Induction of gp120-specific protective immune responses by genetic vaccination with linear polyethylenimine-plasmid complex

The induction of IFN-gamma-secreting CD8+ T cells and neutralizing antibodies to HIV-1 are both key requirements for prevention of viral transmission and clearance of pathogenic HIV. Although DNA vaccination has been shown to induce both humoral and cellular immune responses against HIV antigens, the magnitude of the immune responses has always been disappointing. In this report, we analyze the ability of polyethylenimine (PEI)-DNA complex expressing an HIV-glycoprotein 120 (gp120) antigen (PEI-pgp120) to induce systemic CD8+ T cell and humoral responses to the gp120 antigen. The administration of PEI-plasmid complex resulted in rapid elevation of serum levels of IL-12 and IFN-gamma. Furthermore, a single administration of PEI-pgp120 complex elicits a number of gp120-specific CD8+ T cells 20 times higher than that elicited by three intramuscular injections of naked DNA. Interestingly, we found that systemic vaccination with PEI-pgp120 induced protective immune responses against both systemic and mucosal challenges with a recombinant vaccinia virus expressing a gp120 antigen. The data also demonstrated that the depletion of macrophages with liposome-encapsulated clodronate completely abolished gp120-specific cellular response. Overall, our results showed that a single administration of PEI-pgp120 complexes, eliciting strong immune responses, is an effective vaccination approach to generate protection against systemic and mucosal viral infections

Adeno-associated viral vectors serotype 8 for cell-specific delivery of therapeutic genes in the central nervous system

Adeno-associated viruses (AAVs) have become highly promising tools for research and clinical applications in the central nervous system (CNS). However, specific delivery of genes to the cell type of interest is essential for the success of gene therapy and therefore a correct selection of the promoter plays a very important role. Here, AAV8 vectors carrying enhanced green fluorescent protein (eGFP) as reporter gene under the transcriptional control of different CNS-specific promoters were used and compared with a strong ubiquitous promoter. Since one of the main limitations of AAV-mediated gene delivery lies in its restricted cloning capacity, we focused our work on small-sized promoters. We tested the transduction efficacy and specificity of each vector after stereotactic injection into the mouse striatum. Three glia-specific AAV vectors were generated using two truncated forms of the human promoter for glial fibrillar acidic protein (GFAP) as well as a truncated form of the murine GFAP promoter. All three vectors resulted in predominantly glial expression; however we also observed eGFP expression in other cell-types such as oligodendrocytes, but never in neurons. In addition, robust and neuron-specific eGFP expression was observed using the minimal promoters for the neural protein BM88 and the neuronal nicotinic receptor β2 (CHRNB2). In summary, we developed a set of AAV vectors designed for specific expression in cells of the CNS using minimal promoters to drive gene expression when the size of the therapeutic gene matters

CRISPR/Cas9-mediated glycolate oxidase disruption is an efficacious and safe treatment for primary hyperoxaluria type I

CRISPR/Cas9 technology offers novel approaches for the development of new therapies for many unmet clinical needs, including a significant number of inherited monogenic diseases. However, in vivo correction of disease-causing genes is still inefficient, especially for those diseases without selective advantage for corrected cells. We reasoned that substrate reduction therapies (SRT) targeting non-essential enzymes could provide an attractive alternative. Here we evaluate the therapeutic efficacy of an in vivo CRISPR/Cas9-mediated SRT to treat primary hyperoxaluria type I (PH1), a rare inborn dysfunction in glyoxylate metabolism that results in excessive hepatic oxalate production causing end-stage renal disease. A single systemic administration of an AAV8-CRISPR/Cas9 vector targeting glycolate oxidase, prevents oxalate overproduction and kidney damage, with no signs of toxicity in Agxt1(-/-) mice. Our results reveal that CRISPR/Cas9-mediated SRT represents a promising therapeutic option for PH1 that can be potentially applied to other metabolic diseases caused by the accumulation of toxic metabolites

