Cellular Immune Responses to Recombinant Mycobacterium bovis BCG Constructs Expressing Major Antigens of Region of Difference 1 of Mycobacterium tuberculosis

Shaban, Kholoud; Amoudy, Hanady A.; Mustafa, Abu Salim

doi:10.1128/cvi.00090-12

Cited by 20 publications

(35 citation statements)

References 40 publications

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“…1), the number of peptides and their amino acid sequences are shown for PE35, according to standard procedures [23]. For cytokine estimations, spleenocytes were re-stimulated in vitro with the peptide pool of PE35 and its individual peptides, and the culture supernatants were tested for the concentration of IFN-γ, IL-5 and IL-10 using enzyme-linked immunosorbent assays [24]. The recombinant plasmids capable of transforming mycobacteria were generated by PCR-amplification of pe35, esat-6 and cfp10 genes from the genomic DNA of M. tuberculosis and cloning into shuttle plasmid pDE22 [24].…”

Section: Materials and Methodologymentioning

confidence: 99%

“…For cytokine estimations, spleenocytes were re-stimulated in vitro with the peptide pool of PE35 and its individual peptides, and the culture supernatants were tested for the concentration of IFN-γ, IL-5 and IL-10 using enzyme-linked immunosorbent assays [24]. The recombinant plasmids capable of transforming mycobacteria were generated by PCR-amplification of pe35, esat-6 and cfp10 genes from the genomic DNA of M. tuberculosis and cloning into shuttle plasmid pDE22 [24]. The recombinant plasmids (pDE22-PE35, pDE22-ESAT-6 and pDE22-CFP10) were propagated in E. coli and purified to obtain sufficient quantities to transform mycobacteria.…”

Section: Materials and Methodologymentioning

confidence: 99%

“…The recombinant plasmids (pDE22-PE35, pDE22-ESAT-6 and pDE22-CFP10) were propagated in E. coli and purified to obtain sufficient quantities to transform mycobacteria. The presence of each M. tuberculosis-specific gene in the respective purified recombinant plasmids was confirmed by DNA sequencing [24].…”

Section: Materials and Methodologymentioning

confidence: 99%

“…rBCG-pDE22-PE35, rBCG-pDE22-ESAT-6 and rBCG-pDE22-CFP10 [24]. Separate groups of mice were immunized with these strains of rBCG and, after appropriate time intervals, spleenocytes were isolated from the immunized mice to study antigen specific cellular immune responses (cellular proliferation, and secretion of IFN-γ, IL-5 and IL-10) [24].…”

Section: Materials and Methodologymentioning

confidence: 99%

See 3 more Smart Citations

Immune Responses to Candidate Vaccine Antigens Delivered Through Naked Plasmid and Mycobacterial Vectors

Mustafa¹

2016

Open Conf. Proceed. J.

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Tuberculosis (TB) is a major global health problem and has been declared "a global emergency" by the World Health Organization. The failures of the currently available vaccine against TB, i.e. Mycobacterium bovis BCG, to impart consistent protection against TB, have led to the need for alternative vaccines. The low molecular weight major antigenic proteins, i.e. PE35, CFP10 and ESAT6, encoded by Mycobacterium tuberculosis-specific region of difference-1 (RD1) are among the antigens considered important to develop new TB vaccines. To deliver these antigens, two DNA vaccine vectors (pUMVC6 and pUMVC7) and three live mycobacterial species (M. bovis BCG, M. vaccae and M. smegmatis) were evaluated for the induction of antigenspecific cellular immune responses in animals. DNA corresponding to pe35, esat6 and cfp10 genes were amplified using polymerase chain reaction (PCR) from the genomic DNA of M. tuberculosis and cloned into plasmids pUMVC6 and pUMVC7 to prepare DNA vaccine constructs. Furthermore, the PCR-amplified DNA were cloned into a shuttle plasmid (pDE22), and the recombinant shuttle plasmids (pDE22-PE35, pDE22-CFP10 and pDE22-ESAT6) were electroporated into mycobacteria. The induction of antigen specific cellular immune responses was studied by immunizing mice and guinea-pigs with the recombinant constructs. The results with all the recombinant constructs and both animal models showed the consistent induction of antigen-specific cellular immune responses (spleen cell proliferation and secretion of protective T helper 1 cytokine interferon-γ in mice, and delayed-type hypersensitivity skin responses in guinea-pigs) only with the recombinant constructs expressing PE35 protein. These results suggest the superiority of PE35 antigen in inducing protective cellular immune responses in animals.

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Section: Materials and Methodologymentioning

confidence: 99%

Section: Materials and Methodologymentioning

confidence: 99%

Section: Materials and Methodologymentioning

confidence: 99%

Section: Materials and Methodologymentioning

confidence: 99%

See 2 more Smart Citations

Immune Responses to Candidate Vaccine Antigens Delivered Through Naked Plasmid and Mycobacterial Vectors

Mustafa¹

2016

Open Conf. Proceed. J.

