Mycobacterium bovis BCG as a Delivery System for the dtb Gene Antigen from Diphtheria Toxin

Diphtheria is a fulminant bacterial disease caused by toxigenic strains of Corynebacterium diphtheriae whose local and systemic manifestations are due to the action of the diphtheria toxin (DT). The vaccine which is used to prevent diphtheria worldwide is a toxoid obtained by detoxifying DT. Although associated with high efficacy in the prevention of disease, the current anti-diphtheria vaccine, one of the components of DTP (diphtheria, tetanus and pertussis triple vaccine), may present post vaccination effects such as toxicity and reactogenicity resulting from the presence of contaminants in the vaccine that originated during the process of production and/or detoxification. Therefore, strategies to develop a less toxic and at the same time economically viable vaccine alternatives are needed to improve existing vaccines in use worldwide. In this study, the Moreau substrain of BCG which is used in Brazil as a live vaccine against human tuberculosis was genetically modified to carry and express the gene encoding for the diphtheria toxin fragment B (DTB). As such, the DNA sequence encoding the dtb gene was cloned into the pUS977 shuttle vector for cytoplasmic expression and successfully introduced into BCG cells by electroporation. Mice immunized with recombinant BCG expressing DTB showed seroconversion with the detection of specific antibodies against DTB. Also, rBCGs stably expressing DTB persisted up to 60 days in the absence of selective pressure in mice and cell viability did not change significantly during the period tested. Finally, immune sera from BALB/c mice vaccinated with rBCGpUS977dtbPW8 were preliminarily tested for their capacity of neutralizing the diphtheria toxin in the Vero Cells assay. How to cite this paper: Nascimento, D.V., Dellagostin, O.A., Matos, D.C.S., McIntosh, D., Hirata Jr., R., Pereira, G.M.B., Mattos-Guaraldi, A.L. and Armôa, G.R.G. (2017) Mycobacterium bovis BCG as a Delivery System for the dtb Gene Antigen from Diphtheria Toxin. American Journal of Molecular Biology, 7, 176-189. https://doi.org/10.4236/ajmb.2017.74014 Received: April 17, 2017 Accepted: September 27, 2017 Published: September 30, 2017 Copyright © 2017 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/


Introduction
Corynebacterium diphtheriae, one of 59 described species in the Corynebacterium genus, causes the highly contagious infection diphtheria in humans [1] [2] [3].C. diphtheriae produces diphtheria toxin (DT) and a pseudomembrane adherent to the tonsils, pharynx, and/or nose.The local and systemic manifestations of diphtheria are mainly related to the action of DT, which is the most studied virulence factor of this species [4] [5] [6] [7].DT is a polypeptide of 535 amino acids with a molecular weight of approximately 58.3 kDa, which is proteolytically cleaved after secretion into two fragments with distinct activities.The aminoterminal fragment A (DTA) is responsible for toxicity and the nontoxic carboxyl fragment (DTB) with 341 amino acids is responsible for adherence and internalization of the DT [8] [9].
Mycobacterium bovis Bacillus Calmette-Guerin (BCG) is used as a vaccine for the control of tuberculosis, is currently the most widely used vaccine in the world and has been given to more than three billion people since 1921 [10] [11].For this reason, BCG is among the best live vector candidates for delivery of protective antigens in vivo, especially in developing countries where the cost of vaccines is a major issue.BCG is able to elicit potent Th1-mediated immune responses and requires no additional adjuvant components in its formulation to evoke protective immunity, as demonstrated in several animal models of infectious diseases [12] [13] [14].Additionally, several other advantages have been associated with the potential use of BCG as an antigen-presenting system such as the ability to induce a long lasting type 1 helper T cell (Th1) immune response and CD8+T-cell triggering with just one dose.BCG can also be given at birth, is one of the most thermostable vaccines to date and can induce mucosal immunity by oral or nasal administration [15] [16].Up to this date, recombinant BCG (rBCG) vaccines expressing a variety of parasite, bacterial, or viral antigens have been shown to induce protective immune responses in murine and primate challenge models [12] [14] [17] [18] [19] [20] [21].The major purpose of our study is the development of a safer, less reactogenic and cheaper diphtheria vaccine prototype based on the use of BCG, the vaccine against tuberculosis, as a live delivery vector.In this report we present data on the construction of a new rBCG strain prototype vaccine against diphtheria based on the Brazilian Moreau vaccine substrain of M. bovis BCG, expressing the C. diphtheriae gene dtb PW8 by means of the non-integrative plasmid vector pUS977.Analysis of dtb expression by BCG, structural stability of the pUS977dtb PW8 after vaccination, ability to induce a specific humoral response against the diphtheria toxoid and a prelimi-nary evaluation of the neutralization power of sera from mice immunized with the rBCGpUS977dtb PW8 prototype was carried out.

