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.

scholarly journals Detection of Anthrax Toxin in the Serum of Animals Infected with Bacillus anthracis by Using Engineered Immunoassays

2006 ◽  
Vol 13 (6) ◽  
pp. 671-677 ◽  
Author(s):  
Robert Mabry ◽  
Kathleen Brasky ◽  
Robert Geiger ◽  
Ricardo Carrion ◽  
Gene B. Hubbard ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Systemic Circulation ◽  
Rapid Identification ◽  
Anthrax Toxin ◽  
Soluble Form ◽  
Before And After

ABSTRACT Several strategies that target anthrax toxin are being developed as therapies for infection by Bacillus anthracis. Although the action of the tripartite anthrax toxin has been extensively studied in vitro, relatively little is known about the presence of toxins during an infection in vivo. We developed a series of sensitive sandwich enzyme-linked immunosorbent assays (ELISAs) for detection of both the protective antigen (PA) and lethal factor (LF) components of the anthrax exotoxin in serum. The assays utilize as capture agents an engineered high-affinity antibody to PA, a soluble form of the extracellular domain of the anthrax toxin receptor (ANTXR2/CMG2), or PA itself. Sandwich immunoassays were used to detect and quantify PA and LF in animals infected with the Ames or Vollum strains of anthrax spores. PA and LF were detected before and after signs of toxemia were observed, with increasing levels reported in the late stages of the infection. These results represent the detection of free PA and LF by ELISA in the systemic circulation of two animal models exposed to either of the two fully virulent strains of anthrax. Simple anthrax toxin detection ELISAs could prove useful in the evaluation of potential therapies and possibly as a clinical diagnostic to complement other strategies for the rapid identification of B. anthracis infection.

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.

The Humoral Immune Response to Various Domains of Protective Antigen of Bacillus anthracis in Cutaneous Anthrax Cases in India

2016 ◽  
Vol 66 (6) ◽  
pp. 645 ◽  
Author(s):  
Anshul Varshney ◽  
Nidhi Puranik ◽  
M. Kumar ◽  
A.K. Goel
Keyword(s):  
Immune Response ◽  
Bacillus Anthracis ◽  
Humoral Immune Response ◽  
Vaccine Development ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Serum Samples ◽  
Public Health Importance ◽  
Humoral Immune ◽  
Cutaneous Anthrax

Anthrax, caused by Bacillus anthracis is known to occur globally since antiquity. Besides being an important biothreat agent, it is an important public health importance pathogen also in countries like India. B. anthracis secretes three distinct toxins, namely protective antigen (PA), lethal factor (LF) and edema factor (EF). PA is the central moiety of the anthrax toxin complex and therefore has been a molecule of choice for vaccine development. PA has four different domains with different functions. In this study, the major domains of PA were cloned and expressed in bacterial system. The purified recombinant proteins were used to determine the humoral immune response by ELISA using 43 human cutaneous anthrax serum samples. The maximum immunoreactivity was observed with the whole PA protein followed by domain 2, 4 and 1. The study corroborated that in addition to full PA, individual domain 2 and 4 can also be good target for vaccine development as well as for serodiagnostic assays for cutaneous anthrax

scholarly journals Functional Analysis of Bacillus anthracis Protective Antigen by Using Neutralizing Monoclonal Antibodies

2004 ◽  
Vol 72 (11) ◽  
pp. 6313-6317 ◽  
Author(s):  
Fabien Brossier ◽  
Martine Lévy ◽  
Annie Landier ◽  
Pierre Lafaye ◽  
Michèle Mock
Keyword(s):  
Functional Analysis ◽  
Monoclonal Antibodies ◽  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Cell Receptor ◽  
Lethal Challenge ◽  
Edema Factor

ABSTRACT Protective antigen (PA) is central to the action of the lethal and edema toxins produced by Bacillus anthracis. It is the common cell-binding component, mediating the translocation of the enzymatic moieties (lethal factor [LF] and edema factor) into the cytoplasm of the host cell. Monoclonal antibodies (MAbs) against PA, able to neutralize the activities of the toxins in vitro and in vivo, were screened. Two such MAbs, named 7.5 and 48.3, were purified and further characterized. MAb 7.5 binds to domain 4 of PA and prevents the binding of PA to its cell receptor. MAb 48.3 binds to domain 2 and blocks the cleavage of PA into PA63, a step necessary for the subsequent interaction with the enzymatic moieties. The epitope recognized by this antibody is in a region involved in the oligomerization of PA63; thus, MAb 48.3 does not recognize the oligomer form. MAbs 7.5 and 48.3 neutralize the activities of anthrax toxins produced by B. anthracis in mice. Also, there is an additive effect between the two MAbs against PA and a MAb against LF, in protecting mice against a lethal challenge by the Sterne strain. This work contributes to the functional analysis of PA and offers immunotherapeutic perspectives for the treatment of anthrax disease.

scholarly journals Contributions of Histamine, Prostanoids, and Neurokinins to Edema Elicited by Edema Toxin from Bacillus anthracis

2007 ◽  
Vol 75 (4) ◽  
pp. 1895-1903 ◽  
Author(s):  
Jeffrey Tessier ◽  
Candace Green ◽  
Diana Padgett ◽  
Wei Zhao ◽  
Lawrence Schwartz ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Cell Types ◽  
Vascular Leakage ◽  
Target Cells ◽  
Edema Factor ◽  
Edema Toxin ◽  
Neurokinin 1

