- Open Access
Immunogenicity of recombinant Mycobacterium bovis bacille Calmette–Guèrin clones expressing T and B cell epitopes of Mycobacterium tuberculosis antigens
© Mohamud et al; licensee BioMed Central Ltd. 2013
- Published: 25 February 2013
Recombinant Mycobacterium bovis bacille Calmette–Guèrin (rBCG) expressing three T cell epitopes of Mycobacterium tuberculosis (MTB) Ag85B antigen (P1, P2, P3) fused to the Mtb8.4 protein (rBCG018) or a combination of these antigens fused to B cell epitopes from ESAT-6, CFP-10 and MTP40 proteins (rBCG032) were used to immunize Balb/c mice. Total IgG responses were determined against Mtb8.4 antigen and ESAT-6 and CFP-10 B cell epitopes after immunization with rBCG032. Mice immunized with rBCG032 showed a significant increase in IgG1 and IgG2a antibodies against ESAT-6 and MTP40 (P1) B cell epitopes and IgG3 against both P1 and P2 B cell epitopes of MPT40. Splenocytes from mice immunized with rBCG018 proliferated against Ag85B P2 and P3 T cell epitopes and Mtb8.4 protein whereas those from mice-immunized with rBCG032 responded against all Ag85B epitopes and the ESAT-6 B cell epitope. CD4+ and CD8+ lymphocytes from mice immunized with rBCG018 produced primarily Th1 type cytokines in response to the T cell epitopes. Similar pattern of recognition against the T cell epitopes were obtained with rBCG032 with the additional recognition of ESAT-6, CFP-10 and one of the MTP40 B cell epitopes with the same pattern of cytokines. This study demonstrates that rBCG constructs expressing either T or T and B cell epitopes of MTB induced appropriate immunogenicity against MTB.
- Cell Epitope
- Stimulation Index
- Mycobacterium Bovis
- Strong Humoral Immune Response
- Subclass Response
Tuberculosis (TB) is one of the most prevalent diseases in developing countries. WHO estimates that 8.7 million new cases and approximately 1.6 million deaths occur annually[1, 2]. The current vaccine against Mycobacterium tuberculosis, M. bovis bacille Calmette–Guèrin (BCG), is the most widely used vaccine in preventing TB disease especially in childhood[3, 4]. However, it has also been established that the protection afforded by BCG is highly controversial. Thus, a more effective TB vaccine is urgently needed.
It has been reported that there was only a slight decrease in the protective efficacy of BCG after 50 years. The intrinsic advantage of BCG as a live vector is its capability to replicate in host macrophages. In addition, BCG can stimulate both cellular and antibody responses and has the possibility to stimulate prolonged memory T cell responses. Moreover, BCG is safe, stable and inexpensive and thus is an attractive target for genetic manipulation in order to improve its protective capability against TB. BCG also represents an effective vehicle for delivery of heterologous antigens due to its intracellular replication in macrophages and dendritic cells.
Despite the dominant dogma that the cellular immune mechanism is primarily responsible for protection against TB, recent evidences suggest a protective role of specific antibodies in the defense against intracellular microorganisms such as MTB[10–14]. There is also an agreement that no single mycobacterial protein or antigen will be able to evoke all the required immune response for effective protection. Vaccine formulations encoding multivalent mixtures of antigens are necessary to increase the chances for covering populations with different MHC.This also may stimulate the innate immune system in providing adequate cytokine and costimulatory molecules. Thus, the multiantigen or multiepitope approach is an important strategy that needs to be explored.
In this study, T and B cell epitopes of MTB were cloned into BCG. The immunogenicity of the rBCG expressing three T cell epitopes of the Ag85B antigen of MTB fused to the entire sequence of MTB8.4 protein and the same construct fused to B cell epitopes of ESAT-6, CFP-10 and MTP-40 proteins of MTB were evaluated to determine its ability to induce specific humoral and cellular immune responses against these epitopes.
Design of the multiepitope MTB gene fragments
The sequences of the MTB proteins were obtained from the UNIPROT database . Linear MTB B cell epitopes were predicted from CFP-10 , ESAT-6 , and MTP40  proteins using the BCEPRED software . T cell epitopes were selected from MTB Ag85B protein based on a previous report  and analyzed using the prediction program PredBalb/c .
Construction of rBCG clones
Two multiepitopic constructs were designed: one containing three T cell epitopes of Ag85B protein of MTB fused to the Mtb8.4 protein, cloned into the shuttle plasmid pNMN018 and another with the addition of the selected B cell epitopes upstream to the previous construct, contained in the shuttle plasmid pNMN032. Both constructs were generated using preferred mycobacterium codon usage.
