http://www.gvt-journal.com The most accessed research articles published by Genetic Vaccines and Therapy 2012-04-20T00:00:00Z Background: Mucopolysaccharidosis type I (MPSI) is caused by a deficiency in alpha-L iduronidase (IDUA), which leads to lysosomal accumulation of the glycosaminoglycans (GAGs) dermatan and heparan sulfate. While the currently available therapies have good systemic effects, they only minimally affect the neurodegenerative process. Based on the neuroprotective and tissue regenerative properties of mesenchymal stem cells (MSCs), we hypothesized that the administration of MSCs transduced with a murine leukemia virus (MLV) vector expressing IDUA to IDUA KO mouse brains could reduce GAG deposition in the brain and, as a result, improve neurofunctionality, as measured by exploratory activity. Methods: MSCs infected with an MLV vector encoding IDUA were injected into the left ventricle of the brain of 12- or 25-month-old IDUA KO mice. The behavior of the treated mice in the elevated plus maze and open field tests was observed for 1 to 2 months. Following these observations, the brains were removed for biochemical and histological analyses. Results: After 1 or 2 months of observation, the presence of the transgene in the brain tissue of almost all of the treated mice was confirmed using PCR, and a significant reduction in GAG deposition was observed. This reduction was directly reflected in an improvement in exploratory activity in the open field and the elevated plus maze tests. Despite these behavioral improvements and the reduction in GAG deposition, IDUA activity was undetectable in these samples. Overall, these results indicate that while the initial level of IDUA was not sustainable for a month, it was enough to reduce and maintain low GAG deposition and improve the exploratory activity for months. Conclusions: These data show that gene therapy, via the direct injection of IDUA-expressing MSCs into the brain, is an effective way to treat neurodegeneration in MPSI mice. http://www.gvt-journal.com/content/10/1/2 Flávia Helena da Silva Vanessa Gonçalves Pereira Eduardo Yasumura Lígia Zacchi Tenório Leonardo Pinto de Carvalho Bianca Cristina Garcia Lisboa Priscila Keiko Matsumoto Roberta Sessa Stilhano Vivian Samoto Bruno Frederico Aguilar Calegare Leticia de Campos Brandão Vania D´Almeida Thaís Filippo Marimelia Porcionatto Leny Toma Helena Bonciani Nader Valderez Bastos Vastos Melissa Camassola Nance Beyer Nardi Sang Won Han Genetic Vaccines and Therapy 2012, null:2 2012-04-20T00:00:00Z doi:10.1186/1479-0556-10-2 /content/figures/1479-0556-10-2-toc.gif Genetic Vaccines and Therapy 1479-0556 ${item.volume} 2 2012-04-20T00:00:00Z PDF Background: Hepatitis C virus (HCV) is one of the leading causes of viral hepatitis worldwide and its genotype 3a is predominant in vast areas of Pakistan.FindingsThe present study reports the first full sequence of HCV 3a isolate PK-1 from Pakistan. This nucleotide sequence was compared with six other HCV genotype 3a full length sequences from different regions of the world by using statistical methods of phylogenetic analysis. Conclusion: The nucleotide difference of these seven sequences shows that HCV genotype 3a of phylogenetically distinct origin is circulating in Pakistan. http://www.gvt-journal.com/content/9/1/2 Irshad-ur Rehman Muhammad Idrees Muhammad Ali Liaqat Ali Sadia Butt Abrar Hussain Haji Akbar Samia Afzal Genetic Vaccines and Therapy 2011, null:2 2011-01-06T00:00:00Z doi:10.1186/1479-0556-9-2 /content/figures/1479-0556-9-2-toc.gif Genetic Vaccines and Therapy 1479-0556 ${item.volume} 2 2011-01-06T00:00:00Z XML Retroviral vector-mediated gene transfer has been central to the development of gene therapy. Retroviruses have several distinct advantages over other vectors, especially when permanent gene transfer is the preferred outcome. The most important advantage that retroviral vectors offer is their ability to transform their single stranded RNA genome into a double stranded DNA molecule that stably integrates into the target cell genome. This means that retroviral vectors