A Vector Expressing PML-RARα Fused to GM-CSF Is an Effective DNA Vaccine for Inducing Specific Immune Response to APL Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4882-4882
Author(s):  
Yangqiu Li ◽  
Dongzhi Cen ◽  
Gang Hu ◽  
Yubing Zhou ◽  
Shaohua Chen ◽  
...  
Keyword(s):  
Immune Response ◽  
Acute Leukemia ◽  
Dna Vaccine ◽  
Tumor Antigens ◽  
Dna Vaccines ◽  
Fusion Gene ◽  
Gene Segment ◽  
A549 Cells ◽  
Nb4 Cells ◽  
Gm Csf

Abstract Despite combinations of different therapeutic strategies have significantly increased survival, acute leukemia is still not curable. To further improve outcome, specific immunotherapy might be one of the best choice. DNA vaccines have been showed leading to strong and persistent cell-mediated and humoral immune response to the antigen encoded by the plasmid. However, little data exist regarding the DNA vaccines in acute leukemia, there are few studies reported that PML-RARα DNA vaccines were developed, but the host immune response were weakly, due to the weak immunogenicity of tumor antigens. In order to improve the effect of DNA vaccine for acute promyelocytic leukemia (APL) therapy, we have used a full-length human GM-CSF (hGM-CSF) sequence fused to PML-RARα breakpoint-drived sequence and develop a vector coexpressing PML-RARα gene and hGM-CSF gene, which was expected to to promote T cells response in host. PML-RARα fusion gene segment and the hGM-CSF gene were amplified from NB4 cells or pORF-hGM-CSF plasmid. Both PCR products were cloned into PIRES plasmid respectively to construct a recombinant plasmid PML-RARα-IRES-hGM-CSF. The recombinant plasmids were then transfected into K562 or A549 cells respectively. The expression of the PML-RARα/GM-CSF mRNA and protein in transfected cells were identified by RT-PCR, dot blotting, ELISA and Western-Blot respectively. By in vivo assays, BALB/c mice were vaccinated at 6–8 week of age with a total of 200 μg DNA in normal saline, injected into two sites in the quadriceps muscles on day 0, 7 and 21. The plasmid containing the same PML-RARα segment and blank plasmid served as controls. Two weeks after the final DNA boost, both PML-RARα/GM-CSF mRNA and protein, serum INF-γ and anti-NB4 cells specific cytotoxicity of splenocytes following 7 days of stimulation in vitro with freeze thawing NB4 cells and recombinant human IL-2 were assessed by ELISA and LDH assays. The results showed that the sequence of the fragments inserted in multi-clone site (MCS) A and MCS B of PIRES plasmid were absolutely correct by double restriction enzyme cutting analysis (Xba I/Sal I) and sequence analysis, the PML-RARα/GM-CSF mRNA and protein could be identified in transfected K562 or A549 cells and in mice quadriceps muscles. The level of serum INF-γ and cytotoxicity of splenocytes against NB4 cells from immunized mice was significant increased than that from control groups. In conclusions, the vector expressing PML-RARα and hGM-CSF was successfully constructed, which can more effective immune response and anti-APL cells effect in animal models than that from plasmid containing single PML-RARα segment. It could be farther used in the research as PML-RARα DNA vaccine for APL.

Development of a PML-RARα-IL-2 Recombinant Plasmid DNA for APL.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4881-4881
Author(s):  
Yubing Zhou ◽  
Gang Hu ◽  
Dongzhi Cen ◽  
Lijian Yang ◽  
Shaohua Chen ◽  
...  
Keyword(s):  
Immune Response ◽  
Dna Vaccine ◽  
Recombinant Plasmid ◽  
Dna Vaccines ◽  
Gene Segment ◽  
Interleukin 2 ◽  
A549 Cells ◽  
Jurkat Cells ◽  
Active Immunotherapy ◽  
Nb4 Cells

