. The HPV-6 L1 protein was further purified by anion-exchange chromatography and hydrophobic chromatography. The HPV-6 L1 protein could self-assemble into VLPs with a diameter of approximately 50-60 nm after removal of the reductant dithiothreitol (DTT). The final purified HPV-6 L1 VLPs product was characterized to estimate yield and purity, and exceeds the requirements for pharmaceutical-grade VLP vaccine. Immunization of mice demonstrated that the vaccine could elicit high titer neutralizing antibodies in vivo. This study confirms the feasibility of producing pharmaceutical-grade HPV type-6 L1 VLPs on an industrial scale for clinical trials.
Subject(s)
Capsid Proteins/metabolism , Industrial Microbiology/methods , Papillomavirus Infections/virology , Papillomavirus Vaccines/metabolism , Animals , Baculoviridae/genetics , Baculoviridae/metabolism , Bioreactors , Capsid Proteins/chemistry , Capsid Proteins/genetics , Capsid Proteins/isolation & purification , Female , Humans , Immunization , Industrial Microbiology/instrumentation , Mice , Mice, Inbred BALB C , Papillomavirus Vaccines/chemistry , Papillomavirus Vaccines/genetics , Papillomavirus Vaccines/isolation & purification , SpodopteraABSTRACT
Introducción: La Comunidad Valenciana inició en octubre del 2008 el programa de vacunación contra el virus del papiloma humano (VPH) en niñas de 14 años. El objetivo de este estudio es evaluar los conocimientos sobre la infección por VPH y su vacuna en madres de adolescentes e identificar los factores asociados a la predisposición de vacunar a sus hijas. Material y métodos: Estudio observacional transversal mediante cuestionario dirigido a madres de alumnas nacidas en 1995 matriculadas en centros de secundaria de la provincia de Valencia durante 2010-2011. Muestra aleatoria estratificada por conglomerados (n=1.279). Análisis estadístico: porcentajes, intervalos de confianza, OR, contrastes chi al cuadrado y regresión logística multivariante. Resultados: Ochocientos treinta y tres cuestionarios completados (65,1%). El 76,6% de las madres habían vacunado a sus hijas contra el VPH. El 93,8% conocía la vacuna, sobre todo a través de la televisión (71,5%). El 78,5% recibió consejo favorable de un profesional sanitario, lo que mejoró la vacunación de sus hijas (OR: 2,4). Los conocimientos globales sobre la infección por VPH y la vacuna fueron bajos. La confianza de las madres en las vacunas como método preventivo mejora la vacunación contra VPH (OR: 3,8). El miedo a los efectos adversos (45,6%) fue el primer motivo de rechazo. Conclusiones: No parece que los medios de comunicación influyan en la decisión de vacunar. Sería conveniente minimizar la percepción de riesgo ante esta vacuna. El consejo del profesional sanitario actúa a favor de la vacunación si este interviene activamente en sentido positivo. Existe una brecha entre nivel de conocimientos y toma de decisión para vacunar (AU)
Introduction: In October 2008, Valencian Community started its human papillomavirus (HPV) vaccination schedules for 14 year-old girls. The aim of this study is to assess knowledge about HPV infection and its vaccine among the mothers of these girls, and to identify factors associated with the willingness to vaccinate their daughters. Material and methods: Cross-sectional study by means of a questionnaire to mothers of girls born in 1995, and attending secondary schools in the province of Valencia during 2010-2011. Cluster stratified random sample (n=1279). Statistical analysis: percentages, confidence intervals, OR, Chi-squared and multivariate logistic regression contrasts. Results: A total of 833 (65.1%) questionnaires were completed. The results obtained showed that, 76.6% of mothers had vaccinated their daughters against HPV; 93.8% knew about the vaccine, particularly through television (71.5%); and 78.5% received positive advice from a health professional which increased the vaccination of their daughters (OR: 2.4). There was low overall knowledge about HPV infection and vaccination. Confidence of the mothers in vaccines as a preventative method increases the HPV vaccination (OR: 3.8). The first reason for refusal was the fear of adverse events (45.6%). Conclusions: Apparently, the media does not influence the willingness to vaccinate. It would be desirable to minimize the perception of risk of the vaccine. Positive health advice from a health professional can have a positive effect on vaccination. There is a gap between the level of knowledge and decision-making to vaccinate (AU)
Subject(s)
