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 ~  Abstract
 ~ Introduction
 ~  Materials and Me...
 ~ Results
 ~ Discussion
 ~  References
 ~  Article Figures

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ORIGINAL ARTICLE
Year : 2013  |  Volume : 31  |  Issue : 1  |  Page : 3-9
 

Evaluation and characterisation of A and B fragments of Corynebacterium diphtheriae toxin towards recombinant diphtheria vaccine


1 Department of Research and Development, VACSERA, Cairo, Egypt
2 Department of Microbiology and Immunology, Helwan University, Cairo, Egypt

Date of Submission26-May-2012
Date of Acceptance04-Oct-2012
Date of Web Publication15-Mar-2013

Correspondence Address:
R El-Domany
Department of Microbiology and Immunology, Helwan University, Cairo
Egypt
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DOI: 10.4103/0255-0857.108702

PMID: 23508421

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 ~ Abstract 

Background: Diphtheria is a highly communicable disease caused by toxin-producing strains of Corynebacterium diphtheriae. Objectives: To evaluate the efficacy of A and B subunits of diphtheria toxin (DT-A, DT-B) as potential vaccines against C. diphtheriae. A culture of C. diphtheriae (strain PW 8) was grown on Loeffler plates while Lingood medium was used for production of diphtheria toxin (DT). Materials and Methods: DT was purified and digested to obtain pure DT-A and DT-B and detoxified to obtain diphtheria toxin. Four groups of mice were immunised with different antigens (Ag) of C. diphtheriae. Results: The antibody (Ab) titres were significantly increased with immunised groups subsequent to three injections. On the other hand, Ab titres were estimated after the three immunisations and the levels of different Ab isotypes were comparatively measured. The levels of various isotypes immune responses showed variation between immunised groups where the IgG subclasses were significantly increased mainly with DPT immunised group. The IgM and IgA were significantly increased with DT-A more than others. Additionally, the evaluation of the cellular immune responses demonstrated that spleen cells from DPT and DT-A groups gave highly significant proliferative response with production of high levels of IL-2 and IFN-γ (Th1/Th2). Separation and purification of DT gene were performed using polymerase chain reaction (PCR) and sub-cloned in pGEM-T vector, for further studying of recombinant vaccine. Conclusion: Our results showed the possibility to prepare a potent recombinant vaccine containing whole DT gene or DT-A against C. diphtheriae or could be used in treatment of cancer as it give high levels of IL-2 and IFN-γ.


Keywords: Corynebacterium diphtheriae, IFN-γ, IL-2, lymphocyte proliferation, polymerase chain reaction


How to cite this article:
Abulmagd S, Emara M, Aziz S, El-Domany R. Evaluation and characterisation of A and B fragments of Corynebacterium diphtheriae toxin towards recombinant diphtheria vaccine. Indian J Med Microbiol 2013;31:3-9

How to cite this URL:
Abulmagd S, Emara M, Aziz S, El-Domany R. Evaluation and characterisation of A and B fragments of Corynebacterium diphtheriae toxin towards recombinant diphtheria vaccine. Indian J Med Microbiol [serial online] 2013 [cited 2014 Nov 21];31:3-9. Available from: http://www.ijmm.org/text.asp?2013/31/1/3/108702



 ~ Introduction Top


Diphtheria is a highly communicable bacterial disease that spreads like common cold, it usually originates in mucosal areas of the respiratory tract and secretes DT which is absorbed into the circulatory system and disseminated. [1] It is a severe and sometimes fatal disease caused by toxin-producing strains of Corynebacterium diphtheriae. Protection against diphtheria is obtained by the presence of significant levels of antibodies against DT. However, antibodies that have been developed subsequent to disease do not usually give rise to a full protection against clinical diphtheria on re-infection, which may cause severe and even lethal disease. After mass vaccination programmes against diphtheria have been established, the disease became very rare in industrialised countries. [2] Changes in the epidemiology of diphtheria, particularly the shift from young children to adolescent and adults, appear to be occurring in both developed and developing countries. [3] The conventional vaccine has a poor ability to induce long-lasting memory where the use of multiple boosters represents a pre-requisite, while the failure to induce local secretory immunity in the respiratory tract has been related to persistent carrier status. [4] In the current study, we evaluated the two subunits of DT to be recruited as vaccine against C. diphtheriae.