can be used to permanently modify the host cell nuclear genome. Recently, retroviral vector-mediated gene transfer, as well as the broader gene therapy field, has been re-invigorated with the development of a new class of retroviral vectors which are derived from lentiviruses. These have the unique ability amongst retroviruses of being able to infect non-cycling cells. Vectors derived from lentiviruses have provided a quantum leap in technology and seemingly offer the means to achieve significant levels of gene transfer in vivo.The ability of retroviruses to integrate into the host cell chromosome also raises the possibility of insertional mutagenesis and oncogene activation. Both these phenomena are well known in the interactions of certain types of wild-type retroviruses with their hosts. However, until recently they had not been observed in replication defective retroviral vector-mediated gene transfer, either in animal models or in clinical trials. This has meant the potential disadvantages of retroviral mediated gene therapy have, until recently, been seen as largely, if not entirely, hypothetical. The recent clinical trial of γc mediated gene therapy for X-linked severe combined immunodeficiency (X-SCID) has proven the potential of retroviral mediated gene transfer for the treatment of inherited metabolic disease. However, it has also illustrated the potential dangers involved, with 2 out of 10 patients developing T cell leukemia as a consequence of the treatment. A considered review of retroviral induced pathogenesis suggests these events were qualitatively, if not quantitatively, predictable. In addition, it is clear that the probability of such events can be greatly reduced by relatively simple vector modifications, such as the use of self-inactivating vectors and vectors derived from non-oncogenic retroviruses. However, these approaches remain to be fully developed and validated. This review also suggests that, in all likelihood, there are no other major retroviral pathogenetic mechanisms that are of general relevance to replication defective retroviral vectors. These are important conclusions as they suggest that, by careful design and engineering of retroviral vectors, we can continue to use this gene transfer technology with confidence. http://www.gvt-journal.com/content/2/1/9 Donald Anson Genetic Vaccines and Therapy 2004, null:9 2004-08-13T00:00:00Z doi:10.1186/1479-0556-2-9 /content/figures/1479-0556-2-9-toc.gif Genetic Vaccines and Therapy 1479-0556 ${item.volume} 9 2004-08-13T00:00:00Z XML Background: Enterovirus 71 (EV71) is a major causative viral agent responsible for large outbreaks of hand, foot and mouth disease (HFMD), a common rash illness in children and infants. There is no effective antiviral treatment for severe EV71 infections and no vaccine is available. The objectives of this study were to design and construct a DNA vaccine against Enterovirus 71 using the viral capsid protein (VP1) gene of EV71 and to verify the functionality of the DNA vaccine in vitro and in vivo. Methods: The VP1 gene of EV71 from two local outbreak isolates were amplified using PCR and then inserted into a eukaryotic expression vector, pVAX1. The 3.9 kb recombinant constructs were transformed into competent E. coli cells and the positive clones were screened and selected using PCR analysis, restriction digestion analysis and DNA sequencing. The constructs were then tested for protein expression in Vero cells. Subsequently, in the in vivo studies, female Balb/c mice were immunized with the DNA vaccine constructs. Enzyme Linked Immunosorbent Assay (ELISA) and virus neutralizing assay were performed to detect the presence of anti-VP1 IgG in mice and its neutralizing effect against the EV71. Results: The pVAX1 vector was successfully cloned with the VP1 gene from each of the isolate (S2/86/1 and 410/4) in the correct orientation and in-frame. The DNA vaccine constructs with the VP1 gene were shown to be expressed in a cell-free in vitro expression system. The VP1 protein was successfully expressed in the mammalian cell line and was detected