Browsing by Author "Rakesh Bhatnagar"

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A combined subunit vaccine comprising BP26, Omp25 and L7/L12 against brucellosis
(Oxford University Press, 2019) Sonal Gupta; Damini Singh; Manish Gupta; Rakesh Bhatnagar
The current vaccines against brucellosis, namely Brucella abortus strains 19 and RB51, prevent infection in animals but pose potential risks like virulence and attenuation reversal. In this milieu, although subunit vaccination using a single potent immunogen of B. abortus, e.g. BP26 or Omp25 or L7/L12 etc., appears as a safer alternative, nonetheless it confers inadequate protection against the zoonosis compared to attenuated vaccines. Hence, we have investigated the prophylactic potential of a combined subunit vaccine (CSV) comprising the BP26, Omp25 and L7/L12 antigens of B. abortus, in mice model. Sera obtained from CSV immunized mice groups showed heightened IgG titers against all the three components and exhibited specificity upon immunoblotting, reiterating their authenticity. Further, the IgG1/IgG2a ratio obtained against each antigen revealed a predominant Th2 immune response in CSV immunized mice group. However, on assessing the levels of Th1-dependent (IFN-γ and TNF-α) and Th2-dependent (IL-4 and IL-10) cytokines in different formulations, prominent IFN-γ levels were elicited in CSV immunized mice. Further, upon infection with virulent B. abortus 544, the combined subunit vaccinated mice displayed superior degree of protection (Log10 reduction) than the individual vaccines; however, B. abortus S19 showed the highest protection. Altogether, this study suggests that co-immunization of three B. abortus immunogens as a CSV complements and triggers a mixed Th1/Th2 immune response leading to superior degree of protection against pathogenic B. abortus 544 infection. © FEMS 2020.
Anthrax prevention through vaccine and post-exposure therapy
(Taylor and Francis Ltd., 2020) Manish Manish; Shashikala Verma; Divya Kandari; Parul Kulshreshtha; Samer Singh; Rakesh Bhatnagar
Introduction: Vaccines and therapeutic antibodies are the most crucial components of anthrax prophylaxis (pre- and post-exposure) and treatment. The improvement in the availability and safety profile of vaccines and the therapeutic antibodies has helped immensely in reducing the worldwide burden of anthrax. Areas covered: Current recommendations for anthrax prophylaxis and control, vaccines and therapeutic antibodies, the recent endeavors, particularly, made after 2010 toward making them safer and more efficacious along with our opinion on its future course. Primarily, PubMed and Europe PMC were searched to cover the recent developments in the above-indicated areas. Expert opinion: Some key existing lacunae in our understanding of the working of biologicals-based anthrax-control measures, i.e., vaccines and therapeutic antibodies, should be addressed to improve their overall stability, safety profile, and efficacy. The identification of novel inhibitors targeting different key-molecules and vital-steps contributing to the overall anthrax pathophysiology could make a difference in anthrax control. © 2020 Informa UK Limited, trading as Taylor & Francis Group.
Antibody engineering and its therapeutic applications
(Taylor and Francis Ltd., 2023) Divya Kandari; Rakesh Bhatnagar
As a natural function, antibodies defend the host from infected cells and pathogens by recognizing their pathogenic determinants. Antibodies (Abs) gained wide acceptance with an enormous impact on human health and have predominantly captured the arena of bio-therapeutics and bio-diagnostics. The scope of Ab-based biologics is vast, and it is likely to solve many unmet clinical needs in future. The majority of attention is now devoted to developing innovative technologies for manufacturing and engineering Abs, better suited to satisfy human needs. The advent of Ab engineering technologies (AET) led to phenomenal developments leading to the generation of Abs-/Ab-derived molecules with desirable functional properties proportional to their expanding requirements. Evolution brought by AET, from the naturally occurring Ab forms to several advanced Ab formats and derivatives, was much needed as it is of great interest to the pharmaceutical industry. Thus, numerous advancements in AET have propelled success in therapeutic Ab development, along with the potential for ever-increasing improvements. Unique characteristics of Abs, such as its diversity, specificity, structural integrity and an array of possible applications, together inspire continuous innovation in the field. Overall, the AET could assist in conquer of several limitations of Abs in terms of their applicability in the field of therapeutics, diagnostics and research; AET has so far led to the production of next-generation Abs, which have revolutionized these arenas. Here in this review, we discuss the various distinguished engineering platforms for Ab development and the progress in modern therapeutics by the so-called “next-generation Abs.”. © 2021 Taylor & Francis Group, LLC.