 ~ Materials and Methods Top


Preparation of diphtheria toxin

C. diphtheriae
Park William strain No. 8, CN 2000 (ATCC 13812) was used for vaccine production, cultured in Lingood media and incubated at 35°C for 48 hour with shaking at 140 rpm.

Purification using ammonium sulphate precipitation

The whole toxin was re-precipitated using a saturated ammonium sulphate solution with a concentration of 20-40%. The saturated ammonium sulphate was added drop wise to each tube and rotated for 1-2 hour on ice.

Preparation of DT-A and DT-B

DT was diluted to 1 mg/ml in 0.05 M Tris-HCl (pH 8.2), 5% glycerol and 0.05 M dithiothreitol "DTT" and was warmed to 37°C. Freshly dissolved trypsin was added (0.001 mg/ml) and after 15, 30 and 45 min, the digestion was stopped by addition of reducing sample buffer. The preparation was applied to a Sephadex G-100 column equilibrated with 10 mM phosphate buffer (pH 7.2) containing 0.1% SDS and 1 mM DTT. Two peaks were obtained representing the separated proteins containing DT-B and DT-A, as judged by SDS-PAGE analyses on fractions collected and separated on 12% polyacrylamide gel.

Immunogenicity

Female BALB/c mice (8-week old) weighting 15-18 g were divided into five groups (six mice/group). On day 0, mice were immunized intraperitoneally with 200 μl of Phosphate Buffered Saline {PBS} (negative control group), 100 μl of DT-A (100 μg/100 μl), DT-B (100 μg/100 μl) and ST DTx (10 μg/100 μl) emulsified with equal volume of complete Freund's adjuvant (CFA) (Sigma Aldrich, St. Louis/MO, USA) for 1 st injection, incomplete Fruend's adjuvant (IFA) (Sigma) for 2 nd injection. On day 28, the 3 rd injection was administered without adjuvant.

Assessment of DT neutralising antibodies

Mice were sacrificed on days a week after each injection (7, 21 and 35), blood was centrifuged at 3000g for 15 min and the serum was collected and stored at −70°C. Nunc-Immuno plates (Nunc International, Roskilde, Denmark) were used in all enzyme-linked immunosorbant assays (ELISAs) to measure the levels of various antibodies against DT-A, DT-B, ST DTx and DPT. Each well was coated overnight with 100 μl of diluted antigen (5 μg/ml) of standard DTx in carbonate/bicarbonate buffer pH 9.6 at room temperature (RT), according to the manufacturer's protocol.

Preparation of spleen-cell suspension

Mice were sacrificed 15 days after the booster immunisation and single-cell suspensions were prepared from the spleen. The aseptically collected spleens were stored in RPMI-1640 (Sigma Aldrich, St. Louis/MO, USA) supplemented with penicillin (100 μg/ml) and streptomycin (100 μg/ml). Single-cell suspension was prepared by passing each spleen through a 70 μm cell strainer (Falcon; Labora AB, Sollentuna, Sweden). The single-cell suspensions were then lysed of RBCs and centrifuged at 1000 rpm for 10 min at 4°C, and the mononuclear cell layers were collected. After repeated washes in RBMI-1640 medium, the cells were re-suspended in culture medium at a concentration of 5 × 10 6 viable cell/ml. The culture medium used for all experiments consisted of RPMI-1640, supplemented with 5% human heat inactivated serum, 1% penicillin and streptomycin, 10 mM Na-Pyruvate, 15-25 mM HEPES and 2% sodium bicarbonate.

