Evaluation of DNA vaccine encoding BCSP31 surface protein of Brucella abortus for protective immunity

Accepted Manuscript Evaluation of DNA vaccine encoding BCSP31 surface protein of Brucella abortus for protective immunity Waqar Imtiaz, Ahrar Khan, Shafia Tehseen Gul, Muhammad Saqib, Muhammad Kashif Saleemi, Asim Shahzad, Jianbao Dong, Riaz Hussain, Meiyan Shen, Xiaoxia Du PII:

S0882-4010(18)31228-2

DOI:

10.1016/j.micpath.2018.10.016

Reference:

YMPAT 3210

To appear in:

Microbial Pathogenesis

Received Date: 7 July 2018 Revised Date:

11 October 2018

Accepted Date: 11 October 2018

Please cite this article as: Imtiaz W, Khan A, Gul ST, Saqib M, Saleemi MK, Shahzad A, Dong J, Hussain R, Shen M, Du X, Evaluation of DNA vaccine encoding BCSP31 surface protein of Brucella abortus for protective immunity, Microbial Pathogenesis (2018), doi: https://doi.org/10.1016/ j.micpath.2018.10.016. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

EVALUATION OF DNA VACCINE ENCODING BCSP31 SURFACE PROTEIN OF

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BRUCELLA ABORTUS FOR PROTECTIVE IMMUNITY

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Waqar Imtiaza, Ahrar Khana,b,*, Shafia Tehseen Gula, Muhammad Saqiba,

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Muhammad Kashif Saleemia, Asim Shahzada, Jianbao DONGb,

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Riaz Hussainc, Meiyan SHENb and Du Xiaoxiab

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Faculty of Veterinary Science, University of Agriculture, Faisalabad-38040, Pakistan.

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b

Shandong Vocational Animal Science and Veterinary College, 88 Eastern Shengli Street,

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Weifang 261061, China

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c

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Bahawalpur-63100, Pakistan.

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University College of Veterinary and Animal Sciences, The Islamia University of Bahawalpur,

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Corresponding Author:

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*Ahrar Khan (

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Faculty of Veterinary Science, University of Agriculture,

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Faisalabad-38040, Pakistan

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https://orcid.org/0000-0001-5492-426)

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*Present Address:

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Shandong Vocational Animal Science and Veterinary College,

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88 Eastern Shengli Street, Weifang 261061, China

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E-mail: [email protected]; [email protected]

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ACCEPTED MANUSCRIPT Abstract

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Brucellosis is a highly contagious and zoonotic disease and has a considerable impact on

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animal health and economy of a country, principally in Pakistan, where rural income largely

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depends upon livestock farming and dairy products. The disease burden is more in

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underdeveloped/developing countries due to the low economy and limited access to the

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diagnostic facilities. In Pakistan, the prevalence of Brucella abortus is very high, so it is the

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need of the hour to control this disease through more advanced methods. This study was

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designed with the aim to construct the DNA based vaccine of genes encoding antigenic

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surface protein (BCSP31). For this purpose, the BCSP31 gene was amplified, purified and

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ligated in pTZ57R/T (cloning vector). Dubbed BCSP31-pTZ57R/T vector was transformed

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into competent cells (DH5α). After plasmid extraction, the plasmid and pET-28a vector was

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restricted with EcoRI and BamHI. Again, ligation was done and dubbed pET-28a-BCSP31

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transformed in E. coli (BL21). After expression, the protein was purified and used for

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evaluation of immunogenic response. The protective and immunogenic efficacy of the

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vaccine was evaluated in rabbits (n = 20). The rabbits were divided into four equal groups.

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Groups A-C were given purified protein diluted in normal saline @ 750, 1,500 and 3,000

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µg/0.2 mL, respectively through intraconjunctival route. Group D was given 0.2 mL normal

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saline through intraconjunctival route. Specific immunoglobulin G (IgG) responses were

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measured through indirect ELISA on a weekly basis. Moreover, log units of protection

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produced by DNA based vaccine in the rabbits (3.02) also indicated the protective efficacy of

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the DNA vaccine against B. abortus challenge. The response of this vaccine in rabbit

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suggested its potential effectiveness against Brucella abortus in large animals.

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Keywords: Brucella, DNA Vaccine, BCSP31 Surface Protein, Protective Immunity

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1.

Introduction Brucellosis is a contagious disease of domestic animals. The other names used for

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brucellosis are contagious abortion or Bang’s disease [1]. It can also affect humans and

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causes undulant fever or Malta fever in humans. It is an economically important disease of

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large animals [2]. The damages done by this disease in animals include weight loss, loss of

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young ones, decreased milk production and infertility. In Pakistan, Brucella abortus has been

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identified as the primary cause of bovine brucellosis in dairy animals including cattle and

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buffaloes [3,4].

