Mycobacterium Bovis Strain BCG Research Articles

Challenging the gold standard: the limitations of molecular assays for detection of Mycobacterium tuberculosis heteroresistance

ObjectivesHeteroresistant infections are defined as infections in which a mixture of drug-resistant and drug-susceptible populations are present. In Mycobacterium tuberculosis (M. tb), heteroresistance poses a challenge in diagnosis and has...

PCR Development for Analysis of Some Type II Toxin-Antitoxin Systems, relJK, mazEF3, and vapBC3 Genes, in Mycobacterium tuberculosis and Mycobacterium bovis.

Toxin-Antitoxin (TA) systems are some small genetic modules in bacteria that play significant roles in resistance and tolerance development to antibiotics. Whole genome sequencing (WGS) is an effective method to analyze TA systems in pathogenic Mycobacteria. However, this study aimed to use a simple and inexpensive PCR-Sequencing approach to investigate the type II TA system. Using data from the WGS of Mycobacterium tuberculosis (M. tuberculosis) strain H37Rv and Mycobacterium bovis (M. bovis) strain BCG, primers specific to the relJK, mazEF3, and vapBC3 gene families were designed by Primer3 software. Following that, a total of 90 isolates were examined using the newly developed PCR assay, consisting of 64M. tuberculosis and 26M. bovis isolates, encompassing both 45 rifampin-sensitive and 45 rifampin-resistant strains. Finally, 28 isolates (including 14 rifampin-resistant isolates) were sent for sequencing, and their sequences were aligned and compared to the mentioned reference sequences. The amplicons size of mazEF3, relJK, and vapBC3 genes were 825, 875, and 934 bp, respectively. Furthermore, all tested isolates showed the specific amplicons for these TA families. To evaluate the specificity of the primers, PCR was performed on S. aureus and E.coli isolates. None of the examined samples had the desired amplicons. Therefore, the primers had acceptable specificity. The results indicated that the developed PCR-Sequencing approach can be used to effectively investigate certain types of TA systems. Considering high costs of WGS and difficulty in interpreting its results, such a simple and inexpensive method is beneficial in the evaluation of TA systems in Mycobacteria.

Galleria mellonella as an infection model for the virulent Mycobacterium tuberculosis H37Rv

ABSTRACT Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a leading cause of infectious disease mortality. Animal infection models have contributed substantially to our understanding of TB, yet their biological and non-biological limitations are a research bottleneck. There is a need for more ethically acceptable, economical, and reproducible TB infection models capable of mimicking key aspects of disease. Here, we demonstrate and present a basic description of how Galleria mellonella (the greater wax moth, Gm) larvae can be used as a low cost, rapid, and ethically more acceptable model for TB research. This is the first study to infect Gm with the fully virulent MTB H37Rv, the most widely used strain in research. Infection of Gm with MTB resulted in a symptomatic lethal infection, the virulence of which differed from both attenuated Mycobacterium bovis BCG and auxotrophic MTB strains. The Gm-MTB model can also be used for anti-TB drug screening, although CFU enumeration from Gm is necessary for confirmation of mycobacterial load reducing activity of the tested compound. Furthermore, comparative virulence of MTB isogenic mutants can be determined in Gm. However, comparison of mutant phenotypes in Gm against conventional models must consider the limitations of innate immunity. Our findings indicate that Gm will be a practical, valuable, and advantageous additional model to be used alongside existing models to advance tuberculosis research.

Vaccination of Calves with the Mycobacterium bovis BCG Strain Induces Protection against Bovine Tuberculosis in Dairy Herds under a Natural Transmission Setting.