Adolescent , Female , Humans , Papillomavirus Vaccines/administration & dosage , Papillomavirus Vaccines , Spain/ethnology , Communications Media/trends , Uterine Cervical Neoplasms/metabolism , Uterine Cervical Neoplasms/pathology , Papillomavirus Vaccines/metabolism , Papillomavirus Vaccines/supply & distribution , 50230 , Observational StudyABSTRACT
Cellular immunity against cancer can be achieved with viral vector- and DNA-based immunizations. In preclinical studies, cancer vaccines are very potent, but in clinical trials these potencies are not achieved yet. Thus, a rational approach to improve cancer vaccines is warranted. We previously demonstrated that the relatively low intrinsic immunogenicity of DNA vaccines could be enhanced by inclusion of endoplasmic reticulum (ER) targeting and universal helper epitopes within the vaccine. We now evaluated whether an optimal antigen format, as defined in DNA vaccines, can further enhance the effectiveness of recombinant Semliki Forest virus (rSFV) vaccines. To this purpose, we generated, characterized and evaluated the efficacy of rSFV replicon particles expressing human papillomavirus E6 and/or E7 proteins fused to several helper T-cell epitopes and an ER targeting signal. Here, we show that inclusion of a helper cassette and an ER targeting signal enhanced protein stability and markedly augmented the frequencies of human papillomavirus-specific T cells. Even at an immunization dose of as low as 10(5) replicon particles, this novel vaccine achieved tumor regression and protection. Thus, even highly effective viral vector vaccines can benefit from an improved antigen format, based on the inclusion of defined helper epitopes and ER targeting.
Subject(s)
Antigens, Viral/immunology , Cancer Vaccines/immunology , Oncogene Proteins, Viral/immunology , Papillomavirus E7 Proteins/immunology , Papillomavirus Vaccines/immunology , Animals , Antigens, Viral/administration & dosage , Antigens, Viral/genetics , Cancer Vaccines/administration & dosage , Cricetinae , Epitopes, T-Lymphocyte/chemistry , Female , Humans , Kidney/cytology , Mice, Inbred C57BL , Oncogene Proteins, Viral/administration & dosage , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Papillomavirus E7 Proteins/administration & dosage , Papillomavirus E7 Proteins/genetics , Papillomavirus E7 Proteins/metabolism , Papillomavirus Vaccines/genetics , Papillomavirus Vaccines/metabolism , Semliki forest virus/genetics , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes, Cytotoxic/metabolism , Uterine Cervical Neoplasms/prevention & control , Vaccines, DNA/administration & dosage , Vaccines, DNA/immunologyABSTRACT
Antibodies specific for neutralizing epitopes in either Human papillomavirus (HPV) capsid protein L1 or L2 can mediate protection from viral challenge and thus their accurate and sensitive measurement at high throughput is likely informative for monitoring response to prophylactic vaccination. Here we compare measurement of L1 and L2-specific neutralizing antibodies in human sera using the standard Pseudovirion-Based Neutralization Assay (L1-PBNA) with the newer Furin-Cleaved Pseudovirion-Based Neutralization Assay (FC-PBNA), a modification of the L1-PBNA intended to improve sensitivity towards L2-specific neutralizing antibodies without compromising assay of L1-specific responses. For detection of L1-specific neutralizing antibodies in human sera, the FC- PBNA and L1-PBNA assays showed similar sensitivity and a high level of correlation using WHO standard sera (nâ=â2), and sera from patients vaccinated with Gardasil (nâ=â30) or an experimental human papillomavirus type 16 (HPV16) L1 VLP vaccine (nâ=â70). The detection of L1-specific cross-neutralizing antibodies in these sera using pseudovirions of types phylogenetically-related to those targeted by the L1 virus-like particle (VLP) vaccines was also consistent between the two assays. However, for sera from patients (nâ=â17) vaccinated with an L2-based immunogen (TA-CIN), the FC-PBNA was more sensitive than the L1-PBNA in detecting L2-specific neutralizing antibodies. Further, the neutralizing antibody titers measured with the FC-PBNA correlated with those determined with the L2-PBNA, another modification of the L1-PBNA that spacio-temporally separates primary and secondary receptor engagement, as well as the protective titers measured using passive transfer studies in the murine genital-challenge model. In sum, the FC-PBNA provided sensitive measurement for both L1 VLP and L2-specific neutralizing antibody in human sera. Vaccination with TA-CIN elicits weak cross-protective antibody in a subset of patients, suggesting the need for an adjuvant.
