Pyroptosis-inducing active caspase-1 as a genetic adjuvant in anti-cancer DNA vaccination.

Pyroptosis is a recently discovered form of inflammatory programmed necrosis characterized by caspase-1-mediated and gasdermin D-dependent cell death leading to the release of pro-inflammatory cytokines such as Interleukin-1 beta (IL-1ß). Here, we evaluated whether pyroptosis could be exploited in DNA vaccination by incorporating a constitutively active variant of caspase-1 to the antigen-expressing DNA. In vitro, transfection with constitutively active caspase-1 DNA induced pro-IL-1ß maturation and IL-1ß release as well as gasdermin D-dependent cell death. To test active caspase-1 as a genetic adjuvant for the induction of antigen-specific T cell responses, mice were vaccinated intradermally with a DNA vaccine consisting of the active caspase-1 plasmid together with a plasmid encoding an ovalbumin-derived CD8 T cell epitope. Active caspase-1 accelerated and amplified antigen-specific CD8 T cell responses when administered simultaneously with the DNA vaccine at an equimolar dose. Moreover, upon challenge with melanoma cells expressing ovalbumin, mice vaccinated with the antigen vaccine adjuvanted with active caspase-1 showed significantly better survival compared to the non-adjuvanted group. In conclusion, we have developed a novel genetic adjuvant that for the first time employs the pyroptosis pathway to improve DNA vaccination against cancer.

A poly-neoantigen DNA vaccine synergizes with PD-1 blockade to induce T cell-mediated tumor control.

The combination of immune-stimulating strategies has the potency to improve immunotherapy of cancer. Vaccination against neoepitopes derived from patient tumor material can generate tumor-specific T cell immunity, which could reinforce the efficacy of checkpoint inhibitor therapies such as anti-PD-1 treatment. DNA vaccination is a versatile platform that allows the inclusion of multiple neoantigen-coding sequences in a single formulation and therefore represents an ideal platform for neoantigen vaccination. We developed an anti-tumor vaccine based on a synthetic DNA vector designed to contain multiple cancer-specific epitopes in tandem. The DNA vector encoded a fusion gene consisting of three neoepitopes derived from the mouse colorectal tumor MC38 and their natural flanking sequences as 40 amino acid stretches. In addition, we incorporated as reporter epitopes the helper and CTL epitope sequences of ovalbumin. The poly-neoantigen DNA vaccine elicited T cell responses to all three neoantigens and induced functional CD8 and CD4 T cell responses to the reporter antigen ovalbumin after intradermal injection in mice. The DNA vaccine was effective in preventing outgrowth of B16 melanoma expressing ovalbumin in a prophylactic setting. Moreover, the combination of therapeutic DNA vaccination and anti-PD-1 treatment was synergistic in controlling MC38 tumor growth whereas individual treatments did not succeed. These data demonstrate the potential of DNA vaccination to target multiple neoepitopes in a single formulation and highlight the cooperation between vaccine-based and checkpoint blockade immunotherapies for the successful eradication of established tumors.

Adenovirus based HPV L2 vaccine induces broad cross-reactive humoral immune responses.

Oncogenic high-risk human papillomavirus (HPV) infections cause a substantial number of genital and non-genital cancers worldwide. Approximately 70% of all cervical cancers are caused by the high-risk HPV16 and 18 types. The remaining 30% can be attributed to twelve other high-risk HPV-types. Highly efficacious 2-valent, 4-valent and 9-valent L1 protein based prophylactic HPV vaccines are available however with limited cross-protection. To further increase the coverage, development of a multivalent cross-protective HPV vaccine is currently focused on the conserved N-terminus of HPV's L2 protein. We have developed a vaccine candidate based on the rare human adenovirus type 35 (HAdV35) vector that displays a concatemer of L2 protein epitopes from four different HPV-types via protein IX (pIX). A mix of two heterologous HAdV35 pIX-L2 display vectors present highly conserved linear epitopes of nine HPV-types. Each HAdV35 pIX-L2 display vector exhibits a good manufacturability profile. HAdV35 pIX-L2 display vaccine vectors were immunogenic and induced neutralizing antibodies against HPV-types included in the vaccine and cross-neutralizing antibodies against distant a HPV-type not included in the vaccine in mice. The HAdV35 pIX-L2 display vectors offer an opportunity for a multivalent HAdV-based prophylactic HPV vaccine.

