Genetic Vaccines and Therapy - Latest Articles

Combined vascular endothelial growth factor-A and fibroblast growth factor 4 gene transfer improves wound healing in diabetic mice

Agnieszka Jazwa — 2010-08-30T00:00:00Z

Background: Impaired wound healing in diabetes is related to decreased production of growth factors. Hence, gene therapy is considered as promising treatment modality. So far, efforts concentrated on single gene therapy with particular emphasis on vascular endothelial growth factor-A (VEGF-A). However, as multiple proteins are involved in this process it is rational to test new approaches. Therefore, the aim of this study was to investigate whether single AAV vector-mediated transfer of VEGF-A and fibroblast growth factor 4 (FGF4) coding sequences will improve the wound healing over the effect of VEGF-A in diabetic (db/db) mice. Methods: Leptin receptor-deficient db/db mice were randomized to receive intradermal injections of PBS or AAVs carrying beta-galactosidase gene (AAV-LacZ), VEGF-A (AAV-VEGF-A), FGF-4 (AAV-FGF4-IRES-GFP) or both therapeutic genes (AAV-FGF4-IRES-VEGF-A). Wound healing kinetics was analyzed until day 21 when all animals were sacrificed for biochemical and histological examination. Results: Complete wound closure in animals treated with AAV-VEGF-A was achieved earlier (day 19) than in control mice or animals injected with AAV harboring FGF4 (both on day 21). However, the fastest healing was observed in mice injected with bicistronic AAV-FGF4-IRES-VEGF-A vector (day 17). This was paralleled by significantly increased granulation tissue formation, vascularity and dermal matrix deposition. Mechanistically, as shown in vitro, FGF4 stimulated matrix metalloproteinase-9 (MMP-9) and VEGF receptor-1 expression in mouse dermal fibroblasts and when delivered in combination with VEGF-A, enhanced their migration. Conclusion: Combined gene transfer of VEGF-A and FGF4 can improve reparative processes in the wounded skin of diabetic mice better than single agent treatment.

Liposomal delivery of p-ialB and p-omp25 DNA vaccines improves immunogenicity but fails to provide full protection against B. melitensis challenge

Nicola Commander — 2010-07-16T00:00:00Z

Background: We have previously demonstrated protective efficacy against B. melitensis using formulations of naked DNA vaccines encoding genes ialB and omp25. The present study was undertaken to further understand the immune response generated by the protective vaccination regimens and to evaluate cationic liposome adsorption as a delivery method to improve vaccine utility. Methods: The protective efficacy and immunogenicity of vaccines delivered as four doses of naked DNA, a single dose of naked DNA or a single dose of DNA surface adsorbed to cationic liposomes were compared using the BALB/c murine infection model of B. melitensis. Antigen-specific T cells and antibody responses were compared between the various formulations. Results: The four dose vaccination strategy was confirmed to be protective against B. melitensis challenge. The immune response elicited by the various vaccines was found to be dependent upon both the antigen and the delivery strategy, with the IalB antigen favouring CD4+ T cell priming and Omp25 antigen favouring CD8+. Delivery of the p-ialB construct as a lipoplex improved antibody generation in comparison to the equivalent quantity of naked DNA. Delivery of p-omp25 as a lipoplex altered the profile of responsive T cells from CD8+ to CD4+ dominated. Under these conditions neither candidate delivered by single dose naked DNA or lipoplex vaccination methods was able to produce a robust protective effect. Conclusions: Delivery of the p-omp25 and p-ialB DNA vaccine candidates as a lipoplex was able to enhance antibody production and effect CD4+ T cell priming, but was insufficient to promote protection from a single dose of either vaccine. The enhancement of immunogenicity by lipoplex delivery is a promising step toward improving the practicality of these two candidate vaccines, and suggests that this lipoplex formulation may be of value in situations where improvements to CD4+ responses are required. However, in the case of Brucella vaccine development it is suggested that further modifications to the candidate vaccines and delivery strategies will be required in order to deliver sustained protection.