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Brucellae are anaerobic, non-motile, gram-negative coccobacilli and facultative

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intracellular pathogens [5]. Brucellosis is caused by various species of genus Brucella those

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are categorized into six terrestrial and two marine species [6]. In humans, the transmission of

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brucellosis from animals occurs through the consumption of infected raw milk and milk

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products like cheese, yogurt and ice cream [7,8]. The disease is mainly transmitted through

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aborted fetuses, uterine secretions and fetal membranes [9]. In infected males, orchitis,

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epididymitis, and seminal vesiculitis are common entities and may lead to permanent sterility

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[10,11]. In female animals, deaths may occur as a consequence of retained fetal membranes

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or acute metritis [12].

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In developing countries, the impact of the disease is very high in animals due to poor

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health and management conditions [13-15]. Brucellosis has been eradicated from most of the

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parts of the world (Japan, New Zealand, Australia, Canada, USA, and Europe), however, it is

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still uncontrolled and endemic in Asia, Middle East, Africa and Latin America [16]. The

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highest incidence reported in bovines around the world ranges from 0.85-76 % [17-19]. There

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are four provinces in Pakistan, i.e., Punjab, Khyber Pakhtunkhwa (KPK), Balochistan and

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Sindh. Overall, seroprevalence of brucellosis in Pakistan ranged from zero to 76% in all

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livestock species [4,14,19-26]. The highest seroprevalence (76%) of brucellosis was reported

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ACCEPTED MANUSCRIPT in goats followed by bulk tank milk samples (42%) and buffalo samples (15%) from Punjab,

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Pakistan [19]. During the initial screening, it has been reported that seroprevalence of

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brucellosis in different species as 38.88 %, 26.19 %, 3.41 %, 0.23 % and 0.00 % in buffaloes,

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cattle, camels, goats, and sheep, respectively [27]. From Sindh and KPK, 21 % and 11 %

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brucellosis prevalence has been reported, respectively [23,24]. The overall prevalence of

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brucellosis in Baluchistan in large ruminants was 20 % [28] while in small ruminants 3.40 %

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[29]. Prevalence of brucellosis was affected by many factors like different climatic

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conditions, sex, age, species, geography, and diagnostic test applied [14,19,30].

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To control brucellosis, the S19 vaccine was the first vaccine used for cattle. It was

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live attenuated. When the live Brucella vaccine was administered, it produced protective

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immunity by the action of cytotoxic-T-lymphocytes. The main difficulty was to distinguish

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between vaccinated and infected cattle due to its identical serological response [31]. The

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RB51 vaccine also lacks its stability [32]. Currently, to control the brucellosis in livestock,

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the live attenuated Brucella vaccines are being used, but the problems related to their safety

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in humans, are the main factor of hindrance of their wide application [33-36].

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Brucella melitensis Rev.1 is a live attenuated vaccine, widely used in livestock to

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control Brucella infection [37]. The presence of smooth lipopolysaccharide (LPS) in the

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vaccine strain Rev1 may interfere with the discrimination between infected and vaccinated

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individuals and impair the test and slaughter strategy [38]. Live attenuated vaccines have the

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disadvantages of being pathogenic for animals and humans, causing abortion in pregnant

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animals, resistant to streptomycin and induction of specific antibodies against their LPS that

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interfere with the diagnostic tests [7,38].

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Alternatively, the subunit vaccines have proven effective and safe against B. abortus

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infection in animals and humans [39]. Various subunit [40], DNA [41,42] or live vector

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vaccines have been developed against B. abortus infection [43,44]. As the protective

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ACCEPTED MANUSCRIPT immunity against Brucella infection seems to be mediated both by humoral and cellular

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immunity, though cell-mediated immunity is expected to play a critical role in protection as

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Brucella is an intracellular pathogen. Role of CD4+ and CD8+ T lymphocytes, both of which

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secrete IFN-γ, has been reported in controlling the Brucella infection [45]. Animals

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immunized with DNA vaccine produced both types of immunity (humoral and cellular

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immunity) against many pathogens for different diseases [46,47], however, the efficacy of

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DNA vaccine against B. abortus is enhanced by encoding various genes like BCSP31, SOD

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and L7/L12 [48]. Similarly, DNA vaccine encoding BAB1_0270, BAB1_0278, BAB1_0278a

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[49,50] and recombinant flagellar proteins (FlgJ and FliN) were able to induce a better

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immune response and protection against B. abortus infection [51].