Development of a replication-deficient adenoviral vector-based vaccine candidate for the interception of HPV16- and HPV18-induced infections and disease.

High-risk Human papilloma virus (HPV) types are the causative agents of cervical cancer and several other anogenital malignancies. The viral proteins expressed in the (pre)malignant cells are considered ideal targets for immunological intervention. Many approaches have been evaluated for this purpose, mostly aiming at the induction of HPV16 E7- and/or E6-specific cellular immunogenicity. As clinical success has so far been limited, novel approaches are required. We describe the development and pre-clinical testing of a vaccine candidate consisting of replication-deficient adenovirus type 26 and 35 based vectors for the interception of HPV16- and HPV18-related disease. We developed HPV16- and HPV18-specific antigens consisting of fusion proteins of E2, E6 and E7. The vaccine will be suitable for every disease stage, from incident and persistent infections where E2 is predominantly expressed up to late stages where E6 and E7 expression are upregulated. Importantly E6 and E7 are present as reordered fragments to abrogate the transforming activity of these two proteins. Loss of transforming activity was demonstrated in different in vitro models. Robust T-cell immunogenicity was induced upon immunization of mice with the vaccine candidate. Finally, the developed vaccine vectors showed considerable therapeutic efficacy in the TC-1 mouse model. The absence of transforming activity of the antigens and the favorable immunogenicity profile of the adenovirus based vectors along with the fact that these vectors can be readily produced on a large scale makes this approach attractive for clinical evaluation.

Intradermal vaccination by DNA tattooing.

DNA vaccination is an attractive vaccination method. First, the production of plasmid DNA as a vaccine is considerably more cheap and simple than the production of recombinant protein. Second, the expression cassette of DNA vaccines can readily be modified, making DNA vaccines highly flexible. Finally, in animal models, DNA vaccination is able to induce potent cellular immune responses. Over the past decade, the focus in the DNA vaccination field has in large part moved from intramuscular immunization towards dermal administration. As a natural "porte d'entrée" for pathogens, the skin is rich in antigen-presenting cells, which are required for generating an efficient antigen-specific immune response. This chapter describes a DNA vaccination protocol that utilizes a simple tattooing device for the dermal delivery of plasmid DNA. This technique, called DNA tattooing, is capable of generating high frequencies of antigen-reactive T cells in mice and macaques.

Rational design of DNA vaccines for the induction of human papillomavirus type 16 E6- and E7-specific cytotoxic T-cell responses.

Many DNA vaccine candidates have been developed for the treatment of human papillomavirus type 16 (HPV16)-induced malignancies. Most of these vaccines consist of a fusion of E7 with a "carrier-protein" that functions to increase the potency of the vaccine. The nature of these carrier-proteins varies widely, and the mechanisms proposed to explain the enhanced immunogenicity of such fusions are often linked to the biological function of the carrier-protein. However, the potentiating effect of these carrier-proteins might also be explained by more general mechanisms, such as the provision of CD4+ T-cell help, increased antigen stability, or altered subcellular localization of the antigen. To assess whether these more generic mechanisms could suffice to generate highly immunogenic DNA vaccines, we evaluated a series of modular HPV16 E7 DNA vaccines in which the presence of CD4+ T-cell help, the presence of an endogenous carrier-protein, and the subcellular localization of the antigen could be systematically altered. Using this approach, we demonstrate that the addition of an element that provides CD4+ T-cell help, elements that enforce endoplasmic reticulum (ER) localization/retention are both necessary and sufficient to create markedly effective HPV16 E7-directed DNA vaccines. Importantly, the resulting design rules also apply to an HPV16 E6-directed DNA vaccine. The developed "HELP(ER)" HPV DNA vaccines encode only very limited additional sequences besides the antigen, thereby reducing the risk of antigenic competition and/or autoimmunity.

