Address: B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Department of Cell and Molecular Biology, Off S. G. Highway, Thaltej, Ahmedabad, Gujarat-380054, India
International Journal of Peptide Research and Therapeutics, 2021
Multidrug resistant Shigella is one of the leading causes of mortality in children and infants. A... more Multidrug resistant Shigella is one of the leading causes of mortality in children and infants. Availability of vaccine could prevent the Shigella infection and reduce the mortality. Conventional approaches of vaccine development against shigellosis have not resulted in desirable vaccine. As shigellosis may be caused by multiple strains and serotypes, there is a need to develop a multivalent vaccine, capable of providing protection against multiple Shigella strains. To develop broad spectrum vaccine, we had previously derived a pool of conserved epitopes against Shigella by using multiple immunoinformatic tools. In this study, the identified conserved epitopes derived from the Outer Membrane Proteins A and C of Shigella were chemically synthesized, and the EpiMix made up of 5 epitopes coupled to a carrier protein, ovalbumin was developed and validated for its immunogenicity. The intramuscular immunization with EpiMix in Balb/c mice led to increase in EpiMix specific serum IgG, and s...
Immunoinformatic plays a pivotal role in vaccine design and development. While traditional method... more Immunoinformatic plays a pivotal role in vaccine design and development. While traditional methods are exclusively depended on immunological experiments, they are less effective, relatively expensive, and time-consuming. However, recent advances in the field of immunoinformatics have provided innovative tools for the rational design of vaccine candidates. This approach allows the selection of immunodominant regions from the sequence of whole genome of a pathogen. The identified immunodominant region could be used to develop potential vaccine candidates that can trigger protective immune responses in the host. At present, epitope-based vaccine is an attractive concept which has been successfully trailed to develop vaccines against a number of pathogens. In this chapter, we outline the methodology and workflow of how to deploy immunoinformatics tools in order to identify immunodominant epitopes using Shigella as a model organism. The immunodominant epitopes, derived from S. flexneri 2...
The non-invasive food grade Lactococcus lactis (L. lactis) represents a safe and attractive alter... more The non-invasive food grade Lactococcus lactis (L. lactis) represents a safe and attractive alternative to invasive pathogens for the delivery of plasmid DNA at mucosal sites. We have earlier shown the DNA delivery potential of r-L. lactis harboring DNA vaccine reporter plasmid; pPERDBY in vitro. In the present work, we examined in vivo delivery potential of food grade non-invasive r-L. lactis::pPERDBY (LacVax DNA-I) in BALB/c mice. Moreover, using EGFP as a model antigen, we also characterized and compared the immune response elicited by LacVax DNA-I with other conventional vaccination approaches using protein and naked DNA immunization. The presence of antigen-specific serum IgG and fecal secretory IgA (sIgA) antibodies demonstrated in vivo DNA delivery and immune elicitation potential of the developed LacVax DNA-I. As compared with intramuscular injection, oral delivery of pPERDBY via L. lactis resulted in a significantly rapid increase in IgG and higher sIgA titers, indicating t...
PD-1 blockade combined with vaccination induced anti-SIV immunity and reduced SIV viral reservoir... more PD-1 blockade combined with vaccination induced anti-SIV immunity and reduced SIV viral reservoirs in lymphoid tissue.
Vaccination with plasmid DNA offers several advantages over traditional vaccination methods. Rece... more Vaccination with plasmid DNA offers several advantages over traditional vaccination methods. Recent novel approach for genetic immunization includes use of attenuated pathogens such as Shigella, Yersinia, Listeria, Salmonella or Mycobaterium. However, reverting to wild type phenotype is of great threat. Hence, use of food grade Lactococcus lactis as a DNA delivery vehicle is an attractive alternative for genetic immunization. Various advantages like immunostimulatory property, extremely safe profile and great advance in L. lactis genetic manipulation tools played an important role in adopting L. lactis as a readily amenable delivery vector. Here, we have constructed a new E. coli-L. lactis shuttle vector, pPERDBY, harbouring eukaryotic expression cassette for genetic immunization. pPERDBY was constructed by fusion of two plasmids i.e. pSEC:Nuc and pEGFP-N1. Salient features of pPERDBY includes presence of I) a eukaryotic region, containing the CytoMegaloVirus promoter (pCMV), a mult...