Measurement of serum levels of IL-2, IL-10 and IFN-γ

Individual spleen-cell suspensions (100 μl, 5 × 10 6 /ml in culture medium) were plated in 96-well round-bottom microtitre plates and stimulated in triplicates. Cells were incubated at 37°C in 5% CO 2 for 3 days and the secretion of IFN-γ, IL-2 and IL-10 by spleen cells subsequent to antigen re-stimulation in vitro was determined by a cell-based ELISA technique. Individual spleen-cell suspensions were prepared, re-stimulated and cultured. After 3 days of culture, the cells were re-suspended and transferred to Maxisorp flat-bottom 96-well ELISA plates (Nunc-Immunoplates, Kamstrup, Denmark). The ELISA plates that had previously been coated with IFN-γ, IL-2 and IL-10 capture antibodies were included in commercial sandwich ELISA kits, washed 3 times with PBS, blocked with Bovine Serum Albumin (BSA, 200μl/well) for at least 1 hour at RT, and finally washed once again with PBS. The standard cytokines included in the ELISA kits were added to their respective plates in accordance with the manufacturer's instructions (Pharmingen San Diego, USA).

Isolation of genomic DNA from gram-positive bacteria

Wizard genomic DNA purification kit was used to isolate genomic DNA from C. diphtheriae (promega, USA). One ml of overnight bacterial culture was centrifuged for 2 min at 13,000g and the supernatant was discarded. The cell pellets were re-suspended in 480 μl of 50 mM EDTA and 150 μl of lyzozyme was added and gently mixed. The sample was incubated at 37°C for 60 min, centrifuged for 2 min at 13,000g and the supernatant was discarded, 600 μl nuclei lysis solution was then added and gently mixed with re-suspended cells. The solution was incubated at 80°C, allowed to cool at RT for 5 min and then 3 μl of RNase solution was added to the cell lysate. Afterwards, the cell lysate was incubated at 37°C for 60 min and 200 μl of protein precipitation solution was added to the cell lysate and incubated on ice for 5 min. The cell lysate was centrifuged at 13,000 g for 3 min and the supernatant was transferred to a 600 μl of isopropanol. Thereafter, the mixture was centrifuged for 2 min at 13,000 g and 600 μl of 70% ethanol was added to the cell pellet, mixed and centrifuged for 2 min at 13,000 g. The ethanol was aspirated and subsequently the DNA pellets were rehydrated in 50 μl of rehydration solution for 1 hour at 65°C or overnight at 4°C and the DNA was stored at 2-8°C.

Polymerase chain reaction

A set of primers targeting the DT gene was used to detect C. diphtheria: Forward primer 5 / ATG AGT CCT GGT AAG GGG ATA CG 3 / and backward primer 5 / TCA GCT TTT GAT TTC AAA AAA TAG C 3 / (Metabion, Martinsried, Germany). The PCR amplification was performed using Gene Amp PCR System 9600 (Perkin-Elmer Waltham, Massachusetts, USA). The reaction mixture consisted of 2 μl of DNA solution, 25 μl master mix (Go Tag green master mix of Promega, USA), 1 μl of each primer (1:3, 100 μmol) and 21 μl of H 2 O. The mixture was de-naturated initially at 95°C for 2 min; subsequent de-naturation was for 30 seconds, annealed at 50°C for 30 sec for 30 cycles and extended at 72°C for 2 min. A final extension was for 5 min at 72°C and then was stored at 4°C. To verify the amplification, 15 μl of the amplified product was electrophoresed on agarose gel for 1 hour at 150 V. The gel was stained with ethidium bromide and visualised on a UV transilluminator.

DNA purification and extraction from gel

QIA quick gel extraction kit (Qiagen, Maryland, USA) was used according to the manufacturer's instructions. The DNA fragment was excised from the agarose gel with a clean, sharp scalpel and the gel slice was weighed in a colourless tube. Three volumes of QG buffer were added to 1 volume of gel (100 mg to 100 μl) and were incubated at 50°C for 10 min or until the gel slice has completely dissolved and 1 gel volume of isopropanol was added to the sample and mixed. The QIA quick spin column was placed in a provided 2 ml collection tube and the sample was applied and centrifuged for 1 min. The flow-through was discarded and 0.5 ml of QG buffer was added to the column and was centrifuged for 1 min. Afterwards, 0.75 ml of PE buffer was added, centrifuged for 1 min and the flow-through was discarded and the QIA quick column was centrifuged for 1 min at 13,000 rpm. Subsequently, the QIA quick column was placed into a clean 1.5 ml microcentrifuge tube and DNA was eluted by adding 50 μl of EB buffer (10 mM Tris·Cl, pH 8.5) or water (pH 7.0-8.5) to the centre of the QIA quick membrane and the column was centrifuged for 1 min. Purified DT gene PCR product was sub-cloned to pGEM vector (Promega) as described in manufacturer's protocol.


