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Pdf 695.43 K Iran. J Biotechnol. January 2021;19(1): e2618 DOI: 10.30498/IJB.2021.2618 Research Article Efficient Immunization of BALB/c Mice against Pathogenic Brucella melitensis and B. ovis: Comparing Cell-Mediated and Protective Immune Responses Elicited by pCDNA3.1 and pVAX1 DNA Vaccines Coding for Omp31 of Brucella melitensis Naser Harzandi1*, Haniyeh Aghababa2,3, Nima Khoramabadi2, Termeh Tabaraie4 1Department of Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran. 2Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. 3Department of Molecular Medicine and Pathology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand. 4Department of Cardiology, Charité Medical University of Berlin, Berlin, Germany. *Corresponding author: Naser Harzandi, - Department of Microbiology, Karaj Branch, Islamic Azad University, Karaj, Iran. Tel:+98-2634182637, Fax:+98-2634418156, E-mail: [email protected] Background: Brucella spp. are intracellular pathogens, therefore cell-mediated immunity is the main response to inhibit survival and growth of the bacteria in vertebrate host. Objective: Many eukaryotic plasmid vectors are being used in setting up DNA vaccines which may show different efficiencies in same conditions. This is important in designing the vaccines and immunization strategies. We looked into the probable differences of immune responses induced by different eukaryotic DNA plasmid vectors (pcDNA3.1 and pVAX1) harboring the same Omp31 gene of B. melitensis. Materials and Methods: Female BALB/c mice were immunized with pcDNA-omp31 and pVAX-omp31 and further boosted with recombinant Omp31. Subclasses of specific serum IgG against the rOmp31 were measured by ELISA. Cytokines responses to rOmp31 in Splenocyte cultures of the immunized mice were evaluated by measuring the production of IL- 4, IL-10, IL-12 and IFN-γ. Protective responses of the immunized mice were evaluated by intraperitoneal challenge with pathogenic Brucella melitensis 16M and Brucella ovis PA76250. Results: Both DNA vaccine candidates conferred potent Th1-type responses with higher levels of cytokines and immunoglobulins observed in mice immunized with pVAX-omp31. Although pcDNA-omp31 and pVAX-omp31 both elicited protective immunity, mice immunized with the latter showed a higher protection against both B. melitensis and B. ovis PA76250. Conclusion: The results of this study highlight the significant differences between efficiency of diverse plasmid backbones in DNA vaccines which code for an identical antigen. Comparing various plasmid vectors should be considered as an essential part of the studies aiming construction of DNA vaccines for intracellular pathogens. Keywords: Brucella, DNA vaccine, Omp31, pCDNA3.1, pVAX1 1. Background after consumption of contaminated dairy or food or by Brucella spp. are Gram-negative, facultative contacts with infected animals (5). There is no licensed intracellular pathogens and cause brucellosis in human vaccines available for prevention of human brucellosis and animals. This pathogen is mainly localized at the and disease prevention principally relies on control reticuloendothelial system of vertebrate hosts (1-3). of animal brucellosis by vaccination (6, 7). The most B. melitensis is the most pathogenic member of the widely used vaccines for control of animal brucellosis genus and responsible for severe disease in humans, are the live attenuated strains, B. melitensis Rev1, and B. although the preferred hosts are goats, sheep, cows, abortus S19 (8). Live attenuated vaccines are pathogenic dogs and camels (4). Brucella infections occur mainly for humans; they also cause serological interference Copyright © 2021 The Author(s); Published by National Institute of Genetic Engineering and Biotechnology. This is an open access article, distributed under the terms of the Creative Commons Attribution-NonCommercial 4.0 International License (http://creativecommons.org/licenses /by-nc/4.0/) which permits others to copy and redistribute material just in noncommercial usages, provided the original work is properly cited. Harzandi N et al. with diagnostic methods in vaccinated animals (9, 10). collection at Pasteur Institute of Iran. E. coli DH5α routinely Brucella pathogens can escape recognition by the host is cultured using LB medium. Brucella melitensis Rev1, innate immune responses and further use sophisticated B. melitensis 16M and B. ovis PA76250 were stored in strategies to avoid intracellular destruction after being culture collection at Department of Bacteriology (Tarbiat phagocytosed by host macrophages. This, enables them Modares University). Brucella strains were cultured to survive and establish a persistent infection (2, 11, routinely on Brucella agar (Merck) and