Functional Characterization and Evaluation of In Vitro Protective Efficacy of Murine Monoclonal Antibodies BURK24 and BURK37 against Burkholderia pseudomallei

Burkholderia pseudomallei, the causative agent of melioidosis has been recognized by CDC as a category B select agent. Although substantial efforts have been made for development of vaccine molecules against the pathogen, significant hurdles still remain. With no licensed vaccines available and high relapse rate of the disease, there is a pressing need for development of alternate protection strategies. Antibody-mediated passive protection is promising in this regard and our primary interest was to unravel this frontier of specific mAbs against Burkholderia pseudomallei infections, as functional characterization of antibodies is a pre-requisite to demonstrate them as protective molecules. To achieve this, we designed our study on in vitro-based approach and assessed two mAbs, namely BURK24 and BURK37, reactive with outer membrane proteins and lipopolysaccharide of the pathogen respectively, for their ability to manifest inhibitory effects on the pathogenesis mechanisms of B. pseudomallei including biofilm formation, invasion and induction of apoptosis. The experiments were performed using B. pseudomallei standard strain NCTC 10274 and a clinical isolate, B. pseudomallei 621 recovered from a septicemia patient with diabetic ailment. The growth kinetic studies of the pathogen in presence of various concentrations of each individual mAb revealed their anti-bacterial properties. Minimal inhibitory concentration and minimal bactericidal concentration of both the mAbs were determined by using standards of Clinical and Laboratory Standards Institute (CLSI) and experiments were performed using individual mAbs at their respective bacteriostatic concentration. As an outcome, both mAbs exhibited significant anti-Burkholderia pseudomallei properties. They limited the formation of biofilm by the bacterium and completely crippled its invasion into human alveolar adenocarcinoma epithelial cells. Also, the mAbs were appreciably successful in preventing the bacterium to induce apoptosis in A549 cells. The present study design revealed the protection attributes possessed by BURK24 and BURK37 that has to be further substantiated by additional in vivo studies.


Introduction
Burkholderia pseudomallei infections, collectively termed as melioidosis, are endemic in Southeast Asia and northern Australia. The disease is fatal with a mortality rate of about 40% among treated patients [1]. Clinical manifestations in case of melioidosis vary from acute febrile illness, chronic localized pathologic symptoms to chronic septicaemia resulting in septic shock [1,2]. In Thailand, B. pseudomallei infections are predominantly manifested as community-acquired septicaemia. The infection recommends longer course of intensive antibiotic therapy that comprises of intravenous administration of ceftazidime for 10 days, followed by oral antimicrobial therapy comprising of trimethoprim and sulfamethoxazole for 3-6 months [3]. However, despite of adequate antibiotic therapy the pathogen is capable of undergoing latency for decades and relapse in later stages of immune suppressed conditions [4]. Furthermore, intrinsic resistance of B. pseudomallei to multiple drugs and emergence of resistant strains against the above mentioned antibiotic regimen has become a challenge for curing melioidosis [5,6,7,8]. No substitutional protecting molecules or human vaccines for the disease currently exist. Therefore, there is a high priority for development of alternative antimicrobial molecules that can efficiently confer passive protection against the disease among the susceptible hosts. Also, generation of immediate protection in infected hosts is required in order to overcome B. pseudomallei infections.
To combat the aforementioned challenge, passive antibodymediated protection would be a better alternative as antibodies upon passive administration confer immediate and specific immunity to the susceptible and/or infected host. Additionally, antibodies have minimal toxicity as they are natural in origin. Monoclonal antibodies (mAbs) hold great promise in this view, since the antimicrobial activity conferred by them shall be highly specific. Thus, they are also growing faster as new class of therapeutic and passive protection molecules [9,10].
Pathogenesis mechanisms of B. pseudomallei are being explored intensively both in vitro and in vivo. As a pathogen, it has several strategies to survive and establish pathogenesis in host system. The key factor of its pathogenesis is the ability to survive intracellularly in both phagocytic and non-phagocytic cells [11]. This feature makes the pathogen successful in escaping host immune responses, establish chronic infections, latency and multi-drug resistance. Once invaded into host cells, B. pseudomallei induces cell-to-cell fusion resulting in formation of multinucleated giant cells (MNGC) [12]. This is followed by the induction of apoptosis in infected cells by unknown mechanism [13]. The inherent ability of the pathogen to form biofilm is also likely to be responsible for persistence of infection [14]. B. pseudomallei has demonstrated resistance to antimicrobial drugs when grown in conditions that induce biofilm formation [15]. Development of specific antibodies against the bacterial components involved in adherence and invasion into host cells might possibly prevent the pathogen to establish pathogenesis and in turn confer passive protection in susceptible and infected hosts. Earlier works reported on similar lines implied the promise of structural virulent factors present on the surface of bacteria for the purpose, since location of the antigens plays an important role in determining the effectiveness of the antibodies [16,17,18]. Further, the antibodies, when bound to the surface-expressed antigens may enhance phagocytosis [19] or trigger complement fixation resulting in subsequent killing of internalized bacteria [20]. Targeting a virulence factor that plays a key role in the former mentioned pathogen-mechanisms appears to be promising.
However, according to a recent review, passive immunization against B. pseudomallei using mAbs raised against surface expressed molecules including capsular polysaccharide (CPS), lipopolysaccharide (LPS) and exopolysaccharide provide significant but not complete protection in murine models [21]. Also, using mAbs raised against CPS and LPS of B. pseudomallei in different studies have shown partial protection in murine models [22,23]. The mAb-based passive immunization studies reported earlier failed to confer complete infection sterilization [24] and protection against different challenge routes [25]. Prior to in vivo challenge studies, characterization of mAbs for their functional ability to block the pathogen mechanisms and also elucidation of the mode of protection they confer to the host is considerable in selection of promising monoclonal antibody to fight against B. pseudomallei infections.
With this background, we attempted to develop mAbs possessing protective properties against B. pseudomallei infections. Here, our principal goal was to systematically investigate the ''functional properties'' of specific monoclonal antibodies in conferring protection against B. pseudomallei infections. An extended proof through in vitro-based experimental models was generated to study the effectiveness of mAbs in mounting reasonable anti-Burkholderia pseudomallei activities. Accordingly, two mAbs, namely BURK24 and BURK37, were generated against the crude outer membrane proteins (OMPs) extract of B. pseudomallei and exploited in the present study. A novel indirect immunofluorescence assay was developed to study the binding of mAbs on the bacterial surface. Both the mAbs were studied for their kinetics in bacteriostasis and bacterial killing. In vitro based biofilm and invasion inhibition assays were also performed to examine the anti-Burkholderia pseudomallei properties of BURK24 and BURK37. The mAbs were evaluated for their ability to prevent pathogeninduced apoptosis as well.