Development of avian influenza virus H5 DNA vaccine and MDP-1 gene of Mycobacterium bovis as genetic adjuvant

Babak Jalilian — 2010-05-24T00:00:00Z

Background: Studies have shown that DNA vaccines can induce protective immunity, which demonstrated the high potential of DNA vaccines as an alternative to inactivated vaccines. Vaccines are frequently formulated with adjuvants to improve their release, delivery and presentation to the host immune system. Methods: The H5 gene of H5N1 virus (A/Ck/Malaysia/5858/04) was cloned separately into pcDNA3.1 + vector. The immunogenicity of the cloned H5 DNA vaccine was tested on SPF chickens using two different approaches. First approach was using H5 DNA vaccine (pcDNA3.1/H5) and the second was using H5 DNA vaccine in addition to the pcDNA3.1/MDP1 vaccine. Ten days old chickens inoculated three times with two weeks intervals. The spleen and muscle samples from chickens immunized with H5 (pcDNA3.1/H5) and H5 + MDP1 (pcDNA3.1/H5 + pcDNA3.1/MDP1) vaccines were collected after sacrificing the chickens and successfully expressed H5 and MDP1 RNA transcripts. The sera of immunized chickens were collected prior to first immunization and every week after immunization; and analyzed using enzyme-linked immunosorbent assay (ELISA) and hemagglutination inhibition (HI) test. Results: Results of competitive ELISA showed successful antibody responses two weeks post immunization. The HI test showed an increased in antibody titers during the course of experiment in group immunized with H5 and H5 + MDP1 vaccines. The result showed that the constructed DNA vaccines were able to produce detectable antibody titer in which the group immunized with H5 + MDP1 vaccine produced higher antibody comparing to H5 vaccine alone. Conclusions: This study shows for the first time the usefulness of MDP1 as a genetic adjuvant for H5 DNA vaccine.

Recombinant lambda-phage nanobioparticles for tumor therapy in mice models

Amir Ghaemi — 2010-05-12T00:00:00Z

Lambda phages have considerable potential as gene delivery vehicles due to their genetic tractability, low cost, safety and physical characteristics in comparison to other nanocarriers and gene porters. Little is known concerning lambda phage-mediated gene transfer and expression in mammalian hosts. We therefore performed experiments to evaluate lambda-ZAP bacteriophage-mediated gene transfer and expression in vitro. For this purpose, we constructed recombinant λ-phage nanobioparticles containing a mammalian expression cassette encoding enhanced green fluorescent protein (EGFP) and E7 gene of human papillomavirus type 16 (λ-HPV-16 E7) using Lambda ZAP- CMV XR vector. Four cell lines (COS-7, CHO, TC-1 and HEK-239) were transduced with the nanobioparticles. We also characterized the therapeutic anti-tumor effects of the recombinant λ-HPV-16 E7 phage in C57BL/6 tumor mice model as a cancer vaccine. Obtained results showed that delivery and expression of these genes in fibroblastic cells (COS-7 and CHO) are more efficient than epithelial cells (TC-1 and HEK-239) using these nanobioparticles. Despite the same phage M.O.I entry, the internalizing titers of COS-7 and CHO cells were more than TC-1 and HEK-293 cells, respectively. Mice vaccinated with λ-HPV-16 E7 are able to generate potent therapeutic antitumor effects against challenge with E7- expressing tumor cell line, TC-1 compared to group treated with the wild phage. The results demonstrated that the recombinant λ-phages, due to their capabilities in transducing mammalian cells, can also be considered in design and construction of novel and safe phage-based nanomedicines.

Protection against the allergic airway inflammation depends on the modulation of spleen dendritic cell function and induction of regulatory T cells in mice

