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

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.

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Enhanced immunogenicity of a synthetic DNA vaccine expressing consensus SARS-CoV-2 Spike protein using needle-free immunization

10.1101/2021.02.01.429219 ◽

2021 ◽

Author(s):

Sawsan S Alamri ◽

Khalid A Alluhaybi ◽

Rowa Y Alhabbab ◽

Abdullah Algaissi ◽

Sarah Almahboub ◽

...

Keyword(s):

Immune Response ◽

Dna Vaccine ◽

Mammalian Cells ◽

Cellular Response ◽

Igg Antibodies ◽

Free System ◽

Mammalian Expression ◽

Specific Igg ◽

Needle Injection ◽

Specific Igg Antibodies

AbstractThe ongoing global pandemic of Coronavirus Disease 2019 (COVID-19) calls for an urgent development of effective and safe prophylactic and therapeutic measures. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) glycoprotein is a major immunogenic and protective protein, and plays a crucial role in viral pathogenesis. In this study, we successfully constructed a synthetic codon-optimized DNA-based vaccine as a countermeasure against SARS-CoV-2; denoted as VIU-1005. The design was based on the synthesis of codon-optimized coding sequence for optimal mammalian expression of a consensus full-length S glycoprotein. The successful construction of the vaccine was confirmed by restriction digestion and sequencing, and the protein expression of the S protein was confirmed by western blot and immunofluorescence staining in mammalian cells. The immunogenicity of the vaccine was tested in two mouse models (BALB/c and C57BL/6J). Th1-skewed systemic S-specific IgG antibodies and neutralizing antibodies (nAbs) were significantly induced in both models four weeks post three injections with 100 μg of the VIU-1005 vaccine via intramuscular needle injection but not intradermal or subcutaneous routes. Importantly, such immunization induced long-lasting IgG response in mice that lasted for at least 6 months. Interestingly, using a needle-free system, we showed an enhanced immunogenicity of VIU-1005 in which lower doses such as 25-50 μg or less number of doses were able to elicit significantly high levels of Th1-biased systemic S-specific IgG antibodies and nAbs via intramuscular immunization compared to needle immunization. Compared to the intradermal needle injection which failed to induce any significant immune response, intradermal needle-free immunization elicited robust Th1-biased humoral response similar to that observed with intramuscular immunization. Furthermore, immunization with VIU-1005 induced potent S-specific cellular response as demonstrated by the significantly high levels of IFN-γ, TNF and IL-2 cytokines production in memory CD8+ and CD4+ T cells in BALB/c mice. Together, our results demonstrate that the synthetic VIU-1005 candidate DNA vaccine is highly immunogenic and capable of inducing long-lasting and Th1-skewed immune response in mice. Furthermore, we show that the use of needle-free system could enhance the immunogenicity and minimize doses needed to induce protective immunity in mice, supporting further preclinical and clinical testing of this candidate vaccine.

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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.

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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.

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A plant protein signal sequence improved humoral immune response to HPV prophylactic and therapeutic DNA vaccines

Human Vaccines & Immunotherapeutics ◽

10.1080/21645515.2017.1264766 ◽

2017 ◽

Vol 13 (2) ◽

pp. 271-282 ◽

Cited By ~ 9

Author(s):

Silvia Massa ◽

Francesca Paolini ◽

Gianfranca Curzio ◽

Marcelo Nazario Cordeiro ◽

Elena Illiano ◽

...

Keyword(s):

Immune Response ◽

Humoral Immune Response ◽

Dna Vaccines ◽

Signal Sequence ◽

Plant Protein ◽

Humoral Immune

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Plasmid Chemokines and Colony-Stimulating Factors Enhance the Immunogenicity of DNA Priming-Viral Vector Boosting Human Immunodeficiency Virus Type 1 Vaccines

Journal of Virology ◽

10.1128/jvi.77.16.8729-8735.2003 ◽

2003 ◽

Vol 77 (16) ◽

pp. 8729-8735 ◽

Cited By ~ 97

Author(s):

Dan H. Barouch ◽

Paul F. McKay ◽

Shawn M. Sumida ◽

Sampa Santra ◽

Shawn S. Jackson ◽

...