Ethics Statement
BALB/c mice used in this study were obtained from Central Animal Facility, Defence Food Research Laboratory (DFRL), and housed under pathogen free condition. All the animal experiments were performed with the approval and written consent of the Animal Welfare and Research Ethics Committee of DFRL.

Bacterial Strains and Media
A standard strain of Burkholderia pseudomallei NCTC 10274 used in this study was received from Defence Research and Development Establishment, Gwalior, India. A clinical isolate of the bacterium, isolated from a case of melioidosis in a diabetic patient [26] was obtained from Dr. RD Kulakarni, Head, Dept. of Microbiology, SDM Medical College and Hospital, Dharwad, India. The mentioned clinical isolate is referred as B. pseudomallei 621 in the present study. Rest of the Burkholderia isolates used in this study were isolated from environmental samples including soil and water using Ashdown's selective medium, as described previously [27]. All other bacteria listed in Table 1 were the standard strains obtained from ATCC (USA), MTCC (Chandigarh, India) and NCIM (Imtech, Pune, India). Cultures were maintained in trypticase soy broth (TSB), Luria Bertani (LB) broth and Mueller Hinton broth (MHB) and Mueller Hinton agar (MHA) (Himedia, India); adjusted to 0.5 McFarland standard when required. Antibiotics were used at the final concentration of 4 mg/ml for gentamicin and 100 mg/ml for ampicillin.

Cell Line
Human alveolar adenocarcinoma epithelial cells -A549, were procured from National Cell Centre for Science, (Pune, India) and grown in Dulbecco's modified Eagle medium (Sigma, India) supplemented with 10% fetal bovine serum (FBS) (Sigma, India) and 16 working concentration of Penicillin-Streptomycin solution (Sigma, India) at 37uC in a humidified atmosphere of 5% CO 2 , as recommended by the supplier. The cells were seeded in tissue culture flasks (Greiner bio-one, US) and allowed to grow to 80-90% confluency. Cells were dislodged with 0.025% (w/v) trypsin/ 0.01% (w/v) EDTA (Sigma, India), counted using a heamocytometer and seeded into 24 well plates (56I0 5 cells/well) for invasion and apoptosis inhibition assays; and 6 well plates (1610 6 cells/well) containing sterile 12 mm diameter glass coverslips coated with 0.1% gelatin for immunofluorescence assay. Cells were incubated to reach 80-90% confluency at 37uC in a humidified atmosphere of 5% CO 2 .

Outer Membrane Proteins Preparation
OMPs were extracted from Burkholderia pseudomallei NCTC 10274 as described by Crosa and Hodges [28]. Briefly, the bacterium was grown overnight aerobically in 200 ml of LB broth and cells were harvested by centrifugation at 50006 g for 30 min at 4uC. Harvested cells were re-suspended in 3 ml of 10 mM Tris amino methane buffer containing 0.3% (w/v) NaCl (pH-8.0). The suspension was treated with 1% (w/v) thiomersol and 1 mM phenylmethyl sulfonyl fluoride (PMSF) and sonicated with a Vibra cell sonicator (Sonics & Materials Inc., Newtown, CT) for 3 times at 10 mM amplitude for 15 minutes with 30 sec interval for every 5 min. Lyzed suspension was centrifuged at 100006 g for 2 min and the supernatant collected was further centrifuged for 1 h at 170006 g at 4uC. The pellets of cell envelop suspension thus obtained were incubated overnight at 4uC with 3% (w/v) sodium lauroyl sarcosinate (sarkosyl) in 10 mM Tris buffer. OMPs were obtained by centrifugation at 170006 g for 1 h and washed twice with distilled water. Resulting OMPs pellet was re-suspended in sterile 10 mM Tris amino methane buffer containing 0.3% NaCl (pH-8.0) and concentration of protein preparation was quantified by Lowry method [29]. The outer membrane proteins profile was analyzed by SDS-PAGE according to standard procedure by Laemmli et al [30].