Complications after plate fixation and elastic stable intramedullary nailing of dislocated midshaft clavicle fractures: a retrospective comparison.

PURPOSE: The incidence of operative treatment of dislocated midshaft clavicle fractures (DMCF) is rising due to unsatisfactory results after non-operative treatment. Knowledge of complications is important for selection of the surgical technique and preoperative patient counselling. The aim of this study is to compare complications after plate fixation and elastic stable intramedullary nailing (ESIN) with a titanium elastic nail (TEN) for DMCF. METHODS: A retrospective analysis of our surgical database was performed. From January 2005 to January 2010, 90 patients with DMCF were treated with plate fixation or ESIN. Complications were evaluated in both treatment groups and subsequently compared. RESULTS: Seven implant failures occurred in six patients (14 %) of the plate group and one implant failure (2.1 %) was seen in the ESIN group (p = 0.051). Major revision surgery was performed in five cases in the plate group (11.6 %) and in one case (2.1 %) in the ESIN group (p = 0.100). Three refractures (7.0 %) were observed in the plate group after removal of the implant against none in the ESIN group (p = 0.105). Six minor revisions (13 %) were reported in the ESIN group and none were reported in the plate group (p = 0.027). CONCLUSIONS: Compared to other studies we report higher rates of refracture (7.0 %), major revision surgery (11.6 %) and implant failure (14.0 %) after plate fixation. The frequency of implant failures differed almost significantly for patients treated with plate fixation compared to ESIN. Furthermore, a tendency towards refracture after implant removal and major revision surgery after plate fixation was observed.

Doença celíaca é super-representada em pacientes com constipação / Celiac disease is overrepresented in patients with constipation

OBJETIVO: Tem sido sugerido que pacientes com constipação sejam triados para doença celíaca. Da mesma forma, recomenda-se a investigação desses pacientes para hipotiroidismo e hipercalcemia. Contudo, nenhuma evidência para essas recomendações está disponível até o momento. Assim, propusemos-nos determinar a prevalência de doença celíaca, hipotiroidismo e hipercalcemia em crianças com constipação. MÉTODOS: Estudo de coorte prospectivo com 370 pacientes consecutivos que preencheram os critérios de Roma III para constipação. Esses pacientes foram encaminhados por um clínico geral a um pediatra devido ao fracasso no tratamento com laxantes. RESULTADOS: A biópsia comprovou doença celíaca em sete desses pacientes. Isso é significativamente mais alto (p < 0,001) do que a prevalência de 1:198 de doença celíaca nos Países Baixos. Dois pacientes tinham tiroidite autoimune. Nenhum paciente tinha hipercalcemia. CONCLUSÕES: Conclui-se que a doença celíaca é significativamente super-representada em pacientes com constipação encaminhados por um clínico geral a um pediatra devido ao fracasso no tratamento com laxantes. Todos esses pacientes devem, portanto, ser triados para doença celíaca.

OBJECTIVE: It is suggested that patients with constipation should be screened for celiac disease. Similarly, it is recommended to investigate these patients for hypothyroidism and hypercalcemia. However, no evidence for these recommendations is available so far. We therefore set out to determine the prevalence of celiac disease, hypothyroidism, and hypercalcemia in children with constipation. METHODS: Prospective cohort study of 370 consecutive patients who met the Rome III criteria for constipation. These patients were referred by a general practitioner to a pediatrician because of failure of laxative treatment. RESULTS: Seven of these patients had biopsy-proven celiac disease. This is significantly higher (p < 0.001) than the 1:198 prevalence of celiac disease in the Netherlands. Two patients had auto-immune thyroiditis. No patient had hypercalcemia. CONCLUSIONS: We conclude that celiac disease is significantly overrepresented in patients with constipation who are referred by a general practitioner to a pediatrician because of failure of laxative treatment. All such patients should, therefore, be screened for celiac disease.

Celiac disease is overrepresented in patients with constipation.