Bacterial Strains, Plasmid, and Culture Conditions
The C. diphtheriae strain Park Williams 8 (ATCC 13812) was obtained from the Institute Butantan (São Paulo, Brazil) as a source of genomic DNA.The DTP vaccine was obtained from Bio-Manguinhos/FIOCRUZ and the plasmid vector pUS977 from Dr. M.A. Medeiros (Institute of Bio-Manguinhos/FIOCRUZ, Rio de Janeiro, RJ, Brazil).C. diphtheriae was grown in liquid Brain Heart Infusion (Difco, Detroit, MI, USA).Escherichia coli strain DH5α, grown either on solid or in liquid Luria-Bertani medium, was used for amplification of plasmids throughout the study.The Moreau substrain of M. bovis BCG used in vaccine production was obtained from the Fundação Ataulfo de Paiva (Rio de Janeiro, Brazil) and was typically grown either in Middlebrook 7H9 broth (Difco) supplemented with 10% albumin-dextrose-catalase (ADC), 0.2% glycerol, and 0.05% Tween 80, or in Middlebrook 7H10 agar (Difco) supplemented with ADC.When necessary, kanamycin was added to the mycobacteria media at a concentration of 25 µg/mL for selection of recombinant bacteria.

Construction of the pUS977 Expression Vector pUS977dtb pw8
A DNA fragment representing the entire dtb gene subunit was amplified from C. diphtheriae genomic DNA using the primers dtb forward A2633B07 5' CGT CTA GAA GGT AGC TCA TTG TC 3' and dtb A2633B08 reverse 5' GCT CTA GAC CCC ACT ACC TTT C 3', each containing a Xbal restriction site.The resulting 0.965 kb fragment was digested with Xbal and cloned in frame in pUS977, previously cut with the same enzyme, to create the construct pUS977dtb PW8.This construct was amplified in E. coli DH5α and used to transform BCG by electroporation using standard methods [22].Transformants were later selected by their resistance to kanamycin.

Western Blotting and Localization of Heterologous Proteins in rBCG
After transformation, recombinant BCG strains were selected on 7H10 Middlebrook medium containing kanamycin 25 µg/mL.Several colonies were tested for their plasmid content by the electroduction method described by [23] and for expression of DTB by Western blotting.For Western blotting analysis, one or more kanamycin-resistant BCG transformants were grown individually in 5 mL 7H9 Middlebrook liquid cultures supplemented with kanamycin.After growth at 37˚C, 2.0 mL cultures were harvested at mid-log phase by centrifugation, resuspended in lysis buffer (10 mM Tris pH 8.0; 1 mM EDTA; 2 mM PMSF (phenyl methyl sulfonyl fluoride) with 50 mg of glass beads (0.1 mm diameter) and then mixed three times (one minute each time) and left to rest on ice.Then the mycobacterial lysate was centrifuged and the resulting supernatant and pellet were separated and resuspended in a sample buffer 1:1 (100 mM Tris-HCL, 200 mM dithiothreitol, 4% SDS, 0.2% bromophenol blue, 20% glycerol).Samples were subsequently boiled for 10 minutes and then loaded onto a 12% SDS-PAGE.After separation, proteins in the SDS-PAGE gel were electrotransferred onto a 0.45 µm nitrocellulose membrane (Bio-Rad, Hercules, CA, USA), which was incubated overnight in 3% nonfat dry milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (vol/vol) (Sigma, St Louis, MO, USA).On the following day, the membrane was incubated with a 1:1000 dilution of antidiphtheria toxoid polyclonal primary antibodies produced in-house.After washing out the primary antibody, the membrane was incubated with alkaline phosphatase-conjugated anti-mouse IgG (Sigma) diluted in PBS-T as the secondary antibody.Antibody binding was detected with NBT and BCIP (nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt) (Sigma) as a substrate.