Immunization
Two female BALB/c mice (one six weeks old weighing 19 g and one eight weeks old weighing 20 g) were subcutaneously immunized with purified crude OMPs of Burkholderia pseudomallei. Briefly, primary immunization was done with 100 mg of purified OMPs in emulsion with Freund's complete adjuvant (Sigma, USA). Two booster doses of 50 mg protein with Freund's incomplete adjuvant (Sigma, USA) were followed on days 14 and 28. The serum was collected from mice prior to each immunization and stored at 220uC. The antibody titer of immunized mice sera was measured by indirect plate ELISA using crude OMPs as antigen. Upon confirmation for desired titer, a final immunization with 100 mg of the antigen in 16 sterile phosphate-buffered saline (7 mM Na 2 HPO 4 , 3 mM NaH 2 PO 4 and 130 mM NaCl at pH 7.4) i.e., PBS, intraperitoneally was done a week prior to sacrifice for generation of hybridomas.

Mouse Monoclonal Antibodies Generation
Hybridoma fusions were performed to generate monoclonal antibodies (mAbs), according to Kohler and Milstein [31]. Briefly, upon achievement of the desired antibody titer, spleen cells from immunized BALB/c mice were fused with sp2/0-Ag.14-1 cells (ATCC, VA) using 50% w/v PEG MW 2000 (Sigma, India). Fused cells were grown in selective HAT-containing Dulbecco's modified Eagle's media (DMEM, Sigma, India) and 10% v/v fetal bovine serum (Sigma, India). The generated hybrid clones were screened for their reactivity against the antigen by indirect plate  [32] to determine the antigenantibody binding pattern and biomolecular nature of epitope. Either crude OMPs preparation or whole-cell bacterial lysate of B. pseudomallei NCTC 10274 was used as antigen. Lysate was prepared by boiling bacteria, grown on blood agar plates, in Laemmli sample buffer. Proteins were separated on sodium dodecyl sulphate-polyacrylamide gels by electrophoresis, electroblotted onto nitrocellulose paper, probed with mAbs: BURK24 and BURK37, followed by horse radish peroxidase-conjugated goat anti-mouse IgG (Sigma, India). Blots were developed with 0.3% diaminobenzidine tetrahydrochloride (Sigma, USA) in 70 mM sodium acetate, pH 6.2 or 3,39,5,59 tetramethylbenzidine with 0.03% H 2 O 2 (v/v) (Sigma, USA) and signals were captured by luminescent image analyzer (Fujifilm, India). In order to determine the biomolecular nature of epitope, an aliquot of OMPs preparation was subjected to protein digestion using 0.02 unit of proteinase K for 30 min at 55uC. Resulting antigen was immunoblotted as described above. (c) Isotyping. Isotyping of mAbs BURK24 and BURK37 was performed using commercially available ELISA based isotyping kit (Sigma, USA) as described by the manufacturer. Briefly, 96-well immunosorbent plates (Nalge Nunc International, NY) were coated with specific goat anti-mouse immunoglobulin Fc subclass antibodies and blocked with 1% bovine serum albumin (BSA). Hybridoma culture supernatants were added and incubated in the plates for 1 h at room

Purification of mAbs
In order to obtain large concentrations of mAbs, for further studies, ascitic fluids of monoclonal antibodies BURK24 and BURK37 were generated produced in 2,6,10,14-tetramethyl pentadecane i.e., Pristane (Sigma, India) primed retired breeder BALB/c mice, by injecting BURK24 and BURK37 hybridomas as described by R B Westerman et al. [33] and purified using mAbTrap kit and HiTrap IgM Purification HP (GE Healthcare, UK) respectively as per the manufacturer's instructions. The purified mAbs were lyophilized, quantified and reconstituted in sterile 16 PBS according to the required concentrations of each study.

Indirect Immunofluorescence Binding Assay
The binding specificity of BURK24 and BURK37 mAbs onto the surface of invading B. pseudomallei NCTC 10274 into a human alveolar epithelial carcinoma cell line A549, was deduced by indirect immunofluorescence binding assay. Briefly, A549 mammalian cells were grown on to the 12 mm 0.1% gelatin coated glass coverslips in 6 wells tissue culture plates (Nalge Nunc International, NY). Cells were grown overnight in DMEM tissue culture medium supplemented with 10% FBS till 80-90% confluency before performing the assay. The coverslips coated with A549 mammalian cells were washed thrice with 16 sterile PBS and infected with overnight grown culture (OD at 600 nm of 0.6) of B. pseudomallei NCTC 10274 at a multiplicity of infection (MOI) of 50:1. After 30 min of incubation, the coverslips were washed with 16 PBS and air dried. Infected cells on to the coverslips were fixed with 3.7% formaldehyde in 16 PBS for 15 min. One percent BSA (Sigma, India) was employed for blocking the cells for duration of 30 min to minimize non-specific binding of antibodies before proceeding further. The formaldehyde fixed cells were incubated with 50 mg of mAb BURK24 and BURK37 separately at 37uC for 1 h. Later, the coverslips were washed twice with 16 sterile PBS, air dried and further incubated for 1 h with 1 ml of 1:500 dilution of anti-mouse immunoglobulin in goat, conjugated with fluorescein isothiocyanate (FITC) (Sigma, USA) at RT as recommended by the manufacturers. Again the coverslips were washed twice with 16 sterile PBS; air dried and coated with Flouroshield (Sigma, USA) for microscopic examination of BURK24 and BURK37 associated bacteria onto the surface of infected A549 human alveolar epithelial carcinoma cell line under fluorescence microscope (Nikon Eclipse Ni, Japan). Non-invasive strain of E. coli, control serum from naive BALB/c mouse and conjugate alone were used as negative controls in the experiment.