Simple SummaryBovine tuberculosis (bTB) is a zoonotic disease caused mainly by Mycobacterium bovis, of which control is based on culling infected animals and, without official compensations, is associated with major economic losses for milk and meat producers. The vaccination of cattle with the M. bovis Bacillus Calmette-Guerin (BCG) strain, as a strategy for bTB control, might attenuate this situation. The objective of this trial was to assess the efficacy of the BCG Russia strain in a cohort study performed under field conditions, with the vaccination of 501 calves in seven dairy farms, including 441 non-vaccinated control animals. Peripheral blood was collected at 6, 12 and 18 months post-vaccination, and infection status was determined using a diagnostic procedure which discriminates the infected amongst vaccinated animals. On average, the BCG vaccine showed a low but significant level of protection (22.4%) at the end of the trial, although diverse levels of protection and duration of immunity were observed between trial herds, suggesting that the efficacy of the BCG vaccination could be influenced by the general health condition of calves and their exposition to non-tuberculous mycobacteria. These results support the use of BCG as a complementary tool in the control of the disease in high prevalence areas.Bovine tuberculosis (bTB) is a zoonotic disease caused mainly by Mycobacterium bovis, which is associated with major economic losses for milk and meat producers. The objective of this trial was to assess the efficacy of the BCG Russia strain in a cohort study performed under field conditions, with the vaccination of calves in seven dairy farms from a high prevalence area in central Chile. The trial was performed with 501 animals, subcutaneously vaccinated with 2–8 × 105 colony-forming units of BCG, whilst 441 matched control animals received a saline placebo. Peripheral blood was collected at 6, 12 and 18 months post-vaccination, and infection status was determined using the IFNγ release assay in conjunction with the DIVA (Detecting Infected amongst Vaccinated Animals) antigens ESAT-6, CFP-10 and Rv3615c. The BCG vaccine showed a low but significant level of protection of 22.4% (95% CI 4.0 to 36.4) at the end of the trial. However, diverse levels of protection and a variable duration of immunity were observed between trial herds. This diverse outcome could be influenced by the general health condition of calves and their exposition to non-tuberculous mycobacteria. These results suggest that BCG vaccination of dairy calves in a natural transmission setting confers variable protection to animals against bTB in a high prevalence area.

TonB-dependent receptor epitopes expressed in M. bovis BCG induced significant protection in the hamster model of leptospirosis.

Leptospirosis is an emerging infectious disease caused by pathogenic Leptospira spp. A universal vaccine against leptospirosis is likely to require highly conserved epitopes from pathogenic leptospires that are exposed on the bacterial surface and that generate a protective and sterilizing immune response. Our group recently identified several genes predicted to encode TonB-dependent receptors (TBDR) in Leptospira interrogans using a reverse vaccinology approach. Three leptospiral TBDRs were previously described and partially characterized as ferric-citrate, hemin, and cobalamin transporters. In the current study, we designed a fusion protein composed of predicted surface-exposed epitopes from three conserved leptospiral TBDRs. Based on their three-dimensional structural models and the prediction of immunogenic regions, nine putative surface-exposed fragments were selected to compose a recombinant chimeric protein. A Mycobacterium bovis BCG strain expressing this chimeric antigen encoded in the pUP500/PpAN mycobacterial expression vector was used to immunize Syrian hamsters. All animals (20/20) vaccinated with recombinant BCG survived infection with an endpoint dose of L. interrogans (p < 0.001). No animal survived in the negative control group. Immunization with our recombinant BCG elicited a humoral immune response against leptospiral TBDRs, as demonstrated by ELISA and immunoblot. No leptospiral DNA was detected by lipL32 qPCR in the kidneys of vaccinated hamsters. Similarly, no growth was observed in macerated kidney cultures from the same animals, suggesting the induction of a sterilizing immune response. Design of new vaccine antigens based on the structure of outer membrane proteins is a promising approach to overcome the impact of leptospirosis by vaccination.Key points• Predicted surface-exposed epitopes were identified in three leptospiral TBDRs.• An M. bovis BCG strain expressing a chimeric protein (rTBDRchi) was constructed.• Hamsters vaccinated with rBCG:TBDRchi were protected from lethal leptospirosis.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s00253-021-11726-9.

Attempt of thyX gene silencing and construction of a thyX deleted clone in a Mycobacterium bovis BCG.

Mycobacterium tuberculosis, the causative agent of tuberculosis, possess flavin-dependent thymidylate synthase, ThyX. Since thyX is absent in humans and was shown to be essential for M. tuberculosis normal growth, ThyX is thought to be an attractive novel TB drug target. This study assessed thyX essentiality in Mycobacterium bovis BCG strains using CRISPR interference based gene silencing and found that thyX is not essential in an M. bovis BCG Tokyo derivative strain. A thyX deletion mutant strain was successfully constructed from that strain, which reinforces the non-essentiality of thyX under a certain genetic background.