Keyword(s):

Immune Responses ◽

Dna Vaccine ◽

Plasmid Dna ◽

Viral Vectors ◽

Dna Vaccines ◽

Viral Vector ◽

Human Populations ◽

Colony Stimulating Factors ◽

Boost Vaccine ◽

Vector Boosting

ABSTRACT Heterologous “prime-boost” regimens that involve priming with plasmid DNA vaccines and boosting with recombinant viral vectors have been shown to elicit potent virus-specific cytotoxic T-lymphocyte responses. Increasing evidence, however, suggests that the utility of recombinant viral vectors in human populations will be significantly limited by preexisting antivector immunity. Here we demonstrate that the coadministration of plasmid chemokines and colony-stimulating factors with plasmid DNA vaccines markedly increases the immunogenicity of DNA prime-recombinant adenovirus serotype 5 (rAd5) boost and DNA prime-recombinant vaccinia virus (rVac) boost vaccine regimens in BALB/c mice. In mice with preexisting anti-Ad5 immunity, priming with the DNA vaccine alone followed by rAd5 boosting elicited only marginal immune responses. In contrast, cytokine-augmented DNA vaccine priming followed by rAd5 vector boosting was able to generate potent immune responses in mice with preexisting anti-Ad5 immunity. These data demonstrate that plasmid cytokines can markedly improve the immunogenicity of DNA prime-viral vector boost vaccine strategies and can partially compensate for antivector immunity.

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

Blood ◽

10.1182/blood.v110.11.4881.4881 ◽

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.

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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.

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Faculty Opinions recommendation of Genetic Immunization With In Vivo Dendritic Cell-targeting Liposomal DNA Vaccine Carrier Induces Long-lasting Antitumor Immune Response.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.726015290.793515733 ◽

2016 ◽

Author(s):

Miguel Vicente-Manzanares

Keyword(s):

Immune Response ◽

Dendritic Cell ◽

Dna Vaccine ◽

Antitumor Immune Response ◽

Cell Targeting ◽

Genetic Immunization ◽

Vaccine Carrier

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Analyses of Lipid A Diversity in Gram-Negative Intestinal Bacteria Using Liquid Chromatography–Quadrupole Time-of-Flight Mass Spectrometry

Metabolites ◽

10.3390/metabo11040197 ◽

2021 ◽

Vol 11 (4) ◽

pp. 197

Author(s):

Nobuyuki Okahashi ◽

Masahiro Ueda ◽

Fumio Matsuda ◽

Makoto Arita

Keyword(s):

Mass Spectrometry ◽

Immune Response ◽

Liquid Chromatography ◽

Mammalian Cells ◽

Lipid A ◽

Acyl Chain ◽

Time Of Flight ◽

Intestinal Bacteria ◽

Gram Negative ◽

Flight Mass Spectrometry

Lipid A is a characteristic molecule of Gram-negative bacteria that elicits an immune response in mammalian cells. The presence of structurally diverse lipid A types in the human gut bacteria has been suggested before, and this appears associated with the immune response. However, lipid A structures and their quantitative heterogeneity have not been well characterized. In this study, a method of analysis for lipid A using liquid chromatography–quadrupole time-of-flight mass spectrometry (LC-QTOF/MS) was developed and applied to the analyses of Escherichia coli and Bacteroidetes strains. In general, phosphate compounds adsorb on stainless-steel piping and cause peak tailing, but the use of an ammonia-containing alkaline solvent produced sharp lipid A peaks with high sensitivity. The method was applied to E. coli strains, and revealed the accumulation of lipid A with abnormal acyl side chains in knockout strains as well as known diphosphoryl hexa-acylated lipid A in a wild-type strain. The analysis of nine representative strains of Bacteroidetes showed the presence of monophosphoryl penta-acylated lipid A characterized by a highly heterogeneous main acyl chain length. Comparison of the structures and amounts of lipid A among the strains suggested a relationship between lipid A profiles and the phylogenetic classification of the strains.

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