In vitro Antimicrobial Studies
(a) Antibody susceptibility test. To examine the antimicrobial property of BURK24 and BURK37, microtiterbased minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests were done as described in guidelines for antimicrobial susceptibility testing [34,35]

Effect of mAbs on Biofilm Formation
The amount of biofilm inhibition induced by the mAbs of the study was determined by a semi-quantitative microtiter plate based assay as described elsewhere [36,37] with few modifications. An overnight culture of B. pseudomallei standard strain grown at 37uC in TSB with 50 mM glucose was adjusted to a bacteria load of 5610 5 CFU/ml and 100 ml of the culture was mixed with equal volume of earlier deduced bacteriostatic concentrations of BURK24 and BURK37. This mixture was transferred to a flatbottomed microtiter plate and incubated aerobically at 37uC for 24 h without shaking. After incubation period, the culture was removed and microtiter plate was washed three times with 16 sterile PBS to remove non adherent bacterial cells and dried in inverted position. Adherent dry biofilm was fixed with 250 ml of methanol for 15 min. The wells were then emptied and air dried. For staining, 250 ml of 1% (w/v) crystal violet was added to each well and incubated for 5 min. The wells were then washed under running tap water and air dried. The adherent bacterial cells in the wells were re-solubilized by adding 250 ml of 33% (v/v) glacial acetic acid per well. The optical density (OD) of each well was measured at 570 nm using an automated ELISA plate reader (InfiniteM200, Tecan, Austria). A negative control containing B. pseudomallei standard strain in TSB with 50 mM glucose in the absence of mAbs was included. The assay was performed twice in triplicates. With the obtained OD values, percent inhibition of biofilm formation was also determined using the following formula.

Invasion Inhibition Assay
The ability of BURK24 and BURK37 to inhibit the invasion of B. pseudomallei into a human alveolar epithelial carcinoma cell line A549 was examined in this experiment. A549 cells were grown in 24-well tissue culture plates containing DMEM tissue culture medium supplemented with 10% FBS. The assay was performed in triplicate using B. pseudomallei NCTC 10274 (OD at 600 nm of 0.6) mixed with BURK24 and BURK37 separately at earlier determined bacteriostatic concentration. The bacterial cells were harvested from the mixture after 30 min, re-suspended in 200 ml of DMEM tissue culture medium supplemented with 2% FBS and transferred to the 24-well tissue culture plates at an MOI of 50:1. The infected confluent monolayers of eukaryotic cells were incubated at 37uC in a humidified atmosphere of 5% CO 2 for 1 h to allow bacterial entry. After invasion, all the wells were washed with 16 sterile PBS for three times and incubated with DMEM supplemented with 2% FBS containing 150 mg/ml tetracycline for 2 h at 37uC in a humidified atmosphere of 5% CO 2 to kill the extracellular bacteria. After incubation, the cell monolayers were washed thrice with 16sterile PBS and lyzed with 0.25% Triton X-100. Enumeration of the intracellular bacteria was done by plating the serial dilution of the lysate on TSB containing 4 mg/L of gentamicin and percentage of invasion in each case was determined using the formula given below. A positive control of non invasive E. coli strain was used in the experiment and in the negative control; monolayers of eukaryotic cells were infected with B. pseudomallei NCTC 10274 in the absence of mAbs. All quantitative invasion inhibition assays were performed in triplicate. The formula to determine percentage of invasion is as follows: (CFU=ml recovered from lysed cells=CFU applied to cells)|100

Statistical Analysis
Statistical analyses were performed using Multivariate Analysis of Variance (MANOVA) and Repeated measure Analysis of Variance (RANOVA) to analyze statistical difference exhibited by B. pseudomallei in terms of growth and biofilm formation in presence and absence of BURK24 and BURK37. Bonferroni post hoc test was performed where applicable. Data are expressed as the mean 6 standard deviation. Differences were considered statistically significant at a p-value#0.05. All statistical analyses were performed using Statistical Package for Social Science (SPSS) version 16.