Immunization Procedures
Immunization experiments with 4 -6 week-old male BALB/c mice were conducted in compliance with The Ethical Principles in Animal Experimentation established by the Brazilian College of Animal Experimentation and approved by the Fundação Oswaldo Cruz-Animal Use Ethical Committee CEUA (P0163-03).Briefly, to evaluate the immune response against the rBCG strain, groups of 5 mice (BCG, BCGpUS977, rBCG pUS977dtb PW8 and DTP), were immunized intraperitoneally (i.p.) with 10 6 CFU/0.1 mL in phosphate buffered saline (PBS) containing 0.05% Tween 80 (vol/vol) (Sigma, St Louis, MO) (PBS-T) at day 0 and boosted 9 weeks later.As a control, each animal in a group of 5 received 50 µL of the conventional DPT vaccine.All animals were bled at different time points after priming at the retro-orbital plexus and sera obtained from blood at each point were pooled and later analyzed by enzyme-linked immunosorbent assays (ELISA), which was performed for detection and quantitation of anti-rDTB PW8 mouse antibodies.Sera were collected 4 weeks after immunization, pooled, and tested by ELISA for the presence of antibodies against DTB.

Evaluation of the Structural Stability of the pUS977dtb PW8 Construct from rBCGs Recuperated from Mice
After immunization, recombinant BCGs were recovered out of the spleens of vaccinated mice for evaluation of the structural stability of the pUS977dtb PW8 construct.Thus, constructs were then directly transferred from spleen reisolated rBCGs to E. coli DH5α by electroduction [23] and subsequently recuperated, purified and digested with restriction endonucleases (XbaI or KpnI).The presence (or absence) of structural changes in the construct was evaluated by comparing it with the banding pattern generated by the plasmid preparation used to produce the original transformant, through electrophoresis in 1% agarose gel.

Analysis of the Humoral Immune Response Induced by rBCG pUS977dtb PW8
Serum antibody responses were quantified by ELISA.Briefly, Maxisorp 96-well plates (Nunc International, Rochester, NY) were coated with the diphtheria toxoid (100 µL; 0.05 µg/mL in 0.2 M carbonate buffer/0.2M bicarbonate pH 9.6; 4˚C overnight), then washed five times with PBS-T, blocked with 4% nonfat dry milk in PBS, and finally incubated with serial dilutions of mouse sera in PBS.
After 1 hour at 37˚C, the plates were washed as described above and incubated with HRP-conjugated goat anti-mouse IgG (1:4,000) (Southern Biotechnology Associates, Inc.) in PBS at 37˚C for 1 hour.After another round of washings, antibodies were visualized by adding TMB substrate (100 µL; 10 mg/mL 3, 3', 5, 5' tetramethylbenzidine in citrate phosphate buffer, containing 0.01% hydrogen peroxide.After adding the substrate, the plates were sealed and incubated in the dark at room temperature for 10 min, after which the reaction was stopped by adding 50 µL of a 20% sulphuric acid solution, and the optical density (OD) of the yellow-orange color developed was measured at 450 -492 nm in a spectrophotometer (Biorad).Absorbance values were plotted against serum dilutions.

Vero Cell Method Potency Test
The in vitro Vero cell (CCL-81) method chosen for the purposes of this study is based on the protocol described by the WHO [25] and was used for titration of the diphtheria antitoxin.Briefly, sera in twofold dilutions in 96-well plates were incubated in the presence of 0.01 Lf/mL of DT (Sigma), which is usually neutralized by 0.031 IU/mL of standard anti-DT per mL in cell medium RPMI (Gibco), without bovine serum for an hour at room temperature in the dark.Vero cells in suspension (2.5 × 10 5 cells/mL) were added to each well of the plates containing medium 199 with Earl's salt's (Gibco) with 4.4% sodium bicarbonate, 4 mg/mL gentamicin and 5% fetal bovine serum.Plates were incubated for 96 h at 37˚C in a 5% CO 2 incubator and 90% relative humidity.The cells were later fixed with 10% formaldehyde for an hour and stained with 1% crystal violet for thirty minutes.After that, plates were washed in water to eliminate the excess stain.After the plates had dried completely, the Vero cells were examined visually on the microscope by observing the holes with approximately 50% of color (hole 50% of cells stained) multiplied by the reference serum titer [26] [27].The titer of neutralizing antibodies in the pooled sera was calculated by multiplying the inverse of its endpoint dilution with that of the standard antitoxin, expressed in IU/ml.Each pool of sera was analyzed in duplicate, and controls for DT and standard anti-DT were included in all experiments.