The most promising solution to the current energy and environmental crisis lies in the use of bio... more The most promising solution to the current energy and environmental crisis lies in the use of biofuel. Various characteristics make Jatropha the preferred choice for source of biodiesel. The enzyme, Diacyl Glycerol Acyl Transferase (DGAT) catalyzes the rate limiting reaction in triacylglycerol bio-assembly. Hence increasing the amount of this enzyme by overexpression of its gene may increase triacylglycerol biosynthesis thus leading to increase in oil content per seed. For enabling the genetic modification of Jatropha plant it is essential to generate the transformed Agrobacterium and further co-cultivate them with leaves and cotyledonary leaves to generate transgenic callus and transgenic somatic embryos. Agrobacterium mediated transformation of binary vector pE172 into Jatropha was also successfully carried out using already standardized protocol of the lab, which were confirmed by antibiotic resistance and for further confirmation DNA isolation from Transformed and Untransformed ...
The increase in energy demand coupled with the decrease in global supply of fossil fuels is a mat... more The increase in energy demand coupled with the decrease in global supply of fossil fuels is a matter of great concern. In addition, there are omnipresent problems associated with the use of fossil fuel like pollution, increase in greenhouse gases and most important is its non-renewable nature. Hence there is an urgent need to address this crisis. The most promising solution is to use biofuels. Since Jatropha plant is the preferred choice of source of biofuel, the study aims to generate transgenic embryos of Jatropha curcas overexpressing DGAT2 (Diacyl Glycerol Acyl Transferase-2), a rate limiting enzyme of oil biosynthesis pathway. This strategy involves increase in metabolic flux towards oil production in Jatropha plant. In order to achieve the same we used plasmid pE172 with VfDGAT2 gene to transform Agrobacterium tumefaciens. We have successfully transformed A tumefaciens with pE172-VfDGAT using Triparental mating. Recombinant A tumefaciens was confirmed by plasmid isolation and ...
Indian Journal of Endocrinology and Metabolism, 2017
Fluorosis is controlled by the duration of fluoride exposure and calcium and Vitamin D nutrition ... more Fluorosis is controlled by the duration of fluoride exposure and calcium and Vitamin D nutrition status. To examine (a) prevalence of dental and skeletal fluorosis in adolescents from upper, middle, and lower socioeconomic strata (SES) and (b) association of fluorosis with calcium intake and Vitamin D status. A cross-sectional study conducted in 10-13.9 years apparently healthy adolescents (n = 90), from different SES of Patan (Gujarat, India). Dental fluorosis was graded as mild, moderate, and severe. Radiographs of the right hand and wrist were examined and graded. Serum 25 hydroxyvitamin D3 (25OHD) and parathyroid hormone concentrations were measured. Diet was recorded (24 h recall) and calcium intake was computed (C-diet V-2.1, 2013, Xenios Technologies Pvt. Ltd). Generalized linear model was used to analyze relationships between fluorosis, SES, serum 25OHD concentration, and calcium intake. Fluorosis was predominant in lower SES (17% had both dental and radiological features whereas 73% had dental fluorosis); no skeletal deformities were observed. Mean 25OHD concentrations and dietary calcium were 26.3 ± 4.9, 23.4 ± 4.7, and 18.6 ± 4 ng/ml and 441.2 ± 227.6, 484.3 ± 160.9, and 749.2 ± 245.4 mg/day, respectively, for lower, middle, and upper SES (P < 0.05). Fluorosis and SES showed a significant association (exponential β = 2.5, P = 0.01) as compared to upper SES, middle SES adolescents were at 1.3 times while lower SES adolescents were at 2.5 times higher risk. Serum 25OHD concentrations (P = 0.937) and dietary calcium intake (P = 0.825) did not show a significant association with fluorosis. Fluorosis was more common in lower SES adolescents, probably due to the lack of access to bottled water. Relatively adequate calcium intake and serum 25OHD concentrations may have increased the efficiency of dietary calcium absorption, thus preventing severe fluorosis.