Immunization and Generation of mAbs
Gradual increase in antibody titer was observed in BALB/c mice sera after each immunization, as revealed by indirect plate ELISA. Comparatively no reactivity was registered in the pre-immune serum against the outer membrane proteins. This indicated that the OMPs extract used was immunogenic resulting in antibody response in immunized mice. The desired antibody titer of 1:64000 was achieved after second booster immunization. The polysera showed reactivity with crude OMPs extract, whole cell lysate and intact cells of B. pseudomallei NCTC 10274 (data not shown). This implied that the polysera harbored antibodies with ability to recognize native epitope. Since the reactivity of polysera was promising, a panel of monoclonal antibodies were generated using the sensitized spleenocytes from BALB/c mice by standard hybridoma technique. A total of one hundred and eight clonal hybridomas were initially screened by an indirect plate ELISA (data not shown) for their reactivity with the OMPs preparation. This resulted in narrowing down to a total of nine mAbs (Table 1). To elaborate, among nine mAbs, 6 demonstrated a reactivity of 9.09%, one mAb showed 72.72% reactivity and two mAbs designated as BURK24 and BURK37 demonstrated a reactivity of 100% with all the eleven strains of B. pseudomallei tested (Table 1, Figure 1). The two mAbs with 100% reactivity for B. pseudomallei strains tested additionally showed cross-reactivity with B. cepacia MTCC 1617 (Table 1, Figure 1). Hence the two mAbs were selected for assessment of their anti-B. pseudomallei properties.

Characterization of mAbs
For better understanding about BURK24 and BURK37, Western blot analysis and isotyping were performed. Both the mAbs -BURK24 and BURK37, were able to recognize the antigen and the biomolecular nature of their epitope was determined in Western blot assay (Figure 2). BURK24 was found to react with protein antigens (approximately 70 kDa, 55 kDa, 50 kDa and majorly at 26 kDa), indicating the presence of the epitope in multiple copies. Whereas, BURK37 revealed a typical LPS-ladder pattern [38,39,40,41] ranging from 34-95 kDa. Since the crude extract of outer membrane proteins is known to have lipopolysaccharide components of bacterial cell wall, it was speculated that the antibodies raised against OMPs might include   the one which reacts with the lipopolysaccharide also. To evaluate that the epitope of BURK37 was non-protein, Western blot was carried out using the proteinase K digested crude OMPs of B. pseudomallei NCTC 10274. The resulting immunoblot pattern of both proteinase K treated and untreated revealed identical immunoblot pattern inferring that the epitope is non-proteinaceous compound, i.e., lipopolysaccharide which has contributed to give raise the mAb -BURK37 (Figure 3). Similar proteinase K treatment experiment study for BURK24 resulted in no immunoblot implying that its epitope is proteinaceous (data not shown). BURK24 and BURK37 were deduced to be IgG1 and IgM respectively upon isotyping. Higher concentrations of mAbs were obtained by producing ascitic fluids in BALB/c mice, purified, concentrated by lyophilization and quantified by Lowry method. The concentration of mAbs BURK24 and BURK37 was 2.5 and 2.3 mg/ml respectively.

Interaction of BURK24 and BURK37 with B. pseudomallei
Before proceeding with the studies on determining the functional properties of BURK24 and BURK37 towards the anti-B. pseudomallei pathogenesis, the mAbs were confirmed for the interaction with intact B. pseudomallei cells by a novel indirect immunofluorescence based assay. Human alveolar epithelial carcinoma cell lines -A549 were employed in the assay for the direct visualization of the result. The processed coverslips when observed under microscope at 525 nm wavelength, a strong green visual signal at the periphery of A549 cells was recorded highlighting the B. pseudomallei infected host cells (Figure 4). BURK24 and BURK37 were found to specifically bind onto the invading B. pseudomallei NCTC 10274 on the A549 mammalian cell lines. In the context of indirect ELISA, BURK24 and BURK37 was found to be significantly consistent in terms of their reactivity and specificity. In this study, to prevent the loss of intactness of bacterial cells we have used mAbs in not more than bacteriostatic concentration and also permeabilizing reagents like Tween 20 were not used to retain the intactness of host cells. Resulting immunofluorescence when observed under fluorescent microscope in the presence of specific mAbs confirmed the surface localization of antigens on B. pseudomallei NCTC 10274. No fluorescence was observed when non-invasive strain of E. coli, control serum and conjugate alone were used as negative controls. This implied that the immunofluorescence was solely due to the specificity of the mAbs for B. pseudomallei.

