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

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.

Download Full-text

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

Blood ◽

10.1182/blood.v110.11.4882.4882 ◽

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.

Download Full-text

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

Proceedings of the National Academy of Sciences of Belarus Biological Series ◽

10.29235/1029-8940-2018-63-4-419-425 ◽

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.

Download Full-text

Immunogenicity and Biodistribution of Anthrax DNA Vaccine Delivered by Intradermal Electroporation

Current Drug Delivery ◽

10.2174/1567201817666200414144550 ◽

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.

Download Full-text

Adjuvanting a DNA vaccine with a TLR9 ligand plus Flt3 ligand results in enhanced cellular immunity against the simian immunodeficiency virus

Journal of Experimental Medicine ◽

10.1084/jem.20071211 ◽

2007 ◽

Vol 204 (11) ◽

pp. 2733-2746 ◽

Cited By ~ 58

Author(s):

Marcin Kwissa ◽

Rama R. Amara ◽

Harriet L. Robinson ◽

Bernard Moss ◽

Sefik Alkan ◽

...

Keyword(s):

T Cells ◽

Dna Vaccine ◽

Dna Vaccines ◽

Rhesus Macaques ◽

Interleukin 2 ◽

Flt3 Ligand ◽

Viral Loads ◽

Immunodeficiency Virus ◽

Ifn Γ

DNA vaccines offer promising strategies for immunization against infections. However, their clinical use requires improvements in immunogenicity. We explored the efficacy of Toll-like receptor (TLR) ligands (TLR-Ls) on augmenting the immunogenicity of a DNA prime–modified vaccinia virus Ankara (MVA) boost vaccine against SIV. Rhesus macaques were injected with Fms-like tyrosine kinase 3 (Flt3)–ligand (FL) to expand dendritic cells (DCs) and were primed with a DNA vaccine encoding immunodeficiency virus antigens mixed with ligands for TLR9 or TLR7/8. Subsequently, the animals were boosted with DNA and twice with recombinant MVA expressing the same antigens. TLR9-L (CpG DNA) mediated activation of DCs in vivo and enhanced the magnitude of antigen-specific CD8+ interferon (IFN) γ+ T cells and polyfunctional CD8+ T cells producing IFN-γ, tumor necrosis factor α, and interleukin 2. Although this trial was designed primarily as an immunogenicity study, we challenged the animals with pathogenic SIVmac251 and observed a reduction in peak viremia and cumulative viral loads in the TLR9-L plus FL-adjuvanted group relative to the unvaccinated group; however, the study design precluded comparisons between the adjuvanted groups and the group vaccinated with DNA/MVA alone. Viral loads were inversely correlated with the magnitude and quality of the immune response. Thus, the immunogenicity of DNA vaccines can be augmented with TLR9-L plus FL.

Download Full-text

Specific Immune Response Induced by PML-Rarα-hIL-2 Vaccine in BALB/C Mice

Blood ◽

10.1182/blood.v112.11.4009.4009 ◽

2008 ◽

Vol 112 (11) ◽

pp. 4009-4009

Author(s):

Yangqiu Li ◽

Dongzhi Cen ◽

Yubing Zhou ◽

Lijian Yang ◽

Gang Hu ◽

...

Keyword(s):

Immune Response ◽

T Cells ◽

Injection Site ◽

Dna Vaccines ◽

Interleukin 2 ◽

Specific Immune Response ◽

Rt Pcr ◽

Control Groups ◽

Specific Cytotoxicity ◽

Cellular Immune

Abstract Specific active immunotherapy with DNA vaccines offers the hope for a natural nontoxic alternative to eradicate minimal residual disease in cancer patients. However, little data are regarding the DNA vaccines in leukemia. Our previous study has successfully developed a vector containing PML-RARα segment fused to human Interleukin- 2 (hIL-2) gene. In the present study, we analyzed the specific immune response by immunization with the recombinant plasmid in BALB/c mice. The recombinant plasmids were injected into BALB/C mice with a total of 200 μg DNA in normal saline at 14-day intervals for three cycles. The plasmid containing the same sequence of PML-RARα segment or hIL-2 gene alone and blank plasmid served as controls. Two weeks after the final DNA boost, the transcription and expression of PML-RARα and hIL-2 in injection site were detected by RT-PCR and immunohistological methods; the specific cytotoxicity of spleen cells from the vaccinated BALB/C mice was detected by LDH release assay; interferon-γ secretion was measured by ELISA; antibodies against human PML-RARα were detected by indirect immunofluorescene analysis (flow cytometry); and T-cell receptor rearrangement excision circles (TRECs) in DNA from mice thymic cells were detected by real-time quantitative RT-PCR (TaqMan), thereby to evaluate the content of naïve T cells. After immunization, the PML-RARα/hIL-2 mRNA and protein could be identified in the injection site of PML-RARα- hIL-2 vaccinated mice. Time dependent antibody production and an increase in INF–γ could be detected in vaccinated mice serum (p<0.05). Specific cytotoxicity on NB4 cells was significantly higher than that from control groups (p=0.012). TRECs level form PML-RARα-hIL-2 vaccine group was significantly higher than that from PML-RARα, hIL-2 vaccine or control groups (p<0.05). In conclusions, the reconstructed PML-RARα plasmids might display the common feature of DNA vaccine, which can induce specific humoral and cellular immune responses in BALB/c mice in vivo and can induce the increase of naïve T cells in mice thymus, it might relate to stronger cellular immune response, it is to our knowledge, the first description on the change of naïve T cells in response to vaccine.