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In Pakistan, earlier studies were mainly based on the epidemiology of the disease in

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various species and association of risk factors, but no plan/method was developed to control

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the disease. As the test and slaughter control policy cannot be adopted in poor nations, so

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there is a dire need to control this disease through more advanced methods. The present study

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was designed with the aim to construct the DNA outer membrane protein vaccine of genes

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encoding antigenic surface protein (BCSP31) of B. abortus to enhance its efficacy for the

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better control of brucellosis. To evaluate the effectiveness of the newly developed DNA

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vaccine, the humoral immune response was measured in an animal model (rabbit).

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2.

Materials and methods

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2.1.

DNA based vaccine construction Brucella abortus strain 19 was obtained from the Veterinary Research Institute,

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Lahore, Pakistan for production of DNA based vaccine. According to restriction mapping of

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M20404 reported complete sequence of B. abortus encoding the antigenic protein of BCSP31

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following forward primer (AK-417: ATG AAA TTC GGA AGC AAA ATC CG and reverse

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primer (AK-418: TTA TTT CAG CAC GCC CGC TTC CT) were designed. DNA was

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extracted from B. abortus strain 19 utilizing DNA extraction kit (Favorgen®; Favorgen

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Biotech Corp, Ping-Tung, Taiwan). The coding regions for the gene of BCSP31 protein from

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B. abortus chromosomal DNA were amplified by using forward (AK-417) and reverse (AK-

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418) primers through conventional PCR. The reaction mixture (25 µL) was prepared by

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adding PCR master mix (12.5 µL), forward primer (1 µL), reverse primer (1 µL), DNA

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sample (2 µL) and water (8.5 µL). Then the samples were placed in a thermocycler

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(BioRad®) under cycling conditions as initial denaturation at the 95 oC for 3 mins, followed

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by 35 cycles of denaturation at the 95 oC for 30 secs, annealing at the 57 oC for 30 secs and

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extension at the 72 oC for 60 secs, final extension was carried out at the 72 oC for 10 mins.

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The primers amplified the BCSP31 gene and furnished an expected product of 990 bps

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visualized through gel electrophoresis. Positive amplicons were purified using PCR

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purification kit (GeneJET PCR Purification Kit, Catalog number:

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Scientific® Waltham, MA USA) following manufacturer protocol.

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K0701; Thermo

2.1.1. Cloning of BCSP31 gene in pTZ57R/T vector

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Using InsTAclone PCR Cloning Kit following the manufacturer protocol purified

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BCSP31 gene was ligated in pTZ57R/T cloning vector (InsTAclone PCR Cloning Kit,

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Thermo Fisher Scientific®, Waltham, MA USA). According to InsTAclone PCR Cloning Kit

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ACCEPTED MANUSCRIPT manufacturer protocol BCSP31 gene was inserted into pTZ57R/T (cloning vector) following

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the recommended protocol. The ligation mixture was prepared by using pTZ57R/T vector (3

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µL), Ligation buffer (6 µL), Amplified PCR product (4 µL), Water (16 µL) and DNA Ligase

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(1 µL) provided in the InsTAclone PCR Cloning Kit. To get the maximum number of

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transforms, overnight incubation was given at 4 °C. From the above-prepared ligation

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mixture, 3 µL was used for bacterial transformation.

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2.1.2. Transformation of BCSP31- pTZ57R/T Vector in E. coli-DH5α

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The ligation product (3 µL) was used for the transformation of E. coli DH5α

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competent cells (50 µL) prepared through the fresh culture of the E. coli DH5α using

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manufacturer protocol (InsTAclone PCR Cloning Kit, Thermo Fisher Scientific®, Waltham,

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MA USA). The transformed cells were cultured on LB agar plates supplemented with

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ampicillin and X-gal/IPTG at 37 ºC overnight. The culture was screened for blue-white

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colonies and white colonies were selected as probable clones. The white colonies were

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subjected to colony PCR and positive colonies were cultured in liquid broth for plasmid

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extraction with plasmid extraction kit (GeneJET Plasmid Miniprep Kit® Cat # K0502;

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Thermo Fisher Scientific®, Waltham, MA USA) and recombinant plasmid was dubbed as

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BCSP31 - pTZ57R/T cloning vector.

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2.1.3. Subcloning of BCSP31 gene in the expression vector The BCSP31 - pTZ57R/T cloning vector was restricted with BamHI and EcoRI that

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will liberate the band of 990 bps. Likewise, the pET-28a (+) expression vector was also

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restricted with same restriction enzymes. The reaction mixture for digestion of plasmid

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BCSP31 - pTZ57R/T and pET-28a (+) expression vector with EcoRI and BamHI was

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performed through adding 5 µL sample (vector/DNA), 1 µL restriction enzyme (EcoRI), 1

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ACCEPTED MANUSCRIPT µL of restriction enzyme (BamH1), 2 µL of buffer (10X), 11 µL of water (nuclease free). The

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mixture was incubated for 1 h at 37 ºC. By using electrophoresis on 1 % agarose gel, the

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digested vectors (BCSP31 - pTZ57R/T and pET-28a) were analyzed. Restricted DNA and

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restricted pET vector were extracted by using the gel extraction kit (GeneJET Gel Extraction

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Kit, Thermo Fisher Scientific®, Waltham, MA USA) from the gel. Utilizing InsTAclone PCR

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cloning kit the purified product was ligated into the restricted pET-28a vector following the

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manufacturer’s instruction making the recipe of ligation by adding pET-28a vector (3 µL),

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ligation buffer (6 µL), purified BCSP31 gene (4 µL), water (16 µL) and DNA ligase (1 µL).