Subject(s)
Antibodies, Neutralizing/blood , Furin/metabolism , Neutralization Tests/methods , Papillomavirus Vaccines/immunology , Papillomavirus Vaccines/metabolism , Vaccination , Virion/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/blood , Antibodies, Viral/immunology , Antibody Specificity , Capsid Proteins/immunology , Capsid Proteins/metabolism , Human Papillomavirus Recombinant Vaccine Quadrivalent, Types 6, 11, 16, 18 , Humans , Neutralization Tests/standards , Oncogene Proteins, Viral/immunology , Oncogene Proteins, Viral/metabolism , Papillomaviridae/immunology , Proteolysis , Reference StandardsABSTRACT
Gardasil® is a quadrivalent human papillomavirus (HPV) protein-based vaccine containing genotype-specific L1 capsid proteins of HPV-16, HPV-18, HPV-6 and HPV-11 in the form of virus-like-particles (VLPs) as the active ingredient. The VLPs are produced by a DNA recombinant technology. It is uncertain if the residual HPV L1 gene DNA fragments in the vaccine products are considered contaminants or excipients of the Gardasil® vaccine. Because naked viral DNA fragments, if present in the vaccine, may bind to the insoluble amorphous aluminum hydroxyphosphate sulfate (AAHS) adjuvant which may help deliver the foreign DNA into macrophages, causing unintended pathophysiologic effects, experiments were undertaken to develop tests for HPV L1 gene DNA fragments in the final products of Gardasil® by polymerase chain reaction (PCR) and direct DNA sequencing. The results showed that while the HPV-11 and HPV-18 L1 gene DNA fragments in Gardasil® were readily amplified by the common GP6/MY11 degenerate consensus primers, the HPV-16 L1 gene DNA may need specially designed non-degenerate PCR primers for amplification at different regions of the L1 gene and different stringency conditions for detection. These variable melting profiles of HPV DNA in the insoluble fraction of the Gardasil® vaccine suggest that the HPV DNA fragments are firmly bound to the aluminum AAHS adjuvant. All methods developed for detecting residual HPV DNA in the vaccine Gardasil® for quality assurance must take into consideration the variable melting profiles of the DNA to avoid false negative results.
Subject(s)
DNA/analysis , Papillomavirus Vaccines/genetics , Adjuvants, Immunologic/chemistry , Base Sequence , Capsid Proteins/biosynthesis , Capsid Proteins/genetics , Capsid Proteins/immunology , DNA/metabolism , DNA Primers/metabolism , Human Papillomavirus Recombinant Vaccine Quadrivalent, Types 6, 11, 16, 18 , Humans , Nucleic Acid Denaturation , Papillomavirus Vaccines/immunology , Papillomavirus Vaccines/metabolism , Polymerase Chain Reaction , Sequence Analysis, DNAABSTRACT
Human papillomavirus is known to be the major pathogen of cervical cancer. Here, we report the efficacy of a bivalent human papillomavirus type 16 and 18 DNA vaccine system following repeated dosing in mice and pigs using a recombinant baculovirus bearing human endogenous retrovirus envelope protein (AcHERV) as a vector. The intramuscular administration of AcHERV-based HPV16L1 and HPV18L1 DNA vaccines induced antigen-specific serum IgG, vaginal IgA, and neutralizing antibodies to levels comparable to those achieved using the commercially marketed vaccine Cervarix. Similar to Cervarix, AcHERV-based bivalent vaccinations completely blocked subsequent vaginal challenge with HPV type-specific pseudovirions. However, AcHERV-based bivalent vaccinations induced significantly higher cell-mediated immune responses than Cervarix, promoting 4.5- (HPV16L1) and 3.9-(HPV18L1) fold higher interferon-γ production in splenocytes upon stimulation with antigen type-specific pseudovirions. Repeated dosing did not affect the immunogenicity of AcHERV DNA vaccines. Three sequential immunizations with AcHERV-HP18L1 DNA vaccine followed by three repeated dosing with AcHERV-HP16L1 over 11 weeks induced an initial production of anti-HPV18L1 antibody followed by subsequent induction of anti-HPV16L1 antibody. Finally, AcHERV-based bivalent DNA vaccination induced antigen-specific serum IgG immune responses in pigs. These results support the further development of AcHERV as a bivalent human papillomavirus DNA vaccine system for use in preventing the viral infection as well as treating the infected women by inducing both humoral and cell-mediated immune responses. Moreover, the possibility of repeated dosing indicates the utility of AcHERV system for reusable vectors of other viral pathogen vaccines.