 ~ Results Top


Production, identification and purification of DT

Purification of different batches of DT with ammonium sulphate showing bands of the same MW 58 KDa (lanes 1-4) matched with standard DTx (lane 5) as shown in [Figure 1].
Figure 1: Evaluation of DT purifi cation using ammonium sulphate precipitation. Ammonium sulphate purifi cation of different batches of DT demonstrating bands of similar MW 58 KDa (lanes 1-4) as compared with standard DTx (lane 5)

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Digestion of DT

[Figure 2] demonstrates the effect of treatment of DT with reducing sample buffer only, DTT, or digestion by trypsin enzyme resulting in partial fractionation (lanes 1-3, respectively). On the other hand, addition of trypsin resulted in complete fractionation with increased concentration of fractions by time (lanes 4-6, respectively).
Figure 2: Evaluation of DT fractionation. Treatment of DT with reducing sample buffer only (lane 1), DTT (lane 2), or digestion by trypsin (lane 3) resulting in partial fractionation. On the other hand, addition of trypsin leads to complete fractionation with increased MW of fractions over time (lanes 4-6)

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DT fractionation

[Figure 3] shows the elution profile of gel filtration chromatography of DT fractions measured at 280 nm. Results obtained demonstrated the presence of two peaks at different fraction numbers. DT fragment B was obtained at 43-45 fraction numbers with a maximum peak at 44, whereas fragment A was obtained at 50-52 fraction numbers with a maximum peak on 51. Silver-stained SDS-PAGE of the purified concentrated fragments revealed fraction A at 21 KDa, whereas fraction B at 37 KDa (lanes 1 and 2, respectively).
Figure 3: Elution profi le of gel fi ltration chromatography of DT fractions. Elution profi le of gel fi ltration chromatography of DT fractions measured at 280 nm demonstrating the presence of two peaks at different fraction numbers. DT fragment B was obtained at 43-45 fractions numbers with a maximum peak at 44, whereas fragment A was obtained at 50-52 fractions numbers with a maximum peak on 51

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The concentrated purified fragments were Silver-stained on SDS-PAGE [Figure 4]. This has shown two fractions of A at 21 KDa and B at 37 KDa.
Figure 4: Silver-stained SDS-PAGE of concentrated purified fragments. Silver-stained SDS-PAGE of the purifi ed concentrated fragments revealed fraction A at 21 KDa, whereas fraction B at 37 KDa (lanes 1 and 2, respectively)

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Immunogenicity

Effect of boosting dose on the resulting antibody titre

Results obtained demonstrated significant higher IgG titres in vaccinated groups as compared with control groups in second and third injections. No significant differences in titres were observed between the second and third injections as shown in [Figure 5].
Figure 5: Effect of boosting on the resulting antibody titre. Comparison of IgG titres in various vaccinated groups as compared to control groups following three injections. No signifi cant differences in titres were observed between the second and third injections

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Detection of IgG1, IgG2a, IgA and IgM isotype titres

The specific immune response in terms of IgG1, IgG2a, IgA and IgM were assessed by means of ELISA. Significant difference (P < 0.01) in the titre was detected between the vaccinated and control groups. IgG1 is considered the main isotype response for DTx neutralisation while DT-A has been shown to give high response to IgA as shown in [Figure 6].
Figure 6: Detection of IgG1, IgG2a, IgA and IgM against diphtheria preparations (second injection). ELISA results demonstrating specific immune responses in terms of IgG1, IgG2a, IgA and IgM where signifi cant difference (Pƒnƒ¬ƒn0.01) in the titre was detected between the vaccinated and control groups