Danio rerio as model organism for adenoviral vector evaluation

Viral vector use is wide-spread in the field of gene therapy, with new clinical trials starting every year for different human pathologies and a growing number of agents being approved by regulatory agencies. However, preclinical testing is long and expensive, especially during the early stages of development. Nowadays, the model organism par excellence is the mouse (Mus musculus), and there are few investigations in which alternative models are used. Here, we assess the possibility of using zebrafish (Danio rerio) as an in vivo model for adenoviral vectors. We describe how E1/E3-deleted adenoviral vectors achieve efficient transduction when they are administered to zebrafish embryos via intracranial injection. In addition, helper-dependent (high-capacity) adenoviral vectors allow sustained transgene expression in this organism. Taking into account the wide repertoire of genetically modified zebrafish lines, the ethical aspects, and the affordability of this model, we conclude that zebrafish could be an efficient alternative for the early-stage preclinical evaluation of adenoviral vectors

Production of recombinant woodchuck IFNalpha and development of monoclonal antibodies

Interferon alpha (IFNalpha) is the first line treatment for chronic hepatitis B and C. In order to test new IFNalpha delivery systems and investigate the function of this cytokine in the woodchuck model, the best animal model of chronic hepatitis B, we produced and purified recombinant woodchuck IFNalpha and used it to produce monoclonal antibodies. wIFNalpha5 was cloned in a prokaryotic expression system, expressed as His-tagged protein and then purified. The rwIFNalpha5 protein was found to induce STAT-3 phosphorylation, to enhance 2',5'-oligoadenylate synthetase mRNA levels and to possess a potent antiviral activity. Two monoclonal antibodies were obtained through immunization of rats with rwIFNalpha5. Both recognized rwIFNalpha5 in western blot analysis and one was able to neutralize the antiviral activity of the rwIFNalpha5 and lymphoblastoid IFNalpha preparations. Finally, a capture rwIFNalpha5 ELISA was developed using both antibodies. In summary, the tools generated in this study will allow different forms of IFNalpha delivery as well as different combination therapies in woodchuck hepatitis virus infection to be tested, thus providing useful information for the design of new strategies to treat chronic hepatitis B in humans

Production of recombinant woodchuck IFNalpha and development of monoclonal antibodies

Interferon alpha (IFNalpha) is the first line treatment for chronic hepatitis B and C. In order to test new IFNalpha delivery systems and investigate the function of this cytokine in the woodchuck model, the best animal model of chronic hepatitis B, we produced and purified recombinant woodchuck IFNalpha and used it to produce monoclonal antibodies. wIFNalpha5 was cloned in a prokaryotic expression system, expressed as His-tagged protein and then purified. The rwIFNalpha5 protein was found to induce STAT-3 phosphorylation, to enhance 2',5'-oligoadenylate synthetase mRNA levels and to possess a potent antiviral activity. Two monoclonal antibodies were obtained through immunization of rats with rwIFNalpha5. Both recognized rwIFNalpha5 in western blot analysis and one was able to neutralize the antiviral activity of the rwIFNalpha5 and lymphoblastoid IFNalpha preparations. Finally, a capture rwIFNalpha5 ELISA was developed using both antibodies. In summary, the tools generated in this study will allow different forms of IFNalpha delivery as well as different combination therapies in woodchuck hepatitis virus infection to be tested, thus providing useful information for the design of new strategies to treat chronic hepatitis B in humans

Danio rerio as model organism for adenoviral vector evaluation

Viral vector use is wide-spread in the field of gene therapy, with new clinical trials starting every year for different human pathologies and a growing number of agents being approved by regulatory agencies. However, preclinical testing is long and expensive, especially during the early stages of development. Nowadays, the model organism par excellence is the mouse (Mus musculus), and there are few investigations in which alternative models are used. Here, we assess the possibility of using zebrafish (Danio rerio) as an in vivo model for adenoviral vectors. We describe how E1/E3-deleted adenoviral vectors achieve efficient transduction when they are administered to zebrafish embryos via intracranial injection. In addition, helper-dependent (high-capacity) adenoviral vectors allow sustained transgene expression in this organism. Taking into account the wide repertoire of genetically modified zebrafish lines, the ethical aspects, and the affordability of this model, we conclude that zebrafish could be an efficient alternative for the early-stage preclinical evaluation of adenoviral vectors