Immunization of Balb/c mice with rBCG
Balb/c mice (n = 5 per group) were immunized intraperitoneally (i.p.) with 2 x 106 CFU of either rBCG018 or rBCG032 in 200 μl PBS containing 0.1% Tween 80 (PBS-T80). A control group of mice were injected with 2 x 106 CFU of the parental BCG strain in 200 μl PBS-T80. After 30 and 60 days, the same amount of either rBCG or parental BCG was injected i.p. as boosters. Sera were collected before each injection from the tail vein and kept at -20 °C for IgG antibodies measurement. Subsequently, three weeks after the last immunization the mice were sacrificed and the spleens were asseptically removed to assess the cellular immune response.
Construction of peptides
The peptides corresponding to the T and B cell epitopes were commercially prepared (Peptron, Korea) and the Mtb8.4 antigen was produced in E. coli.
Enzyme-linked immunosorbent assay (ELISA)
Total IgG and IgG1, IgG2a, IgG2b and IgG3 (only against B cell epitopes) were measured from sera of immunized mice by ELISA as described elsewhere. The results were analysed using One-way ANOVA test and the differences between pairs were determined by the Multiple Range test. p values less than 0.05 were considered statistically significant.
Lymphocyte proliferation assay
T and B cell epitopes or recombinant Mtb8.4 protein were added to the cultures at a final concentration of 10 μg/ml. RPMI medium was used as negative control and the T cell mitogen, concanavalin A (10 μg/ml) was used as positive control. Results were calculated as stimulation index (SI) (SI = cpm of stimulated culture/cpm of non-stimulated culture). SI of more than 2 was considered as positive.
Analysis of intracellular cytokines
10 μg/ml of each peptide corresponding to the T and B cell epitopes or recombinant Mtb8.4 protein were cultured with 2 × 106 spleen cells/ml. The cells were then stained with 20 μl (20 μg /ml) anti-mouse IL-2, IL-4 or IFN-γ labelled with PE (BD Pharmingen). The increase in the percentage of intracellular cytokine levels in lymphocytes of rBCG-immunized mice was compared to those of BCG-immunized mice and scored as high, moderate, or weak/absent corresponding to ≥ 3 fold, ≥ 2 fold, < 2 fold increase respectively.
Antibody response against T and B cell epitopes
Mice receiving rBCG018, did not produce any antibody responses against these epitopes. ESAT-6 and CFP-10 have previously been reported to be immunogenic in miceand in non-human primates. Mtb8.4 did not elicit significant IgG response in mice immunized with either rBCG018 or rBCG032, despite containing several B cell epitopes. Individual IgG subclass response against CFP-10 did not reach significant levels despite the elevated levels of total IgG produced against this epitope. We are uncertain why this discrepancy was observed but this may highlight the importance of measuring both total and subclass IgG to cater for subtle but significant changes in antibody response.
The subclass response observed against the individual B cell epitope suggest that both, the TH1 and TH2 arms of the immune response maybe operative in this study.
Cellular immune response against T and B cell epitopes
Proliferative response against Ag85B T cell epitopes
Splenocytes isolated from rBCG018-immunized mice showed increased proliferative response after stimulation with Ag85B (P2) epitope (SI = 4.9); (P3) epitope (SI = 2.5) as well as with Mtb8.4 protein (SI = 3.2), whereas no response was observed against Ag85B (P1).
Intracellular cytokine expression against T and B cell epitopes and Mtb8.4 protein in animals immunized with rBCG018 or rBCG032
Summary of the percentage of cells expressing selected cytokines as determined by flow cytometry. Response (fold increase) of CD4+ and CD8+ lymphocytes from rBCG-immunized mice (rBCG018, rBCG032) was compared to those of BCG-immunized mice (BCG).
CD4 + /IL-4
CD4 + /IFN-γ
CD8 + /IL-2
CD8 + /IL-4
CD8 + /IFN-γ
We can conclude that some T cell epitopes may induce the production of Th2 instead of Th1 cytokines in Balb/c mice, and that the construction of a multi-epitope vaccine need to ensure that the overall potential “protection” that was intended is not compromised. In addition, it would be necessary to test the candidate vaccines in other strains of mice to ensure a more “universal” conclusion to be drawn.
The ability to elicit a predominant Th1 immune response is a desirable characteristic for any vaccine against MTB. The two vaccine candidates studied in the present work fulfilled this characteristic. In general, both rBCG018 and rBCG032 stimulated strong cellular response to the T cell epitopes and for the latter strong humoral immune response as well. Our findings suggest that co-expression of T and B cell epitopes of MTB in BCG stimulated both arms of the immune response although the protective capability of rBCG expressing both T and B cell epitopes needs further investigation.