Use of food grade Lactococcus lactis (L. lactis) is fast emerging as a safe alternative for deliv... more Use of food grade Lactococcus lactis (L. lactis) is fast emerging as a safe alternative for delivery of DNA vaccine. To attain efficient DNA delivery, L. lactis, a non-invasive bacterium is converted to invasive strain either by expressing proteins like Internalin A (InlA) or Fibronectin binding protein A (FnBPA) or through chemical treatments. However the safety status of invasive L. lactis is questionable. In the present report, we have shown that non-invasive L. lactis efficiently delivered the newly constructed reporter plasmid pPERDBY to mammalian cells without any chemical enhancers. The salient features of the vector are; I) Ability to replicate in two different hosts; Escherichia coli (E. coli) and Lactic Acid Bacteria (LAB), II) One of the smallest reporter plasmid for DNA vaccine, III) Enhanced Green Fluorescence Protein (EGFP) linked to Multiple Cloning Site (MCS), IV) Immunostimulatory CpG motifs functioning as an adjuvant. Expression of EGFP in pPERDBY transfected CHO-K...
Potential use of Lactococcus lactis (L. lactis) as a heterologous protein expression host as well... more Potential use of Lactococcus lactis (L. lactis) as a heterologous protein expression host as well as for delivery of multiple therapeutic proteins has been investigated extensively using Nisin Inducible Controlled Expression (NICE) system. Optimum inducible expression of heterologous protein by NICE system in L. lactis depends on multiple factors. To study the unexplored role of factors affecting heterologous protein expression in L. lactis using NICE, the present study outlines the optimization of various key parameters such as inducer concentration, host's proteases and precipitating agent using Outer membrane protein A (OmpA). For efficient expression and secretion of OmpA, pSEC:OmpA vector was successfully constructed. To circumvent the troubles encountered during detection of expressed OmpA, the precipitating agent was switched from TCA to methanol. Nevertheless, detection was achieved accompanied by degraded protein products. Speculating the accountability of observed degradation at higher inducer concentration, different nisin concentrations were evaluated. Lower nisin concentrations were found desirable for optimum expression of OmpA. Consistently observed degradation was eliminated by incorporation of protease inhibitor cocktail which inhibits intracellular proteases and expression in VEL1153 (NZ9000 ΔhtrA) strain which inhibits extracellular protease leading to optimum expression of OmpA. Versatility and complexity of NICE system in L. lactis requires fine-tuning of target protein specific parameters for optimum expression.
Vaccination with plasmid DNA offers several advantages over traditional vaccination methods. Rece... more Vaccination with plasmid DNA offers several advantages over traditional vaccination methods. Recent novel approach for genetic immunization includes use of attenuated pathogens such as Shigella, Yersinia, Listeria, Salmonella or Mycobaterium. However, reverting to wild type phenotype is of great threat. Hence, use of food grade Lactococcus lactis as a DNA delivery vehicle is an attractive alternative for genetic immunization. Various advantages like immunostimulatory property, extremely safe profile and great advance in L. lactis genetic manipulation tools played an important role in adopting L. lactis as a readily amenable delivery vector. Here, we have constructed a new E. coli-L. lactis shuttle vector, pPERDBY, harbouring eukaryotic expression cassette for genetic immunization. pPERDBY was constructed by fusion of two plasmids i.e. pSEC:Nuc and pEGFP-N1. Salient features of pPERDBY includes presence of I) a eukaryotic region, containing the CytoMegaloVirus promoter (pCMV), a mult...
International Journal of Peptide Research and Therapeutics, 2021
Multidrug resistant Shigella is one of the leading causes of mortality in children and infants. A... more Multidrug resistant Shigella is one of the leading causes of mortality in children and infants. Availability of vaccine could prevent the Shigella infection and reduce the mortality. Conventional approaches of vaccine development against shigellosis have not resulted in desirable vaccine. As shigellosis may be caused by multiple strains and serotypes, there is a need to develop a multivalent vaccine, capable of providing protection against multiple Shigella strains. To develop broad spectrum vaccine, we had previously derived a pool of conserved epitopes against Shigella by using multiple immunoinformatic tools. In this study, the identified conserved epitopes derived from the Outer Membrane Proteins A and C of Shigella were chemically synthesized, and the EpiMix made up of 5 epitopes coupled to a carrier protein, ovalbumin was developed and validated for its immunogenicity. The intramuscular immunization with EpiMix in Balb/c mice led to increase in EpiMix specific serum IgG, and s...