OBJECTIVE: It is suggested that patients with constipation should be screened for celiac disease. Similarly, it is recommended to investigate these patients for hypothyroidism and hypercalcemia. However, no evidence for these recommendations is available so far. We therefore set out to determine the prevalence of celiac disease, hypothyroidism, and hypercalcemia in children with constipation. METHODS: Prospective cohort study of 370 consecutive patients who met the Rome III criteria for constipation. These patients were referred by a general practitioner to a pediatrician because of failure of laxative treatment. RESULTS: Seven of these patients had biopsy-proven celiac disease. This is significantly higher (p < 0.001) than the 1:198 prevalence of celiac disease in the Netherlands. Two patients had auto-immune thyroiditis. No patient had hypercalcemia. CONCLUSIONS: We conclude that celiac disease is significantly overrepresented in patients with constipation who are referred by a general practitioner to a pediatrician because of failure of laxative treatment. All such patients should, therefore, be screened for celiac disease.

Preclinical development of highly effective and safe DNA vaccines directed against HPV 16 E6 and E7.

To allow vaccination irrespective of HLA type, DNA vaccines encoding full-length antigens are required. However, here, we demonstrate that the immunogenicity of DNA vaccines encoding the full-length human papillomavirus (HPV) type 16 E7 and E6 proteins is highly reduced compared to vaccines encoding only the immunodominant epitope. Furthermore, the low remaining immunogenicity is essentially lost for both E7 and E6 when a nononcogenic "gene-shuffled" variant is utilized. To address these issues, we tested whether alterations in transgene design can restore the immunogenicity of full-length and gene-shuffled DNA vaccines. Remarkably, genetic fusion of E7 with tetanus toxin fragment C (TTFC) resulted in a dramatic increase in immunogenicity both for the full-length and the gene-shuffled version of E7. Moreover, the TTFC fusion vaccines were more immunogenic than a vaccine encoding a fusion of E7 and mycobacterial heat shock protein-70, which has recently been tested in a clinical trial. Interestingly, vaccination with these TTFC fusion vaccines also resulted in extremely persistent T-cell responses. The E7-specific CD8(+) T cells induced by TTFC fusion vaccines were functional in terms of IFN-γ production, formation of immunological memory, in vivo cytolytic activity and tumor eradication. Finally, we show that genetic fusion with TTFC also improves the immunogenicity of a gene-shuffled E6 DNA vaccine. These data demonstrate that genetic fusion with tetanus toxin fragment C can dramatically improve the immunogenicity of full-length and gene-shuffled DNA vaccines. The DNA fusion vaccines developed here will be evaluated for the treatment of HPV-positive carcinomas in future studies.

DNA vaccination in oncology: current status, opportunities and perspectives.

After almost 20 years of research, DNA vaccination is still a relatively young technique in the vaccine-toolbox. DNA vaccines can easily be modified by conventional cloning techniques, are relatively easy to produce and might be particularly useful for therapeutic vaccination against intracellular pathogens and cancer. After the early pre-clinical successes, DNA vaccination moved into the clinic and numerous trials have been performed thus far. In the oncology field, these trials aimed for the induction of cellular immunity directed against tumor specific antigens. Although DNA vaccines proved to be well tolerated, and elicited some immune activation in patients, robust immune activation followed by clinical responses has not been observed yet. Nevertheless, several promising strategies are currently under development to increase the performance of the current generation DNA vaccines. Future research has to demonstrate whether these strategies are able to give DNA vaccination a defined position in cancer treatment.

Shielding the cationic charge of nanoparticle-formulated dermal DNA vaccines is essential for antigen expression and immunogenicity.

Nanoparticle-formulated DNA vaccines hold promise for the design of in vivo vaccination platforms that target defined cell types in human skin. A variety of DNA formulations, mainly based on cationic liposomes or polymers, has been investigated to improve transfection efficiency in in vitro assays. Here we demonstrate that formulation of DNA into both liposomal and polymeric cationic nanoparticles completely blocks vaccination-induced antigen expression in mice and ex vivo human skin. Furthermore, this detrimental effect of cationic nanoparticle formulation is associated with an essentially complete block in vaccine immunogenicity. The blocking of DNA vaccine activity may be explained by immobilization of the nanoparticles in the extracellular matrix, caused by electrostatic interactions of the cationic nanoparticles with negatively charged extracellular matrix components. Shielding the surface charge of the nanoparticles by PEGylation improves in vivo antigen expression more than 55 fold. Furthermore, this shielding of cationic surface charge results in antigen-specific T cell responses that are similar as those induced by naked DNA for the two lipo- and polyplex DNA carrier systems. These observations suggest that charge shielding forms a generally applicable strategy for the development of dermally applied vaccine formulations. Furthermore, the nanoparticle formulations developed here form an attractive platform for the design of targeted nanoparticle formulations that can be utilized for in vivo transfection of defined cell types.

