Brucella melitensis VirB12 recombinant protein is a potential marker for serodiagnosis of human brucellosis

The numerous drawbacks of current serological tests for diagnosis of brucellosis which mainly results from cross reactivity with LPS from other gram-negative bacteria have generated an increasing interest to find more specific non-LPS antigens. Previous studies had indicated that Brucella VirB12 protein, a cell surface protein and component of type IV secretion system, induces antibody response during animal infection. However, this protein has not yet been tested as a serological diagnostic marker in human brucellosis. Recombinant VirB12 protein was prepared and evaluated the efficacy of it in an indirect enzyme-linked immunosorbent assay (ELISA) for brucellosis with sera collected from different region of Iran and the results were compared with a commercial ELISA kit. Sera from human brucellosis patients strongly reacted to the purified recombinant VirB12. The sensitivity, specificity, accuracy, negative predictive value and positive predictive value of recombinant VirB12-based ELISA related to the commercial-ELISA method were 87.8, 94, 90, 80 and 96.6% respectively. We concluded that antigenic VirB12 have a property value that can be considered as a candidate for using in serodiagnostic tests for human brucellosis.


Background
Brucella spp. is a facultative intracellular pathogen that can be involved with many tissues and organs leading to a chronic infection, Brucellosis, in animals and humans [1][2][3][4][5]. Brucellosis is caused by several species of the genus Brucella including Brucella abortus, Brucella melitensis, Brucella suis, Brucella canis, Brucella ovis, and Brucella neotomae [6][7][8][9]. B. melitensis is the most frequently isolated species which is endemic in many developing countries [10,11]. Clinical manifestations of brucellosis are very similar to other febrile diseases; therefore, the clinical diagnosis of this disease remains a challenge [12]. Common laboratory tests include either bacteriological culture of the pathogen or serological titration of anti-Brucella antibody. Although the gold standard test is said to be bacteriological isolation, the success rate of blood cultures is around 70-80% of cases in acute disease produced by B. melitensis [13]. This rate is very much lower in chronic cases. Various factors including disease duration, isolation method, and prior antibiotic intake can drastically impact the bacteriological diagnosis [2]. Serum agglutination serological tests often rely on detection of antibody against smooth lipoplysaccharide (LPS) present on bacterial cell surface. Due to existence of extensive cross reactivity with LPS from other gram negative bacteria, the specificity of these approaches are poorly suited for use in general diagnostic laboratories [2,[14][15][16][17][18]. The drawbacks of these classical serological tests have generated an increasing interest in finding more  [2,19,20]. In this regard, the outer membrane proteins of Brucella species have been proposed as appropriate candidate for antigenic component. Rolan et al. [21] noted that Brucella VirB12 protein, a component of type IV secretion system, which is situated on bacterial cell surface, is expressed during infection and induces an antibody response in cattle. However, they had not looked at any possible humoral response in humans during active or chronic infections. In a previous communicate we reported cloning of the virB12 gene of B. melitensis [22]. In the present study, we are reporting expression and purification of the recombinant VirB12 protein. Furthermore, the seroreactivity of the purified recombinant virB12 of B. melitensis was evaluated with human serum samples in an indirect enzyme-linked immunosorbent assay (ELISA) for brucellosis.

Preparation and recognition of Brucella VirB12 recombinant protein
The plasmid construct pET28a-VirB12 was purified from an overnight culture of Escherichia coli DH5α cell.

Immunoreactivity of recombinant Brucella purified rVirB12 to human sera using western blotting
Recombinant protein was subjected to 12% gradient SDS-PAGE with the molecular protein marker and was transferred from the unstained polyacrylamide gel onto 0.45 µm nitrocellulose membrane. The blotted membrane blocked using 5% skim milk in TSBST 1%. After washing with TSBT, transferred proteins were immunostained with serum obtained from human brucellosis infection at a dilution of 1/1000. Secondary antibody conjugated to horseradish peroxidase was used in the assay. The reaction was visualized with enhanced chemiluminescence (ECL) and ECL system (GE Healthcare. Uppsala, Sweden).

Production of polyclonal anti-virB12 recombinant protein in rabbit
A mature white New Zealand rabbit was immunized with purified VirB12 recombinant protein. Immunization was performed according to the protocol of Hay et al. [23].
In the first i.m injection, mixture of 250 µg recombinant protein with the same volume of Freund's complete adjuvant was injected. For second injection the rabbit was injected with 125 µg recombinant protein with the same volume of Freund's incomplete adjuvant, 1, 2 weeks later. Finally, 2 weeks after the last immunization, blood was collected and sera separated.

ELISA test with commercial kit
To investigate the serological status of the samples from human and evaluate the quality of the detection method, all of 100 serum samples were subjected to ELISA kit (IBL, Germany

Evaluation of ELISA method against commercial ELISA
The sensitivity, specificity, positive predictive value and negative predictive value of recombinant VirB12 ELISA for serodiagnosis of brucellosis were evaluated in comparison to commercial ELISA kit.