Asp 187 and Phe 190 residues in lethal factor are required for the expression of anthrax lethal toxin activity
(2002) Aparna Singh; Vibha Chauhan; Ajit Sodhi; Rakesh Bhatnagar
Anthrax toxin consists of three proteins, protective antigen, lethal factor, and edema factor. Protective antigen translocates lethal factor and edema factor to the cytosol of mammalian cells. The amino-termini of lethal factor and edema factor have several homologous stretches. These regions are presumably involved in binding to protective antigen. In the present study we have determined the role of one such homologous stretch in lethal factor. Residues 187AspLeuLeuPhe190 were replaced by alanine. Asp187Ala and Phe190Ala were found to be non-toxic in combination with protective antigen. Their protective antigen-binding ability was drastically reduced. We propose that Asp187 and Phe190 are crucial for the expression of anthrax lethal toxin activity. © 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
ATP-binding cassette (ABC) import systems of Mycobacterium tuberculosis: target for drug and vaccine development
(Taylor and Francis Ltd., 2020) Dharmendra Kumar Soni; Suresh Kumar Dubey; Rakesh Bhatnagar
Nutrient procurement specifically from nutrient-limiting environment is essential for pathogenic bacteria to survive and/or persist within the host. Long-term survival or persistent infection is one of the main reasons for the overuse of antibiotics, and contributes to the development and spread of antibiotic resistance. Mycobacterium tuberculosis is known for long-term survival within the host, and develops multidrug resistance. Before and during infection, the pathogen encounters various harsh environmental conditions. To cope up with such nutrient-limiting conditions, it is crucial to uptake essential nutrients such as ions, sugars, amino acids, peptides, and metals, necessary for numerous vital biological activities. Among the various types of transporters, ATP-binding cassette (ABC) importers are essentially unique to bacteria, accessible as drug targets without penetrating the cytoplasmic membrane, and offer an ATP-dependent gateway into the cell by mimicking substrates of the importer and designing inhibitors against substrate-binding proteins, ABC importers endeavour for the development of successful drug candidates and antibiotics. Alternatively, the production of antibodies against substrate-binding proteins could lead to vaccine development. In this review, we will emphasize the role of M. tuberculosis ABC importers for survival and virulence within the host. Furthermore, we will elucidate their unique characteristics to discover emerging therapies to combat tuberculosis. © 2020, © 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group, on behalf of Shanghai Shangyixun Cultural Communication Co., Ltd.
Co-administration of aluminium hydroxide nanoparticles and protective antigen domain 4 encapsulated non-ionic surfactant vesicles show enhanced immune response and superior protection against anthrax
(MDPI AG, 2020) Himanshu Gogoi; Rajesh Mani; Anshu Malik; Parveen Sehrawat; Rakesh Bhatnagar
Aluminium salts have been the adjuvant of choice in more than 100 licensed vaccines. Here, we have studied the synergistic effect of aluminium hydroxide nanoparticles (AH np) and non-ionic surfactant-based vesicles (NISV) in modulating the immune response against protective antigen domain 4 (D4) of Bacillus anthracis. NISV was prepared from Span 60 and cholesterol, while AH np was prepared from aluminium chloride and sodium hydroxide. AH np was co-administered with NISV encapsulating D4 (NISV-D4) to formulate AHnp/NISV-D4. The antigen-specific immune response of AHnp/NISV-D4 was compared with that of commercial alhydrogel (alhy) co-administered with NISV-D4 (alhydrogel/NISV-D4), NISV-D4, AHnp/D4, and alhydrogel/D4. Co-administration of NISV-D4 with AH np greatly improved the D4-specific antibody titer as compared to the control groups. Based on IgG isotyping and ex vivo cytokine analysis, AHnp/NISV-D4 generated a balanced Th1/Th2 response. Furthermore, AH np/NISV-D4 showed superior protection against anthrax spore challenge in comparison to other groups. Thus, we demonstrate the possibility of developing a novel combinatorial nanoformulation capable of augmenting both humoral and cellular response, paving the way for adjuvant research. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
Common garlic (Allium sativum L.) has potent Anti-Bacillus anthracis activity
(Elsevier Ireland Ltd, 2021) Rajinder Kaur; Atul Tiwari; Manish Manish; Indresh K. Maurya; Rakesh Bhatnagar; Samer Singh