The humoral immune response elicited upon immunization with recombinant BCG vaccines protects against hRSV and hMPV in a murine preclinical model

Abstract Human respiratory syncytial virus (hRSV) and human metapneumovirus (hMPV) are among the main etiological agents responsible of acute lower respiratory tract infections in vulnerable populations worldwide. Despite their clinical relevance, the licensing of a safe and effective vaccine has not been achieved. Considering this, our laboratory developed two different recombinant Mycobacterium bovis BCG strains as vaccine candidates. These recombinant BCGs express either the hRSV-Nucleoprotein (rBCG-N) or the hMPV-Phosphoprotein (rBCG-P). Both vaccines prototypes were able to promote cellular protection against their respective virus. However, the humoral response elicited upon immunization with these vaccines has not been characterized. Here we show that, upon immunization and challenge of mice with the respective rBCG and virus, the induced humoral response was able to protect against the viral infection, promoting the secretion of specific antibodies against each virus and several of their proteins, as measured in sera samples by ELISA assays. We also described that immunization and challenge promoted the induction of antibodies with enhanced neutralizing capacities, as seen during in vitro assays. Moreover, we show that sera transferred from immunized and challenged mice into naïve mice is enough to protect from the infection, when these naïve mice were challenged with the viruses. This protection was characterized by a decreased in the viral replication and pulmonary disease. These results support the notion that the use of rBCG vectors could be an effective vaccine platform against these two major respiratory pathogens and places our vaccine candidates as suitable approaches for the treatment against both hRSV and hMPV.

In silico Studies, Synthesis and Antitubercular Activity of Some Novel Quinoline - Azitidinone Derivatives.

Diarylquinolines like Bedaquiline have shown promising antitubercular activity by their action of Mycobacterial ATPase. The structural features necessary for a good antitubercular activity for a series of quinoline derivatives were explored through computational chemistry tools like QSAR and combinatorial library generation. In the current study, 3-Chloro-4-(2-mercaptoquinoline-3-yl)-1- substitutedphenylazitidin-2-one derivatives have been designed and synthesized based on molecular modeling studies as anti-tubercular agents. 2D and 3D QSAR analyses were used to designed compounds having a quinoline scaffold. The synthesized compounds were evaluated against active and dormant strains of Mycobacterium tuberculosis (MTB) H37 Ra and Mycobacterium bovis BCG. The compounds were also tested for cytotoxicity against MCF-7, A549 and Panc-1 cell lines using MTT assay. The binding affinity of designed compounds was gauged by molecular docking studies. Statistically significant QSAR models generated by the SA-MLR method for 2D QSAR exhibited r2 = 0.852, q2 = 0.811, whereas 3D QSAR with SA-kNN showed q2 = 0.77. The synthesized compounds exhibited MIC in the range of 1.38-14.59(μg/ml). These compounds showed some crucial interaction with MTB ATPase. The present study has shown some promising results which can be further explored for lead generation.

Phylogenomic and epidemiological insights into two clinical Mycobacterium bovis BCG strains circulating in South Africa

BackgroundMycobacterium bovis BCG is a live, attenuated tuberculosis vaccine. While the vaccine protects infants from tuberculosis, complications including disseminated infections have been reported following vaccination. Genetically diverse BCG sub-strains now exist following continuous passaging of the original Pasteur strain for vaccine manufacture. This genetic diversity reportedly influences the severity of disseminated BCG infections and the efficacy of BCG immunization. MethodsM. bovis BCG was isolated from infants suspected of being infected with tuberculosis. The whole genome of the clinical isolates and BCG Moscow were sequenced using Illumina Miseq and the sequences were analysed using CLC Genomics Workbench 7.0, PhyResSE v1.0, and Parsnp. Results and conclusionsGenetic variations between the clinical strains and the reference BCG Copenhagen were identified. The clinical strains shared only one mutation in a secretion protein. Mutations were identified in various antibiotic resistance genes in the BCG isolates, which suggests their potential as multidrug-resistant (MDR) phenotypes. Phylogenetic analysis showed that the two isolates were distantly related, and the M1_S48 clinical isolate was closely related to M. bovis BCG Moscow. The phylogenomics results imply that two different BCG strains may be circulating in South Africa. However, it is difficult to associate the BCG vaccine strain administered and the BCG strain supplied with specific adverse events, as BCGiosis is under-reported. This study presents background genomic information for future surveillance and tracking of the distribution of BCGiosis-associated mycobacteria. It is also the first to report on the genomes of clinical BCG strains in Africa.