Yaoli Wang — 2010-03-24T00:00:00Z

Background: Allergen-induced imbalance of specific T regulatory (Treg) cells and T helper 2 cells plays a decisive role in the development of immune response against allergens.ObjectiveTo evaluate effects and potential mechanisms of DNA vaccine containing ovalbumin (OVA) and Fc fusion on allergic airway inflammation. Methods: Bronchoalveolar lavage (BAL) levels of inflammatory mediators and leukocyte infiltration, expression of CD11c+CD80+ and CD11c+CD86+ co-stimulatory molecules in spleen dendritic cells (DCs), circulating CD4+ and CD8+ T cells, Foxp3+ in spleen CD4+ T cells and spleen CD4+ T cells were measured in OVA-sensitized and challenged animals pretreated with pcDNA, OVA-pcDNA, Fc-pcDNA, and OVA-Fc-pcDNA. Results: OVA-Sensitized and challenged mice developed airway inflammation and Th2 responses, and decreased the proliferation of peripheral CD4+and CD8+ T cells and the number of spleen Foxp3+ Treg. Those changes with increased INF-γ production and reduced OVA-specific IgE production were protected by the pretreatment with OVA-Fc-pcDNA. Conclusion: DNA vaccine encoding both Fc and OVA showed more effective than DNA vaccine encoding Fc or OVA alone, through the balance of DCs and Treg.

Optimised electroporation mediated DNA vaccination for treatment of prostate cancer

Sarfraz Ahmad — 2010-02-05T00:00:00Z

Background: Immunological therapies enhance the ability of the immune system to recognise and destroy cancer cells via selective killing mechanisms. DNA vaccines have potential to activate the immune system against specific antigens, with accompanying potent immunological adjuvant effects from unmethylated CpG motifs as on prokaryotic DNA. We investigated an electroporation driven plasmid DNA vaccination strategy in animal models for treatment of prostate cancer. Methods: Plasmid expressing human PSA gene (phPSA) was delivered in vivo by intra-muscular electroporation, to induce effective anti-tumour immune responses against prostate antigen expressing tumours. Groups of male C57 BL/6 mice received intra-muscular injections of phPSA plasmid. For phPSA delivery, quadriceps muscle was injected with 50 μg plasmid. After 80 seconds, square-wave pulses were administered in sequence using a custom designed pulse generator and acustom-designed applicator with 2 needles placed through the skin central to the muscle. To determine an optimum treatment regimen, three different vaccination schedules were investigated. In a separate experiment, the immune potential of the phPSA vaccine was further enhanced with co- administration of synthetic CpG rich oligonucleotides. One week after last vaccination, the mice were challenged subcutaneously with TRAMPC1/hPSA (prostate cancer cell line stably expressing human PSA) and tumour growth was monitored. Serum from animals was examined by ELISA for anti-hPSA antibodies and for IFNγ. Histological assessment of the tumours was also carried out. In vivo and in vitro cytotoxicity assays were performed with splenocytes from treated mice. Results: The phPSA vaccine therapy significantly delayed the appearance of tumours and resulted in prolonged survival of the animals. Four-dose vaccination regimen provided optimal immunological effects. Co - administration of the synthetic CpG with phPSA increased anti-tumour responses, preventing tumour occurrence in 54% of treated animals. Vaccination with phPSA resulted in anti-hPSA Abs production and a significant production of IFNγ was observed in immunised animals (p < 0.05). Immune responses were tumour specific and were transferable in adoptive T cell transfer experiments. Conclusions: This phPSA plasmid electroporation vaccination strategy can effectively activate tumour specific immune responses. Optimisation of the approach indicated that a four-dose regimen provided highest tumour protection. In vivo electroporation mediated vaccination is a safe and effective modality for the treatment of prostate cancer and has a potential to be used as a neo-adjuvant or adjuvant therapy.

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

Alice Trinh — 2009-12-30T00:00:00Z

Background: Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone. Methods: A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection. Results: Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene. Conclusion: These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.

Combined therapy with cyclophosphamide and DNA preparation inhibits the tumor growth in mice