Ethnopharmacological relevance: Gastrointestinal anthrax, a disease caused by Bacillus anthracis, remains an important but relatively neglected endemic disease of animals and humans in remote areas of the Indian subcontinent and some parts of Africa. Its initial symptoms include diarrhea and stomachache. In the current study, several common plants indicated for diarrhea, dysentery, stomachache or as stomachic as per traditional knowledge in the Indian subcontinent, i.e., Aegle marmelos (L.) Correa (Bael), Allium cepa L. (Onion), Allium sativum L. (Garlic), Azadirachta indica A. Juss. (Neem), Berberis asiatica Roxb. ex DC. (Daruharidra), Coriandrum sativum L. (Coriander), Curcuma longa L. (Turmeric), Cynodon dactylon (L.) Pers. (Bermuda grass), Mangifera indica L. (Mango), Morus indica L. (Black mulberry), Ocimum tenuiflorum L. (Ocimum sanctum L., Holy Basil), Ocimum gratissimum L. (Ram Tulsi), Psidium guajava L. (Guava), Zingiber officinale Roscoe (Ginger), were evaluated for their anti-Bacillus anthracis property. The usage of Azadirachta indica A. Juss. and Curcuma longa L. by Santals (India), and Allium sp. by biblical people to alleviate anthrax-like symptoms is well documented, but the usage of other plants is traditionally only indicated for different gastrointestinal disturbances/conditions. Aim of the study: Evaluate the above listed commonly available edible plants from the Indian subcontinent that are used in the traditional medicine to treat gastrointestinal diseases including those also indicated for anthrax-like symptoms for the presence of potent anti-B. anthracis activity in a form amenable to use by the general population in the endemic areas. Materials and methods: Aqueous extracts made from fourteen plants indicated above were screened for their anti-B. anthracis activity using agar-well diffusion assay (AWDA) and broth microdilution methods. The Aqueous Garlic Extract (AGE) that displayed most potent anti-B. anthracis activity was assessed for its thermostability, stability under pH extremes encountered in the gastrointestinal tract, and potential antagonistic interaction with bile salts as well as the FDA-approved antibiotics used for anthrax control. The bioactive fractions from the AGE were isolated by TLC coupled bioautography followed by their characterization using GC-MS. Results: Garlic (Allium sativum L.) extract was identified as the most promising candidate with bactericidal activity against B. anthracis. It consistently inhibited the growth of B. anthracis in AWDA and decreased the viable colony-forming unit counts in liquid-broth cultures by 6-logs within 6–12 h. The AGE displayed acceptable thermostability (>80% anti-B. anthracis activity retained on incubation at 50 °C for 12 h) and stability in gastric pH range (2–8). It did not antagonize the activity of FDA-approved antibiotics used for anthrax control. GC-MS analysis of the TLC separated bioactive fractions of AGE indicated the presence of previously unreported constituents such as phthalic acid derivatives, acid esters, phenyl group-containing compounds, steroids etc. Conclusion: The Aqueous Garlic Extract (AGE) displayed potent anti-B. anthracis activity. It was better than that displayed by Azadirachta indica A. Juss. (Neem) and Mangifera indica L., while Curcuma longa L. (Turmeric) did not show any activity under the assay conditions used. Further work should be undertaken to explore the possible application of AGE in preventing anthrax incidences in endemic areas. © 2020 Elsevier B.V.
Cross-serotype protection against group A Streptococcal infections induced by immunization with SPy_2191
(Nature Research, 2020) Pooja Sanduja; Manish Gupta; Vikas Kumar Somani; Vikas Yadav; Meenakshi Dua; Emanuel Hanski; Abhinay Sharma; Rakesh Bhatnagar; Atul Kumar Johri
Group A Streptococcus (GAS) infection causes a range of diseases, but vaccine development is hampered by the high number of serotypes. Here, using reverse vaccinology the authors identify SPy_2191 as a cross-protective vaccine candidate. From 18 initially identified surface proteins, only SPy_2191 is conserved, surface-exposed and inhibits both GAS adhesion and invasion. SPy_2191 immunization in mice generates bactericidal antibodies resulting in opsonophagocytic killing of prevalent and invasive GAS serotypes of different geographical regions, including M1 and M49 (India), M3.1 (Israel), M1 (UK) and M1 (USA). Resident splenocytes show higher interferon-γ and tumor necrosis factor-α secretion upon antigen re-stimulation, suggesting activation of cell-mediated immunity. SPy_2191 immunization significantly reduces streptococcal load in the organs and confers ~76-92% protection upon challenge with invasive GAS serotypes. Further, it significantly suppresses GAS pharyngeal colonization in mice mucosal infection model. Our findings suggest that SPy_2191 can act as a universal vaccine candidate against GAS infections. © 2020, The Author(s).