Antimicrobial Activity
Although the mAbs certified their ability to bind on the intact bacterial cells, we questioned whether this property rendered any antibacterial activity to them. To evaluate this, we assessed methodically the antimicrobial activity of purified mAbs on B. pseudomallei. Since, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) determine the optimum bacteriostatic and bactericidal concentration of any antimicrobial agent we proceeded to ascertain the same for BURK24 and BURK37. An experiment was conducted according to the CLSI guidelines [34,35] in microdilution method to deduce the concentrations of BURK24 and BURK37 for the assessment of their functional attributes as protective molecules. The MIC was read as the concentration of mAb at which there was no visible growth. The MBC was read as the concentration of mAb which resulted complete inhibition of bacterial growth upon plating. The results are shown in Table 2. A uniform susceptibility to the mAbs was demonstrated by both the strains of B. pseudomallei tested. Briefly, MIC of both the strains tested, B. pseudomallei NCTC 10274 and B. pseudomallei strain 621, was determined as 30 mg/ml and 62.5 mg/ml for BURK24 and BURK37, respectively. Whereas, MBC was 125 mg/ml and 500 mg/ml for BURK24 and BURK37, respectively. . The respective MBC:MIC ratio of mAbs: BURK24 and BURK37 was 4.1 and 8, indicating a bacteriostatic mode of action on B. pseudomallei. The experiment was repeated (n = 6) and the results were found to be statistically significant (p,0.05) and reproducible.
Our findings suggested that BURK24 and BURK37 possessed specific antibacterial activity. To further characterize this property of both the mAbs, we examined the kinetics of this activity. The study revealed that the range of mAb concentrations used in this experiment included bactericidal, bacteriostatic as well as nonbacteriostatic concentrations (Table 3). Upon comparison, the bacteriostatic and bactericidal concentrations derived in the kinetics study showed 100% concurrency with that of the antibody susceptibility testing. The kinetics study revealed that both the mAbs exhibited bacteriostatic activity within 1 h of the experiment ( Figure 5). This advises that BURK24 and BURK37 might be promising in conferring immediate immunity in infected persons upon administration. The results also suggested that the bacteriostatic activity of BURK24 and BURK37 was concentration dependent as substantiated by statistical analysis. Bacteriostatic activity of BURK24 was statistically significant (p,0.05) at concentrations of 30 mg/ml and 62.5 mg/ml and insignificant (p = 1.000) at the concentration of 15 mg/ml. In case of BURK37, the activity was significant (p,0.05) at concentration of 60 mg/ml and insignificant (p = 1.000) at lower concentrations.
As described earlier in antibody susceptibility test and bacteriostatic kinetic study, BURK24 and BURK37 exhibited bactericidal activity at the concentration of 125 mg/ml and 500 mg/ml respectively. Here, we also studied the kinetics of bacterial killing exhibited by BURK24 and BURK37 in vitro to determine their respective time kill curve. The experimentally measured growth curves for B. pseudomallei NCTC 10274 in presence of 125 mg/ml of BURK24 and in presence of 500 mg/ml BURK37 in comparison with B. pseudomallei NCTC 10274 alone demonstrated significant reduction in growth. The gradual decrease in bacterial load was observed with the increase in time indicating that the killing effect of both the mAbs was time-dependent. And the 3 log10 CFU/ml reduction, which represents the 99.9% killing of bacteria was recorded after incubation for more than 4 h. The curve pattern revealed that both the mAbs demonstrated the bactericidal activity and the effect was persistent throughout the test duration of 24 h ( Figure 6). The killing effect by BURK24 and BURK37 was confirmed to be reproducible and statistically significant (p,0.05) as well. Inspite of their difference in isotype, both the mAbs exhibited similar antimicrobial activity.

Biofilm Inhibition Study
As an analogous study to unravel the anti-B. pseudomallei based functional property of BURK24 and BURK37, we evaluated both the mAbs for their potential in inhibiting the formation of biofilm by B. pseudomallei. Since the biofilm forming potency varies among strains, both B. pseudomallei NCTC 10274 and the B. pseudomallei strain 621 were employed in the study in order to ascertain the inhibitory effect of BURK24 and BURK37 on variable biofilm formers. To begin with this, a microplate based assay was performed as described previously [36]. The rate of biofilm formation was estimated based on the OD 570 (Figure 7) of the test samples and catagorized as highly positive (OD 570 $1), low-grade positive (0.1#OD 570 ,1), or negative (OD 570 ,0.1). B. pseudomallei NCTC 10274 was determined as a highly positive biofilm former with a mean OD 570 of 1.57 whereas the B. pseudomallei strain 621 demonstrated a low-grade biofilm formation with a mean OD 570 of 0.56. The average percentage of inhibition of biofilm formation (Table 4, Figure 8) exhibited by BURK24 on B. pseudomallei NCTC 10274 and B. pseudomallei strain 621 was recorded to be 82.95% and 63.72% respectively. Whereas, that of BURK37 on B. pseudomallei NCTC 10274 and B. pseudomallei strain 621 was 68.21% and 76.02% respectively. Also, statistical analysis based on OD 570 of both the strains in presence and absence of individual mAb revealed significant difference (p,0.05). The results were found to be significantly reproducible upon repetition (n = 6). Though BURK24 and BURK37 exhibited variable activity as biofilm inhibitors on the two strains tested, overall effect was found to be significant.

Blockade of Invasion
For the first time we have evaluated the inhibitory role of mAbs on invasion of B. pseudomallei into the epithelial cell lines. Both mAbs BURK24 and BURK37 at their bacteriostatic concentration showed virtually 100% invasion inhibition after 2 h of infection. To explain, the mean invasion capacity of B. pseudomallei NCTC 10274 was predetermined as to be 33.46%. Whereas, a mean percentage invasion of 0.41 and 0.32 was recorded due to the inhibitory effect of BURK24 and BURK37 respectively ( Figure 9).