Ekaterina Alyamkina — 2009-08-14T00:00:00Z

Background: When cyclophosphamide and preparations of fragmented exogenous genomic double stranded DNA were administered in sequence, the regressive effect on the tumor was synergic: this combined treatment had a more pronounced effect than cyclophosphamide alone. Our further studies demonstrated that exogenous DNA stimulated the maturation and specific activities of dendritic cells. This suggests that cyclophosphamide, combined with DNA, leads to an immune response to the tumors that were grafted into the subjects post treatment. Methods: Three-month old CBA/Lac mice were used in the experiments. The mice were injected with cyclosphamide (200 mkg per 1 kg body weight) and genomic DNA (of human, mouse or salmon sperm origin). The DNA was administered intraperitoneally or subcutaneously. After 23 to 60 days, one million tumor cells were intramuscularly grafted into the mice. In the final experiment, the mice were pre-immunized by subcutaneous injections of 20 million repeatedly thawed and frozen tumor cells. Changes in tumor growth were determined by multiplying the three perpendicular diameters (measured by caliper). Students' t-tests were used to determine the difference between tumor growth and average survival rate between the mouse groups and the controls. Results: An analysis of varying treatments with cyclophosphamide and exogenous DNA, followed by tumor grafting, provided evidence that this combined treatment had an immunizing effect. This inhibitory effect in mice was analyzed in an experiment with the classical immunization of a tumor homogenate. The strongest inhibitory action on a transplanted graft was created through the following steps: cyclophosphamide at 200 mg/kg of body weight administered as a pretreatment; 6 mg fragmented exogenous DNA administered over the course of 3 days; tumor homogenate grafted 10 days following the final DNA injection. Conclusion: Fragmented exogenous DNA injected with cyclophosphamide inhibits the growth of tumors that are grafted to mice after this combined treatment.

Dietary restriction abrogates antibody production induced by a DNA vaccine encoding the mycobacterial 65kDa heat shock protein

Larissa Lumi Watanabe Ishikawa — 2009-07-16T00:00:00Z

Background: Protein-calorie malnutrition (PCM) is the most common type of malnutrition. PCM leads to immunodeficiency and consequent increased susceptibility to infectious agents. In addition, responses to prophylactic vaccines depend on nutritional status. This study aims to evaluate the ability of undernourished mice to mount an immune response to a genetic vaccine (pVAXhsp65) against tuberculosis, containing the gene coding for the heat shock protein 65 from mycobacteria. Methods: Young adult female BALB/c mice were fed ad libitum or with 80% of the amount of food consumed by a normal diet group. We initially characterized a mice model of dietary restriction by determining body and spleen weights, hematological parameters and histopathological changes in lymphoid organs. The ability of splenic cells to produce IFN-gamma and IL-4 upon in vitro stimulation with LPS or S. aureus and the serum titer of specific IgG1 and IgG2a anti-hsp65 antibodies after intramuscular immunization with pVAXhsp65 was then tested. Results: Dietary restriction significantly decreased body and spleen weights and also the total lymphocyte count in blood. This restriction also determined a striking atrophy in lymphoid organs as spleen, thymus and lymphoid tissue associated with the small intestine. Specific antibodies were not detected in mice submitted to dietary restriction whereas the well nourished animals produced significant levels of both, IgG1 and IgG2a anti-hsp65. Conclusion: 20% restriction in food intake deeply compromised humoral immunity induced by a genetic vaccine, alerting, therefore, for the relevance of the nutritional condition in vaccination programs based on these kinds of constructs.

Anti-tumor effects of a human VEGFR-2-based DNA vaccine in mouse models

Ke Xie — 2009-06-21T00:00:00Z

Background: Vascular endothelial growth factor (VEGF) and its receptor, VEGFR-2 (Flk-1/KDR), play a key role in tumor angiogenesis. Blocking the VEGF-VEGFR-2 pathway may inhibit tumor growth. Here, we used human VEGFR-2 as a model antigen to explore the feasibility of immunotherapy with a plasmid DNA vaccine based on a xenogeneic homologue of this receptor. Methods: The protective effects and therapeutic anti-tumor immunity mediated by the DNA vaccine were investigated in mouse models. Anti-angiogenesis effects were detected by immunohistochemical staining and the alginate-encapsulate tumor cell assay. The mechanism of action of the DNA vaccine was primarily explored by detection of auto-antibodies and CTL activity. Results: The DNA vaccine elicited a strong, protective and therapeutic anti-tumor immunity through an anti-angiogenesis mechanism in mouse models, mediated by the stimulation of an antigen-specific response against mFlk-1. Conclusion: Our study shows that a DNA vaccine based on a xenogeneic homologue plasmid DNA induced autoimmunity against VEGFR-2, resulting in inhibition of tumor growth. Such vaccines may be clinically relevant for cancer immunotherapy.