Crystalline and amorphous preparation of aluminum hydroxide nanoparticles enhances protective antigen domain 4 specific immunogenicity and provides protection against anthrax
(Dove Medical Press Ltd., 2020) Himanshu Gogoi; Rajesh Mani; Soumya Aggarwal; Anshu Malik Manoj Munde; Rakesh Bhatnagar
Introduction: Aluminum salts, although they have been used as adjuvants in many vaccine formulations since 1926, exclusively induce a Th2-biased immune response, thereby limiting their use against intracellular pathogens like Mycobacterium tuberculosis. Methods and Results: Herein, we synthesized amorphous and crystalline forms of aluminum hydroxide nanoparticles (AH nps) of 150–200 nm size range. Using Bacillus anthracis protective antigen domain 4 (D4) as a model antigen, we demonstrated that both amorphous and crystalline forms of AH nps displayed enhanced antigen D4 uptake by THP1 cells as compared to commercial adjuvant aluminum hydroxide gel (AH gel). In a mouse model, both amorphous and crystalline AH nps triggered an enhanced D4-specific Th2-and Th1-type immune response and conferred superior protection against anthrax spore challenge as compared to AH gel. Physicochemical characterization of crystalline and amorphous AH nps revealed stronger antigen D4 binding and release than AH gel. Conclusion: These results demonstrate that size and crystallinity of AH nps play important roles in mediating enhanced antigen presenting cells (APCs) activation and potentiating a strong antigen-specific immune response, and are critical parameters for the rational design of alum-based Th1-type adjuvant to induce a more balanced antigen-specific immune response. © 2020 Gogoi et al.
Delineation of the Residues of Bacillus anthracis Zinc Uptake Regulator Protein Directly Involved in Its Interaction with Cognate DNA
(Humana Press Inc., 2021) Divya Kandari; Hemant Joshi; Neetu Tanwar; Manoj Munde; Rakesh Bhatnagar
Zinc uptake regulator (Zur) is a negative transcriptional regulator of bacteria that belongs to the FUR superfamily of proteins and regulates zinc (Zn) homeostasis under extreme Zn conditions. The Zur protein of Bacillus anthracis (BaZur) was though characterized previously, but the residues of this transcriptional regulator, crucial for binding to the consensus Zur box in the cognate DNA, remain unexplored. In this study, we reveal the essential residues of the protein that govern the specific interaction with the cognate DNA, through mutational and binding studies. In silico predicted model of the BaZur protein with the promoter region of one of the regulon candidates was utilized to identify specific residues of the N-terminal domain (NTD), constituting the DNA-binding recognition helix. Our results suggest that two phenylalanine residues, a non-polar aliphatic leucine and a positively charged arginine residue of NTD, are predominantly involved in DNA binding of BaZur. Among these, the arginine residue (Arg58) is conserved among all the Zur proteins and the two Phe residues, namely Phe53 and Phe63, are conserved in the Zur proteins of Staphylococcus aureus and Listeria monocytogenes. Taken together, the current study represents an in-depth investigation into the key DNA-binding residues involved in the BaZur-DNA interaction. © 2020, Springer Science+Business Media, LLC, part of Springer Nature.