Results
The pUS977dtb PW8 expression vector was created on top of the mycobacterial strain resulting from transformation with the construct pUS977dtb PW8 was evaluated in terms of its capacity to express rDTB pw8 in vitro as shown in Figure 1(b).As expected, the lysate of rBCGs carrying the pUS977dtb PW8 separated by SDS-PAGE revealed an immunoreactive band corresponding to an approximately 45-kDa protein, as recognized by anti-diphtheria toxoid polyclonal antibodies.Results published elsewhere (37) of electrophoretic separations of PCR products from rBCGpUS977dtb PW8 cells reisolated from spleens of mice after vaccination with the rBCG prototype, revealed that rBCGs recovered from the spleens of vaccinated mice carried not only the construct but also the functional dtb expression cassette even sixty days after vaccination.In fact, analysis of the structural stability of rBCG constructs recovered from the spleens of mice showed no alteration in the structure of the plasmid, as essayed through the digestion of plasmids rescued from E. coli with either endonuclease XbaI or KpnI.
The structural stability of the recombinant plasmid introduced into the BCG should reflect the functional stability of the prototype vaccine with the expression of the target protein for several generations.Also very important is the evidence that the same rBCGs recovered from the spleen of BALB/c mice 60 days after vaccination, when subjected to six consecutive sub-cultures, showed no difference in the ability to express the DTB, thus demonstrating the persistence of functional stability of the rBCG transformed with pUS977dtb PW8 construct.
The size of the protein expressed by the rescued constructs was exactly as expected, and it was also recognized by anti-diphtheria toxoid polyclonal antibodies in western blottings.The infectivity/persistence study carried out in THP-1 human monocytes was done in two independent experiments (8 and 12 days, respectively), in the presence or absence of kanamycin [24].The rBCGpUS977 dtb pw8 retained full infectivity when compared to non-modified BCG and was able to persist in THP-1 cells up to the maximum time limit tested (12 days) with no plasmid loss in the absence of kanamycin (Figure 2).Additionally, viability counts of both rBCGpUS977dtb pw8 and non-modified BCG originated from infected THP-1 cells were similar at all time points in both experiments, regardless of the presence of kanamycin, which denotes the stability of the rBCGpUS977dtb pw8 inside a human cell as well, even in the absence of selective pressure.Similarly, the genetic integrity of the pUS977dtb pw8 construct was confirmed later by PCR analyses of plasmids electroeluted to E.coli from rBCGs recovered from THP-1 cells.
In humans, BCG and DPT vaccines are administered by different routes, intradermally for BCG and intramuscularly for DPT.In this study, we chose to use the intraperitoneal route for both rBCG and DPT so that we could compare the response induced by the rBCG vaccine prototype and DPT in the same immunization context.We thus verified that BALB/c mice vaccinated with the rBCGpUS977dtb pw8 were able to mount a specific immune response against the diphtheria toxoid.Sera collected from immunized mice and controls after up to booster with high titer of IgG anti-fragment B antibodies and was two-fold higher when compared to the response induced by the classic cellular DTP vaccine (Figure 3).Sera from vaccinated mice were preliminarily tested for evidences of neutralization capacity against DT.So, pooled sera obtained from mice 60 days after immunization with rBCGpUS977dtb PW8, were able to neutralize the cytotoxic effects of DT at dilution 1:4 (equivalent to the titer of 0,062 IU/mL of the reference anti-diphtheria serum, SAD) using the WHO protocol for the Vero Cell assay (Figure 4).These results, although important, need to be confirmed by the rabbit neutralization assay.