Abstract Specific active immunotherapy with DNA vaccines is one of the best approaches to eradicate minimal residual disease in cancer patients, when appropriate tumor-specific antigens are identified for this disease. However, little data are regarding the DNA vaccines in acute leukemia, recent reports has shown that a human PML-RARα breakpoint-drived DNA vaccine can prevent APL in mice model, but the host immune response were weakly, due to the weak immunogenicity. In order to improve the effect of DNA vaccine for acute promyelocytic leukemia (APL) therapy, we develop a vector coexpressing PML-RARα gene fused to human Interleukin- 2 (hIL-2) gene. PML-RARα fusion gene segment and the full-lenght hIL-2 gene were amplified from NB4 cells or Jurkat cells. Both PCR products were cloned into PIRES plasmid respectively to construct a recombinant plasmid PML-RARα-IRES-hIL-2. The recombinant plasmids were then transfected into K562 or A549 cells respectively. The expression of the PML-RARα/hIL-2 mRNA and protein in transfected cells were identified by RT-PCR, dot blotting, ELISA and Western-Blot respectively. By in vivo assays, BALB/c mice were vaccinated at 6–8 week of age with a total of 200 μg DNA in normal saline, injected into two sites in the quadriceps muscles on day 0, 7 and 21. The plasmid containing the same sequence of PML-RARα gene and blank plasmid served as controls. Two weeks after the final DNA boost, both PML-RARα/hIL-2 mRNA and protein, serum INF-γ and anti-NB4 cells specific cytotoxicity of splenocytes following 7 days of stimulation in vitro with freeze thawing NB4 cells and recombinant human IL-2 were assessed by ELISA and LDH assays. The results showed that the sequence of the fragments inserted in multi-clone site (MCS) A and MCS B of PIRES plasmid were absolutely correct by double restriction enzyme cutting analysis (Sal I/Not I) and sequence analysis, the PML-RARα/hIL-2 mRNA and protein could be identified in transfected K562 or A549 cells and in mice quadriceps muscles. The level of serum INF-γ and cytotoxicity of splenocytes against NB4 cells from immunized mice was significant increased than that from control groups. Our results indicated that the recombinant plasmid expressing PML-RARα and hIL-2 was successfully constructed, which can induce more effective immune response and anti-APL cells effect in animal models. The data suggest that effective vaccination approaches should be possible against APL. It could be farther used in the research as PML-RARα DNA vaccine for APL.

scholarly journals Design of genetic construction for creation DNA vaccine against porcine reproductive and respiratory syndrome

2018 ◽  
Vol 63 (4) ◽  
pp. 419-425
Author(s):  
L. M. Kravchenko ◽  
K. V. Kudzin ◽  
U. A. Prakulevich
Keyword(s):  
Immune Response ◽  
Dna Vaccine ◽  
Mammalian Cells ◽  
Dna Vaccines ◽  
Viral Vector ◽  
Signal Sequence ◽  
Regulatory Elements ◽  
Economic Damage ◽  
Chain Gene ◽  
Prrs Virus

The porcine reproductive and respiratory syndrome (PRRS) caused the serious economic damage to swine breeding around the world. It is a viral infective disease against which live attenuated and inactivated vaccines are not always successful. Development of new types of drugs such as DNA vaccines is necessary for improving the protection against the virus. DNA vaccines induce the development of both a cellular and humoral immune response. Such vaccines consist of a plasmid or viral vector with genes of potentially immunogenic proteins. The expression of these genes realized in cells of the vaccinated animal. It leads to the synthesis of antigen proteins triggering the immune response. The purpose of this work is to create a genetic construction that can be used as DNA vaccine against PRRS virus. The construction consists of the commercial vector pVAX1 and open reading frame of two structural proteins of PRRS virus, a lysosomal localization signal sequence of the invariant chain gene and regulatory elements necessary for the expression of cloned genes in mammalian cells.