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Reaction components were vortex and centrifugation for a few seconds. To get the maximum

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number of transforms, the ligation mixture was incubated at 4°C for 24 hours. For the

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bacterial transformation, 2.5 µL mixture was used.

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2.1.4. Transformation of Recombinant BCSP31 - pET-28a into E. coli (BL21) The 2.5 µL of ligated product of BCSP31 - pET-28a was added to 50 µL competent

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cells (BL21) prepared through fresh culture of the E. coli BL21 using manufacturer protocol

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(InsTAclone PCR Cloning Kit, Thermo Fisher Scientific®, Waltham, MA USA) and mixed

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carefully with the pipette tip and 30 mins incubation was given on ice. Heat shock to the cells

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was given for 60 secs at 42 °C in a water bath and cells were chilled immediately on ice for 2

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mins. 1 mL LB medium (without antibiotic) was added in the mixture and incubated for 60

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mins at 37°C with continuous shaking. Pre-warmed LB agar plates having kanamycin (50

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µg/mL) were taken and 200 µL cell suspensions were spread on them. Overnight incubation

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at 37 °C was given to these plates. A positive control (5 µL uncut plasmid with 100 µL

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competent cells) and a negative control (5 µL nuclease-free water with 100 µL competent

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cells) were also included in each transformation experiment. Five white colonies were

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analyzed for the presence of desired DNA insert by using colony PCR.

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2.1.5. Purification of desired protein The transformed cells were cultured in LB broth having kanamycin and allowed to

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grow overnight. The fully-grown culture was cultured in 2xYT broth supplemented with

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kanamycin upon initiation of log phase IPTG was added for induction of BCSP31 gene

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expression. After four hours of induction, the cells were harvested and lysed by sonication for

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assessment and purification of BCSP31 protein production and checked on SDS-PAGE. The

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functional BCSP31 protein was purified through ammonium sulfate precipitation (salting out)

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and chromatography (ion-exchange or hydrophobic interaction). The purity of protein was

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monitored by SDS-PAGE and the amount was quantified by Bradford protein assay. Purified

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protein was stored at -20 ˚C.

Experiments undertaken

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2.2.

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2.2.1. Humoral immune response against DNA vaccine

A total of 20 rabbits almost of the same age (6-8 months) and weight (950 to 1050g)

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were procured and kept in cages. Green fodder and water were available around the clock.

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After 5 days of acclimatization, these rabbits were divided into four equal groups. Groups A-

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C were given purified protein, respectively at 750, 1,500 and 3,000 µg/0.2 mL through

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intraconjunctival route for immunization [48]. Group D was administered 0.20 mL normal

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saline solution through intraconjunctival route and kept as control. Orbital blood was

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collected from each rabbit in each group on a weekly basis for three weeks after

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immunization. The sera were used to analyze the humoral immune response, i.e., specific

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immunoglobulin G (IgG) were measured by using the indirect ELISA [49]. The humoral

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immune response in the serum of immunized rabbits was measured in triplicate wells. By

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using the purified protein BCSP31 at a final concentration of 5 mg/mL in PBS having pH 7,

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the wells of the plates were coated overnight at 4 °C. The plates were saturated after 3 wash

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ACCEPTED MANUSCRIPT cycles with PBS for 2 hours at 25 °C with 150 µL blocking buffer having 2.5% casein. By

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using PBS having 0.1% Tween 20 the plates were washed. Serum samples (50 µL) from

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vaccinated groups were diluted serially in coated plates and incubated for 1 hour at 25 °C.

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The sera from the non-immunized rabbits were taken and used as negative control. Then,

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after 5 washes, 50 µL of goat biotinylated anti-rabbit immunoglobulins (Cat # ab97051;

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Abcam®, Cambridge, MA) was added and the plates were incubated for 60 mins at 25 °C. The

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plates were washed and the 50 µL streptavidin-horseradish peroxidase was added in the

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plates for the detection of IgG isotype and incubated at 25 °C for 60 mins. At the final step to

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check the activity of peroxidase the TMB (50-tetramethylbenzidine) in citrate phosphate

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buffer and 2 mM H2O2 was added in the plates. After 20 mins the reaction was stopped by

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adding stop solution (2 M H2SO4). Optical density was then measured through ELISA reader.