Subject(s)
Endogenous Retroviruses/genetics , Papillomavirus Vaccines/immunology , Retroviridae/genetics , Vaccines, DNA/metabolism , Animals , Baculoviridae/genetics , Endogenous Retroviruses/metabolism , Enzyme-Linked Immunosorbent Assay/methods , Female , Genetic Vectors , Green Fluorescent Proteins/metabolism , Human papillomavirus 16/metabolism , Human papillomavirus 18/metabolism , Humans , Mice , Mice, Inbred BALB C , Neutralization Tests , Papillomavirus Vaccines/metabolism , SwineSubject(s)
Humans , Female , Adolescent , Young Adult , Adult , Middle Aged , Papillomavirus Vaccines/immunology , Papillomavirus Vaccines/metabolism , Papillomavirus Vaccines/therapeutic use , Uterine Cervical Diseases/immunology , Uterine Cervical Diseases/prevention & control , Warts/immunology , Condylomata Acuminata/immunology , Condylomata Acuminata/prevention & control , Uterine Cervical Neoplasms/epidemiology , Uterine Cervical Neoplasms/immunologyABSTRACT
Several studies indicated that biopharmaceuticals based on the recombinant protein E7 of human papillomavirus (HPV) can serve as therapeutic vaccines preventing the development of cancer in women infected with high-risk types of HPV such as HPV16. Here, we report effective extraction and purification of a plant-produced E7GGG-lichenase fusion protein, an HPV16 subunit vaccine candidate, from Nicotiana benthamiana plants, to a high yield. The target contains the modified HPV16 E7 protein internally fused to the surface loop of a truncated, hexa-His- and KDEL-tagged variant of bacterial lichenase, and has been previously shown to possess anti-cancer activity in an animal model. We purified the protein using a combination of immobilized metal-ion affinity chromatography and gel filtration. The achieved purity of the final product was 99% as confirmed by Coomassie or SYPRO Ruby staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by analytical size exclusion chromatography coupled with multi-angle laser light scattering. The overall yield was 50% corresponding to 0.1g of protein per 1 kg plant biomass. Only slight changes in these parameters were observed during the process scale-up from 50 g to 1 kg of processed leaf biomass.
Subject(s)
Human papillomavirus 16/chemistry , Papillomavirus E7 Proteins/chemistry , Papillomavirus Vaccines/chemistry , Recombinant Fusion Proteins/chemistry , Blotting, Western , Buffers , Chromatography, Gel , Chromatography, Ion Exchange , Electrophoresis, Polyacrylamide Gel , Glycoside Hydrolases/metabolism , Human papillomavirus 16/immunology , Molecular Weight , Papillomavirus E7 Proteins/immunology , Papillomavirus E7 Proteins/isolation & purification , Papillomavirus Vaccines/metabolism , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/isolation & purification , Nicotiana/chemistry , Nicotiana/virology , Vaccines, Subunit/chemistry , Vaccines, Subunit/immunologyABSTRACT
Human papillomavirus (HPV) causes cervical cancer in women worldwide, which is currently prevented by vaccines based on virus-like particles (VLPs). However, these vaccines have certain limitations in their availability to developing countries, largely due to elevated costs. Concerning the highest burden of disease in resource-poor countries, development of an improved mucosal and cost-effective vaccine is a necessity. As an alternative to VLPs, capsomeres have been shown to be highly immunogenic and can be used as vaccine candidate. Furthermore, coupling of an adjuvant like Escherichia coli heat-labile enterotoxin subunit B (LTB) to an antigen can increase its immunogenicity and reduce the costs related to separate co-administration of adjuvants. Our study demonstrates the expression of two pentameric proteins: the modified HPV-16 L1 (L1_2xCysM) and LTB as a fusion protein in tobacco chloroplasts. Homoplasmy of the transplastomic plants was confirmed by Southern blotting. Western blot analysis showed that the LTB-L1 fusion protein was properly expressed in the plastids and the recombinant protein was estimated to accumulate up to 2% of total soluble protein. Proper folding and display of conformational epitopes for both LTB and L1 in the fusion protein was confirmed by GM1-ganglioside binding assay and antigen capture ELISA, respectively. However, all transplastomic lines showed chlorosis, male sterility and growth retardation, which persisted in the ensuing four generations studied. Nevertheless, plants reached maturity and produced seeds by pollination with wild-type plants. Taken together, these results pave the way for the possible development of a low-cost adjuvant-coupled vaccine with potentially improved immunogenicity against cervical cancer.