Immunogenicity and Biodistribution of Anthrax DNA Vaccine Delivered by Intradermal Electroporation

2020 ◽  
Vol 17 (5) ◽  
pp. 414-421
Author(s):  
Na Young Kim ◽  
Won Rak Son ◽  
Jun Young Choi ◽  
Chi Ho Yu ◽  
Gyeung Haeng Hur ◽  
...  
Keyword(s):  
Immune Response ◽  
Bacillus Anthracis ◽  
Dna Vaccine ◽  
Fluorescent Protein ◽  
Protective Antigen ◽  
Dna Vaccines ◽  
Bacterial Disease ◽  
Intradermal Administration ◽  
Bacterium Bacillus

Purpose: Anthrax is a lethal bacterial disease caused by gram-positive bacterium Bacillus anthracis and vaccination is a desirable method to prevent anthrax infections. In the present study, DNA vaccine encoding a protective antigen of Bacillus anthracis was prepared and we investigated the influence of DNA electrotransfer in the skin on the induced immune response and biodistribution. Methods and Results: The tdTomato reporter gene for the whole animal in vivo imaging was used to assess gene transfer efficiency into the skin as a function of electrical parameters. Compared to that with 25 V, the transgene expression of red fluorescent protein increased significantly when a voltage of 90 V was used. Delivery of DNA vaccines expressing Bacillus anthracis protective antigen domain 4 (PAD4) with an applied voltage of 90 V induced robust PA-D4-specific antibody responses. In addition, the in vivo fate of anthrax DNA vaccine was studied after intradermal administration into the mouse. DNA plasmids remained at the skin injection site for an appropriate period of time after immunization. Intradermal administration of DNA vaccine resulted in detection in various organs (viz., lung, heart, kidney, spleen, brain, and liver), although the levels were significantly reduced. Conclusion: Our results offer important insights into how anthrax DNA vaccine delivery by intradermal electroporation affects the immune response and biodistribution of DNA vaccine. Therefore, it may provide valuable information for the development of effective DNA vaccines against anthrax infection.

Variable effects of the co-administration of a GM-CSF-expressing plasmid on the immune response to flavivirus DNA vaccines in mice

2014 ◽  
Vol 162 (1) ◽  
pp. 140-148 ◽  
Author(s):  
Hui Chen ◽  
Na Gao ◽  
Jiangman Wu ◽  
Xiaoyan Zheng ◽  
Jieqiong Li ◽  
...  
Keyword(s):  
Immune Response ◽  
Dna Vaccines ◽  
Gm Csf

scholarly journals DNA Vaccines: Developing New Strategies against Cancer

2010 ◽  
Vol 2010 ◽  
pp. 1-16 ◽  
Author(s):  
Daniela Fioretti ◽  
Sandra Iurescia ◽  
Vito Michele Fazio ◽  
Monica Rinaldi
Keyword(s):  
Clinical Trials ◽  
Dna Vaccine ◽  
Tumor Antigens ◽  
Cell Function ◽  
Immune Cell ◽  
Dna Vaccines ◽  
Basic Research ◽  
Antigen Expression ◽  
Routes Of Administration ◽  
Processing Pathways

Due to their rapid and widespread development, DNA vaccines have entered into a variety of human clinical trials for vaccines against various diseases including cancer. Evidence that DNA vaccines are well tolerated and have an excellent safety profile proved to be of advantage as many clinical trials combines the first phase with the second, saving both time and money. It is clear from the results obtained in clinical trials that such DNA vaccines require much improvement in antigen expression and delivery methods to make them sufficiently effective in the clinic. Similarly, it is clear that additional strategies are required to activate effective immunity against poorly immunogenic tumor antigens. Engineering vaccine design for manipulating antigen presentation and processing pathways is one of the most important aspects that can be easily handled in the DNA vaccine technology. Several approaches have been investigated including DNA vaccine engineering, co-delivery of immunomodulatory molecules, safe routes of administration, prime-boost regimen and strategies to break the immunosuppressive networks mechanisms adopted by malignant cells to prevent immune cell function. Combined or single strategies to enhance the efficacy and immunogenicity of DNA vaccines are applied in completed and ongoing clinical trials, where the safety and tolerability of the DNA platform are substantiated. In this review on DNA vaccines, salient aspects on this topic going from basic research to the clinic are evaluated. Some representative DNA cancer vaccine studies are also discussed.

A phase I, open-label, dose-escalation and cohort expansion study evaluating the safety and immune response to autologous dendritic cells transduced with AdGMCA9 in patients with metastatic renal cell carcinoma.

