Genetic Vaccines and Therapy最新文献

Development of various vaccines for prostate cancer (PCa) is becoming an active research area. PCa vaccines are perceived to have less toxicity compared with the available cytotoxic agents. While various immune-based strategies can elicit anti-tumour responses, DNA vaccines present increased efficacy, inducing both humoural and cellular immunity. This immune activation has been proven effective in animal models and initial clinical trials are encouraging. However, to validate the role of DNA vaccination in currently available PCa management paradigms, strong clinical evidence is still lacking. This article provides an overview of the basic principles of DNA vaccines and aims to provide a summary of preclinical and clinical trials outlining the benefits of this immunotherapy in the management of PCa.

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Background: Liver fluke can infect cattle and sheep, and is also emerging as a human pathogen in developing countries. Cathepsin B (Cat B2) is a major cysteine protease secreted by the juvenile flukes. To enhance the immune responses of Cat B2, the cDNA sequence was fused with four different DNA vaccine vectors. The induced cellular and antibody responses were compared in vaccinated mice.

Methods: The following recombinant DNA vaccine constructs were constructed: empty vector VR1012 as negative control, cytoplasmic construct pVR1012 Cat B2, secretory construct pVR1020 Cat B2, chemokine-fused construct pMCP3 Cat B2 and lymph node targeting construct pCTLA-4 Cat B2. Plasmids were constructed using standard procedures, and positive constructs screened and selected using restriction digestion analysis followed by sequence analysis. The constructs were then tested in Cos-7 cells for in vitro expression, which was analysed using immunoblotting. Subsequently, female BALB/c mice were immunised with DNA constructs as vaccines. Elicited antibody responses were measured using ELISA. The ratio between IgG1 and IgG2a antibody responses was estimated among different vaccine groups. IgG antibody avidity assay was performed and the relative avidity index was calculated. The induced cytokine production from splenocytes of vaccinated animals was estimated using ELISPOT.

Results: DNA vaccine constructs carrying Cat B2 were expressed in Cos-7 cell lines and encoded protein was recognised using western blotting using rat anti- cathepsin B antibody. DNA vaccines elicited high Cat B2- specific IgG, IgG1, IgE and also modest IgG2a antibody responses. Cat B2 specific IL-4 T cell responses were also observed in Cat B2 vaccinated mice. The comparison of immunogenic potential in each of these constructs was demonstrated as enhanced antibody responses on the lymph-node targeting vector pCTLA-4 Cat B2, the high antibody avidity of chemo-attractant pMCP3 Cat B2 and stronger T cellular responses of non-secretory DNA vaccine pVR1012 Cat B2 in vaccinated animals.

Conclusion: This study showed that the targeting DNA vaccine strategies enhanced specific immune responses to juvenile fluke Cat B2. The results of our current study have demonstrated that a gene-based vaccine as an immunotherapeutic approach to combat Fasciola infection may be feasible.

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Background: Malaria causes immense human morbidity and mortality globally. The plasmodium species vivax and falciparum cause over 75 % clinical malaria cases. Until now, gene-based strategies against malaria have only been applied to plasmodium species and their mosquito-vector. Merozoites of these two respective plasmodium species target and invade red blood cells (RBCs) by using the duffy antigen receptor for chemokines (DARC), and Sialic Acid (SLC4A1) residues of the O-linked glycans of Glycophorin A. RBCs of naturally selected duffy-negative blacks are resistant to P.vivax tropism. We hypothesized that artificial aberration of the host-pathway by target mutagenesis of either RBC -receptors, may abolish or reduce susceptibility of the host to malaria. As a first step towards the experimental actualization of these concepts, we aimed to identify zinc finger arrays (ZFAs) for constructing ZFNs that target genes of either wild-type host-RBC- receptors.

Methods: In-Silico Gene & Genome Informatics

Results: Using the genomic contextual nucleotide-sequences of homo-sapiens darc and glycophorin-a, and the ZFN-consortia software- CoDA-ZiFiT-ZFA and CoDA-ZiFiT-ZFN: we identified 163 and over 1,000 single zinc finger arrays (sZFAs) that bind sequences within the genes for the two respective RBC-receptors. Second, 2 and 18 paired zinc finger arrays (pZFAs) that are precursors for zinc finger nucleases (ZFNs) capable of cleaving the genes for darc and glycophorin-a were respectively assembled. Third, a mega-BLAST evaluation of the genome-wide cleavage specificity of this set of ZFNs was done, revealing alternate homologous nucleotide targets in the human genome other than darc or glycophorin A.

Conclusions: ZFNs engineered with these ZFA-precursors--with further optimization to enhance their specificity to only darc and glycophorin-a, could be used in constructing an experimental gene-based-malaria vaccine. Alternatively, meganucleases and transcription activator-like (TAL) nucleases that target conserved stretches of darc and glycophorin-a DNA may serve the purpose of abrogating invasion of RBCs by falciparam and vivax plasmodia species.

