RESUMO
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.
Assuntos
Antígenos Virais/imunologia , Vacinas Anticâncer/imunologia , Proteínas Oncogênicas Virais/imunologia , Proteínas E7 de Papillomavirus/imunologia , Vacinas contra Papillomavirus/imunologia , Animais , Antígenos Virais/administração & dosagem , Antígenos Virais/genética , Vacinas Anticâncer/administração & dosagem , Cricetinae , Epitopos de Linfócito T/química , Feminino , Humanos , Rim/citologia , Camundongos Endogâmicos C57BL , Proteínas Oncogênicas Virais/administração & dosagem , Proteínas Oncogênicas Virais/genética , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/administração & dosagem , Proteínas E7 de Papillomavirus/genética , Proteínas E7 de Papillomavirus/metabolismo , Vacinas contra Papillomavirus/genética , Vacinas contra Papillomavirus/metabolismo , Vírus da Floresta de Semliki/genética , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/metabolismo , Neoplasias do Colo do Útero/prevenção & controle , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologiaRESUMO
Over the past two decades, DNA vaccination has been developed as a method for the induction of immune responses. However, in spite of high expectations based on their efficacy in preclinical models, immunogenicity of first generation DNA vaccines in clinical trials was shown to be poor, and no DNA vaccines have yet been licensed for human use. In recent years significant progress has been made in the development of second generation DNA vaccines and DNA vaccine delivery methods. Here we review the key characteristics of DNA vaccines as compared to other vaccine platforms, and recent insights into the prerequisites for induction of immune responses by DNA vaccines will be discussed. We illustrate the development of second generation DNA vaccines with the description of DNA tattooing as a novel DNA delivery method. This technique has shown great promise both in a small animal model and in non-human primates and is currently under clinical evaluation.
Assuntos
Infecções Bacterianas/prevenção & controle , DNA/imunologia , Derme/imunologia , Injeções Intradérmicas/métodos , Células de Langerhans/imunologia , Vacinação/métodos , Vacinas de DNA , Viroses/prevenção & controle , Animais , Infecções Bacterianas/imunologia , Ensaios Clínicos como Assunto , DNA/genética , Derme/citologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Eletroporação , Humanos , Imunidade Celular , Imunidade Humoral , Injeções a Jato , Células de Langerhans/citologia , Camundongos , Agulhas , Plasmídeos/genética , Plasmídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Tatuagem , Vacinas de DNA/administração & dosagem , Vacinas de DNA/imunologia , Viroses/imunologiaRESUMO
BACKGROUND: Shouldice repair for primary inguinal hernia is reported to have better results than classic Bassini-type repairs. The indirect inguinal hernia with a firm posterior wall is often assumed to be adequately treated by high ligation and ring narrowing. STUDY DESIGN: This double randomized controlled trial compared high ligation and ring narrowing with Bassini-Stetten repair for the indirect inguinal hernia with a firm posterior wall, and Shouldice with Bassini-Stetten repair for the inguinal hernia with a weakened posterior wall, direct or indirect. This report focuses on longterm (12-15 years) recurrence rates. RESULTS: From July 1980 to May 1983, 102 indirect primary inguinal hernias with a firm posterior wall (group I) and 263 primary inguinal hernias with a weakened posterior wall (group II) were included. By 1995, 89 patients with 100 hernia repairs had died, and for 30 repairs the patients could not be located. In 41 hernia repairs, a recurrence had been established previously. Of the remaining 194 hernia repairs, followup was updated by physical examination in 179 (92%) and by telephone interview in 15 (8%). A total of 83 recurrences were recorded, 42% of which were asymptomatic at the time of diagnosis. Seventy-three percent of the recurrences happened > 2 years after the operation. The life-table method showed the following longterm (12-15 years) recurrence rates: group I, Bassini-Stetten 33% versus ring narrowing 34%; group II, Bassini-Stetten 32% versus Shouldice 15% (p = 0.033). CONCLUSIONS: The Shouldice is the best type of hernia repair, although the 15% recurrence rate is high. Bassini-Stetten and high ligation with ring narrowing are inadequate repairs, regardless of the type of hernia.
Assuntos
Hérnia Inguinal/cirurgia , Técnicas de Sutura , Idoso , Seguimentos , Humanos , Tábuas de Vida , Ligadura/métodos , Masculino , Pessoa de Meia-Idade , Recidiva , Resultado do TratamentoRESUMO
DNA vaccination is an attractive method for therapeutic vaccination against intracellular pathogens and cancer. This review provides an introduction into the DNA vaccination field and discusses the pre-clinical successes and most interesting clinical achievements thus far. Furthermore, general attributes, mechanism of action and safety of DNA vaccination will be discussed. Since clinical results with DNA vaccination so far show room for improvement, possibilities to improve the delivery and immunogenicity of DNA vaccines are reviewed. In the coming years, these new developments should show whether DNA vaccination is able to induce clinically relevant responses in patients.