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ODs were read at 450 nm with an automatic microplate reader. The endpoint titer was

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defined as the highest dilution of serum that gave an absorbance value that exceeded an

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optical density of 0.050 and was twofold greater than that of the matched dilution of

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unvaccinated rabbit sera.

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2.2.2. Virulence strain challenge experiment

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Another group of 20 rabbits (divided into four equal groups) was kept for virulence

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strain challenge experiment. All the management conditions were the same as mentioned in

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the previous experiment. These rabbits were immunized with purified protein, respectively at

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750, 1,500 and 3,000 µg/0.2 mL through intraconjunctival route for immunization. Group D

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was administered 0.20 mL normal saline solution through intraconjunctival route. After 2

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weeks of immunization, each rabbit in each group was given infection with B. abortus (live

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culture procured from Veterinary Research Institute, Lahore, Pakistan) through conjunctiva

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@ 0.02 mL/animal having the bacterial concentration of 1 x 108/mL. All this work was

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ACCEPTED MANUSCRIPT carried out under strict biosafety measures. Infected animals were sacrificed 2 weeks after the

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challenge. Spleen from individual rabbit was homogenized in phosphate buffered saline

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(PBS), 10-fold serially diluted and plated in triplicate on Brucella Broth Agar. Plates were

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incubated at 37ºC in the air containing 5% carbon dioxide (CO2), and bacterial CFU was

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counted visually after 3 days of culture.

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2.3.

Statistical analysis

The data thus collected were analyzed using analysis of variance and different means

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of vaccinated and non-vaccinated groups at different times after inoculation were compared

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by Tukey’s test (p ≤ 0.05). A statistical analysis software Statistix 10® (Analytical Software,

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Tallahassee, FL, USA) was used for analysis.

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3.

Results

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3.1.

Vaccine construction A 990 bps BCSP31 gene fragment was amplified from the bacterial DNA by using

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primers (AK-417 and AK-418) visualized in gel documentation system after gel

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electrophoresis (Figure 1). Amplified gene was cropped from the gel and purified through the

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gel purification kit. Purified gene was ligated in cloning vector pTZ57R/T and recombinant

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vector (BCSP31-pTZ57R/T) was transformed in competent E. coli (DH5α). Ampicillin

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resistant transformed cells having recombinant vector (BCSP31-pTZ57R/T) grow as white

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colored colonies on LB plate under the action of X-Gal/IPTG after overnight incubation at 37

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°C. This showed the successful transformation of recombinant plasmid (BCSP31-pTZ57R/T)

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in white colonies of E. coli (DH5α).

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Approximately 90% colonies were white, out of these, five white colonies were

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analyzed for the presence of 990 bps insert. Four of five analyzed colonies contained the

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plasmid with the insert (Figure 2). Positive colonies were grown overnight in LB broth and

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recombinant vector was extracted through plasmid extraction kit and extracted recombinant

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vector (BCSP31-pTZ57R/T) was visualized on gel showing >3kb size vector (Figure 3).

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3.1.1. Sub cloning of cloned vector in expression vector

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The pET-28a (expression vector) and isolated plasmids (BCSP31-pTZ57R/T) were

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restricted with EcoR1 and BamHI restriction enzymes, which cut the pET-28a and flank the

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insert in the cloned vector (Figure 4). Restricted pET-28a and flanked insert was purified

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from the gel and ligated using a cloning kit. The ligated plasmid carrying insert (BCSP31-

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pET-28a) was transformed in BL21 competent cells. Transformed cells were then spread on

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pre-warmed LB agar plates having kanamycin (50µg/ml) and incubated overnight at 37 °C.

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Under the action of X-Gal/IPTG transformed cells were grown as white colonies on LB agar

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after overnight incubation. E. coli BL21 cells transformed with BCSP31-pET-28a plasmid was used for protein

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production. The transformed cells were cultured in 1-Liter broth with X-Gal/IPTG. After 16

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hours of incubation the cells were harvested by centrifugation. The cells were lysed by

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sonication and maximum protein was found insoluble while limited protein was found in

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soluble fraction. The insoluble fraction was subjected to urea denaturation for proper

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solubilizing. According to the Ni+2 affinity chromatography the maximum polyhistidine-

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tagged recombinant protein was eluted by 200 mM imidazole. According to post dialysis

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analysis of eluted protein maximum was found soluble. The soluble protein was stored at -

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20ºC (Figure 5).

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3.2.