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Detection IgG2b and IgG3 isotype titres

The specific immune response in terms of total IgG2b and IgG3 was identified by ELISA. Significant difference (P < 0.01) in the titre was detected between the vaccinated and control groups, as well as between groups each other as depicted in [Figure 7].
Figure 7: Detection of IgG2b and IgG3 titres against diphtheria preparations (after second injection). ELISA results showing the specific immune response in terms of total IgG2b and IgG3. Significant difference (P<0.01) in the titre was detected between the vaccinated and control groups, as well as between groups each other

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Determination of IL-2, IL-10 and IFN-γ levels in lymphocyte culture

The specificity of the T-cell response induced by purified DT was compared with the response obtained subsequent to immunisation with different Ag. Our results showed that significant levels of IL-2 and IFN-γ were recorded in all vaccinated groups of BALB/c mice as compared with controls. Fragment A showed higher cytokine levels as compared with groups immunised with fragment B and ST toxoid; however, such differences did not reach statistical significance as demonstrated in [Figure 8].
Figure 8: Concentration of various interleukin (IL-2, IL-10 and IFN-γ). The specifi city of the T-cell response induced by purified diphtheria toxin was compared with the response obtained subsequent to immunisation with different Ag. Our results showed that significant levels of IL-2 and IFN-γ were recorded in all vaccinated groups of BALB/c mice as compared with controls. Fragment A showed higher cytokine levels as compared with groups immunised with fragment B and ST toxoid; however, such differences did not reach statistical signifi cance as demonstrated in Figure 8

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Molecular biology

Amplification of DT gene by PCR

The gene encoding DT was amplified by direct PCR utilising genomic DNA extracted from C. diphtheriae strain as a template. The resulting PCR was 1.8 Kb [Figure 9].
Figure 9: Amplifi cation of 1.8 Kb diphtheria toxin gene. M: Molecular size marker, 1: The complete diphtheria genome, 2 → 4: DT gene PCR products

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DT gene purification

DT gene was extracted from 1% agarose gel and was further purified by QIA quick gel extraction kit [Figure 10], 1% agarose gel represents pure complete gene of DT, this gene will be cloned.
Figure 10: Purity of DT gene. DT ge ne was extracted from 1% agarose gel and was purifi ed by QIA quick gel extraction kit

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 ~ Discussion Top


In this study, the main objective is the evaluation of the use of DT fragments A and B as a potential vaccine for C. diphtheria and the possibility of starting up a trial production of fragments A and B to be used in recombinant DNA technology methods.

Here, C. diphtheriae strain used for production of DT was PW8 CN 2000 which contains ωtox + Phage in agreement with Wahby, et al.[5] LF value is the first international reference reagent used for evaluation of culture and production of DT as described by Preneta-Blanc et al. [6] it was about 200 lf/ml at the end of culture as expected and in concordance with Sundaran, et al.[7] Purification of crude DT (culture filtrate) was performed by stepwise ammonium sulphate precipitation at concentration ranging from 25 to 34%. Different acrylamide gel concentrations were used for detection of the largest MW range for the examined protein samples, one migrating band appeared at MW of 58 KDa (the expected MW of DT molecule), and compared with standard DT. Formaldehyde treatment converts the wild toxin into non-toxic immunogenic toxoid as DT contains some sensitive sites that react very easily with formaldehyde, even at low concentration converting the toxin into non-toxic toxoid.