Assuntos
Neoplasias , Vacinas de DNA , Humanos , VacinaçãoRESUMO
We report an unexpected contamination during clinical manufacture of a Human Papilomavirus (HPV) 16 E6 encoding plasmid DNA (pDNA) vaccine, with a transposon originating from the Escherichia coli DH5 host cell genome. During processing, presence of this transposable element, insertion sequence 2 (IS2) in the plasmid vector was not noticed until quality control of the bulk pDNA vaccine when results of restriction digestion, sequencing, and CGE analysis were clearly indicative for the presence of a contaminant. Due to the very low level of contamination, only an insert-specific PCR method was capable of tracing back the presence of the transposon in the source pDNA and master cell bank (MCB). Based on the presence of an uncontrolled contamination with unknown clinical relevance, the product was rejected for clinical use. In order to prevent costly rejection of clinical material, both in-process controls and quality control methods must be sensitive enough to detect such a contamination as early as possible, i.e. preferably during plasmid DNA source generation, MCB production and ultimately during upstream processing. However, as we have shown that contamination early in the process development pipeline (source pDNA, MCB) can be present below limits of detection of generally applied analytical methods, the introduction of "engineered" or transposon-free host cells seems the only 100% effective solution to avoid contamination with movable elements and should be considered when searching for a suitable host cell-vector combination.
Assuntos
Elementos de DNA Transponíveis , Contaminação de Medicamentos , Escherichia coli/genética , Vacinas contra Papillomavirus/biossíntese , Vacinas de DNA/biossíntese , DNA Bacteriano/química , Fermentação , Vetores Genéticos , Limite de Detecção , Proteínas Oncogênicas Virais/genética , Vacinas contra Papillomavirus/genética , Plasmídeos , Reação em Cadeia da Polimerase , Controle de Qualidade , Proteínas Repressoras/genética , Mapeamento por Restrição , Análise de Sequência de DNA , Vacinas de DNA/genéticaRESUMO
Persistent infection with high-risk human papillomaviruses (hrHPV) can result in the formation of anogenital cancers. As hrHPV proteins E6 and E7 are required for cancer initiation and maintenance, they are ideal targets for immunotherapeutic interventions. Previously, we have described the development of DNA vaccines for the induction of HPV16 E6 and E7 specific T cell immunity. These vaccines consist of 'gene-shuffled' (SH) versions of HPV16 E6 and E7 that were fused to Tetanus Toxin Fragment C domain 1 (TTFC) and were named TTFC-E6SH and TTFC-E7SH. Gene-shuffling was performed to avoid the risk of inducing malignant transformation at the vaccination site. Here, we describe the preclinical safety evaluation of these candidate vaccines by analysis of their transforming capacity in vitro using established murine fibroblasts (NIH 3T3 cells) and primary human foreskin keratinocytes (HFKs). We demonstrate that neither ectopic expression of TTFC-E6SH and TTFC-E7SH alone or in combination enabled NIH 3T3 cells to form colonies in soft agar. In contrast, expression of HPV16 E6WT and E7WT alone or in combination resulted in effective transformation. Similarly, retroviral transduction of HFKs from three independent donors with both TTFC-E6SH and TTFC-E7SH alone or in combination did not show any signs of immortalization. In contrast, the combined expression of E6WT and E7WT induced immortalization in HFKs from all donors. Based on these results we consider it justified to proceed to clinical evaluation of DNA vaccines encoding TTFC-E6SH and TTFC-E7SH in patients with HPV16 associated (pre)malignancies.
Assuntos
Transformação Celular Neoplásica , Proteínas Oncogênicas Virais/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Vacinas contra Papillomavirus/efeitos adversos , Proteínas Repressoras/metabolismo , Vacinas de DNA/efeitos adversos , Animais , Células Cultivadas , Embaralhamento de DNA , Expressão Gênica , Humanos , Camundongos , Proteínas Oncogênicas Virais/genética , Proteínas E7 de Papillomavirus/genética , Vacinas contra Papillomavirus/administração & dosagem , Vacinas contra Papillomavirus/genética , Proteínas Repressoras/genética , Toxoide Tetânico/efeitos adversos , Toxoide Tetânico/genética , Transdução Genética , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/efeitos adversos , Vacinas Sintéticas/genéticaAssuntos
Varizes/cirurgia , Adulto , Fatores Etários , Idoso , Feminino , Humanos , Masculino , Métodos , Pessoa de Meia-Idade , Fatores SexuaisRESUMO
Recently, DNA tattooing was introduced as novel intradermal administration technique for plasmid DNA (pDNA) vaccines. The aim of this study was to determine if tattooing affects the integrity of pDNA (reduction in supercoiled (SC) content) and whether a change in pDNA topology would affect antigen expression and immune response. We show that 1.) in vitro tattooing of pDNA solutions results in minor damage to pDNA (