IL12-mediated liver inflammation reduces the formation of AAV transcriptionally active forms but has no effect over preexisting AAV transgene expression

Recombinant adenoassociated viral vectors (rAAV) have proven to be excellent candidates for gene therapy clinical applications. Recent results showed that cellular immunity to AAV represents a major challenge facing the clinical use of systemic administration of these vectors. Interestingly, no preclinical animal model has previously fully reproduced the clinical findings. The aim of the present work was to enhance the T cell immune response against AAV capsid in mice by the administration of a rAAV expressing the immunostimulatory cytokine IL-12. Our results indicate that although IL-12 expression enhanced the AAV capsid-specific immune response it failed to eliminate transduced hepatocytes and long-term expression was achieved. We found that AAV-mediated transgene expression is altered by IL-12-induced liver inflammation. However, IL-12 expression has no effect over preexisting AAV-mediated transgene expression. IL-12 down-regulates AAV mediated transgene expression via induction of IFN-γ production by NK and T cells, but without altering the transduction efficiency measured by viral genomes. Our results indicate that liver inflammation affects the formation of transcriptionally active AAV vector genomes through an unknown mechanism that can be avoided by the use of DNA-demethylating or anti-inflammatory agents

Identification of CD4+ and CD8+ T cell epitopes of woodchuck hepatitis virus core and surface antigens in BALB/c mice

A therapeutic vaccine against chronic hepatitis B virus (HBV) infection requires the development of a strong and multispecific Th1 cell immune response. Woodchucks chronically infected with the woodchuck hepatitis virus (WHV) closely resemble HBV infection and represent the best animal model for this hepadnavirus-induced disease. Using the BIMAS "HLA Peptide Binding Predictions" program, we have identified and further characterized novel H-2(d)-restricted CD8+ epitopes within the WHV core (peptides C#12-21, C#18-32, C#19-27, C#61-69) and surface antigens (peptides preS2#10-18, preS2#27-35, S#76-84, S#133-140 and S#257-265), respectively. These peptides bind to H-2(d) with high efficiency and upon immunization of mice with peptide and Freund's adjuvant they induce the development of IFN-gamma producing T cells. More importantly, WHV core peptides C#19-27 and C#61-69 and WHV surface peptides S#133-140 and S#257-265 were also recognized by CD8+ T cells after immunization of mice with DNA/PEI nanoparticles. Direct stimulation of splenocytes obtained from such DNA-immunized mice with peptides C#18-32, S#76-84, and S#257-265 resulted in significant production of IFN-gamma. Thus, we have identified T cell determinants in mice from WHV core and surface antigens that have important value for designing and evaluating an effective vaccine against hepadnavirus infection

Identification of CD4+ and CD8+ T cell epitopes of woodchuck hepatitis virus core and surface antigens in BALB/c mice

A therapeutic vaccine against chronic hepatitis B virus (HBV) infection requires the development of a strong and multispecific Th1 cell immune response. Woodchucks chronically infected with the woodchuck hepatitis virus (WHV) closely resemble HBV infection and represent the best animal model for this hepadnavirus-induced disease. Using the BIMAS "HLA Peptide Binding Predictions" program, we have identified and further characterized novel H-2(d)-restricted CD8+ epitopes within the WHV core (peptides C#12-21, C#18-32, C#19-27, C#61-69) and surface antigens (peptides preS2#10-18, preS2#27-35, S#76-84, S#133-140 and S#257-265), respectively. These peptides bind to H-2(d) with high efficiency and upon immunization of mice with peptide and Freund's adjuvant they induce the development of IFN-gamma producing T cells. More importantly, WHV core peptides C#19-27 and C#61-69 and WHV surface peptides S#133-140 and S#257-265 were also recognized by CD8+ T cells after immunization of mice with DNA/PEI nanoparticles. Direct stimulation of splenocytes obtained from such DNA-immunized mice with peptides C#18-32, S#76-84, and S#257-265 resulted in significant production of IFN-gamma. Thus, we have identified T cell determinants in mice from WHV core and surface antigens that have important value for designing and evaluating an effective vaccine against hepadnavirus infection