We thank Jamaruddin Mat Asan for his technical assistance in flow cytometry and Dr. Rapeah Suppian for her assistance in the design of the composite gene. This work was supported by the Ministry of Higher Education Top Down Fundamental Research Grant Scheme (FRGS) [No. 203/PPSK/6170010],the Ministry of Science, Technology and Innovation, Malaysia (07-01-05-MEB007, 10-01-05 MEB002) and Ministry of Science and Technology, Cuba. National Science Fellowship (NSF) supported Rohimah Mohamud and USM Pasca Fellowship supported Maryam Azlan.
This article has been published as part of BMC Immunology Volume 14 Supplement 1, 2013: Proceedings from Delivery Systems and Current strategies to drug design. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcimmunol/supplements/14/S1.
- Young DB, Perkins MD, Duncan K, Barry CE: Confronting the scientific obstacles to global control of tuberculosis. J Clin Invest. 2008, 118 (4): 1255-65. 10.1172/JCI34614.PubMed CentralView ArticlePubMedGoogle Scholar
- Dye C: Tuberculosis 2000-2010: control, but not elimination. Int J Tuberc Lung Dis. 2000, 4 (12 Suppl 2): S146-52.PubMedGoogle Scholar
- al-Kassimi FA, al-Hajjaj MS, al-Orainey IO, Bamgboye EA: Does the protective effect of neonatal BCG correlate with vaccine-induced tuberculin reaction?. Am J Respir Crit Care Med. 1995, 152 (5 Pt 1): 1575-8.View ArticlePubMedGoogle Scholar
- Norazmi MN, Sarmiento ME, Acosta A: Recent advances in tuberculosis vaccine development. Current Respiratory Medicine Reviews. 2005, 1 (2): 109-116. 10.2174/1573398054023000.View ArticleGoogle Scholar
- Brewer TF: Preventing tuberculosis with bacillus Calmette-Guérin vaccine: a meta-analysis of the literature. Clin Infect Dis. 2000, 31 (Suppl 3): S64-67.View ArticlePubMedGoogle Scholar
- Aronson NE, Santosham M, Comstock GW, Howard RS, Moulton LH, Rhoades ER, Harrison LH: Long-term efficacy of BCG vaccine in American Indians and Alaska Natives: A 60-year follow-up study. JAMA. 2004, 291 (17): 2086-91. 10.1001/jama.291.17.2086.View ArticlePubMedGoogle Scholar
- Rapeah S, Norazmi MN: Immunogenicity of a recombinant Mycobacterium bovis bacille Calmette-Guèrin expressing malarial and tuberculosis epitopes. Vaccine. 2006, 24 (17): 3646-53. 10.1016/j.vaccine.2006.01.053.View ArticlePubMedGoogle Scholar
- Bao L, Chen W, Zhang H, Wang X: Virulence, immunogenicity, and protective efficacy of two recombinant Mycobacterium bovis bacillus Calmette-Guérin strains expressing the antigen ESAT-6 from Mycobacterium tuberculosis. Infect Immun. 2003, 71 (4): 1656-61. 10.1128/IAI.71.4.1656-1661.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Giri PK, Schorey JS: Exosomes derived from M. bovis BCG infected macrophages activate antigen-specific CD4+ and CD8+ T cells in vitro and in vivo. PLoS ONE. 2008, 3 (6): e2461-10.1371/journal.pone.0002461.PubMed CentralView ArticlePubMedGoogle Scholar
- Dietrich J, Weldingh K, Andersen P: Prospects for a novel vaccine against tuberculosis. Vet Microbiol. 2006, 112 (2-4): 163-69. 10.1016/j.vetmic.2005.11.030.View ArticlePubMedGoogle Scholar
- Williams A, Reljic R, Naylor I, Clark SO, Falero-Diaz G, Singh M, Challacombe S, Marsh PD, Ivanyi J: Passive protection with immunoglobulin A antibodies against tuberculous early infection of the lungs. Immunology. 2004, 111 (3): 328-33. 10.1111/j.1365-2567.2004.01809.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Olivares N, León A, López Y, Puig A, Cádiz A, Falero G, Martínez M, Sarmiento ME, Fariñas M, Infante JF, Sierra G, Solís RL, Acosta A: The effect of the administration of human gamma globulins in a model of BCG infection in mice. Tuberculosis (Edinb). 2006, 86 (3-4): 268-72. 10.1016/j.tube.2006.01.006.View ArticleGoogle Scholar