To evaluate the purification and detoxification of DT in comparison with standard DT and DTx, the electrophoretic analysis revealed three major differences that results from the reaction with formaldehyde. First, a shift in the toxin bands; second, a change in the ratio of nicked toxoid form as two fragments of 21.0 [A fragment] and 37.3 KDa [B fragment] to apparently intact toxoid (58 KDa) and finally the protein bands became more diffuse. This effect has been attributed to cross-linking formed within the toxin and/or between amino acids present in the toxoid and the toxin Metz, et al.[8] Analysis of the reducibility of purified DT molecules has revealed that using 2-mercaptoethanol as a reducing agent, gave incomplete cleavage of the inter-chain disulphide bond thus, producing three protein bands namely, intact DT molecule, A and B fragments like as Drazin, et al.[9] Fragments A and B were obtained by digestion of the purified toxin with different trypsin concentrations until complete digestion was achieved as detected by SDS-PAGE. Goor, et al. reported that fragments dimer may be formed from two monomeric units as demonstrated by SDS-PAGE. [10] Trypsin enzyme and DTT successfully digest the whole toxin molecule into two bands at 21 KDa, which represents A fragment and 37 KDa for B fragment which is in line with Metz, et al. [8]

Gel filtration chromatography using sephadex G-100 column has the advantages of fractionation and separation of protein molecules according to their MW, which retain the protein molecules in the reactive form without any changes in their structure or reactivity. Sephadex G-100 separated the toxin molecule fragments after trypsin digestion as described by Rolf and Eidels. [11]

In this study, we tested the hypothesis that whether fragment A or B of DT could replace the toxoid produced by formaldehyde treatment in the vaccination against diphtheria infection. DT fragments A and B have been prepared and used in the vaccination of BALB/c mice and the resulting immune response was estimated in terms of antibody (Ab) response (total IgG and isotypes) as well as production of cytokines. This view was supported by the observation of previous studies indicated that both fragments A and B are immunogenic. Moreover, it has been suggested that the immunological reactions of spontaneously dissociated fragments of DT may account for the presence of some multiple antigenic determinants that are unlikely to be present in the toxoid as described by Usuwanthim, et al.[12]

Our results have demonstrated that mice that have been immunised by fragments A, B, standard toxoid and DPT (as positive control), induced specific IgG responses. No significant difference was observed between the Ab titre of the different vaccinated groups and the positive control groups while the vaccinated groups of mice produced significantly higher titres of total IgG as compared with the negative control groups. Both IgG1 and IgG2a isotypes were produced in the immunised groups following the first immunisation and the titres have been raised significantly after the second immunisation. The resulting immune response was Th2 dominant response characterized by the induction of IgG1. This observation was confirmed by the Th1/Th2 ratio and the pattern of produced cytokines. The pattern of cytokines plays a critical role in the development of specific immune responses towards infectious agents' DTx induced mixed TH1/TH2 response is in agreement with McNeela, et al.[13]

Our data indicated that fragments A, B, standard toxoid and DPT induced potent systemic cell-mediated and humoral-mediated immune response, which are characterised by neutralising antibodies. The neutralising Ab response is characterised by the production of IFN-γ, IgG1 and IgG2a. These results are in accordance with the pattern previously described by Aguila, et al.[14] In this study, we investigated the induction of systemic T-cell subtypes in mice and the correlation between antigen-specific T-cell cytokine production and levels of toxin-neutralising antibodies in the mice sera. There was no correlation as expected and is in concordance with McNeela, et al.[13]

These results are in line with that indicated by Miyagi, et al.,[15] who stated that the immunity against diphtheria is Th2 dominant and characterised by the induction of specific IgG1 against DT. Although Th1 responses may not be necessary for protection against diphtheria itself, the ability to stimulate a balanced pattern of immune responsiveness may be particularly useful for any mucosal combined vaccine directed at multiple antigens as described by Aguila, et al.[14] The utilisation of DT fragments with Freund's adjuvant instead of the toxoid may help in minimisisng the problem that arises with the currently used vaccine. For example, vaccination of DT in alum generally cannot stimulate mucosal immune response which limits their ability to infect the mucosal tissues such as the respiratory tract and resulting in the emerging limitation of the current vaccine schedule against diphtheria and this is in agreement with Clements and Griffiths. [4] The Ab subclass profile in the immunised mice has shown a shift in Th1/Th2 cytokine balance. This shows that IgG2a responses were driven by Th1 cells secreting IFN-γ, whereas IgG1 responses were driven by Th2 cells secreting IL-4 as described by Bδckstrφm and Dahlgren. [16]