Dengue virus infection is a serious health problem infecting 2.5 billion people worldwide. Dengue is now endemic in more than 100 countries, including Pakistan. Each year hundreds of people get infected with dengue in Pakistan. Currently, there is no vaccine available for the prevention of Dengue virus infection due to four viral serotypes. Dengue infection can cause death of patients in its most severity, meanwhile many antiviral compounds are being tested against dengue virus infection to eradicate this disease but still there is a need to develop an efficient, low-cost and safe vaccine that can target all the four serotypes of dengue virus. This review summarizes dengue molecular virology, important drug targets, prevalence in Pakistan, diagnosis, treatment and medicinal plant inhibitors against dengue.

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Background: The use of optimized delivery devices has been shown to enhance the potency of DNA vaccines. However, further optimization of DNA vaccine delivery is needed for this vaccine modality to ultimately be efficacious in humans.

Methods: Herein we evaluated antigen expression and immunogenicity after intradermal delivery of different doses of DNA vaccines by needle or by the Biojector jet-injection device, with or without the addition of electroporation (EP).

Results: Neither needle injection augmented by EP nor Biojector alone could induce higher magnitudes of immune responses after immunizations with a high dose of DNA. After division of a defined DNA dose into multiple skin sites, the humoral response was particularly enhanced by Biojector while cellular responses were particularly enhanced by EP. Furthermore, a close correlation between in vivo antigen expression and cell-mediated as well as humoral immune responses was observed.

Conclusions: These results show that two optimized DNA vaccine delivery devices can act together to overcome dose restrictions of plasmid DNA vaccines.

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Background: Gp41 is an envelope glycoprotein of human immune deficiency virus (HIV). HIV viral glycoprotein gp41, present in complex with gp120, assists the viral entry into host cell. Over eighty thousands individuals are HIV infected in Pakistan which makes about 0.2% of 38.6 million infected patients worldwide. Hence, HIV gp41 protein sequences isolated in Pakistan were analyzed for the CD4 and CD8 T cells binding epitopes.

Results: Immunoinformatics tools were applied for the study of variant region of HIV gp41envelope protein. The protein nature was analyzed using freely accessible computational software. About 90 gp41 sequences of Pakistani origin were aligned and variable and conserved regions were found. Four segments were found to be conserved in gp41 viral protein. A method was developed, involving the secondary structure, surface accessibility, hydrophobicity, antigenicity and molecular docking for the prediction and location of epitopes in the viral glycoprotein. Some highly conserved CD4 and CD8 binding epitopes were also found using multiple parameters. The predicted continuous epitopes mostly fall in the conserved region of 1-12; 14-22 and 25-46 and can be used as effective vaccine candidates.

Conclusions: The study revealed potential HIV subtype a derived cytotoxic T cell (CTL) epitopes from viral proteome of Pakistani origin. The conserved epitopes are very useful for the diagnosis of the HIV 1 subtype a. This study will also help scientists to promote research for vaccine development against HIV 1 subtype a, isolated in Pakistan.

Background: The appropriate tropism of adeno-associated virus (AAV) vectors that are systemically injected is crucial for successful gene therapy when local injection is not practical. Acidic oligopeptides have been shown to enhance drug delivery to bones.

Methods: In this study six-L aspartic acids (D6) were inserted into the AAV2 capsid protein sequence between amino acid residues 587 and 588. 129SVE mice were injected with double-stranded wild-type- (WT-) or D6-AAV2 mCherry expression vectors (3.24 x 1010 vg per animal) via the superficial temporal vein within 24 hours of birth.

Results: Fluorescence microscopy and quantitative polymerase chain reaction confirmed higher levels of mCherry expression in the paraspinal and gluteus muscles in the D6-AAV2 injected mice. The results revealed that although D6-AAV2 was less efficient in the transduction of immortalized cells stronger mCherry signals were detected over the spine and pelvis by live imaging in the D6-AAV2-injected mice than were detected in the WT-AAV2-injected mice. In addition, D6-AAV2 lost the liver tropism observed for WT-AAV2.

Conclusions: An acidic oligopeptide displayed on AAV2 improves axial muscle tropism and decreases liver tropism after systemic delivery. This modification should be useful in creating AAV vectors that are suitable for gene therapy for diseases involving the proximal muscles.

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.

Background: Countrywide 5.9 million, 0-11 Month old children are immunized annually by EPI (Expended Program on Immunization) against 8 vaccine preventable diseases including measles and so on. Unfortunately the basic immunity centers are not uniform throughout the country. Each center provides services to about 27000 people which is inadequate. The purpose of this study was to explore the development of EPI Pakistan in terms of immunization of measles.

Methods: Nucleotide sequences were analyzed by neighbor joining method (bootstrap test) using Bio- edit and MEGA-5 software to find evolutionary relationship between wild type measles strain and vaccine strain (Edmonston strain) used in Pakistan. For statistical analysis of data SPSS 16 was used.

Results: Currently 1.3 vaccinators are working at each U C (union council) which according to national EPI policy should be at least 2. About 56% and 44% children of age 0-11 months did not received second dose of measles in the last two years respectively. Out of these 4231 cases which were reported last year, 1370 have received their first dose of measles vaccine.

Conclusion: Seroconversion and seroprevalence study of the vaccine and field strain of measles virus is needed to confirm whether its failure is due to service unavailability or vaccine in-affectivity.