Subject(s)
Capsid Proteins/immunology , Oncogene Proteins, Viral/immunology , Papillomavirus Vaccines/genetics , Adjuvants, Immunologic/genetics , Antigens, Viral/genetics , Bacterial Toxins/chemistry , Bacterial Toxins/genetics , Bacterial Toxins/metabolism , Capsid Proteins/chemistry , Capsid Proteins/genetics , Chloroplasts/genetics , Chloroplasts/metabolism , Enterotoxins/chemistry , Enterotoxins/genetics , Enterotoxins/metabolism , Epitopes , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Female , G(M1) Ganglioside/metabolism , Humans , Oncogene Proteins, Viral/chemistry , Oncogene Proteins, Viral/genetics , Papillomavirus Vaccines/immunology , Papillomavirus Vaccines/metabolism , Phenotype , Plant Infertility/genetics , Plants, Genetically Modified/genetics , Plastids/genetics , Pollination , Protein Conformation , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Seeds/physiology , Nicotiana/genetics , Nicotiana/growth & development , Nicotiana/metabolism , Uterine Cervical Neoplasms/immunologyABSTRACT
We investigated the potential of Nicotiana benthamiana to express the E7 protein of human papillomavirus 8 (HPV-8), a paradigm genotype among cutaneous HPVs. The protein, modified in its putative pRb-binding domain (E7(QGD)), was transiently expressed in leaves following infiltration with agrobacteria carrying either a binary vector combined with silencing suppressor constructs or replicating tobacco mosaic virus (TMV)-based vectors with different targeting signals. HPV-8 E7(QGD) yields ranged from 250 ng to 4.6 mg per gram of fresh leaf tissue. The highest yields were obtained with TMV-based vectors targeting the antigen to the apoplast. HPV8-CER (H2(q)) mice transformed with the complete early region of HPV-8 showed a delay in the onset of skin papillomatous lesions and produced E7-specific immunoglobulins G when inoculated subcutaneously with leaf extracts expressing E7(QGD). Furthermore, we demonstrated that the plant-made HPV-8 E7(QGD) induced a specific cytotoxic response in C57BL/6 (H2(b)) mice.
Subject(s)
Oncogene Proteins, Viral/immunology , Papilloma/prevention & control , Papillomavirus Vaccines/immunology , Skin Neoplasms/prevention & control , Animals , Antibodies, Viral/blood , Cytotoxicity, Immunologic , Disease Models, Animal , Immunoglobulin G/blood , Injections, Subcutaneous , Mice , Mice, Inbred C57BL , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Papilloma/immunology , Papillomavirus Vaccines/genetics , Papillomavirus Vaccines/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , Skin , Skin Neoplasms/immunology , Nicotiana/genetics , Nicotiana/metabolismABSTRACT
Commercially available prophylactic HPV vaccines for cervical cancer prevention have limited use in women with previous viral exposure. Therefore, a therapeutic HPV vaccine would benefit patients with HPV-associated genital diseases. Being developed by Cancer Research Technology Ltd, under license from Xenova Group plc, TA-CIN (Tissue Antigen - Cervical Intraepithelial Neoplasia) is a fusion protein vaccine comprising the HPV16 viral proteins L2, E6 and E7 for the treatment of HPV16-associated genital diseases. In mouse models, TA-CIN induced dose-dependent HPV16-specific CD4 and CD8 T-cell responses, which were enhanced when boosted with the vaccinia-based vector vaccine TA-HPV (Therapeutic Antigen - HPV). A phase I clinical trial of TA-CIN in healthy volunteers reported no serious adverse events and HPV16-specific cellular immune responses. Phase II trials in patients with anogenital and vulval intraepithelial neoplasia investigated heterologous prime/boost strategies with TA-CIN/TA-HPV and TA-HPV/TA-CIN, but neither of the regimens offered advantages over single-agent TA-HPV. A recent phase II trial investigating imiquimod/TA-CIN in patients with vulval intraepithelial neoplasia demonstrated significant infiltration of CD4 and CD8 T-cells in lesion responders and complete lesion regression in 63% of patients. More comprehensive case-controlled trials are needed to define responders to immunotherapy with TA-CIN and verify its prophylactic and therapeutic properties.