using RT-PCR, Indirect Immunofluorescence Assay (IFA) and western blotting. The anti-VP1 IgG levels in mice immunized with the DNA vaccine constructs increased after the first booster but declined following the second booster. The anti-VP1 IgG in the mice immunized with the DNA vaccine constructs exhibited neutralising activity against EV71. Conclusion: The promising results obtained in the present study have prompted further testing to improve the expression and immunogenicity of this potential EV71 DNA vaccine. http://www.gvt-journal.com/content/5/1/6 Wong Siew Tung Sazaly Abu Bakar Zamberi Sekawi Rozita Rosli Genetic Vaccines and Therapy 2007, null:6 2007-04-19T00:00:00Z doi:10.1186/1479-0556-5-6 /content/figures/1479-0556-5-6-toc.gif Genetic Vaccines and Therapy 1479-0556 ${item.volume} 6 2007-04-19T00:00:00Z XML DNA vaccination is a relatively recent development in vaccine methodology. It is now possible to undertake a rational step-by-step approach to DNA vaccine design. Strategies may include the incorporation of immunostimulatory sequences in the backbone of the plasmid, co-expression of stimulatory molecules, utilisation of localisation/secretory signals, and utilisation of the appropriate delivery system, for example. However, another important consideration is the utilisation of methods designed to optimise transgene expression. In this review we discuss the importance of regulatory elements, kozak sequences and codon optimisation in transgene expression. http://www.gvt-journal.com/content/1/1/2 Helen Garmory Katherine Brown Richard Titball Genetic Vaccines and Therapy 2003, null:2 2003-09-16T00:00:00Z doi:10.1186/1479-0556-1-2 DNA vaccines: improving expression of antigens. /content/figures/1479-0556-1-2-toc.gif Genetic Vaccines and Therapy 1479-0556 ${item.volume} 2 2003-09-16T00:00:00Z XML The complexity of parasitic infections requires novel approaches to vaccine design. The versatility of DNA vaccination provides new perspectives. This review discusses the use of prime-boost immunizations, genetic adjuvants, multivalent vaccines and codon optimization for optimal DNA vaccine design against parasites. http://www.gvt-journal.com/content/2/1/17 Catherine Ivory Kris Chadee Genetic Vaccines and Therapy 2004, null:17 2004-12-03T00:00:00Z doi:10.1186/1479-0556-2-17 /content/figures/1479-0556-2-17-toc.gif Genetic Vaccines and Therapy 1479-0556 ${item.volume} 17 2004-12-03T00:00:00Z XML Tattooing is one of a number of DNA delivery methods which results in an efficient expression of an introduced gene in the epidermal and dermal layers of the skin. The tattoo procedure causes many minor mechanical injuries followed by hemorrhage, necrosis, inflammation and regeneration of the skin and thus non-specifically stimulates the immune system. DNA vaccines delivered by tattooing have been shown to induce higher specific humoral and cellular immune responses than intramuscularly injected DNA. In this study, we focused on the comparison of DNA immunization protocols using different routes of administrations of DNA (intradermal tattoo versus intramuscular injection) and molecular adjuvants (cardiotoxin pre-treatment or GM-CSF DNA co-delivery). For this comparison we used the major capsid protein L1 of human papillomavirus type 16 as a model antigen. L1-specific immune responses were detected after three and four immunizations with 50 μg plasmid DNA. Cardiotoxin pretreatment or GM-CSF DNA co-delivery substantially enhanced the efficacy of DNA vaccine delivered intramuscularly by needle injection but had virtually no effect on the intradermal tattoo vaccination. The promoting effect of both adjuvants was more pronounced after three rather than four immunizations. However, three DNA tattoo immunizations without any adjuvant induced significantly higher L1-specific humoral immune responses than three or even four intramuscular DNA injections supported by molecular adjuvants. Tattooing also elicited significantly higher L1-specific cellular immune responses than intramuscularly delivered DNA in combination with adjuvants. In addition, the