incubated at 37 12). Since these pathogens survive in macrophages, °C. These pathogenic bacteria were handled according to cell-mediated immunity is necessary for activation of biosafety level 2 practices and regulations. infected macrophages and clearance of the pathogen and formation of active CD8+ cytotoxic T-lymphocytes 3.3. Recombinant Omp31 (2, 13). Many reports are available on using subunit Recombinant Omp31 (rOmp31) was previously vaccines consisting of Brucella outer membrane produced in our lab and preserved in -80 °C as a proteins and their ability to elicit Th1-type responses concentrated stock of ~20±1 mg.mL-1. and partial protection against pathogenic strains (14- 16). Many protein antigens including outer membrane 3.4. Construction and Preparation of DNA Vaccines proteins and intracellular ones reported to induce The complete coding sequence of Omp31 was inserted potent cytokine and antibody responses especially between BamHI and XhoI recognition sequences in IFN-γ, IL-12 and IgG2a - immunological mediators pVAX1TM (InvitrogenTM, V260-20) and pcDNATM3.1 which are important for inhibiting Brucella infection (InvitrogenTM, V790-20). Specific primers including in hosts (17). The need for induction of more potent restriction sites at the 5’ ends were used (bmO31dnF- cell-mediated immunity has led some researchers to 5’-AAT GGA TCC ACC ACC ATG AAC TCC GTA use DNA vaccines as they are believed to elicit Th1- ATT TTG GCG TCC ATC-5› and bmO31dnR- 5’-ACA type responses (18-22). A neglected factor remains to CTC GAG TTA GAA CTT GTA GTT CAG ACC GAC be investigated which is the influence of the plasmid GCG-5›) with the Kozak consensus sequence considered vector used for constructing the DNA vaccine. Different before the ATG codon (underlined) (26). Recombinant plasmid vectors are used to compose DNA vaccines and plasmids named as pVAX-omp31 and pcDNA-omp31 they may have variable efficiencies in stimulating host were confirmed by sequencing. Recombinant plasmids immune system. This could be of crucial importance to were amplified inE. coli DH5α host and were extracted enlighten whether it is necessary to compare different and purified by EndoFree Plasmid Giga Kit (QIAGEN, DNA plasmid backbones for a single antigen or not. Catalog no. 12391) according to the manufacturer instructions. Quality and quantity of the purified plasmids 2. Objectives were assessed using NanoDrop spectrophotometer This study was aimed to present a research model for (NanoDropTM 2000; Thermo Sientific). To confirm the exploring the efficiency of different eukaryotic DNA recombinant plasmids can express the inserted omp31 plasmids (pVAX1TM and pcDNATM3.1) for eliciting cell- gene, they were separately transfected to the COS-7 mediated responses against Omp31 (23, 24) – a well- cell line (ATCC CRL-1651, Pasteur Institute of Iran) by studied antigen of Brucellae – as DNA vaccine. In this Lipofectamine® 2000 reagent (Life Technologies) and model, the immunogenic Omp31 is the constant antigen protein expression was traced through western blotting to study the effect of plasmid vector backbone selection as described previously (24, 27). Rabbit polyclonal on immunological responses elicited in BALB/c mice. antiserum against rOmp31 (Anti-Omp31; produced in our laboratory) (1:5000) was used for tracing Omp31 3. Materials and Methods expression in transfected COS-7 cells. Membranes were then treated with goat anti-rabbit IgG (Sigma) 3.1. Animal Model and after being washed thoroughly, developed with Six- to eight-weeks old female BALB/c mice were enhanced chemiluminescent (ECL) substrate under acquired from Laboratory Animal Production Center standard conditions. Appearance of fluorescent bands (Pasteur Institute, Iran). Mice were maintained under was recorded on radiographic films (28). standard laboratory conditions and kept one week for adaptation before experiments (25). 3.5. Immunization of Mice Six- to eight-week old female BALB/c mice were 3.2. Bacterial Strains and Culture Conditions anesthetized with Ketamine/Xylazine intraperitoneally Escherichia coli DH5α was obtained from the culture and immunized intramuscularly with 100μg of Iran. J Biotechnol. January 2021;19(1): e2618 41 Harzandi N et al. pcDNA-omp31 (Group I), pVAX-omp31 (Group II), 24-well plates were examined by specific ELISA for intact pcDNA3.1 (Group III) or pVAX1 (Group IV) in mouse IL-4, IL-10, IL-12 and IFN-γ (R&D) as triplicate separate groups of 15 mice. Mice were injected with for each mouse. the same plasmids four times in two-week intervals (24). Thereafter, mice which were immunized with 3.8. Protection Assessment pcDNA-omp31 (Group I) and pVAX-omp31 (Group Four weeks after the last booster dose, 8 mice from II) were boosted
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