Humoral responses generated after immunization

Rabbit immunization was carried out by vaccination of rabbit through

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intraconjunctival injection. Antibody titers against three different concentrations of the

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purified protein were determined to evaluate the humoral immune response in sera harvested

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from immunized rabbits at different time intervals after injection. The titer of specific

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immunoglobulins (IgG) against the antigen was significantly (p < 0.05) higher in vaccinated

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groups A-C as compared to group D (control group) in a dose dependent manner during

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different periods (day 7, 14 and 21) of the experiment (Table 1). The titer was the highest on

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day 21 of the inoculation in group C vaccinated with 3,000 µg/0.2 mL of purified protein.

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Specific antibodies showed an increasing trend after the first, second, and the third week in

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rabbits vaccinated with DNA vaccine with respect to the concentration of the vaccine.

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Rabbits vaccinated with low concentration of vaccine (750 µg/0.2 mL) dissolved in saline

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produced significantly (p < 0.05) lower antibody responses as compared to the rabbits of

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group C injected with high concentration (3,000 µg/0.2 mL) of vaccine. The rabbits of group

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D which were given the saline injection (control group) produced no antibodies during the

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whole duration of the experiment.

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3.3.

Virulence strain challenge experiment

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DNA based vaccine protected rabbits of vaccinated groups against Brucella infection.

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The levels of infection were evaluated by measuring CFU in the spleen (Table 2). Log10

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CFU (6.46 ± 0.20) was significantly (p < 0.05) higher in group injected normal saline 0.2 mL

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then given infection as compared to groups A, B and C those received DNA based vaccine @

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750, 1,500 and 3,000 µg/0.2 mL, respectively, then infection. Vaccinated rabbits displayed a

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significantly (p < 0.05) higher level of protection in a dose dependent manner than rabbits

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injected with normal saline (3.02 log units higher). DNA vaccine provided significantly (p <

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0.05) higher protection log units i.e., 3.02, 2.96 and 2.12, respectively in group C (3,000

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µg/0.2 mL), B (1,500 µg/0.2 mL) and A (750 µg/0.2 mL) as compared to control positive

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group (Table 2). These results indicated that the developed DNA vaccine has significantly (p

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< 0.05) higher degree of protection against Brucella infection.

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4.

Discussion Brucellosis is a highly contagious and zoonotic disease in the world [50]. Besides

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considerable impact on animal health it poses drastic economic losses through abortion and

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decreased milk production [19]. The disease affects all mammalian species, including

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domestic animals, marine animals [55] and freshwater animals and also the wildlife species

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[56,57].

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Brucellosis is more common in the developing countries, including Pakistan owning

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to insufficient measures for animal health programs and less availability of specific

334

diagnostic methods [1,58-61]. In a number of developed countries including Japan, New

335

Zealand, Australia, Canada, USA, and Europe the disease is eradicated. But the disease is still

336

uncontrolled and is endemic in Asia, Middle East, Africa and Latin America [15]. In most

337

parts of the world, the highest incidence was reported in bovines and the prevalence of this

338

disease in bovines in the world ranges from 0.85-76 %. Prevalence of brucellosis was

339

affected by many factors like different climatic conditions, sex, lactation, age, species,

340

geography and diagnostic test applied [1].

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To control brucellosis, S19 vaccine was the first vaccine used in cattle. It contains a

342

live attenuated virus [42] which produces protective immunity by the action of Cytotoxic-T-

343

lymphocytes [62]. The main difficulty was to distinguish between vaccinated and infected

344

cattle due to its identical serological response [30]. Similarly, RB51 vaccine, which is also

345

used to control the brucellosis lacks its stability and is cost effective [31]. Currently used

346

vaccines against brucellosis in animals are mainly live attenuated vaccines, but the main

347

hindrance of their wide application is the safety of humans and health related risks [62].

348

However, live attenuated vaccines have the disadvantages of being pathogenic for humans,

349

causing abortion in pregnant animals, resistant to streptomycin and induction of specific

350

antibodies against their LPS that interfere with the diagnostic tests [38,59]. Moreover, the

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available vaccines against Brucella infection are considered to have limited value because

352

they did not produce required protection in animals against Brucella infection [64-66]. In contrast, subunit vaccines are safe, well defined, nonviable and avirulent.

354

Therefore, they are the promising vaccine candidates against Brucella [67-69]. Immunized

355

animals with DNA vaccine produced both (humoral and cellular immunity) type of immunity

356

[46,47,70,71]. Different studies and experiments are going to develop a subunit or DNA

357

vaccines against Brucella infection in animals and humans which are safe and effective

358

against brucellosis [36,39,72].