Subject(s)
Genital Diseases, Female/drug therapy , Genital Diseases, Female/virology , Genital Diseases, Male/drug therapy , Genital Diseases, Male/virology , Papillomavirus Infections/drug therapy , Papillomavirus Vaccines/therapeutic use , Recombinant Fusion Proteins/therapeutic use , Animals , Clinical Trials, Phase I as Topic , Clinical Trials, Phase II as Topic , Female , Genital Diseases, Female/immunology , Genital Diseases, Male/immunology , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Papillomavirus Infections/immunology , Papillomavirus Infections/virology , Papillomavirus Vaccines/adverse effects , Papillomavirus Vaccines/genetics , Papillomavirus Vaccines/metabolism , Randomized Controlled Trials as Topic , Recombinant Fusion Proteins/adverse effects , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolismABSTRACT
AIM: To develop a recombinant protein vaccine with epitopes on L1 protein for prevention of HPV16 infection. METHODS: One B cell epitope and one T cell epitope from HPV16 L1 protein were chosen to construct genes encoding different recombinant proteins. Then all the 3D structures of the proteins were predicted by Geno3D in internet. An optimal protein named RH was chosen in which three B epitopes' location simulates that in the real HPV16 L1. Gene X (consisting of B+T+T) and gene Y (consisting of B+T) were constructed by multiple PCR. RH gene (consisting of X+X+X+X+Y+Y+Y) was constructed, sub-cloned into pFastbac1, and expressed in Sf9. RESULTS: RH protein with epitopes on HPV16 L1 was designed and predicted by Geno3D. RH gene was constructed and sub cloned into pFastbac1. Finally, it was successfully expressed in baculovirus expression system. CONCLUSION: A HPV16 recombinant vaccine is successfully constructed and expressed.
Subject(s)
Capsid Proteins/genetics , Computer-Aided Design , Oncogene Proteins, Viral/genetics , Papillomavirus Vaccines/genetics , Protein Engineering , Animals , Capsid Proteins/chemistry , Capsid Proteins/metabolism , Cell Line , Gene Expression , Oncogene Proteins, Viral/chemistry , Oncogene Proteins, Viral/metabolism , Papillomavirus Vaccines/chemistry , Papillomavirus Vaccines/metabolism , Protein Structure, TertiaryABSTRACT
The amino (N) terminus of the human papillomavirus (HPV) minor capsid protein L2 can induce low-titer, cross-neutralizing antibodies. The aim of this study was to improve immunogenicity of L2 peptides by surface display on highly ordered, self-assembled virus-like particles (VLP) of major capsid protein L1, and to more completely characterize neutralization epitopes of L2. Overlapping peptides comprising amino acids (aa) 2 to 22 (hereafter, chimera or peptide 2-22), 13 to 107, 18 to 31, 17 to 36, 35 to 75, 75 to 112, 115 to 154, 149 to 175, and 172 to 200 of HPV type 16 (HPV16) L2 were genetically engineered into the DE surface loop of bovine papillomavirus type 1 L1 VLP. Except for chimeras 35-75 and 13-107, recombinant fusion proteins assembled into VLP. Vaccination of rabbits with Freund's adjuvanted native VLP induced higher L2-specific antibody titers than vaccination with corresponding sodium dodecyl sulfate-denatured proteins. Immune sera to epitopes within residues 13 to 154 neutralized HPV16 in pseudovirion neutralization assays, whereas chimera 17-36 induced additional cross-neutralization to divergent high-risk HPV18, -31, -45, -52, and -58; low-risk HPV11; and beta-type HPV5 (titers of 50 to 10,000). Aluminum hydroxide-monophosphoryl lipid A (Alum-MPL)-adjuvanted VLP induced similar patterns of neutralization in both rabbits and mice, albeit with 100-fold-lower titers than Freund's adjuvant. Importantly, Alum-MPL-adjuvanted immunization with chimeric HPV16L1-HPV16L2 (peptide 17-36) VLP induced neutralization or cross-neutralization of HPV16, -18, -31, -45, -52, and -58; HPV6 and -11; and HPV5 (titers of 50 to 100,000). Immunization with HPV16 L1-HPV16 L2 (chimera 17-36) VLP in adjuvant applicable for human use induces broad-spectrum neutralizing antibodies against HPV types evolutionarily divergent to HPV16 and thus may protect against infection with mucosal high-risk, low-risk, and beta HPV types and associated disease.