2018 ◽  
Vol 36 (6_suppl) ◽  
pp. 653-653
Author(s):  
Izak Faiena ◽  
Nazy Zomorodian ◽  
Begonya Comin-Anduix ◽  
Ankush Sachadeva ◽  
Adrian Bot ◽  
...  
Keyword(s):  
Immune Response ◽  
Dendritic Cells ◽  
Dose Escalation ◽  
Mononuclear Cells ◽  
Clear Cell ◽  
Fusion Gene ◽  
Recombinant Adenovirus ◽  
Antitumor Effects ◽  
Gm Csf

653 Background: We developed a fusion gene construct, GM-CSF + CAIX, transduced by a replication deficient adenovirus into autologous dendritic cells (DC) that are injected in patients with metastatic RCC (mRCC) in this phase 1 study targeting CAIX overexpressed on RCC tumors. Methods: A recombinant adenovirus encoding the GMCSF-CAIX fusion gene (AdGMCAIX) manufactured per GMP in collaboration with the NCI Rapid Access to Intervention Development (RAID) program. The final product was produced using DCs produced ex-vivo from patients’ peripheral blood mononuclear cells (PBMC), by culturing with GM-CSF & IL-4, then engineered with AdGMCAIX prior to intradermal injection. The injected transduced DCs were expected to stimulate an antigen specific immune response against CAIX expressing RCC. Three dose escalation cohorts (5, 15, and 50 X 106 cells/administration) were injected based on 3+3 design. DC-AdGMCAIX was given intradermally q2wkX3 doses. The primary aim is safety. Secondary aims are to evaluate immune responses & antitumor effects per RECIST 1.1. Eligibility criteria included patients with clear cell mRCC with ECOG 0-1, measurable disease, and adequate organ function. Results: Fifteen patients with clear cell mRCC were enrolled. Nine patients received all 3 planned vaccine doses, comprising DC expressing CAIX, CD11c and other relevant markers. No serious adverse events (SAEs) were seen. Grade 1/2 AEs include fatigue (3/1), leukopenia (1/1) and flu-like symptoms (0/1). Of the 9 patients who received treatment, 1 expired of progressive disease (PD), 2 patients were lost to follow-up and 6 patients are alive. Of the 6 patients, 5 have PD and are currently receiving standard-of-care therapies, and 1 has completed treatment with stable disease at 6 mon follow up and is being evaluated for retreatment. Conclusions: These early data show that autologous DC transduced by Ad-GMCAIX vector can be safely given to mRCC patients without any SAEs noted at the doses tested. These data support further development of Ad-GMCAIX vaccine strategies, either alone, or in combination with approved therapies. Clinical trial information: NCT01826877.

scholarly journals Boosting Immune Response to Hepatitis B DNA Vaccine by Coadministration of Prothymosin α-Expressing Plasmid

2005 ◽  
Vol 12 (12) ◽  
pp. 1364-1369 ◽  
Author(s):  
Yanwen Jin ◽  
Cheng Cao ◽  
Ping Li ◽  
Xuan Liu ◽  
Wei Huang ◽  
...  
Keyword(s):  
Immune Response ◽  
Hepatitis B ◽  
Dna Vaccine ◽  
Dna Vaccines ◽  
Hepatitis B Surface Antigen ◽  
Antibody Responses ◽  
Prothymosin Α ◽  
Vaccine Potency ◽  
Antibody Titers ◽  
Cellular Immune

ABSTRACT DNA vaccines induce protective humoral and cell-mediated immune responses in several animal models. However, compared to conventional vaccines, DNA vaccines usually induce poor antibody responses. In this study, we report that coadministration of a hepatitis B virus (HBV) DNA vaccine with prothymosin α as an adjuvant improves antibody responses to HBV S antigen. We also observed higher seroconversion rates and higher antibody titers. Prothymosin α appears to increase the number and affinity of hepatitis B surface antigen-specific, gamma interferon-secreting T cells and to enhance cellular immune response to the PreS2S DNA vaccine. Interestingly, administering the DNA separately from the prothymosin α plasmid abrogated the enhancement of DNA vaccine potency. The results suggest that prothymosin α may be a promising adjuvant for DNA vaccines.

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