Development of a novel multiepitope chimeric vaccine against anthrax
(Springer Verlag, 2019) Somya Aggarwal; Vikas Kumar Somani; Sonal Gupta; Rajni Garg; Rakesh Bhatnagar
Abstract: Bacillus anthracis (BA), the etiological agent of anthrax, secretes protective antigen (PA), lethal factor (LF), and edema factor (EF) as major virulence mediators. Amongst these, PA-based vaccines are most effective for providing immunity against BA, but their low shelf life limits their usage. Previous studies showed that B-cell epitopes, ID II and ID III present in PA domain IV possess higher toxin neutralization activity and elicit higher antibody titer than ID I. Moreover, N-terminal region of both LF and EF harbors PA-binding sites which share 100% identity with each other. Here, in this study, we have developed an epitope-based chimeric vaccine (ID–LFn) comprising ID II–ID III region of PA and N-terminal region of LF. We have also evaluated its protective efficacy as well as stability and found it to be more stable than PA-based vaccine. Binding reactivities of ID–LFn with anti-PA/LF/EF antibodies were determined by ELISA. The stability of chimeric vaccine was assessed using circular dichroism spectroscopy. ID–LFn response was characterized by toxin neutralization, lymphocyte proliferation isotyping and cytokine profiling. The protective efficacy was analyzed by challenging ID–LFn-immunized mice with B. anthracis (pXO1 + and pXO2 + ). ID–LFn was found to be significantly stable as compared to PA. Anti-ID–LFn antibodies recognized PA, LF as well as EF. The T-cell response and the protective efficacy of ID–LFn were found to be almost similar to PA. ID–LFn exhibits equal protective efficacy in mice and possesses more stability as compared to PA along with the capability of recognizing PA, LF and EF at the same time. Thus, it can be considered as an improved vaccine against anthrax with better shelf life. Graphical abstract: [Figure not available: see fulltext.] ID-LFn, a novel multiepitope chimeric anthrax vaccine: ID-LFn comprises of immunodominant epitopes of domain 4 of PA and N-terminal homologous stretch of LF and EF. The administration of this protein as a vaccine provides protection against anthrax. © 2019, Springer-Verlag GmbH Germany, part of Springer Nature.
Homology modelling and molecular simulation approach to prediction of B-cell and T-cell epitopes in an OMP25 peptide vaccine against Brucella abortus
(Taylor and Francis Ltd., 2023) Dhananjay Jade; Sonal Gupta; Surender Mohan; Sreenivasan Ponnambalam; Michael Harrison; Rakesh Bhatnagar
The live attenuated vaccine composition against brucellosis poses potential risks to recipient animals. Therefore, we have analysed an in-silico approach to design a novel multi-epitope vaccine peptide to elicit a desirable immune response against Brucella abortus infection. This study designed a peptide vaccine based on outer membrane protein from B. abortus. The selected OMP sequence shows 0.7575 antigenic proteins. We predicted the T-cell epitopes of different lengths. Cluster analysis was performed for 180 epitope peptides, showcasing a total of 19 clusters, constituting 14 clusters as Consensus clusters and 5 as Singleton clusters. We select the top three clusters which has 24 peptides showing antigen property. Selected 24-antigen peptides were docked with MHC classes I and II and selected the top eight peptides based on binding energy used for molecular dynamic simulation. Immuno-informatics analysis, Molecular Docking and Molecular simulation indicated that epitope peptide vaccine could work as effective peptides helpful in scheming peptide vaccine against B. abortus infection by developing broad-spectrum peptide vaccine in near future. The use of Omp25 as a vaccine candidate has been supported based on previous experimental studies. Multitope vaccine can be developed which can protect mice against virulent B. abortus challenge. © 2023 Informa UK Limited, trading as Taylor & Francis Group.
Importance of cell wall-associated poly-α-l-glutamine in the biology of pathogenic mycobacteria
(Springer Singapore, 2019) Rajni Garg; Rajesh Mani; Manish Gupta; Deeksha Tripathi; Harish Chandra; Rakesh Bhatnagar; Nirupama Banerjee
Mycobacterium tuberculosis (Mtb), the formidable scourge known to mankind since ancient times, has remained untamed despite vigorous scientific research in the field. In the last several decades, significant advances have been made to study this pathogen; however, a lot more remains in the realm of unknown. The complex and unique cell wall of the bacterium is a major factor contributing to the unrestrained success of the pathogen in infecting millions around the world. Since the discovery of this bacterium, numerous studies have attempted to unravel the complexities of mycobacterial cell envelop to characterize individual constituents and their importance in pathobiology of Mtb. Major components of the cell envelop of mycobacteria such as lipid-linked polysaccharides-lipoarabinomannan (LAM), dimycolyl trehalose (cord factor), sulfolipids, and mycolyl-arabinogalactan-peptidoglycan (mAGP) complex have been investigated extensively. However, a lesser known molecule, poly-α-L-glutamine/glutamate (PLG), that constitutes ~10% of dry weight of cell wall has not attracted as much attention. As early as 1990, Hirschfield et al. isolated PLG as insoluble material and showed its association with the Mtb cell wall. In the last few years, our group has been working to identify enzymes that may play a role in the synthesis/assembly and localization of this polymer in the cell wall of mycobacteria. Our recently published work has shown that PLG by itself is weakly immunogenic in mice, but when combined with protein antigens, it can stimulate different arms of the T helper-mediated responses, demonstrating its potential to act as an adjuvant (Mani et al. 2018). © Springer Nature Singapore Pte Ltd. 2019.