In addition to the systemic immune responses, the local immune response was determined by measuring anti-DT specific IgA titres in serum where fragment A showed a higher IgA titre which may be used in oral vaccines. Th2 cells mediate the protection against extracellular parasites and bacteria by promoting IgA, IgE and possibly toxin-neutralising IgG in agreement with McNeela, et al.[17] On the other hand, Th1 responses are necessary to clear infectious virus and intracellular parasitic bacteria, Th2 responses predominantly promote humoral responses that can effectively prevent toxin-related bacterial infection, particularly in the mucosal surface as described by Bretsche, et al. [18] Alternatively, administration of antigen in modes that primes Th1 responses has been shown to inhibit Th2-mediated mucosal inflammation described by Tang, et al. [19]

Data regarding the spleen cell lymphokine secretion assay provided indirect evidence for the induction of different T-cell subpopulations by C. diphtheriae infection or immunisation. Recent findings have pointed to the existence of a subset of murine helper T-cell, named Th1 and Th2 distinguishable by the array of lymphokines, they secrete. The secretions of high levels of IL-2 and IFN-γ as well as low levels of IL-10 by spleen cells from convalescent mice have shown a typical Th1 response, whereas diphtheria- immunised mice produced a lymphokine secretion profile more typical of a Th1/Th2 response, with high IL-10, IL-2 and IFN-γ levels, which is in agreement with Yano, et al.[20]

Th1 cells are predominantly effective in mediating inflammatory, cytotoxic and delayed-type hypersensitivity responses, whereas Th-2 cells are more efficient in providing help for Ab production. These facts are consistent with our findings that immunisation with DT fragments, DTx and DPT induced strong T-cell proliferation and a high level of IL-10, IL-2 and IFN-γ. Future work will involve the utilisation of PCR-mediated cloning approach to clone the whole gene, as well as different fragments of DT and the expression of these proteins in suitable host. The selection of cloning method to be used is dependent on several factors, including the type of DNA polymerase, the length of the PCR product and the purpose of the cloning experiment. To conclude, our results showed that whole DT gene or DT-A could serve as potential candidates to prepare a potent recombinant vaccine against C. diphtheriae.

 
 ~ References Top

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15.Miyaji EN, Mazzantini RP, Dias WO, Nascimento AL, Marcovistz R, Matos DS, et al. Induction of neutralizing antibodies against diphtheria toxin by priming with recombinant Mycobacterium bovis BCG expressing CRM (197), a mutant diphtheria toxin. Infect Immun 2001;69:869-74.  Back to cited text no. 15
    
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17.McNeela EA, Jabbal-Gill I, Illum L, Pizza M, Rappuoli R, Podda A, et al. Intranasal immunization with genetically detoxified diphtheria toxin induces T cell responses in humans: Enhancement of Th2 responses and toxin-neutralizing antibodies by formulation with chitosan. Vaccine 2004;22:909-14.  Back to cited text no. 17
    
18.Bretsche PA, Ismail N, Menon JN, Power CA, Uzonna J, Wei G. Vaccination against and treatment of tuberculosis, the leishmaniases and AIDS: Perspectives from basic immunology and immunity to chronic intracellular infections. Cell Mol Life Sci 2001;58:1879-96.  Back to cited text no. 18
    
19.Tang C, Inman MD, van Rooijen N, Yang P, Shen H, Matsumoto K, et al. Th type 1-stimulating activity of lung macrophages inhibits Th2-mediated allergic airway inflammation by an IFN-gamma-dependent mechanism. J Immunol 2001;166:1471-81.  Back to cited text no. 19
    
20.Yano A, Komatsu T, Ishibashi M, Udaka K. Potent CTL induction by a whole cell pertussis vaccine in anti-tumor peptide immunotherapy. Microbiol Immunol 2007;51:685-99.  Back to cited text no. 20
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]



 

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2004 - Indian Journal of Medical Microbiology
Published by Medknow

Online since April 2001, new site since 1st August '04