Exploration of the diversity of multi-drug resistant Mycobacterium tuberculosis complex in Lagos, Nigeria using WGS: Distribution of lineages, drug resistance patterns and genetic mutations.

Multidrug-resistant (MDR) or extensively drug-resistant (XDR) Tuberculosis (TB) is a major challenge to global TB control. Therefore, accurate tracing of in-country MDR-TB transmission are crucial for the development of optimal TB management strategies. This study aimed to investigate the diversity of MTBC in Nigeria. The lineage and drug-resistance patterns of the clinical MTBC isolates of TB patients in Southwestern region of Nigeria were determined using the WGS approach. The phenotypic DST of the isolates was determined for nine anti-TB drugs. The sequencing achieved average genome coverage of 65.99X. The most represented lineages were L4 (n = 52, 83%), L1 (n = 8, 12%), L2 (n = 2, 3%) and L5 (n = 1, 2%), suggesting a diversified MTB population. In term of detection of M/XDR-TB, while mutations in katG and rpoB genes are the strong predictors for the presence of M/XDR-TB, the current study also found the lack of good genetic markers for drug resistance amongst the MTBC in Nigeria which may pose greater problems on local tuberculosis management efforts. This high-resolution molecular epidemiological data provides valuable insights into the mechanistic for M/XDR TB in Lagos, Nigeria.

Mass spectrometry-based immunopeptidomics and computational vaccinology strategies for the identification of universal Shigella immunogenic candidates.

Shigella is a Gram-negative bacteria that cause shigellosis. Treatment with antibiotics cannot be sustained to control the bacterial infection due to the risk of antibiotic resistance. Vaccine development against the highly prevalent Shigella serotypes could provide a generous benefit in reducing the occurrence of shigellosis. The present study is aimed to identify the peptides that could be the ideal candidates for the Shigella vaccine development. THP-1 human macrophage cell lines were infected with clinical strains of Shigella flexneri 2a. The bacterial peptides bound on HLA class II molecules of infected THP-1 were analyzed and identified using the immunopeptidomics approach. Following mass spectrometry identification, a total of 14 proteins were predicted by PSORTb, CELLO, and Gneg-mPLoc as outer membrane proteins (OMPs) of Shigella. Of which, 12 OMPs were found to be conserved among Shigella species and had no significance with human proteomes. Outer membrane receptor FepA and TonB-dependent receptor were among the OMPs predicted to possess the high number of immunogenic B- and T-cell epitopes. The epitopes with high antigenicity from FepA and TonB were identified as potential peptide candidates for Shigella vaccine development. The immunoreactivity of the constructed recombinant proteins were determined using the Shigella-infected human and rabbit sera, respectively. Their protective efficacy and immune responses in controlling the Shigella infection will further be investigated in experimental animal models.

Apoptosis Activity of the Mouse Macrophage Cell Line J774A.1 Infected with a Recombinant BCG consisting the C-Terminus of Merozoite Surface Protein-1 of Plasmodium falciparum.

Macrophage apoptosis exerts an efficient mechanism in controlling intracellular infection during innate immune response against various pathogens including malaria parasites. This study was carried out to determine the apoptosis activity in mouse macrophage cell line J774A.1 infected with a Mycobacterium bovis bacille Calmette-Guerin (BCG) clone and a recombinant BCG clone expressing the C-terminus of merozoite surface protein-1 (BCG-MSP1C) of Plasmodium falciparum for 48 h. In this study, a parent BCG cells was used as a control. The nuclear staining with Hoechst 33342 showed that the BCG-MSP1C cells was capable of increasing the nuclear condensation and morphological stages of apoptosis in the infected cells compared to the BCG-infected cells and the lipopolysaccharide (LPS)-stimulated cells. The flow cytometric analysis using Annexin-V and Propidium iodide (PI) staining confirmed that the BCG-MSP1C cells significantly increased the percentage of early apoptotic activity in the infected macrophage higher than the one stimulated by the parent BCG cells and LPS. This apoptotic response corresponded with the reduction of the anti-apoptotic Bcl-2 protein expression and higher p53 expression. The colorimetric assay demonstrated that the BCG cells capable of stimulating higher production of caspase-1, -3, -8 and -9 while the BCG-MSP1C cells stimulated the expression of caspase-1 and -9 in the infected macrophages, suggesting the involvement of mitochondrial-mediated (intrinsic) pathway of apoptosis. In conclusion, both the BCG and BCG-MSP1C cells are capable of inducing macrophage apoptosis activity in the mouse macrophage cell line J774A.1. This mechanism is important for the elimination of pathogens such as malaria parasite during the phagocytosis activity of macrophage. However, the BCG-MSP1C cells showed higher apoptosis activity than those produced by the parent BCG cells.

Genome Sequencing and Annotation of Mycobacterium tuberculosis PR08 strain.