Combined immunodeficiency in a patient with c-Rel deficiency

This study reports a homozygous mutation in REL abrogating c-Rel protein expression in a patient with combined immunodeficiency characterized by susceptibility to Mycobacterium tuberculosis, Salmonella, Cryptosporidium, and cytomegalovirus.

Priming With Recombinant BCG Expressing Novel HIV-1 Conserved Mosaic Immunogens and Boosting With Recombinant ChAdOx1 Is Safe, Stable, and Elicits HIV-1-Specific T-Cell Responses in BALB/c Mice.

BCG is currently the only licensed vaccine against tuberculosis (TB) and confers protection against meningitis and miliary tuberculosis in infants, although pulmonary disease protection in adults is inconsistent. Recently, promising HIV-1 immunogens were developed, such as the T-cell immunogens “tHIVconsvX,” designed using functionally conserved protein regions across group M strains, with mosaic immunogens to improve HIV-1 variant match and response breadth. In this study, we constructed an integrative E. coli-mycobacterial shuttle plasmid, p2auxo.HIVconsvXint, expressing the immunogens HIVconsv1&2. This expression vector used an antibiotic resistance-free mechanism for plasmid selection and maintenance. It was first transformed into a glycine auxotrophic E. coli strain and subsequently transformed into a lysine auxotrophic Mycobacterium bovis BCG strain to generate vaccines BCG.HIVconsv12auxo.int and BCG.HIVconsv22auxo.int. The DNA sequence coding for the HIVconsv1&2 immunogens and protein expression were confirmed and working vaccine stocks were genetically and phenotypically characterized. We demonstrated that BCG.HIVconsv1&22auxo.int in combination with ChAdOx1.tHIVconsv5&6 were well tolerated and induced HIV-1-specific T-cell responses in adult BALB/c mice. In addition, we showed that the BCG.HIVconsv1&22auxo.int vaccine strains were stable in vitro after 35 bacterial generations and in vivo 7 weeks after inoculation. The use of integrative expression vectors and novel HIV-1 immunogens are likely to have improved the mycobacterial vaccine stability and specific immunogenicity and may enable the development of a useful vaccine platform for priming protective responses against HIV-1/TB and other prevalent pediatric pathogens shortly following birth.

Utilisation of the Prestwick Chemical Library to identify drugs that inhibit the growth of mycobacteria.

Tuberculosis (TB) is an infectious bacterial disease that kills approximately 1.3 million people every year. Despite global efforts to reduce both the incidence and mortality associated with TB, the emergence of drug resistant strains has slowed any progress made towards combating the spread of this deadly disease. The current TB drug regimen is inadequate, takes months to complete and poses significant challenges when administering to patients suffering from drug resistant TB. New treatments that are faster, simpler and more affordable are urgently required. Arguably, a good strategy to discover new drugs is to start with an old drug. Here, we have screened a library of 1200 FDA approved drugs from the Prestwick Chemical library using a GFP microplate assay. Drugs were screened against GFP expressing strains of Mycobacterium smegmatis and Mycobacterium bovis BCG as surrogates for Mycobacterium tuberculosis, the causative agent of TB in humans. We identified several classes of drugs that displayed antimycobacterial activity against both M. smegmatis and BCG, however each organism also displayed some selectivity towards certain drug classes. Variant analysis of whole genomes sequenced for resistant mutants raised to florfenicol, vanoxerine and pentamidine highlight new pathways that could be exploited in drug repurposing programmes.