Discussion and Conclusions
Since the first transformation of mycobacteria with foreign DNA, the recombinant BCG technology has been used to evaluate the expression of foreign protective antigens in BCG for the development of new and improved BCG vaccines [13] [14] [15] [16] [24] [28]- [36].Although the expression of DT antigens in different bacterial species has been previously done [8] [37]- [41], in BCG the expressed DT antigens came from the mutant CRM 197 [27] and the diphtheria toxin dtb gene of the PW8 vaccine strain [42].It is well known that DTB is crucial for the adherence of diphtheria toxin to target cell receptors, a fundamental step in the internalization of the DT toxic fragment A. Thus, if we are able to induce the production of antibodies against DTB with an rBCG engineered to do it so, it is expected that the antibodies generated will interfere with adherence and the internalization process of DT and, consequently, will prevent the toxigenic effects of toxin produced by C. diphtheriae strains.
Despite the advances reached so far, a major limitation of the rBCG technology is the stability of the modified BCG [42].In the present study, the promoter P AN of M. paratuberculosis [43] was used with great success to drive the expression of DTB into the cytoplasm of the BCG cells.Data presented here showed that a 40 kDa DTB polypeptide was expressed, well tolerated by BCG and was also recognized by anti-diphtheria toxoid polyclonal antibodies.Moreover, the rBCGpUS977dtb pw8 remained genetically stable and fully functional even after recovery from the spleens of BALB/c mice immunized with the same rBCGs expressing DTB, as demonstrated by the immunoblotting of anti-DT specific sera with rBCG lysates in the laboratory.The rBCGpUS977dtb pw8 is definitely one of the most stable rBCGs ever constructed in our laboratory.
The ability of the rBCGpUS977dtb pw8 to infect and persist in human monocyte cells (THP-1) in addition to its capacity of inducing a specific anti-DT immune response in BALB/c mice was demonstrated.The capability of the live modified bacterial vector to enter and persist was observed in human monocyte (THP-1 lineage) for a given period of time in a fundamental part of the antigen presentation process, rBCGpUS977dtb pw8 .A previous study of our group using the same expression vector demonstrated the long term humoral response (up to 8 months) induced by mice vaccinated with an rBCG strain expressing the S1 subunit of pertussis toxin [30].Our results showed that, contrary to DTP, the response to the rBCGpUS977dtbpw8 was strong, especially in the 12 -20 week interval.Previous results with rBCG showed that the expression of CRM 197 was found to generate a neutralizing response [27].In our study the rBCGpUS977d tbpw8 was also able to generate neutralizing antibodies, albeit at low sera dilu- tions.However, it is important to keep in mind that sera-neutralizing capabilities in mice, guinea pigs, rabbits and humans are not comparable and further studies will have to be done to assure the neutralizing quality of the response generated by the rBCGpUS977dtbpw8.Taken together, the results achieved so far reveal that the new strain rBCGpUS977dtbpw8 developed in our laboratory proved to be genetically stable up to 20 weeks after vaccination and was able to generate a specific humoral response against the target antigen DTB associated to the neutralization of the toxin produced by C. diphtheria.The rBCGpUS977dt bpw8 has a potential application as a vaccine prototype and may as well be use- ful in future studies of neutralizing immune responses and protection against C. diphtheriae.
HEPES, and 10% fetal calf serum (FCS) as a growth medium.THP-1 monolayers were infected with equivalent levels of BCG or rBCG strains at a multiplicity of infection (MOI) of approximately 10 bacterial cells per monocyte.Bacteria necessary for infection was prepared by dilution of log phase cultures to the desired concentration based on direct enumeration by light microscopy.The actual number of viable BCG and rBCG strains used in each infection experiment was verified retrospectively by plating serial dilutions of the culture on Middlebrook 7H10 agar (Difco) with incubation for up to 4 weeks.Samples (4 wells per culture) were collected at 4 h, 24 h, 48 h, 5, 8, 9 and 12 days post-infection exposure.After that, infected THP-1 cells were lysed with 0.1% Tween 80 in distilled water (25 µL/well), serial diluted, and plated onto plain 7H10 agar (BCG control) or onto the same medium with kanamycin (rBCG).The levels of growth recovered at 4 h were used to calculate the relative infective capabilities of the rBCG in comparison to control BCG.The values obtained for colony forming units (CFU) recovered at other time points were used to calculate the degree of intracellular persistence of the rBCG.The functional stability of rBCG was assessed by comparing the CFU values obtained in media with and without kana-

Figure 1 .
Figure 1.Architecture of the plasmid vector pUS977dtbPW8 carrying the cassette for expression of the dtb gene in BCG (a).Western blot analyses of the BCGpUS977dtbPW8 lysate separated by SDS-PAGE, with anti-diphtheria toxoid polyclonal antibodies.Lane M: molecular weight markers (Broad Range: 80, 51 and 36 kDa); lane 1: BCGpUS977dtbPW8 lysate (b).

Figure 2 .
Figure2.Humoral response induced in BALB/c mice after i.p. vaccination with BCGpUS977dtbPW8.Four groups of 5 male mice each (4 to 6 weeks old) were immunized with 10 6 CFU mL of either BCG (non transformed), inactivated BCGpUS977dtbPW8 (BCGpUS977dtbPW8I), BCGpUS977dtbPW8 or the DTP vaccine (positive control).The graphic above displays antibodies against the diphtheria toxoid in pooled sera collected from each group of mice at different time points as quantified by ELISA (A 492nm ) and up to 20 weeks after immunization.

Figure 3 .Figure 4 .
Figure 3. Analyzes of infectivity, persistence, and plasmid maintenance/integrity of BCGpUS977dtbPW8 in human monocytes (THP-1) in the presence of kanamycin (25 µl per well) or not.The graphic displays the kinetics of the intracellular persistence of each strain up to 12 days.CFUs are shown in log10 units.Values recorded at 4 h, 24 h and 5, 9, 12 days represent the number of bacteria which had been internalized after 4 h of contact with cells.