lymphocytes of mice treated with the tattoo device proliferated more strongly after mitogen stimulation suggesting the presence of inflammatory responses after tattooing. The tattoo delivery of DNA is a cost-effective method that may be used in laboratory conditions when more rapid and more robust immune responses are required. http://www.gvt-journal.com/content/6/1/4 Dana Pokorna Ivonne Rubio Martin Muller Genetic Vaccines and Therapy 2008, null:4 2008-02-07T00:00:00Z doi:10.1186/1479-0556-6-4 DNA tattoos boost vaccine delivery The tattoo delivery of DNA vaccines is cost effective, induces a greater immune response, and is a more efficient delivery strategy for laboratory studies than intramuscular injection. /content/figures/1479-0556-6-4-toc.gif Genetic Vaccines and Therapy 1479-0556 ${item.volume} 4 2008-02-07T00:00:00Z XML Background: A recently discovered occult HCV entity reported by various investigators seems to be highly controversial. Especially, the clinical significance of these findings remains uncertain. For optimal outcome of antiviral therapy, investigation of occult HCV needs a broad-based probe in order to investigate the results of viral therapy and its host/viral interaction. The current study was aimed at determining the prevalence of occult HCV in peripheral blood lymphocytes of predominantly genotype 3 HCV-infected patients after completion of antiviral therapy and to investigate long term outcomes in the presence or absence of PBMC positivity.MethodA total of 151 chronic, antiHCV and serum RNA-positive patients were enrolled in the study. Patients with a complete virological response at the end of treatment were screened for the presence of viral RNA in their PBMCs and were followed for up to one year for the presence of serum and PBMC viral genomic RNA. Results: Out of 151 patients, 104 (70%) responded to the prescribed interferon treatment and showed viral-clearance from serum. These were screened for the presence of genomic RNA in their PBMCs. Sixteen samples were PBMC-positive for viral RNA at the end of treatment (EOT). All these patients had also cleared the virus from peripheral blood cells after the 6-12 month follow-up study. Conclusion: True occult hepatitis C virus does not exist in our cohort. Residual viremia at the EOT stage merely reflects a difference in viral kinetics in various compartments that remains a target of immune response even after the end of antiviral therapy and is eventually cleared out at the sustained viral response (SVR). http://www.gvt-journal.com/content/9/1/14 Ambreen Muazzam Saleem Qureshi Atika Mansoor Lubna Ali Musarrat Iqbal Saima Siddiqi Khalid Khan Kehkashan Mazhar Genetic Vaccines and Therapy 2011, null:14 2011-09-06T00:00:00Z doi:10.1186/1479-0556-9-14 /content/figures/1479-0556-9-14-toc.gif Genetic Vaccines and Therapy 1479-0556 ${item.volume} 14 2011-09-06T00:00:00Z XML Background: A number of reports have demonstrated that rodents immunized with DNA vaccines can produce antibodies and cellular immune responses presenting a long-lasting protective immunity. These findings have attracted considerable interest in the field of DNA vaccination. We have previously described the prophylactic and therapeutic effects of a DNA vaccine encoding the Mycobacterium leprae 65 kDa heat shock protein (DNA-HSP65) in a murine model of tuberculosis. As DNA vaccines are often less effective in humans, we aimed to find out how the DNA-HSP65 stimulates human immune responses. Methods: To address this question, we analysed the activation of both human macrophages and dendritic cells (DCs) cultured with DNA-HSP65. Then, these cells stimulated with the DNA vaccine were evaluated regarding the expression of surface markers, cytokine production and microbicidal activity. Results: It was observed that DCs and macrophages presented different ability to uptake DNA vaccine. Under DNA stimulation, macrophages, characterized as CD11b+/CD86+/HLA-DR+, produced high levels of TNF-alpha, IL-6 (pro-inflammatory cytokines), and IL-10 (anti-inflammatory cytokine). Besides, they also presented a microbicidal