Mycobacterium tuberculosis is an acid fast bacterial species in the family Mycobacteriaceae and is the causative agent of most cases of tuberculosis. Here, we report the genomic features of Mycobacterium tuberculosis isolated from the cerebrospinal fluid (CSF) of a patient diagnosed with both pulmonary and extrapulmonary tuberculosis (TB). The isolated strain was identified as Mycobacterium tuberculosis PR08 (MTB PR08). Genomic DNA of the MTB PR08 strain was extracted and subjected to whole genome sequencing using MiSeq (Illumina, CA,USA). The draft genome size of MTB PR08 strain is 4,292,364 bp with a G + C content of 65.2%. This strain was annotated to have 4723 genes and 48 RNAs. This whole genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number CP010895.

Genome sequencing and annotation of multidrug resistant Mycobacterium tuberculosis (MDR-TB) PR10 strain.

Here, we report the draft genome sequence and annotation of a multidrug resistant Mycobacterium tuberculosis strain PR10 (MDR-TB PR10) isolated from a patient diagnosed with tuberculosis. The size of the draft genome MDR-TB PR10 is 4.34 Mbp with 65.6% of G + C content and consists of 4637 predicted genes. The determinants were categorized by RAST into 400 subsystems with 4286 coding sequences and 50 RNAs. The whole genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number CP010968.

Halal pharmaceutical industry: opportunities and challenges.

The expanding global Muslim population has increased the demand for halal pharmaceuticals. However, there are several challenges for this emerging niche industry, foremost of which is the need to establish a proper, well-regulated, and harmonized accreditation and halal management system.

Evaluation of specific humoral immune response and cross reactivity against Mycobacterium tuberculosis antigens induced in mice immunized with liposomes composed of total lipids extracted from Mycobacterium smegmatis.

The development of a new tuberculosis (TB) vaccine has become one of the main objectives of the scientific community. Protein antigens have been widely explored as subunit TB vaccines, however lipid antigens could be equally important to be used or included in such a vaccine. The aim of this study was to demonstrate the potential of a liposome formulation composed of an extract of lipids from Mycobacterium smegmatis (Ms) as a TB vaccine candidate. We evaluated the immunogenicity of this formulation as well as the cross reactive response against antigens from Mycobacterium tuberculosis (MTb) in BALB/c mice. We determined the anti-liposome IgG response in sera from TB patients and from healthy subjects who displayed a positive (PPD+) or negative (PPD-) tuberculin skin test. A significant increase in anti-liposome IgG (p<0.05) was detected in animals immunized with Bacille Calmette-Guérin (BCG) compared with all groups, and in the group immunized with liposomes from Ms (LMs) compared to animals immunized with either LMs adjuvanted with aluminium (LMs-A) or the negative control group (phosphate buffered saline, PBS) respectively. With respect to the cross reactive response against a cocktail of cell wall antigens (CWA) from MTb, significantly higher IgG levels were observed in animals immunized with BCG and LMs compared to negative controls and either, aluminium-adjuvanted liposomes (LMs-A) or montanide (LMs-M) (p<0.05). Furthermore, the anti-liposome IgG response was significantly superior in sera from pulmonary TB patients compared to PPD+ and PPD- healthy subjects (p<0.001) suggesting the expression of these antigens in vivo during active MTb infection. The results obtained provide some evidence for the potential use of liposomes containing total lipid extracts of Ms as a TB vaccine candidate.

Evaluation of the humoral immune response and cross reactivity against Mycobacterium tuberculosis of mice immunized with liposomes containing glycolipids of Mycobacterium smegmatis.

Mycobacterium smegmatis (Ms) is a nonpathogenic mycobacteria of rapid growth, which shares many characteristics with Mycobacterium tuberculosis (MTB), the major causative agent of tuberculosis. MTB has several cell wall glycolipids in common with Ms, which play an important role in the pathogenesis of tuberculosis and the induction of a protective immune response against MTB infection in some animal models. In this study, the humoral immune response and cross reactivity against MTB, of liposomes containing a mixture of cell wall glycolipids of Ms and commercial lipids was evaluated, in order to study its possible use as a component of a vaccine candidate against tuberculosis. Liposomes containing total lipids extracted from Ms, distearoyl phosphatidyl choline and cholesterol were prepared by the dehydration-rehydration technique. Balb/c mice were immunized with the liposomes obtained and the antibody response and cross reactivity against MTB were tested by ELISA. Total lipids extract from Ms showed the presence of several polar glycolipids in common with MTB, such as phosphatidylinositol mannosides. Liposomes that contained glycolipids of Ms were capable of inducing a specific IgG antibody response that allowed the recognition of surface antigens of MTB. The results of this study demonstrated the presence of immunogenic glycolipids in Ms, which could be included to enhance the protective effects of subunit vaccine formulations against tuberculosis.