Targeting wild‐type and drug‐resistant mycobacteria in infected macrophages using drug‐coated nanoparticles

AbstractBACKGROUNDTuberculosis therapeutics is facing a crisis because of the emergence of new multi and total drug‐resistant strains of Mycobacterium tuberculosis. Increasing intra‐bacterial as well as intra‐macrophage drug concentrations through nanoparticles is a promising approach to target resistant bacteria.RESULTSTo target both wild‐type (WT) and resistant Mycobacterium smegmatis (NORr), positively‐charged norfloxacin‐coated iron oxide nanoparticles (NOR@IONP) were synthesized. While these particles are taken up identically in both WT and NORr, drug uptake was up to 4‐fold higher in cells treated with NOR@IONP compared with the corresponding free drug. This increased intracellular drug accumulation also translated to an enhanced rate of bacterial killing, as is observed in the kill kinetics study. The coated nanoparticles also proved to be effective in reducing the minimum inhibitory concentration (MIC) of NOR towards Mycobacterium bovis BCG strain. Further, the effectiveness of NOR@IONP was also studied against differentiated THP1 cells infected with WT or NORr M. smegmatis. Intracellular bacterial clearance studies indicated that the NOR concentration required to kill intracellular WT or drug‐resistant bacteria is 2‐fold lower when treated with NOR@IONP.CONCLUSIONThis antibiotic‐coated nanoparticle formulation is a novel antimicrobial agent with the potential to target both WT and resistant strains of mycobacteria present intracellularly. © 2018 Society of Chemical Industry

Identification and characterization of aspartyl-tRNA synthetase inhibitors against Mycobacterium tuberculosis by an integrated whole-cell target-based approach

Mycobacterium tuberculosis, the causative agent of tuberculosis, has surpassed HIV as the leading cause of death due to an infectious disease worldwide, being responsible for more than 1.5 million deaths in low-income countries. In response to a pandemic threat by drug resistant strains, the tuberculosis research community is searching for new chemical entities with novel mechanisms of action to avoid drug resistance and shorten treatment regimens using combinatorial chemotherapy. Herein, we have identified several novel chemical scaffolds, GSK97C (spiro-oxazolidin-2-one), GSK93A (2-amino-1,3-thiazole, GSK85A and GSK92A (enamides), which target M. tuberculosis aspartyl-tRNA synthetase (Mt-AspRS), an essential component of the protein synthesis machinery of tuberculosis, using a whole-cell target-based screening strategy against a genetically modified Mycobacterium bovis BCG strain. We also provide further evidence of protein inhibition and inhibitor profiling through a classical aminoacylation reaction and a tRNA-independent assay, respectively. Altogether, our results have identified a number of hit new molecules with novel mechanism of action for further development through medicinal chemistry as hits and leads.

Stability attenuation of BCG vaccine strain

As the basis for prevention of tuberculosis is the use of BCG vaccine. The experience of using the tuberculous vaccine has shown quite positive results. Mycobacterium bovis of BCG vaccine strain, which are preserved in laboratories from different countries may differ slightly in their biological properties, including virulence and immunogenicity. Objective of work is to investigate the stability attenuation of BCG vaccine strain. BCG vaccine strain (BCG-Russia) was used for investigations. The morphology, cultural and biochemical properties of mycobacteria determined by means of agreed-upon methods. We used amplifier iCycler iQ5 and a set of reagents for PCR-amplification of DNA with real-time detection for carrying out PCR. The determination of the virulence and sensitivizing properties of mycobacteria was carried out using the biological test. The pieces of the spleen and lungs were taken for histological examination. It was found that Mycobacterium bovis BCG strains formed on the nutrient medium by Mordovskyi the matted colonies of ivory color with a hilly surface and uneven edges (R-forms). In the smears from colonies, after staining by Ziehl-Neelsen, acid-resistant and non-acid resistant sticks were observed. Mycobacteria BCG strain in the medium of Shkolnykova formed microcolonies, which morphologically resembled «cord». Gene-molecular studies have established the presence of the DNA-target in the mycobacteria of the vaccinal strain. In infected Guinea pigs 3–4 weeks later, an ulcer was formed at the place of introduction of the suspension. Mycobacteria did not cause in laboratory animals the death and development of an infectious process characteristic of tuberculosis. At the autopsy of Guinea pigs characteristic macroscopic changes (tubercular nodes) were not observed. In the spleen of all animals, moderate hyperemia, red pulp hyperplasia were observed. Lymphoid follicles had signs of hyperplasia. At the intersection of laboratory animals of the second and third passages macroscopic pathoanatomical changes were not found. Conclusion: BCG strain do not cause macroscopic pathoanatomical changes in Guinea pigs during three «blind» passages, indicating the stability of his attenuation.