activity higher than that observed in DCs after infection with M. tuberculosis. On the other hand, DCs, characterized as CD11c+/CD86+/CD123-/BDCA-4+/IFN-alpha-, produced high levels of IL-12 and low levels of TNF-alpha, IL-6 and IL-10. Finally, the DNA-HSP65 vaccine was able to induce proliferation of peripheral blood lymphocytes. Conclusion: Our data suggest that the immune response is differently activated by the DNA-HSP65 vaccine in humans. These findings provide important clues to the design of new strategies for using DNA vaccines in human immunotherapy. http://www.gvt-journal.com/content/6/1/3 Luis Franco Pryscilla Wowk Celio Silva Ana Trombone Arlete Coelho-Castelo Constance Oliver Maria Jamur Edson Moretto Vania Bonato Genetic Vaccines and Therapy 2008, null:3 2008-01-21T00:00:00Z doi:10.1186/1479-0556-6-3 DNA vaccine provides immunotherapy insights A DNA vaccine against tuberculosis, effective in mice, is shown to stimulate the human immune response, providing clues for new strategies in human immunotherapy. /content/figures/1479-0556-6-3-toc.gif Genetic Vaccines and Therapy 1479-0556 ${item.volume} 3 2008-01-21T00:00:00Z XML Background: Hepatitis C core protein is an attractive target for HCV vaccine aimed to exterminate HCV infected cells. However, although highly immunogenic in natural infection, core appears to have low immunogenicity in experimental settings. We aimed to design an HCV vaccine prototype based on core, and devise immunization regimens that would lead to potent anti-core immune responses which circumvent the immunogenicity limitations earlier observed. Methods: Plasmids encoding core with no translation initiation signal (pCMVcore); with Kozak sequence (pCMVcoreKozak); and with HCV IRES (pCMVcoreIRES) were designed and expressed in a variety of eukaryotic cells. Polyproteins corresponding to HCV 1b amino acids (aa) 1–98 and 1–173 were expressed in E. coli. C57BL/6 mice were immunized with four 25-μg doses of pCMVcoreKozak, or pCMV (I). BALB/c mice were immunized with 100 μg of either pCMVcore, or pCMVcoreKozak, or pCMVcoreIRES, or empty pCMV (II). Lastly, BALB/c mice were immunized with 20 μg of core aa 1–98 in prime and boost, or with 100 μg of pCMVcoreKozak in prime and 20 μg of core aa 1–98 in boost (III). Antibody response, [3H]-T-incorporation, and cytokine secretion by core/core peptide-stimulated splenocytes were assessed after each immunization. Results: Plasmids differed in core-expression capacity: mouse fibroblasts transfected with pCMVcore, pCMVcoreIRES and pCMVcoreKozak expressed 0.22 ± 0.18, 0.83 ± 0.5, and 13 ± 5 ng core per cell, respectively. Single immunization with highly expressing pCMVcoreKozak induced specific IFN-γ and IL-2, and weak antibody response. Single immunization with plasmids directing low levels of core expression induced similar levels of cytokines, strong T-cell proliferation (pCMVcoreIRES), and antibodies in titer 103(pCMVcore). Boosting with pCMVcoreKozak induced low antibody response, core-specific T-cell proliferation and IFN-γ secretion that subsided after the 3rd plasmid injection. The latter also led to a decrease in specific IL-2 secretion. The best was the heterologous pCMVcoreKozak prime/protein boost regimen that generated mixed Th1/Th2-cellular response with core-specific antibodies in titer ≥ 3 × 103. Conclusion: Thus, administration of highly expressed HCV core gene, as one large dose or repeated injections of smaller doses, may suppress core-specific immune response. Instead, the latter is induced by a heterologous DNA prime/protein boost regimen that circumvents the negative effects of intracellular core expression. http://www.gvt-journal.com/content/7/1/7 Ekaterina Alekseeva Irina Sominskaya Dace Skrastina Irina Egorova Elizaveta Starodubova Eriks Kushners Marija Mihailova Natalia Petrakova Ruta Bruvere Tatyana Kozlovskaya Maria Isaguliants Paul Pumpens Genetic Vaccines and Therapy 2009, null:7 2009-06-08T00:00:00Z doi:10.1186/1479-0556-7-7 /content/figures/1479-0556-7-7-toc.gif Genetic Vaccines and Therapy 1479-0556 ${item.volume} 7 2009-06-08T00:00:00Z XML