In-vitro detection of phytopathogenic fungal cell wall by polyclonal sera raised against trimethyl chitosan nanoparticles
(Dove Medical Press Ltd., 2019) Hemant Joshi; Anshu Malik; Soumya Aggarwal; Manoj Munde; Subhrangsu Sundar Maitra; Nidhi Adlakha; Rakesh Bhatnagar
Purpose: The objective of this research was to generate a tool for the first-line detection of fungal infection in plants. Chitin is one of the unique fungal cell wall polysaccharide which is naturally deacetylated to chitosan upon infection. It is said to be involved in the fungal cell wall modulation and plant-pathogen communication. Therefore, detection of chitosan could be potentially helpful in the detection of fungal contamination. Methods: Five different phytopathogenic fungi strains were used for the study. Polyclonal sera were raised in the mice against Trimethylchitosan nanoparticles to generate an enhanced humoral immune response and generate a rich and heterogeneous repertoire of antibodies. The binding affinity of the sera with fungal cell wall was analyzed by ELISA, Langmuir isotherm, confocal microscopy and ITC (Isothermal Calorimetry). Results: The raised polyclonal sera could detect chitosan in the fungal cell wall, as analyzed with the different techniques. However, the detection specificity varied among the strains in proportion to the chitin content of their cell wall. Fusarium oxysporum was detected with the highest affinity while Trichoderma reesei was detected with the least affinity by ELISA. Adsorption isotherm, as well as ITC, revealed the specific and high binding capacity. Confocal microscopy also confirmed the detection of all strains used in the study. Conclusion: This novel technique employing TMC nanoparticulate system could be potentially used as a source to raise sera against chitosan in an inexpensive and less laborious manner. Rapid detection of fungal contamination by the polyclonal antibodies could help in devising a quick solution. The polyclonal sera are expected to detect a span of epitopes and provide precise detection. The detection system could be advanced for future applications such as food quality control, crop protection, and human fungal infection detection and treatment. © 2019 Joshi et al.
Preclinical evidence of nanomedicine formulation to target mycobacterium tuberculosis at its bone marrow niche
(MDPI AG, 2020) Jaishree Garhyan; Surender Mohan; Vinoth Rajendran; Rakesh Bhatnagar
One-third of the world’s population is estimated to be latently infected with Mycobacterium tuberculosis (Mtb). Recently, we found that dormant Mtb hides in bone marrow mesenchymal stem cells (BM-MSCs) post-chemotherapy in mice model and in clinical subjects. It is known that residual Mtb post-chemotherapy may be responsible for increased relapse rates. However, strategies for Mtb clearance post-chemotherapy are lacking. In this study, we engineered and formulated novel bone-homing PEGylated liposome nanoparticles (BTL-NPs) which actively targeted the bone microenvironment leading to Mtb clearance. Targeting of BM-resident Mtb was carried out through bone-homing liposomes tagged with alendronate (Ald). BTL characterization using TEM and DLS showed that the size of bone-homing isoniazid (INH) and rifampicin (RIF) BTLs were 100 ± 16.3 nm and 84 ± 18.4 nm, respectively, with the encapsulation efficiency of 69.5% ± 4.2% and 70.6% ± 4.7%. Further characterization of BTLs, displayed by sustained in vitro release patterns, increased in vivo tissue uptake and enhanced internalization of BTLs in RAW cells and CD271+BM-MSCs. The efficacy of isoniazid (INH)- and rifampicin (RIF)-loaded BTLs were shown using a mice model where the relapse rate of the tuberculosis was decreased significantly in targeted versus non-targeted groups. Our findings suggest that BTLs may play an important role in developing a clinical strategy for the clearance of dormant Mtb post-chemotherapy in BM cells. View Full-Text. © 2020 by the authors.
