Expression of a soluble IL-10 receptor enhances the therapeutic effects of a papillomavirus-associated antitumor vaccine in a murine model.

The presence of IL-10, produced either by tumor cells or immunosuppressive cells, is frequently associated with a poor prognosis for cancer progression. It may also negatively impact anticancer treatments, such as immunotherapies, that otherwise would promote the activation of cytotoxic T cells capable of detecting and destroying malignant cells. In the present study, we evaluated a new adjuvant approach for anticancer immunotherapy using a plasmid vector encoding a soluble form of the IL-10 receptor (pIL-10R). pIL-10R was coadministered to mice with a DNA vaccine encoding the type 16 human papillomavirus (HPV-16) E7 oncoprotein genetically fused with glycoprotein D of herpes simplex virus (HSV) (pgDE7h). Immunization regimens based on the coadministration of pIL-10R and pgDE7h enhanced the antitumor immunity elicited in mice injected with TC-1 cells, which express HPV-16 oncoproteins. The administration of the DNA vaccines by in vivo electroporation further enhanced the anticancer effects of the vaccines, leading to the activation of tumor-infiltrating polyfunctional E7-specific cytotoxic CD8+ T cells and control of the expansion of immunosuppressive cells. In addition, the combination of immunotherapy and pIL-10R allowed the control of tumors in more advanced growth stages that otherwise would not be treatable by the pgDE7h vaccine. In conclusion, the proposed treatment involving the expression of IL-10R enhanced the antitumor protective immunity induced by pgDE7h administration and may contribute to the development of more efficient clinical interventions against HPV-induced tumors.

Branched amphiphilic cationic oligopeptides form peptiplexes with DNA: a study of their biophysical properties and transfection efficiency.

Over the past decade, peptides have emerged as a new family of potential carriers in gene therapy. Peptides are easy to synthesize and quite stable. Additionally, sequences shared by the host proteome are not expected to be immunogenic or trigger inflammatory responses, which are commonly observed with viral approaches. We recently reported on a new class of branched amphiphilic peptide capsules (BAPCs) that self-assemble into extremely stable nanospheres. These capsules are capable of retaining and delivering alpha-emitting radionuclides to cells. Here we report that, in the presence of double stranded plasmid DNA, BAPCs are unable to form. Instead, depending of the peptide/DNA ratios, the peptides either coat the plasmid surface forming nanofibers (high peptide to DNA ratio) or condense the plasmid into nanometer-sized compacted structures (at low peptide to DNA ratios). Different gene delivery efficiencies are observed for the two types of assemblies. The compacted nanometer-sized structures display much higher transfection efficiencies in HeLa cells. This level of transfection is greater than that observed for a lipid-based reagent when the total number of viable transfected cells is taken into account.

Antigen Delivery to DEC205+ Dendritic Cells Induces Immunological Memory and Protective Therapeutic Effects against HPV-Associated Tumors at Different Anatomical Sites.

The generation of successful anticancer vaccines relies on the ability to induce efficient and long-lasting immune responses to tumor antigens. In this scenario, dendritic cells (DCs) are essential cellular components in the generation of antitumor immune responses. Thus, delivery of tumor antigens to specific DC populations represents a promising approach to enhance the efficiency of antitumor immunotherapies. In the present study, we employed antibody-antigen conjugates targeting a specific DC C-type lectin receptor. For that purpose, we genetically fused the anti-DEC205 monoclonal antibody to the type 16 human papillomavirus (HPV-16) E7 oncoprotein to create a therapeutic vaccine to treat HPV-associated tumors in syngeneic mouse tumor models. The therapeutic efficacy of the αDEC205-E7 mAb was investigated in three distinct anatomical tumor models (subcutaneous, lingual and intravaginal). The immunization regimen comprised two doses of the αDEC205-E7 mAb coadministered with a DC maturation stimulus (Polyinosinic:polycytidylic acid, poly (I:C)) as an adjuvant. The combined immunotherapy produced robust antitumor effects on both the subcutaneous and orthotopic tumor models, stimulating rapid tumor regression and long-term survival. These outcomes were related to the activation of tumor antigen-specific CD8+ T cells in both systemic compartments and lymphoid tissues. The αDEC205-E7 antibody plus poly (I:C) administration induced long-lasting immunity and controlled tumor relapses. Our results highlight that the delivery of HPV tumor antigens to DCs, particularly via the DEC205 surface receptor, is a promising therapeutic approach, providing new opportunities for the development of alternative immunotherapies for patients with HPV-associated tumors at different anatomical sites.

A therapeutic DNA vaccine and gemcitabine act synergistically to eradicate HPV-associated tumors in a preclinical model.

Although active immunotherapies are effective strategies to induce activation of CD8+ T cells, advanced stage tumors require further improvements for efficient control. Concerning the burden of cancer-related to Human papillomavirus (HPV), particularly the high incidence and mortality of cervical cancer, our group developed an approach based on a DNA vaccine targeting the HPV-16 E7 oncoprotein (pgDE7h). This immunotherapy is capable of inducing an antitumour CD8+ T cell response but show only partial control of tumors in more advanced growth stages. Here, we combined a chemotherapeutic agent (gemcitabine- Gem) with pgDE7h to overcome immunosuppression and improve antitumour responses in a preclinical mouse tumor model. Our results demonstrated that administration of Gem had synergistic antitumor effects when combined with pgDE7h leading to eradication of both early-stages and established tumors. Overall, the antiproliferative effects of Gem observed in vitro and in vivo provided an optimal window for immunotherapy. In addition, the enhanced antitumour responses induced by the combined therapeutic regimen included enhanced frequencies of antigen-presenting cells (APCs), E7-specific IFN-γ-producing CD8+ T cells, and cytotoxic CD8+ T cells and, concomitantly, less pronounced accumulation of immunosuppressive myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs). These findings demonstrated that the combination of Gem and an active immunotherapy strategy show increased effectiveness, leading to a reduced need for multiple drug doses and, therefore, decreased deleterious side effects avoiding resistance and tumor relapses. Altogether, our results provide evidence for a new and feasible chemoimmunotherapeutic strategy that supports future clinical translation.