Evaluate the Efficiency of A60 Protein from BCG Strain of Mycobacterium Bovis as a Diagnostic Antigen

Evaluate the Efficiency of A60 Protein from BCG Strain of Mycobacterium Bovis as a Diagnostic Antigen

A Combination of Recombinant Mycobacterium bovis BCG Strains Expressing Pneumococcal Proteins Induces Cellular and Humoral Immune Responses and Protects against Pneumococcal Colonization and Sepsis.

Pneumococcal diseases remain a substantial cause of mortality in young children in developing countries. The development of potentially serotype-transcending vaccines has been extensively studied; ideally, such a vaccine should include antigens that are able to induce protection against colonization (likely mediated by interleukin-17A [IL-17A]) and invasive disease (likely mediated by antibody). The use of strong adjuvants or alternative delivery systems that are able to improve the immunological response of recombinant proteins has been proposed but poses potential safety and practical concerns in children. We have previously constructed a recombinant Mycobacterium bovis BCG strain expressing a pneumococcal surface protein A (PspA)-PdT fusion protein (rBCG PspA-PdT) that was able to induce an effective immune response and protection against sepsis in a prime-boost strategy. Here, we constructed two new rBCG strains expressing the pneumococcal proteins SP 0148 and SP 2108, which confer IL-17A-dependent protection against pneumococcal colonization in mouse models. Immunization of mice with rBCG 0148 or rBCG 2108 in a prime-boost strategy induced IL-17A and gamma interferon (IFN-γ) production. The combination of these rBCG strains with rBCG PspA-PdT (rBCG Mix), followed by a booster dose of the combined recombinant proteins (rMix) induced an IL-17A response against SP 0148 and SP 2108 and a humoral response characterized by increased levels of IgG2c against PspA and functional antibodies against pneumolysin. Furthermore, immunization with the rBCG Mix prime/rMix booster (rBCG Mix/rMix) provides protection against pneumococcal colonization and sepsis. These results suggest the use of combined rBCG strains as a potentially serotype-transcending pneumococcal vaccine in a prime-boost strategy, which could provide protection against pneumococcal colonization and sepsis.

Synthesis, antimycobacterial screening and molecular docking studies of 4-aryl-4′-methyl-2′-aryl-2,5′-bisthiazole derivatives

A series of 4-aryl-4′-methyl-2′-aryl-2,5′-bisthiazole derivatives (5a–o) were synthesized and screened for inhibitory activity against Mycobacterium tuberculosis H37Ra (ATCC 25177) and Mycobacterium bovis BCG (ATCC 35743) strains. Five lead compounds (5e, 5f, 5g, 5h, and 5o) were further confirmed from their dose dependent effect against MTB and Bovine–Calmette–Guerin. The most promising compounds 5f (MIC90: 11.32 µg/mL), 5h (MIC90: 11.59 µg/mL), and 5o (MIC90: 23.64 µg/mL) showed strong antitubercular activity against dormant MTB and BCG as well as almost insignificant cytotoxicity up to 100 µg/mL against HeLa, A549, and PANC-1 human cancer cell lines. Further, the synthesized compounds were found to have potential antibacterial activity against Gram-negative bacteria, Escherichia coli, Pseudomonas flurescence and Gram-positive bacteria, Staphylococcus aureus, Bacillus subtilis. Most of the synthesized compounds showed moderate activity against fungal strain Candida albicans. Molecular docking studies of these compounds showed significant interactions with crystal structure of the cytochrome P45014α-sterol demethylase (CYP51) PDB ID: 1E9X. Hydrogen bond interactions with SER261 and VAL395 are important interactions for selective inhibition of designed inhibitors. Compounds 5f, 5h, and 5o showed significant interactions with 1E9X. All the experimental results promote us to consider this series as a starting point for the development of novel, selective and more potent antitubercular agents in the future.