Induction of an Antibody Response against Plasmodium falciparum F2RIIEBA by Heterologous Prime-boost Immunisation.

Heterologous prime-boost immunisation strategies can evoke powerful antibody responses and may be of value in developing an improved malaria vaccine. Herein, we show that an immunisation protocol that primes Balb/c mice with a recombinant Bacille Calmette-Guérin (rBCG) vaccine consisting of a plasmid encoding a synthetic fragment of the ESAT-6 epitope of Mycobacterium tuberculosis, the fragment 2 region II of erythrocyte-binding antigen (F2RIIEBA) and the three repeat sequences of the circumsporozoite protein (NANP)3 of Plasmodium falciparum before subsequently boosting the mice with either two doses of the rBCG clone or with a DNA vaccine expressing the native form of F2RIIEBA generating higher serum anti-F2RIIEBA antibody levels than an immunisation protocol that calls for a homologous prime-boost with two doses of rBCG. These results demonstrate the potential of DNA vaccination in boosting the antibody response to a recombinant vaccine expressing multiple epitopes.

Immunolocalization of insulin-like growth factors and their receptors in the diabetic mouse oviduct and uterine tissues during the preimplantation period.

The aim of the present study was to analyze the immunolocalization of insulin-like growth factor (IGF)-1 and IGF-2 and their receptors in the oviduct and uterus of control and diabetic mice. Sexually mature female ICR mice aged 6-8 weeks were rendered diabetic by streptozotocin (200 mg/kg, administered intraperitoneally). Oviductal and uterine tissues were obtained from the superovulated control and diabetic mice at 48, 72 and 96 h post-human chorionic gonadotropin (hCG) treatment. Localization of IGF-1, IGF-2, IGF-1R and IGF-2R was determined by immunohistochemistry and a semi-quantitative scoring of immunolabelling was performed using a standardized 5-point system. The immunohistochemical scorings for both IGF-1 and IGF-1R were significantly decreased in the oviducts of diabetic mice at 96 h post-hCG treatment. The scores for IGF-2 were significantly increased in the oviducts of diabetic mice at 48 and 72 h post-hCG treatment, and for IGF-2R at 72 h post-hCG treatment. However, there was no significant difference in the scores of IGFs and their receptors in the uterus of control and diabetic mice. In conclusion, the oviductal immunolabelling for IGFs and their receptors was significantly altered by maternal diabetes, which may be of importance in the pathogenesis of preimplantation diabetic embryopathy.

Diabetes alters the mRNA expression of insulin-like growth factors and their receptors in the mouse fallopian tube and uterus during the preimplantation stages.

The possible role of insulin-like growth factors (IGFs) and their receptors (IGFRs) in the pathogenesis of diabetic embryopathy was investigated. Sexually mature female ICR mice of 6-8 weeks old were made diabetic by a single intraperitoneal injection with 200 mg/kg streptozotocin ten days prior to mating. Fallopian tubes and uterine tissues were obtained from the superovulated diabetic and normal mice 48, 72 and 96 hours following human chorionic gonadotropin (hCG) injection. The mRNA expression of IGF-1 and IGF-2 as well as their receptors was determined in the tissues using Real-time Polymerase Chain Reaction (Real-time PCR). The mRNA expression of IGF-1 in the fallopian tube and uterus of the diabetic mice was significantly lower 72 and 96 hours after hCG treatment, respectively, as compared to the controls. The mRNA expression of IGF-1R at 96 hours post-hCG treatment was significantly higher in the fallopian tube and lower in the uterus of the diabetic mice as compared to the controls. The mRNA expression IGF-2 in the fallopian tube was significantly higher 48 and 96 hours after hCG treatment, but was lower in the uterus of diabetic mice 96 hours after hCG treatment as compared to controls. The mRNA expression of IGF-2R in the diabetic mice was significantly higher 48 and 96 hours (the fallopian tube) and 48 hours (uterus) after hCG treatments as compared to the controls. In conclusion, an alteration in mRNA expression of IGFs and their receptors in the diabetic mice as observed in this study could possibly result in diabetic embryopathy.

Approaches towards the development of a vaccine against tuberculosis: recombinant BCG and DNA vaccine.

The last few years have witnessed intense research on vaccine development against tuberculosis. This has been driven by the upsurge of tuberculosis cases globally, especially those caused by multi-drug-resistant Mycobacterium tuberculosis strains. Various vaccine strategies are currently being developed which can be broadly divided into the so-called living and non-living vaccines. Examples are attenuated members of the M. tuberculosis complex, recombinant mycobacteria, subunit proteins and DNA vaccines. Given current developments, we anticipate that recombinant BCG and DNA vaccines are the most promising. Multiple epitopes of M. tuberculosis may need to be cloned in a vaccine construct for the desired efficacy to be achieved. The technique of assembly polymerase chain reaction could facilitate such a cloning procedure.