In vivo electroporation enhances vaccine-mediated therapeutic control of human papilloma virus-associated tumors by the activation of multifunctional and effector memory CD8+ T cells.

In vivo electroporation (EP) has reignited the clinical interest on DNA vaccines as immunotherapeutic approaches to control different types of cancer. EP has been associated with increased immune response potency, but its capacity in influencing immunomodulation remains unclear. Here we evaluated the impact of in vivo EP on the induction of cellular immune responses and therapeutic effects of a DNA vaccine targeting human papillomavirus-induced tumors. Our results demonstrate that association of EP with the conventional intramuscular administration route promoted a more efficient activation of multifunctional and effector memory CD8+ T cells with enhanced cytotoxic activity. Furthermore, EP increased tumor infiltration of CD8+ T cells and avoided tumor recurrences. Finally, our results demonstrated that EP promotes local migration of antigen presenting cells that enhances with vaccine co-delivery. Altogether the present evidences shed further light on the in vivo electroporation action and its impact on the immunogenicity of DNA vaccines.

Bacterial spores as particulate carriers for gene gun delivery of plasmid DNA.

Bacillus subtilis spores represent a suitable platform for the adsorption of proteins, enzymes and viral particles at physiological conditions. In the present work, we demonstrate that purified spores can also adsorb DNA on their surface after treatment with cationic molecules. In addition, we demonstrate that DNA-coated B. subtilis spores can be used as particulate carriers and act as an alternative to gold microparticles for the biolistic (gene gun) administration of plasmid DNA in mice. Gene gun delivery of spores pre-treated with DODAB (dioctadecyldimethylammonium bromide) allowed efficient plasmid DNA absorption and induced protein expression levels similar to those obtained with gold microparticles. More importantly, we demonstrated that a DNA vaccine adsorbed on spores can be loaded into biolistic cartridges and efficiently delivered into mice, which induced specific cellular and antibody responses. Altogether, these data indicate that B. subtilis spores represent a simple and low cost alternative for the in vivo delivery of DNA vaccines by the gene gun technology.

Bacillus subtilis spores as adjuvants for DNA vaccines.

Recently, Bacillus subtilis spores were shown to be endowed with strong adjuvant capacity when co-administered with purified antigenic proteins. In the present study we assessed whether spores possess adjuvant properties when combined with DNA vaccines. We showed that B. subtilis spores promoted the activation of dendritic cells in vitro and induced migration of pro-inflammatory cells after parenteral administration to mice. Likewise, co-administration of spores with a DNA vaccine encoding the human papillomavirus type 16 (HPV-16) E7 protein enhanced the activation of antigen-specific CD8(+) T cell responses in vivo. Mice immunized with the DNA vaccine admixed with spores presented a protective immunity increase to previously implanted tumor cells, capable of expressing HPV-16 oncoproteins. Finally, we observed that the adjuvant effect can vary accordingly to the number of co-administered spores which may be ascribed with the ability to induce. Collectively, the present results demonstrate for the first time that B. subtilis spores can also confer adjuvant effects to DNA vaccines.

Enhanced therapeutic effects conferred by an experimental DNA vaccine targeting human papillomavirus-induced tumors.

Human papillomavirus (HPV) infection is responsible for all cervical cancer cases, other anogenital cancers, and head and neck tumors. The epidemiological relevance of HPV-induced tumors reinforces the need for the development of therapeutic antitumor vaccines. Clinical trials with different vaccine formulations, particularly DNA vaccines, have provided promising results but have still been unable to achieve the immunogenicity required for use in infected patients. In experimental conditions, anticancer HPV-specific vaccines induced E7-specific CD8(+) T-cell responses but did not confer full therapeutic antitumor protection in mice with transplanted HPV-expressing TC-1 cells, which are the most frequently used nonclinical protection correlate for antitumor effects. Our group has developed a DNA vaccine strategy based on the fusion of HPV oncoproteins to the herpes virus gD protein. This vaccine promoted the induction of antigen-specific cytotoxic CD8(+) T-cell responses and partial antitumor therapeutic effects based on the blockade of coinhibitory signals and the enhancement of coactivation mechanisms. In the present study, we report conditions leading to full therapeutic antitumor effects using the TC-1 cell murine model after a single vaccine dose. The combination of a coadministered plasmid encoding IL-2, optimization of the coding sequence for mammalian cells, and the use of different delivery routes resulted in enhancements of the E7-specific cytotoxic CD8(+) T-cell responses and full therapeutic protection under experimental conditions. The combination of these strategies augmented the potency of the DNA vaccine formulation to levels not previously achieved by other therapeutic antitumor vaccines under similar experimental conditions, including some that have been taken to clinical trials.