Effect on Pathogen-Induced Apoptosis
We extended our studies on the functional dimension of BURK24 and BURK37 by examining their ability to nullify the pathogen-induced apoptosis in mammalian cell lines. A comparative study was performed to observe the protection offered by BURK24 and BURK37 to A549 cells in presence of B. pseudomallei NCTC 10274 for duration of 24 h. A549 cells were infected with the bacterium to induce apoptosis and was referred as positive control. Whereas, A549 cells alone grown under defined normal condition in absence of the pathogen was considered as negative control. The study revealed that B. pseudomallei NCTC 10274 adequately induced apoptosis in A549 cells resulting in typical apoptotic morphological changes including decrease in cell volume, rupture of cell membrane and cell-to-cell fusion. On the other hand, no typical apoptotic morphological changes were observed in A549 cells when treated with the pathogen in presence of BURK24 and BURK37 separately was compared to negative control. The cell layer and volume appeared to be integrated. No cell fusions were observed ( Figure 10). Complementary result was obtained in DNA fragmentation assay. Approximately 100 ng of DNA from tests, positive and negative control cells collected at each interval of the experiment was electrophoresed and stained with ethidium bromide for visualization under UV light ( Figure 11). DNA fragmentation of A549 cells infected with B. pseudomallei NCTC 10274 was observed from 16 h of infection, suggesting the appreciable induction of apoptosis by the pathogen. However, DNA of A549 cells infected with B. pseudomallei NCTC 10274 in presence of bacteriostatic concentration of BURK24 and BURK37 were intact in agreement with that of the control cells throughout the experiment duration of 24 h. The result indicated that both the mAbs were protective for A549 cells, by preventing the pathogen to induce apoptosis.