Preparation and recognition of Brucella VirB12 recombinant protein
The  (Fig. 1). The recombinant VirB12 was mostly accumulated in the cytoplasm of E. coli transformant as inclusion bodies, which could only by extracted and purified under denaturing condition using 8 M urea. Following purification of rViB12 by Ni-NTA affinity chromatography, the yield of the purified protein was estimated by Bradford method to be about 0.6 mg/ml of culture (Fig. 2).

Immunoreactivity of recombinant Brucella purified rVirB12 to human sera using western blotting
To evaluate immune reactivity of the purified protein, western blot was performed with sera (1/1000 dilution) from Brucella infected human. The sera from human reacted to purified recombinant VirB12. Antigenicity of the expressed protein was confirmed by western blot analysis using patient sera. The specific antibody response from five patient sera was observed. Serum samples from normal individual was also tested as negative control and no anti VirB12 antibodies were detected. Additionally, there was no reactivity between the expressed pET28a in E. coli Bl21 (DE3) with patient serum (Fig. 3).

Production of polyclonal anti-virB12 recombinant protein in rabbit
Increasing the antibody titers to high level after third boost was confirmed the good immunogenicity of VirB12. The rabbit antiserum was able to recognize the virB12 in Brucella lysate and the purified recombinant VirB12.

Evaluation of recombinant VirB12 ELISA
Immune reactivity of recombinant virB12 was determined using an indirect ELISA. A total 100 serum samples (66 commercial-ELISA positive and 34 commercial-ELISA negative) were collected from different region of the country and were tested by indirect ELISA against the recombinant VirB12 antigen. The VirB12 ELISA was considered positive only if the mean absorbance value was greater than two SDS above the mean value for healthy serum samples. The cut of value was 0.325 (mean, 0.17 SD, 0.077). Out of 100 serum samples tested, 60 (60%) were positive and 40 (40%) were negative by ELISA. The sensitivity, specificity, accuracy, negative predictive value and positive predictive value of recombinant VirB12-based ELISA related to the commercial-ELISA method are shown in Table 1.

Discussion
Despite using vaccination in livestocks, brucellosis remains as an endemic infection in many developing countries such as Iran. Human and animal infection with Brucella inflicts an enormous cost to people and government [24]. Since culture method for diagnosis of Brucellosis is time consuming and has infection risk for laboratory workers, serological tests are commonly used for clinical diagnosis [25]. Due to cross reactions with many other Gram negative bacteria in most routinely used LPS-based serological tests, identification of more specific Brucella spp. protective antigens can be useful for developing serological tests which avoid the drawbacks of classical ones. The outer membrane proteins in Gram negative bacteria have particular significance as a potential immunity target [26]. Brucella VirB12 is one of these structural protein which is expressed during Brucellosis infection. Investigators had demonstrated that VirB12 protein acts as an immunogen and induces partial immunity in animal models [27]. However, there is not any report concerning antigenicity of the recombinant VirB12 protein in human. VirB12 protein is located on the bacterial cell surface and is highly conserved among Brucella isolates. Therefore, VirB12 can be considered as an antigenic candidate for serological diagnosis of brucellosis. His tag sequence in the target protein also provides the possibility for purification through Ni-NTA affinity chromatography. Metal affinity chromatography was subsequently performed to purify VirB12, producing amount 3 mg of pure recombinant protein per liter of bacterial culture. Antigenicity of the purified protein component was evaluated in immunoblotting with human brucellosis sera. Data showed that the recombinant VirB12 protein could be detected as an antigenic component by sera from acute phase of human brucellosis. By using western blot analysis, we showed that the recombinant VirB12 did not shown any cross reactivity with normal human sera. There was also no serological interference related to the fused amino acids. The results, in consist of other researcher showed that there was no interference related to fused amino acids [12]. To examine the practical value of VirB12, 100 positive and negative serum samples from different part of country used in virB12 antigen based indirect ELISA for detection of antibody response against Brucella. The finding demonstrated that the virB12 recombinant protein had good immunogenicity and indicates that the availability of virB12 to the immune system. The Pearson correlation coefficient of the virB12 antigen-based indirect ELISA against commercial ELISA, a test based on LPS antigen was 0.73 (P < 0.001). The sensitivity of the virB12-ELISA was 87.8% and specificity was 94%. The accuracy in all samples reached 90%. The sensitivity and specificity of virB12 ELISA in comparison with commercial ELISA confirmed the fact this system comparable to the commercial assay available as a potential immunogenic marker for screening of brucellosis and needs further evaluation.

Conclusions
In summary, our results showed that VirB12 was expressed at high amounts in E. coli Bl21 and could be purified by Ni-NTA affinity chromatography. This recombinant protein reacted strongly with sera from patient with brucellosis by western blot. Thus we concluded that VirB12 is antigenic and has property to be considered as a suitable candidate for development of more specific diagnostic tests.