Download Full-text

Construction of an encapsulated ESAT-6-based anti-TB DNA vaccine and evaluation of its immunogenic properties

Biological Chemistry ◽

10.1515/bc.2008.063 ◽

2008 ◽

Vol 389 (5) ◽

Cited By ~ 5

Author(s):

Olesya Nosareva ◽

Andrey Nesterov ◽

Alexander Boldyrev ◽

Olga Smirnova ◽

Yurii Tumanov ◽

...

Keyword(s):

Immune Response ◽

Mycobacterium Tuberculosis ◽

T Cell ◽

Dna Vaccine ◽

Murine Model ◽

Recombinant Plasmid ◽

Cell Immune Response ◽

The Core ◽

Immunogenic Properties

AbstractExperimental preparations based on a DNA vaccine encoding the ESAT-6 antigen ofMycobacterium tuberculosishave been obtained (KpONE6) and studied for immunogenic effects in the murine model. The core of the preparation contains DNA of the recombinant plasmid pONE6 encapsulated within a spermidine-polyglucin conjugate, thereby protecting the DNA vaccine from degradation. KpONE6 induces a proliferative T-cell immune response in mice upon intramuscular immunization.

Download Full-text

Humoral and Cellular Immune Responses Induced by 3a DNA Vaccines against Severe Acute Respiratory Syndrome (SARS) or SARS-Like Coronavirus in Mice

Clinical and Vaccine Immunology ◽

10.1128/cvi.00261-08 ◽

2008 ◽

Vol 16 (1) ◽

pp. 73-77 ◽

Cited By ~ 15

Author(s):

Baojing Lu ◽

Ling Tao ◽

Ting Wang ◽

Zhenhua Zheng ◽

Bao Li ◽

...

Keyword(s):

T Cells ◽

Severe Acute Respiratory Syndrome ◽

Dna Vaccine ◽

Vaccine Development ◽

Dna Vaccines ◽

Interleukin 2 ◽

Reading Frame ◽

Cellular Immune Responses ◽

Humoral Responses ◽

Cellular Immune

ABSTRACT Vaccine development for severe acute respiratory syndrome coronavirus (SARS-CoV) has mainly focused on the spike (S) protein. However, the variation of the S gene between viruses may affect the efficacy of a vaccine, particularly for cross-protection against SARS-like CoV (SL-CoV). Recently, a more conserved group-specific open reading frame (ORF), the 3a gene, was found in both SARS-CoV and SL-CoV. Here, we studied the immunogenicity of human SARS-CoV 3a and bat SL-CoV 3a DNA vaccines in mice through electroporation immunization followed by enzyme-linked immunosorbent, enzyme-linked immunospot, and flow cytometry assays. Our results showed that high levels of specific humoral responses were induced by SARS-CoV 3a and SL-CoV 3a DNA vaccines. Furthermore, a strong Th1-based cellular immune response was stimulated by both DNA vaccines. The vaccines stimulated gamma interferon production mainly by CD8+ T cells and interleukin-2 (IL-2) mainly by CD4+ T cells. Of interest, the frequency of IL-2-positive cells elicited by the SARS-CoV 3a DNA vaccine was significantly higher than that elicited by the SL-CoV 3a DNA vaccine. In summary, our study provides a reference for designing cross-protective DNA vaccines based on the group-specific ORFs of CoVs.

Download Full-text

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

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.12.12.1364-1369.2005 ◽

2005 ◽

Vol 12 (12) ◽

pp. 1364-1369 ◽

Cited By ~ 5

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.

Download Full-text

Influence of introduction of interleukin-2 and interleukin-12 into experimental marker DNA-vaccine

Faktori eksperimental'noi evolucii organizmiv ◽

10.7124/feeo.v25.1158 ◽

2019 ◽

Vol 25 ◽

pp. 160-165

Author(s):

Ia. O. Pokholenko ◽

T. P. Gulko ◽

V. A. Kordium

Keyword(s):

Immune Response ◽

Dna Vaccine ◽

Humoral Immune Response ◽

Interleukin 2 ◽

Classical Swine Fever ◽

Interleukin 12 ◽

Chimeric Proteins ◽

Humoral Immune

Aim. To determine the effects of the combined administration of recombinant expression vectors containing genes of murine interleukin-2 and interleukin-12 on humoral immune response, elicited by the experimental marker DNA-vaccine against classical swine fever. Methods. Expression of chimeric proteins in vitro and in vivo was determined by western-blot analysis. The antibodies specific to target antigens in blood serum have been detected by ELISA. Results. A series of recombinant plasmid vectors containing the genes of murine interleukin-2 and chimeric murine interleukin-12 have been developed. It has been shown that target chimeric proteins were expressed from the developed vectors both in vitro in HEK293 and in vivo in murine muscle tissue. The use of combined administration of murine interleukin-2 and interleukin-12 genes resulted in significant enhancement of titer of the anti-E2 and anti-β-galactosidase IgG, induced by vaccination with experimental marker DNA-vaccine against CSF, and model DNA-vaccine respectively. Conclusions. The data obtained show that the introduction of recombinant expressing vectors containing genes of murine interleukin-2 and interleukin-12 into vaccine preparations enhances humoral immune response elicited by the experimental marker DNA-vaccine against CSF and modelDNA-vaccine. Keywords: DNA-vaccine, humoral immune response, interleukin-2, interleukin-12, classical swine fever.

Download Full-text