ABSTRACT Bacillus anthracis edema toxin (ET), composed of protective antigen and an adenylate cyclase edema factor (EF), elicits edema in host tissues, but the target cells and events leading from EF-mediated cyclic-AMP production to edema are unknown. We evaluated the direct effect of ET on several cell types in vitro and tested the possibility that mediators of vascular leakage, such as histamine, contribute to edema in rabbits given intradermal ET. ET increased the transendothelial electrical resistance of endothelial monolayers, a response that is mechanistically inconsistent with the in vivo vascular leakage induced by ET. Screening of several drugs by intradermal treatment prior to toxin injection demonstrated reduced ET-induced vascular leakage with a cyclo-oxygenase inhibitor (indomethacin), agents that interfere with histamine (pyrilamine or cromolyn), or a neurokinin antagonist (spantide). Systemic administration of indomethacin or celecoxib (cyclo-oxygenase inhibitors), pyrilamine, aprepitant (a neurokinin 1 receptor antagonist), or indomethacin with pyrilamine significantly reduced vascular leakage associated with ET. Although the effects of pyrilamine, cromolyn, or aprepitant on ET-induced vascular leakage suggest a possible role for mast cells (MC) and sensory neurons in ET-induced edema, ET did not elicit degranulation of human skin MC or substance P release from NT2N cells in vitro. Our results indicate that ET, acting indirectly or directly on a target yet to be identified, stimulates the production/release of multiple inflammatory mediators, specifically neurokinins, prostanoids, and histamine. These mediators, individually and through complex interactions, increase vascular permeability, and interventions directed at these mediators may benefit hosts infected with B. anthracis.

scholarly journals Association of Bacillus anthracis Capsule with Lethal Toxin during Experimental Infection

2008 ◽  
Vol 77 (2) ◽  
pp. 749-755 ◽  
Author(s):  
J. W. Ezzell ◽  
T. G. Abshire ◽  
R. Panchal ◽  
D. Chabot ◽  
S. Bavari ◽  
...  
Keyword(s):  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Lethal Factor ◽  
Lethal Toxin ◽  
Size Exclusion ◽  
Terminal Phase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Animal Blood

ABSTRACT Bacillus anthracis lethal toxin (LT) was characterized in plasma from infected African Green monkeys, rabbits, and guinea pigs. In all cases, during the terminal phase of infection only the protease-activated 63-kDa form of protective antigen (PA63) and the residual 20-kDa fragment (PA20) were detected in the plasma. No uncut PA with a molecular mass of 83 kDa was detected in plasma from toxemic animals during the terminal stage of infection. PA63 was largely associated with lethal factor (LF), forming LT. Characterization of LT by Western blotting, capture enzyme-linked immunosorbent assay, and size exclusion chromatography revealed that the antiphagocytic poly-γ-d-glutamic acid (γ-DPGA) capsule released from B. anthracis bacilli was associated with LT in animal blood in variable amounts. While the nature of this in vivo association is not understood, we were able to determine that a portion of these LT/γ-DPGA complexes retained LF protease activity. Our findings suggest that the in vivo LT complexes differ from in vitro-produced LT and that including γ-DPGA when examining the effects of LT on specific immune cells in vitro may reveal novel and important roles for γ-DPGA in anthrax pathogenesis.

scholarly journals Protective Immune Response against Bacillus anthracis Induced by Intranasal Introduction of a Recombinant Adenovirus Expressing the Protective Antigen Fused to the Fc-fragment of IgG2a

Acta Naturae ◽  
2014 ◽  
Vol 6 (1) ◽  
pp. 76-84 ◽  
Author(s):  
D. N. Shcherbinin ◽  
I. B. Esmagambetov ◽  
A. N. Noskov ◽  
Yu. O. Selyaninov ◽  
I. L. Tutykhina ◽  
...  
Keyword(s):  
Immune Response ◽  
Bacillus Anthracis ◽  
Protective Antigen ◽  
Recombinant Adenovirus ◽  
Antibody Fragment ◽  
Human Adenovirus ◽  
Skin Lesions ◽  
Aerobic Bacterium ◽  
Protective Properties ◽  
Fc Fragment

Anthrax is a particularly dangerous infectious disease that affects humans and livestock. It is characterized by intoxication, serosanguineous skin lesions, development of lymph nodes and internal organs, and may manifest itsself in either a cutaneous or septic form. The pathogenic agent is Bacillus anthracis, a grampositive, endospore-forming, rod-shaped aerobic bacterium. Efficacious vaccines that can rapidly induce a long-term immune response are required to prevent anthrax infection in humans. In this study, we designed three recombinant human adenovirus serotype-5-based vectors containing various modifications of the fourth domain of the B. anthracis protective antigen (PA). Three PA modifications were constructed: a secretable form (Ad-sPA), a non-secretable form (Ad-cPA), and a form with the protective antigen fused to the Fc fragment of immunoglobulin G2a (Ad-PA-Fc). All these forms exhibited protective properties against Bacillus anthracis. The highest level of protection was induced by the Ad-PA-Fc recombinant adenovirus. Our findings indicate that the introduction of the Fc antibody fragment into the protective antigen significantly improves the protective properties of the Ad-PA-Fc adenovirus against B. anthracis.

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.

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