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Keeping in view the problem of brucellosis in Pakistan, there is a dire need to control

360

the disease through effective control strategies. That is why the present study was designed to

361

construct a cost-effective and safer DNA based recombinant outer membrane protein vaccine

362

for the protection of the animals. The protection efficacy of the vaccine was carried out in

363

rabbit through intraconjuctival injection. The antibody titers against three different

364

concentrations of the vaccine were determined through humoral immune response from the

365

sera of immunized rabbits at different time intervals after injection (weekly basis). The titers

366

of specific immunoglobulins were strongly increased in the vaccinated groups with different

367

concentrations of the recombinant protein in a dose dependent manner as compared with the

368

control group.

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In the present study, we observed that the DNA based vaccine provided 3.02 log units

370

of protection in rabbits indicated the protective efficacy of the DNA vaccine against B.

371

abortus challenge. In other studies, DNA vaccines expressing single antigens have been

372

proven to induce certain levels of protection. Genes including L7/L12 and SOD, respectively,

373

induced 1.26 and 2.16 log units of protection [43,73]. Immunological responses against

374

different proteins encoded by genes, including Cu-Zn SOD [42] and ribosomal L7/L12 gene

375

[69] were studied in a mice model which showed significant protection against B. abortus by

Page 16 of 29

ACCEPTED MANUSCRIPT producing specific antibodies [74,75]. Similarly, recombinant L7/L12-TOmp31 also

377

produced titer of specific antibodies against Brucella infection [74]. Thus, in the published

378

literature, it is proved that DNA vaccine encoding various genes like BCSP31, SOD, L7/L12,

379

BAB1_0270, BAB1_0278, BAB1_0278a and recombinant flagellar proteins (FlgJ and FliN)

380

enhances humoral and cellular immunity against B. abortus [48-51].

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The noteworthy finding of this study is that the DNA vaccine provided high titers of

382

protection in rabbits. DNA vaccines expressing antigens have been proven to induce

383

appropriate cellular immune responses. As DNA vaccination method is a more efficient

384

method of vaccination to enhance the cellular and humoral response in animals and gave

385

better immunity against various pathogens. These data encouraged us to investigate the

386

protective efficacy of the DNA vaccine in other laboratory animals and domestic animals for

387

the better control of brucellosis.

388 389

Conclusions

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Trails of developed vaccine against Brucella abortus for immunization in laboratory

391

animals (rabbits) showed significantly (p < 0.05) increased humoral immune response in sera

392

harvested from immunized rabbits. The titer was the highest on day 21 of the inoculation in

393

group C vaccinated with 3,000 µg/0.2 mL of purified protein. Moreover, the DNA based

394

vaccine provided 3.02 log units of protection in rabbits indicated the protective efficacy of

395

the DNA vaccine against B. abortus challenge. Thus, this developed vaccine can be used in

396

for protection against brucellosis in large animals, however, field trials are suggested before

397

its use. More studies are needed to explain the different immune pathways.

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Conflicts of interest

400

None.

401 402

Funding

403

Financial support vide Grant # 20-1795/R&D/10 of Higher Education Commission,

404

Islamabad, Pakistan is highly acknowledged.

405 406

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vaccine, Mol. Immunol. 49 (2011) 175-184. [70]

W.K. Kim, J.Y. Moon, S. Kim, J. Hur, Comparison between immunization routes of

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live attenuated Salmonella typhimurium strains expressing BCSP31, Omp3b, and

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SOD of Brucella abortus in murine model, Front. Microbiol. 7 (2016) 550.

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[71]

G.Z. Lin, Y.Z. Liu, K.Z. Cai, J.L. Liu, Z.R. Ma, Immunogenicity of recombinant adenovirus co-expressing the L7/L12 and BCSP31 proteins of Brucella abortus,

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Kafkas Univ. Vet. Fak. Derg. 24 (2018) 211-217. [72]

Vaccinol. 1 (2011) 61-88.

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E. Kurar, G.A. Splitter, Nucleic acid vaccination of Brucella abortus ribosomal L7/L12 gene elicits immune response, Vaccine 15 (1997) 1851-1857.

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R. Strugnell, F. Zepp, A. Cunningham, T. Tantawichien, Vaccine antigens, Perspect.

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[74]

C. Beata, N. Walters, T. Thornburg, T. Hoyt, X. Yang, D.W. Pascual, DNA

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vaccination of bison to brucella antigens elicits elevated antibody and IFN-γ

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responses, Bacteriol. Mycol. 47 (2011) 501-510.

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[75]

M. Golshani, S. Rafati, S.D. Siadat, M. Nejati-Moheimani, F. Shahcheraghi, A.

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Arsang, S. Bouzari, Improved immunogenicity and protective efficacy of a divalent

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DNA vaccine encoding Brucella L7/L12-truncated Omp31 fusion protein by a DNA

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priming and protein boosting regimen, Mol. Immunol. 66 (2015) 384-391. Page 24 of 29

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Figure 1:

Photograph of gel showing a fragment of amplified product at 990 bps (lane 1). Lane 2 was negative control and lane 3, 100 bps plus DNA Ladder.