Vaccine Against Entheropathogenic E. colii: A Systematic Review

Enteric diseases are a major cause of childhood death in the developing world, ranking as the second cause of death in children. Enteropathogenic Escherichia coli(EPEC) are important diarrheal pathogens of children under 5 years of age. Due to high mortality, several international organizations such as the WHO and UNICEF have dedicated preventive and control programs for diarrheal diseases. Among the suggested initiatives aiming to prevent infectious diarrhea include vaccine development and improvement in sanitation and water and food supplies. Upon contact with host cells, EPEC delivers an array of virulence protein factors, which integrated actions interferes with the normal adjusting the targets molecular cell functions leading to diarrhea. The locus of entherocyte effacement (LEE)pathogenicity island contains genes encoding synthesis of the EPEC virulence factors membrane adhesin intimin, T3SS (Esc and Sep proteins), chaperones (Ces proteins), translocators (EspA, EspB, and EspD), effector proteins (EspF, EspG, EspH, Map and EspZ), the translocated intimin receptor (Tir), and the regulatory protein Ler (LEEencoded regulator).Bundle-forming pilus (BfpA) is another virulence factor that mediates the initial contact between EPEC and the host cell. BfpA is encoded by a gene localized on a 50–70 MDa plasmid and is designated as EPEC adherence factor (EAF). BfpA, intimin and translocated Tir initiate EPEC infection. This repertoire of virulence factors offers strategic epitopes as vaccine candidates. Employing the proposed technologies for modern vaccines, recombinant Mycobacterium smegmatis (Smeg) and Mycobacterium bovis BCG strains were constructed to express BfpA, intimin and EspA. Recombinant clones were selected based on kanamycin resistance. Recombinant proteins are immunogenic, and the resultant antibodies recognize and block EPEC adhesion onto HEp-2 target cells. In attention to regulatory agency recommendations, kanamycin should not be used to produce rBCG expressing the E. colii EPEC virulence factors BfpA, intimin and EspA. Accordingly, an expression system with a Pasteur auxotrophic BCG mutant for leucine (BCG Pasteur ΔleuD) and a replicative vector (pUP410)should be used. Obtained recombinants will be grown in large scale, and their immunogenic effectivity and human safety submitted to WHO indicated quality controls

Preclinical development of BCG.HIVA2auxo.int, harboring an integrative expression vector, for a HIV-TB Pediatric vaccine. Enhancement of stability and specific HIV-1 T-cell immunity

ABSTRACTOne of the critical issues that should be addressed in the development of a BCG-based HIV vaccine is genetic plasmid stability. Therefore, to address this issue we have considered using integrative vectors and the auxotrophic mutant of BCG complemented with a plasmid carrying a wild-type complementing gene. In this study, we have constructed an integrative E. coli-mycobacterial shuttle plasmid, p2auxo.HIVAint, expressing the HIV-1 clade A immunogen HIVA. This shuttle vector uses an antibiotic resistance-free mechanism for plasmid selection and maintenance. It was first transformed into a glycine auxotrophic E. coli strain and subsequently transformed into a lysine auxotrophic Mycobacterium bovis BCG strain to generate the vaccine BCG.HIVA2auxo.int. Presence of the HIVA gene sequence and protein expression was confirmed. We demonstrated that the in vitro stability of the integrative plasmid p2auxo.HIVAint was increased 4-fold, as compared with the BCG strain harboring the episomal plasmid, and was genetically and phenotypically characterized. The BCG.HIVA2auxo.int vaccine in combination with modified vaccinia virus Ankara (MVA).HIVA was found to be safe and induced HIV-1 and Mycobacterium tuberculosis-specific interferon-γ-producing T-cell responses in adult BALB/c mice. We have engineered a more stable and immunogenic BCG-vectored vaccine using the prototype immunogen HIVA. Thus, the use of integrative expression vectors and the antibiotic-free plasmid selection system based on “double” auxotrophic complementation are likely to improve the mycobacterial vaccine stability in vivo and immunogenicity to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective responses shortly following birth.