- Olivares N, Puig A, Aguilar D, Moya A, Cádiz A, Otero O, Izquierdo L, Falero G, Solis RL, Orozco H, Sarmiento ME, Norazmi MN, Hernández-Pando R, Acosta A: Prophylactic effect of administration of human gamma globulins in a mouse model of tuberculosis. Tuberculosis. 2009, 89 (3): 218-20. 10.1016/j.tube.2009.02.003.View ArticlePubMedGoogle Scholar
- López Y, Yero D, Falero-Diaz G: Induction of a protective response with an IgA monoclonal antibody against Mycobacterium tuberculosis 16kDa protein in a model of progressive pulmonary infection. Int J Med Microbiol. 2009, 299 (6): 447-52. 10.1016/j.ijmm.2008.10.007.View ArticlePubMedGoogle Scholar
- Andersen P, Doherty TM: The success and failure of BCG - implications for a novel tuberculosis vaccine. Nat Rev Microbiol. 2005, 3 (8): 656-62. 10.1038/nrmicro1211.View ArticlePubMedGoogle Scholar
- Britton WJ, Palendira U: Improving vaccines against tuberculosis. Immunol Cell Biol. 2003, 81 (1): 34-45. 10.1046/j.0818-9641.2002.01143.x.View ArticlePubMedGoogle Scholar
- Reed SG, Alderson MR, Dalemans W, Lobet Y, Skeiky YA: Prospects for a better vaccine against tuberculosis. Tuberculosis (Edinb). 2003, 83 (1-3): 213-9. 10.1016/S1472-9792(02)00080-X.View ArticleGoogle Scholar
- Wu CH, Apweiler R, Bairoch A, Natale DA, Barker WC, Boeckmann B, Ferro S, Gasteiger E, Huang H, Lopez R, Magrane M, Martin MJ, Mazumder R, O'Donovan C, Redaschi N, Suzek B: The Universal Protein Resource (UniProt): an expanding universe of protein information. Nucleic Acids Res. 2006, 34 (Database issue): D187-91.PubMed CentralView ArticlePubMedGoogle Scholar
- Guinn KM, Hickey MJ, Mathur SK, Zakel KL, Grotzke JE, Lewinsohn DM, Smith S, Sherman DR: Individual RD1-region genes are required for export of ESAT-6/CFP-10 and for virulence of Mycobacterium tuberculosis. Mol Microbiol. 2004, 51 (2): 359-70. 10.1046/j.1365-2958.2003.03844.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Viana-Niero C, de Haas PE, van Soolingen D, Leão SC: Analysis of genetic polymorphisms affecting the four phospholipase C (plc) genes in Mycobacterium tuberculosis complex clinical isolates. Microbiology. 2004, 150 (Pt 4): 967-78.View ArticlePubMedGoogle Scholar
- Saha S, Raghava GPS: BcePred: prediction of continuous B-cell epitopes in antigenic sequences using physico-chemical properties. Artificial Immune Systems, Third International Conference (ICARIS 2004), LNCS 3239, Italy Catania, Sicily. Edited by: Nicosia, G., Cutello, V., Bentley, P.J., Timis, J. 2004, Berlin/Heidelberg. Springer, 197-204.Google Scholar
- Lee BY, Horwitz MA: T-cell epitope mapping of the three most abundant extracellular proteins of Mycobacterium tuberculosis in outbred guinea pigs. Infect Immun. 1999, 67 (5): 2665-70.PubMed CentralPubMedGoogle Scholar
- Zhang GL, Srinivasan KN, Veeramani A, August JT, Brusic V: PREDBALB/c: a system for the prediction of peptide binding to H2d molecules, a haplotype of the BALB/c mouse. Nucleic Acids Res. 2005, 33 (Web Server issue): W180-83.PubMed CentralView ArticlePubMedGoogle Scholar
- Harboe M, Malin AS, Dockrell HS, Wiker HG, Ulvund G, Holm A, Jørgensen MC, Andersen P: B-cell epitopes and quantification of the ESAT-6 protein of Mycobacterium tuberculosis. Infect Immun. 1998, 66 (2): 717-23.PubMed CentralPubMedGoogle Scholar
- Kanaujia GV, Motzel S, Garcia MA, Andersen P, Gennaro ML: Recognition of ESAT-6 sequences by antibodies in sera of tuberculous nonhuman primates. Clin Diagn Lab Immunol. 2004, 11 (1): 222-26.PubMed CentralPubMedGoogle Scholar
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