Functional inhibition of NF-kappaB signal transduction in alphavbeta3 integrin expressing endothelial cells by using RGD-PEG-modified adenovirus with a mutant IkappaB gene.

In order to selectively block nuclear factor kappaB (NF-kappaB)-dependent signal transduction in angiogenic endothelial cells, we constructed an alphavbeta3 integrin specific adenovirus encoding dominant negative IkappaB (dnIkappaB) as a therapeutic gene. By virtue of RGD modification of the PEGylated virus, the specificity of the cell entry pathway of adenovirus shifted from coxsacki-adenovirus receptor dependent to alphavbeta3 integrin dependent entry. The therapeutic outcome of delivery of the transgene into endothelial cells was determined by analysis of cellular responsiveness to tumor necrosis factor (TNF)-alpha. Using real time reverse transcription PCR, mRNA levels of the cell adhesion molecules E-selectin, vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1, the cytokines/growth factors IL-6, IL-8 and vascular endothelial growth factor (VEGF)-A, and the receptor tyrosine kinase Tie-2 were assessed. Furthermore, levels of ICAM-1 protein were determined by flow cytometric analysis. RGD-targeted adenovirus delivered the dnIkappaB via alphavbeta3 to become functionally expressed, leading to complete abolishment of TNF-alpha-induced up-regulation of E-selectin, ICAM-1, VCAM-1, IL-6, IL-8, VEGF-A and Tie-2. The approach of targeted delivery of dnIkappaB into endothelial cells presented here can be employed for diseases such as rheumatoid arthritis and inflammatory bowel disease where activation of NF-kappaB activity should be locally restored to basal levels in the endothelium.

Differential effects of NF-{kappa}B and p38 MAPK inhibitors and combinations thereof on TNF-{alpha}- and IL-1{beta}-induced proinflammatory status of endothelial cells in vitro.

Endothelial cells actively participate in inflammatory events by regulating leukocyte recruitment via the expression of inflammatory genes such as E-selectin, VCAM-1, ICAM-1, IL-6, IL-8, and cyclooxygenase (COX)-2. In this study we showed by real-time RT-PCR that activation of human umbilical vein endothelial cells (HUVEC) by TNF-alpha and IL-1beta differentially affected the expression of these inflammatory genes. Combined treatment with TNF-alpha and IL-1beta resulted in nonadditive, additive, and even synergistic induction of expression of VCAM-1, IL-8, and IL-6, respectively. Overexpression of dominant-negative inhibitor kappaB protein blocking NF-kappaB signaling confirmed a major role of this pathway in controlling both TNF-alpha- and IL-1beta-induced expression of most of the genes studied. Although dexamethasone exerted limited effects at 1 muM, the thioredoxin inhibitor MOL-294, which regulates the redox state of NF-kappaB, mainly inhibited adhesion molecule expression. Its most pronounced effect was seen on VCAM-1 mRNA levels, especially in IL-1beta-activated endothelium. One micromolar RWJ-67657, an inhibitor of p38 MAPK activity, diminished TNF-alpha- and IL-1beta-induced expression of IL-6, IL-8, and E-selectin but had little effect on VCAM-1 and ICAM-1. Combined treatment of HUVEC with MOL-294 and RWJ-67657 resulted in significant blocking of the expression of E-selectin, IL-6, IL-8, and COX-2. The inhibitory effects were much stronger than those observed with single drug treatment. Application of combinations of drugs that affect multiple targets in activated endothelial cells may therefore be considered as a potential new therapeutic strategy to inhibit inflammatory disease activity.