Subject(s)
Baculoviridae/metabolism , Capsid Proteins/metabolism , Oncogene Proteins, Viral/metabolism , Papillomavirus Vaccines/metabolism , Animals , Capsid/chemistry , Epitopes/chemistry , Mice , Mice, Inbred BALB C , Microscopy, Electron, Transmission/methods , Peptides/chemistry , Protein Structure, Tertiary , Rabbits , Recombinant Fusion Proteins/chemistry , RiskABSTRACT
DNA vaccines have emerged as a potential alternative to current strategies to control cancer for their safety, stability and ease of preparation. We have previously demonstrated that a DNA vaccine encoding calreticulin (CRT) linked to human papillomavirus type 16 (HPV-16) E7 antigen (CRT/E7) can generate significant E7-specific immune responses and antitumor effects in vaccinated mice, thus enhancing DNA vaccine potency. Another strategy to improve DNA vaccine potency is by enhancing the level of expression of the antigen encoded in the vaccine. DNA methylation has been shown to lead to silencing of the genes that would affect the expression of the encoded antigen of the DNA vaccines. In the current study, we reasoned that CRT/E7 DNA vaccination combined with demethylating agent, 5-aza-2'-deoxycytidine (DAC) would lead to upregulation of CRT/E7 expression, resulting in improved DNA vaccine potency. We found that pre-treatment with DAC led to increased CRT/E7 DNA expression, leading to enhanced E7-specific CD8+ T cell immune responses as well as the antitumor effects generated by the CRT/E7 DNA vaccine. Thus, our data suggest that combination of CRT/E7 DNA vaccination with DAC treatment may represent a potentially promising approach to control HPV-associated malignancies. The clinical implications of this study are discussed.
Subject(s)
Azacitidine/analogs & derivatives , Papillomavirus Vaccines/immunology , Papillomavirus Vaccines/metabolism , Vaccines, DNA/immunology , Vaccines, DNA/metabolism , Animals , Azacitidine/metabolism , CD8-Positive T-Lymphocytes/immunology , Decitabine , Female , Gene Expression , Humans , Mice , Survival AnalysisABSTRACT
BACKGROUND: It is important to develop innovative therapies for advanced stage cancers in addition to the conventional therapies including chemotherapy, radiation and surgery. Antigen-specific immunotherapy has emerged as a novel alternate therapy for advanced stage cancers, which may be employed in conjunction with conventional therapies. METHODS: In the current study, we tested the effect of treatment with the chemotherapeutic agent, apigenin in combination with DNA vaccines encoding the HPV-16 E7 antigen linked to heat shock protein 70 (HSP70) in the control of the E7-expressing tumor, TC-1. RESULTS: We observed that treatment with apigenin rendered the TC-1 tumor cells more susceptible to lysis by E7-specific cytotoxic CD8+ T cells. Furthermore, treatment of TC-1 tumor cells with apigenin was found to enhance apoptotic tumor cell death in vitro in a dose-dependant manner. We showed that TC-1 tumor-bearing mice treated with apigenin combined with E7-HSP70 DNA generate highest frequency of primary and memory E7-specific CD8+ T cells, leading to potent therapeutic anti-tumor effects against E7-expressing tumors. CONCLUSION: Thus, apigenin represents a promising chemotherapeutic agent, which may be used in combination with immunotherapy for the treatment of advanced stage cancers. The clinical implications of the current strategy are discussed.
Subject(s)
Antineoplastic Agents/administration & dosage , Apigenin/therapeutic use , Immunotherapy/methods , Animals , Antigens, Neoplasm/metabolism , Apoptosis , Combined Modality Therapy/methods , Female , HSP70 Heat-Shock Proteins/metabolism , Humans , Mice , Mice, Inbred C57BL , Oncogene Proteins, Viral/genetics , Papillomavirus E7 Proteins , Papillomavirus Vaccines/metabolism , Vaccines, DNA/therapeutic useABSTRACT
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