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Figure 2:

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Colony PCR showing desired gene of interest of 990 bps. Lane description from left side: Lane 1 showing Ladder (100 bps), lane 2 indicating negative

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control and lanes 3-4 showed positive transforms through colony PCR.

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Figure 3:

Gel electrophoresis image of BCSP31-pTZ57R/T. Lane description: Lane 1 and 4, 100 bps ladder, lane 2 and 3 showing plasmid of >3kb of cloned vector

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(BCSP31- pTZ57R/T cloned vector).

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Figure 4:

Restriction analysis of cloned BCSP31-pTZ57R/T and pET-28a expression

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vector. Lane description: Lane 1 = 1kb ladder, lane 2 = restricted pET-28a

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vector of 5kb and lane 3 = BCSP31 of 990 bps

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Figure 5:

Coomassie stained 12% polyacrylamide gel displaying purification scheme of BCSP31 protein. M

Scientific, USA)

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Protein Marker (PageRuler™ Plus Prestained Protein Ladder, Thermo

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Total cell lysates of BCSP31-pET-28 recombinant vector containing E. coli BL21(DE3) pLysS cells Page 26 of 29

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2

Total cell lysates of BCSP31-pET-28 recombinant vector containing E. coli BL21(DE3) pLysS cells after 0.4mM IPTG induction

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3

Total insoluble proteins (precipitated portion) of induced cells

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4

Total cytoplasmic protein (soluble portion) of induced cells

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5

Eluted fraction from Ni+2-chromatography representing purified

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protein

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ACCEPTED MANUSCRIPT Table 1: Humoral titers of rabbits vaccinated with DNA vaccine prepared from Groups

Humoral titer at experimental day 7

14

21

A (750 µg/0.2 mL)

0.206 ± 0.028c

0.902 ± 0.160c

1.663 ± 0.075c

B (1,500 µg/0.2 mL)

0.361 ± 0.050b

1.147 ± 0.065b

1.916 ± 0.163b

C (3,000 µg/0.2 mL)

0.533 ± 0.048a

1.488 ± 0.089a

2.445 ± 0.138a

D (Normal saline 0.2 mL)

0.023 ± 0.010d

0.025 ± 0.010d

0.023 ± 0.010d

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Brucella abortus strain 19 was used for DNA based vaccine production. DNA extracted and

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BCSP31 protein from B. abortus chromosomal DNA identified by specific primers and

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amplified. By opting various steps of ligation, transformation, desired DNA insert was

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identified and then purified. Then immunization of rabbits was performed. Groups A-C were

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given purified protein respectively at 750, 1,500 and 3,000 µg/0.2 mL through

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intraconjunctival route. Group D served as control and received 0.20 mL normal saline

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through intraconjunctival route. Blood samples were collected from the rabbits on weekly

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basis and specific immunoglobulins were measured by using the indirect ELISA. Data thus

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collected were analyzed. Values (Mean + SD) bearing different alphabets in a column differ

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significantly (p < 0.05).

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Table 2: Bacterial count in spleen showing protection by the DNA based vaccine against B.

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abortus in rabbits Log10 CFU

Log Units of Protection

A (750 µg/0.2 mL)

4.34 ± 0.30c

2.12

B (1,500 µg/0.2 mL)

3.50 ± 0.15c

C (3,000 µg/0.2 mL)

3.44 ± 0.18b

D (Normal saline 0.2 mL)

6.46 ± 0.20a

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DNA based vaccine of Brucella abortus strain 19 was produced. Rabbits of groups A-C were

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given purified protein respectively at 750, 1,500 and 3,000 µg/0.2 mL through

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intraconjunctival route for immunization. Group D received 0.20 mL normal saline. After 2

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weeks of immunization, rabbits in each group were given infection with live B. abortus

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through conjunctiva. Two weeks post infection, rabbits were sacrificed. Homogenates from

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spleen were cultured on Brucella Broth Agar, then bacterial CFU were counted. Data thus

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collected were analyzed. Values (Mean + SD) bearing different alphabets in a column differ

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significantly (p < 0.05).

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Brucellosis is a highly contagious and zoonotic disease and causes huge economic losses in Pakistan. A DNA based vaccine of genes encoding antigenic surface protein (BCSP31) was produced.

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The protective and immunogenic efficacy of vaccine was evaluated in rabbits.

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Specific immunoglobulin G (IgG) responses measured by indirect ELISA indicated significantly high immunogenicity.

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Log units of protection produced by DNA based vaccine in the rabbits (3.02) also

The response of this vaccine in rabbit suggested its potential effectiveness against

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Brucella abortus in large animals.

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indicated the protective efficacy of the DNA vaccine against B. abortus challenge.