Simultaneous immunization with omp25 and l7/l12 provides protection against brucellosis in mice
(MDPI AG, 2020) Sonal Gupta; Surender Mohan; Vikas Kumar Somani; Somya Aggarwal; Rakesh Bhatnagar
Currently used Brucella vaccines, Brucella abortus strain 19 and RB51, comprises of live attenuated Brucella strains and prevent infection in animals. However, these vaccines pose potential risks to recipient animals such as attenuation reversal and virulence in susceptible hosts on administration. In this context, recombinant subunit vaccines emerge as a safe and competent alternative in combating the disease. In this study, we formulated a divalent recombinant vaccine consisting of Omp25 and L7/L12 of B. abortus and evaluated vaccine potential individually as well as in combination. Sera obtained from divalent vaccine (Omp25+L7/L12) immunized mice group exhibited enhanced IgG titers against both components and indicated specificity upon immunoblotting reiterating its authenticity. Further, the IgG1/IgG2a ratio obtained against each antigen predicted a predominant Th2 immune response in the Omp25+L7/L12 immunized mice group. Upon infection with virulent B. abortus 544, Omp25+L7/L12 infected mice exhibited superior Log10 protection compared to individual vaccines. Consequently, this study recommends that simultaneous immunization of Omp25 and L7/L12 as a divalent vaccine complements and triggers a Th2 mediated immune response in mice competent of providing protection against brucellosis. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.
Structure-based drug repurposing to inhibit the DNA gyrase of Mycobacterium tuberculosis
(Portland Press Ltd, 2020) G.L. Balasubramani; Rinky Rajput; Manish Gupta; Pradeep Dahiya; Jitendra K. Thakur; Rakesh Bhatnagar; Abhinav Grover
Drug repurposing is an alternative avenue for identifying new drugs to treat tuberculosis (TB). Despite the broad-range of anti-tubercular drugs, the emergence of multi-drug-resistant and extensively drug-resistant strains of Mycobacterium tuberculosis (Mtb) H37Rv, as well as the significant death toll globally, necessitates the development of new and effective drugs to treat TB. In this study, we have employed a drug repurposing approach to address this drug resistance problem by screening the drugbank database to identify novel inhibitors of the Mtb target enzyme, DNA gyrase. The compounds were screened against the ATPase domain of the gyrase B subunit (MtbGyrB47), and the docking results showed that echinacoside, doxorubicin, epirubicin, and idarubicin possess high binding affinities against MtbGyrB47. Comprehensive assessment using fluorescence spectroscopy, surface plasmon resonance spectroscopy (SPR), and circular dichroism (CD) titration studies revealed echinacoside as a potent binder of MtbGyrB47. Furthermore, ATPase, and DNA supercoiling assays exhibited an IC50 values of 2.1-4.7 mM for echinacoside, doxorubicin, epirubicin, and idarubicin. Among these compounds, the least MIC90 of 6.3 and 12 μM were observed for epirubicin and echinacoside, respectively, against Mtb. Our findings indicate that echinacoside and epirubicin targets mycobacterial DNA gyrase, inhibit its catalytic cycle, and retard mycobacterium growth. Further, these compounds exhibit potential scaffolds for optimizing novel anti-mycobacterial agents that can act on drug-resistant strains. © 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society
Zur: Zinc‐sensing transcriptional regulator in a diverse set of bacterial species
(MDPI, 2021) Divya Kandari; Hemant Joshi; Rakesh Bhatnagar
Zinc (Zn) is the quintessential d block metal, needed for survival in all living organisms. While Zn is an essential element, its excess is deleterious, therefore, maintenance of its intracellular concentrations is needed for survival. The living organisms, during the course of evolution, devel-oped proteins that can track the limitation or excess of necessary metal ions, thus providing survival benefits under variable environmental conditions. Zinc uptake regulator (Zur) is a regulatory transcriptional factor of the FUR superfamily of proteins, abundant among the bacterial species and known for its intracellular Zn sensing ability. In this study, we highlight the roles played by Zur in maintaining the Zn levels in various bacterial species as well as the fact that in recent years Zur has emerged not only as a Zn homeostatic regulator but also as a protein involved directly or indirectly in virulence of some pathogens. This functional aspect of Zur could be exploited in the ventures for the identification of newer antimicrobial targets. Despite extensive research on Zur, the insights into its overall regulon and its moonlighting functions in various pathogens yet remain to be explored. Here in this review, we aim to summarise the disparate functional aspects of Zur proteins present in various bacterial species. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.