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“…The systematic analysis of these ORFs for immunological reactivity in cell mediated immunity assays lead to the identification of four major antigens, i.e. PE35, PPE68, CFP10 and ESAT-6 [25][26][27][28][29][30][31][32][33][34][35][36]. Among these antigens, CFP10 (ESXB) and ESAT6 (ESXA) proteins belong to ESAT6 family ( Table 1).…”

Section: Introductionmentioning

confidence: 99%

Diagnostic and Vaccine Potentials of ESAT-6 Family Proteins encoded by M. tuberculosis genomic regions absent in M. bovis BCG

Mustafa¹

2013

J Mycobac Dis

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Tuberculosis is a major international health problem and its control requires cost-effective diagnostic reagents and protective vaccines. Among the candidates advocated for both applications is the immunodominant 6 kDa early secreted antigenic target (ESAT-6) of M. tuberculosis. The esat6 gene is located in the M. tuberculosis-specific genomic region of difference 1 (RD1), which is absent in all vaccine strains of M. bovis BCG. In addition to ESAT6, RD1 contains the gene for another immunodominant low molecular weight culture filtrate protein 10 (CFP-10). Both ESAT-6 (ESXA) and CFP10 (ESXB) belong to the ESAT-6 family. The sequences of ESXA and ESXB lack significant homology with each other and any other member of ESAT-6 family. Furthermore, four additional immune dominant proteins belonging to ESAT-6 family have been identified, whose genes are present in M. tuberculosis-specific genomic regions absent in M. bovis BCG, i.e. Rv2346c/ESXO and Rv2347c/ESXP in RD7, and Rv3619c/ESXV and Rv3620c/ESXW in RD9. ESXO and ESXV belong to ESAT-6 subfamily-1 and ESXP and ESXW to ESAT-6 subfamily 2. Each subfamily contains five members and has orthologs in M. bovis BCG. Although, members of subfamily 1 lack sequence identity with members of subfamily 2, each of the five members within a given family share >92% sequence identity. Immunization with RD proteins of ESAT-6 family protects animals against challenge with M. tuberculosis, but due to the immunodominant recognition by the majority of TB-infected and exposed individuals, and the absence in M. bovis BCG, ESXA and ESXB should be reserved for diagnostic applications, and the ESAT-6 subfamily 1 and 2 proteins deserve to be considered as subunit vaccine candidates.

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Distribution of vitamin D‐binding protein/group‐specific component gene subtypes in Kuwaiti population

Al-Shammri

Mustafa

Bhattacharya

2022

Molec Gen & Gen Med

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BackgroundVitamin D‐binding protein or group‐specific component (Gc) is the major plasma carrier protein of Vitamin D. Two single nucleotide polymorphisms, rs7041 (NM_000583.3:c.1296G>T;NP_000574.2:p.Asp432Glu) and rs4588 (c.1307C>A; p.Thr436Lys), in the GC gene result in three major genotypes, that is, GC1F (c.1296T, c.1307C), GC1S (c.1296G, c.1307C), GC2 (c.1296T, c.1307A), and phenotypes such as Gc1F (p.432Asp, p.436Thr), Gc1S (p.432Glu, p.436Thr), and Gc2 (p.432Asp, p.436Lys). Significant variations in the frequencies of GC subtypes (genotypes/phenotypes) are reported in different populations living in different geographical locations, for example, GC1S/Gc1S (c.1296G, c.1307C/p.432Glu, p.436Thr) and GC2/Gc2 (c.1296T, c.1307A/p.432Asp, p.436Lys) are predominant in Caucasians and people living in the northern hemisphere, and GC1F/Gc1F (c.1296T, c.1307C/p.432Asp, p.436Thr) is predominant in Africans. However, frequencies of major GC subtypes are not known in the Kuwaiti population. In this study, we investigated 512 alleles to identify the major GC subtypes in Kuwaiti nationals.MethodsGenomic DNA was isolated from blood samples of 128 healthy subjects. DNA regions covering the targeted mutations were amplified by PCR. Amplified DNAs were sequenced by the Sanger method and analyzed for specific mutations to determine the GC genotypes and phenotypes.ResultsThe results identified the presence of four GC genotypes/phenotypes namely GC1F/Gc1F (c.1296T, c.1307C/p.432Asp, p.436Thr), GC1S/Gc1S (c.1296G, c.1307C/p.432Glu, p.436Thr), GC2/Gc2 (c.1296T, c.1307A/p.432Asp, p.436Lys), and GC3/Gc3 (c.1296G;c.1307A/p.432Glu, p.436Lys). Among the allelic subtypes (n = 512), GC1S (c.1296G; c.1307C) (n = 270, 52.7%) was predominant, followed by GC1F (c.1296T; c.1307C) (n = 138, 27%), GC2 (c.1296T; c.1307A) (n = 72, 14%), and GC3 (c.1296G; c.1307A) (n = 32, 6.3%). Three common subtypes, that is, GC1F (c.1296T; c.1307C), GC1S (c.1296G; c.1307C), and GC2 (c.1296T; c.1307A) are well documented in the literature, but GC3 (c.1296T; c.1307A) is an uncommon variant found in our study subjects.ConclusionWe found that GC subtype distribution was unique in the Kuwaiti population, with some affinity to Caucasians. Several factors including ancestral origin, migration history, and environmental forces such as solar intensity may be responsible for the unique distribution of GC subtypes in this population.

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Cellular Immune Responses to Recombinant Mycobacterium bovis BCG Constructs Expressing Major Antigens of Region of Difference 1 of Mycobacterium tuberculosis

Cited by 20 publications

References 40 publications

Immune Responses to Candidate Vaccine Antigens Delivered Through Naked Plasmid and Mycobacterial Vectors

Immune Responses to Candidate Vaccine Antigens Delivered Through Naked Plasmid and Mycobacterial Vectors

Diagnostic and Vaccine Potentials of ESAT-6 Family Proteins encoded by M. tuberculosis genomic regions absent in M. bovis BCG

Distribution of vitamin D‐binding protein/group‐specific component gene subtypes in Kuwaiti population

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