Discussion
In the present study, we focused on unraveling the functional attributes of monoclonal antibodies as anti-B. pseudomallei molecules. To achieve this, we adopted in vitro based approach on basis of four reasons. Firstly, in vitro experiments negate the possible effect of unknown factors on experiments which usually accom-panies in vivo studies. Secondly, in vitro analysis more directly assess the authenticity of the work in terms of reproducibility. Thirdly, they are beneficial in terms of ethical considerations. Lastly, in vitro-based approach serves best to reduce expenses during preliminary studies as any molecule needs to be evaluated for its protective efficacy by several repetitive experiments.
Selection of an antigen was the primary challenge to generate promising mAbs with possible efficiency of establishing a passive immunity in the susceptible host. Inherent resistance to antibiotics, ability to form biofilm, invasiveness into both pathogocytic and non-phagocytic host cells are addressed for the strengthening pathogenesis of the bacterium. Outer membrane proteins (OMPs) that account for approximately 50% of the outer membrane mass in Gram-negative bacteria [42] seemed to be pertinent in this context. They promote serum resistance to the pathogen [42,43,44,45,46]. They play important role in adhesion [47] and invasion [48] of the bacterium to the host cells. Multi-drug efflux systems which confer multi-drug resistance to the pathogen also represent the OMPs [49]. In totality, OMPs play a key role in the Gram negative bacterial pathogenesis as they favor the pathogen to adapt in host niches [50]. Accordingly, we selected OMPs as the antigen for generation of potential antibodies, resulting in development of an array of mouse mAbs against crude outer membrane proteins extract of B. pseudomallei NCTC 10274 and screened for highly reactivity and promising mAbs. This resulted in encountering of two mAbs, namely BURK24 and BURK37, which were found to possess 100% reactivity for a total of eleven B. pseudomallei strains tested. They also exhibited reactivity with B. cepacia, a species of Burkholderia with clinical importance. BURK24 and BURK37 were deciphered to belong to IgG1 and IgM class of immunoglobulins, respectively. Additionally, Western blot analysis revealed that the epitope for BURK24 was the outer membrane proteins antigen present in multiple copies. The epitope for BURK37 was identified as LPS contaminants of the crude OMPs preparation. It is also well proven that LPS-specific antibodies majorly belong to IgM class of antibodies [51]. The isotyping and Western blot results shed light on two facts. Firstly, BURK24 and BURK37 belong to those immunoglobulin subclasses, which have been proven to have protective efficacy. To elaborate, IgG1, the immunoglobulin with highest biological half-life (21 days) is essayed to possess the activity of pathogen neutralization in tissues. IgM plays a major role in complement activation. Secondly, the epitope for BURK24 is undoubtedly present in multiple copies and that of BURK37 i.e., LPS is one of the major components of bacterial cell membrane. Thus we contemplated that BURK24 and BURK37 might possibly bind the pathogen efficiently throughout the surface and elicit changes in epitope or induce osmotic lysis resulting in the neutralization of pathogen activity and thus in turn demonstrate antimicrobial activity. Also, binding of mAb on the bacterial surface triggers the complement fixation, antibody dependent cell mediated cytotoxicity and opsonic activities on mAb-bacteria complex resulting in much more effective elimination of infection. To confirm this, we advanced to locate the binding region of both the mAbs on B. pseudomallei NCTC 10274 by designing a novel indirect immunofluorescence assay. We inferred a valuable functional property of BURK24 and BURK37 that they efficiently bind onto the surface of live bacteria adhering onto A549 cells resulting in fluorescence. This demonstrated the possible ability of both mAbs to reciprocate the same in vivo in infected hosts.
With this background, we examined BURK24 and BURK37 for their functional attributes as protective molecules in four different anti-Burkholderia pseudomallei activities in vitro. (1) As antimicrobial agents (2) As inhibitors of biofilm formation (3) As interrupting molecules in invasion of the pathogen into host cell (4) As agents to prevent bacterial-induced apoptosis.
When we channelized our work towards assessing the antimicrobial properties of BURK24 and BURK37, we selected the approved standards of CLSI which provides standards and guidelines to determine the in vitro susceptibility of antimicrobial molecules. To the best of our knowledge, this is the first report of applying CLSI standards for evaluating the antimicrobial activity of a monoclonal antibody. Use of monoclonal antibodies as bactericidal and bacteriostatic agent is justified, since both the antimicrobial actions are fundamental mechanisms of a host defence system. Accordingly, a successful ''antibody susceptibility testing'' was carried out to determine MIC and MBC of BURK24 and BURK37. The resulting MBC:MIC ratio revealed that both the mAbs possessed bacteriostatic action on B. pseudomallei, since bacteriostatic activity of any antimicrobial has been defined as a ratio of MBC to MIC of .4. This provides valuable information on the potential antimicrobial action of both the mAbs in vitro. Kinetic study of bacteriostasis and time-kill curve analysis revealed that the antimicrobial activity of BURK24 and BURK37 was both concentration and time-dependent. This is because, a gradual decrease of 3 log 10 in initial bacterial load was observed after fourth hour of the experiment.
Resistance of the biofilm forming B. pseudomallei to antimicrobial agents has been markedly proved [52,53]. This is because biofilm formation aids the bacteria to shield themselves in a polysaccharide matrix [54] and become inaccessible to the antibiotics. Thus biofilm formation results in the relapse of infection inspite of proper diagnosis and treatment. Nullifying the formation of biofilm has a remarkable importance in case of B. pseudomallei infections. As an attempt, BURK24 and BURK37 were studied for their efficacy in inhibiting the biofilm formation. Evaluation of biofilm formation of the strains in present study: B. pseudomallei NCTC 10274 and the B. pseudomallei strain 621 revealed that they possess variable capacity to form biofilm. B. pseudomallei NCTC 10274 was determined as a highly positive biofilm former whereas B. pseudomallei strain 621 was a low-grade biofilm former. However, BURK24 and BURK37 both showed significant inhibitory effect on biofilm formation of B. pseudomallei irrespective of the rate of biofilm formation of the pathogen. Biofilm formation depends on the successful establishment of interactions, between cell-to-cell and also cell-to-surface, which are regulated by various cell surface factors [55]. The inhibition of biofilm formation also indirectly suggests that the antigens of BURK24 and BURK37 i.e., OMPs and LPS might be involved in the biofilm formation of B. pseudomallei.
Currently, very sparse information is available about the mechanism of modulation induced in host cells by the invasive bacterium -B. pseudomallei. The bacterium has been reported to invade both phagocytic and non-phagocytic cells [56]. Adherence of the pathogen on the host cell surface is the primary step in establishment of pathogenesis of majority of infectious diseases. Similarly, as the first step of invasion, B. pseudomallei adheres onto the host cell membrane and further gets internalized [11]. Prevention of B. pseudomallei invasion into the host cells, thus promisingly prevents the pathogenesis. Efficiency of mAbs of this study was evaluated for the same. Prior to performing invasion inhibition assay, antibiotic resistance profile of both the strains, used in this study, was determined by antibiotic disc diffusion method. Among the antibiotics tested, chloramphenicol (30 mg/ ml), ciprofloxacin (5 mg/ml) and tetracycline (30 mg/ml) inhibited the growth of the strains to a considerable degree (data not shown). Similar results were observed by Susan Shahin and David Proll in their study on adhesion and invasion of A549 cells by B. pseudomallei [57]. Accordingly, tetracycline was selected at a concentration of 150 mg/ml to kill extracellular bacterial cells and the killing activity at the mentioned concentration was confirmed for reproducibility (data not shown). The apparent inhibition of invasion of B. pseudomallei into A549 cells, represents a prototypical mode of action of BURK24 and BURK37 on prevention of invasiveness of the pathogen.
Induction of apoptosis by B. pseudomallei in epithelial cell lines has been well studied [14,58]. Similar results were obtained in the present study, where B. pseudomallei NCTC 10274 was found to cause morphological changes in A549 epithelial cells; including reduction in cell volume, cell fusion and rupture of cell membrane. Concurrent DNA damage analysis inferred that the strain could induce DNA damage after 16 h of infection, as demonstrated by different strains of the pathogen in earlier studies [14]. On the other hand, the morphological changes in A549 cells infected with B. pseudomallei NCTC 10274 in presence of BURK24 and BURK37 appeared to be intermediate between control cells and those infected with bacteria alone throughout the experimental duration. They appeared to be under physiological stress. We predict the reason for stress could be the establishment of pathogenesis by a few intracellular bacterial cells (0.41% and 0.32% of infected bacteria in presence of BURK24 and BURK37 respectively, as determined in invasion inhibition assay presented in this study) in the later stage of infection. However, DNA of the epithelial cells remained intact throughout the experiment, revealing the potential of both the mAbs in preventing B. pseudomallei to induce apoptosis in host cells and confer considerable protection.
The results derived in all the in vitro experiments in the current study provides an insight into a possible protective intervention against B. pseudomallei infections -Passive immunization of the susceptible host with the mAbs possessing antibacterial, timedependent bacteriostatic, anti-biofilm, anti-invasive and antiapoptotic attributes i.e., BURK24 and BURK37 possibly provides a complete cure from B. pseudomallei infections. The additional reactivity of the mAbs with B. cepacia also projects the possibility of obtaining cross protection against B. cepacia infections -one of the major challenges in case of cystic fibrosis. This needs additional and intense clinical validation of the results by performing in vivo experiments in murine models by employing mouse, scFv-based and humanized BURK24 and BURK37.