Immunoinformatic plays a pivotal role in vaccine design and development. While traditional method... more Immunoinformatic plays a pivotal role in vaccine design and development. While traditional methods are exclusively depended on immunological experiments, they are less effective, relatively expensive, and time-consuming. However, recent advances in the field of immunoinformatics have provided innovative tools for the rational design of vaccine candidates. This approach allows the selection of immunodominant regions from the sequence of whole genome of a pathogen. The identified immunodominant region could be used to develop potential vaccine candidates that can trigger protective immune responses in the host. At present, epitope-based vaccine is an attractive concept which has been successfully trailed to develop vaccines against a number of pathogens. In this chapter, we outline the methodology and workflow of how to deploy immunoinformatics tools in order to identify immunodominant epitopes using Shigella as a model organism. The immunodominant epitopes, derived from S. flexneri 2...
The non-invasive food grade Lactococcus lactis (L. lactis) represents a safe and attractive alter... more The non-invasive food grade Lactococcus lactis (L. lactis) represents a safe and attractive alternative to invasive pathogens for the delivery of plasmid DNA at mucosal sites. We have earlier shown the DNA delivery potential of r-L. lactis harboring DNA vaccine reporter plasmid; pPERDBY in vitro. In the present work, we examined in vivo delivery potential of food grade non-invasive r-L. lactis::pPERDBY (LacVax DNA-I) in BALB/c mice. Moreover, using EGFP as a model antigen, we also characterized and compared the immune response elicited by LacVax DNA-I with other conventional vaccination approaches using protein and naked DNA immunization. The presence of antigen-specific serum IgG and fecal secretory IgA (sIgA) antibodies demonstrated in vivo DNA delivery and immune elicitation potential of the developed LacVax DNA-I. As compared with intramuscular injection, oral delivery of pPERDBY via L. lactis resulted in a significantly rapid increase in IgG and higher sIgA titers, indicating t...
PD-1 blockade combined with vaccination induced anti-SIV immunity and reduced SIV viral reservoir... more PD-1 blockade combined with vaccination induced anti-SIV immunity and reduced SIV viral reservoirs in lymphoid tissue.
Vaccination with plasmid DNA offers several advantages over traditional vaccination methods. Rece... more Vaccination with plasmid DNA offers several advantages over traditional vaccination methods. Recent novel approach for genetic immunization includes use of attenuated pathogens such as Shigella, Yersinia, Listeria, Salmonella or Mycobaterium. However, reverting to wild type phenotype is of great threat. Hence, use of food grade Lactococcus lactis as a DNA delivery vehicle is an attractive alternative for genetic immunization. Various advantages like immunostimulatory property, extremely safe profile and great advance in L. lactis genetic manipulation tools played an important role in adopting L. lactis as a readily amenable delivery vector. Here, we have constructed a new E. coli-L. lactis shuttle vector, pPERDBY, harbouring eukaryotic expression cassette for genetic immunization. pPERDBY was constructed by fusion of two plasmids i.e. pSEC:Nuc and pEGFP-N1. Salient features of pPERDBY includes presence of I) a eukaryotic region, containing the CytoMegaloVirus promoter (pCMV), a mult...
The most promising solution to the current energy and environmental crisis lies in the use of bio... more The most promising solution to the current energy and environmental crisis lies in the use of biofuel. Various characteristics make Jatropha the preferred choice for source of biodiesel. The enzyme, Diacyl Glycerol Acyl Transferase (DGAT) catalyzes the rate limiting reaction in triacylglycerol bio-assembly. Hence increasing the amount of this enzyme by overexpression of its gene may increase triacylglycerol biosynthesis thus leading to increase in oil content per seed. For enabling the genetic modification of Jatropha plant it is essential to generate the transformed Agrobacterium and further co-cultivate them with leaves and cotyledonary leaves to generate transgenic callus and transgenic somatic embryos. Agrobacterium mediated transformation of binary vector pE172 into Jatropha was also successfully carried out using already standardized protocol of the lab, which were confirmed by antibiotic resistance and for further confirmation DNA isolation from Transformed and Untransformed ...
The increase in energy demand coupled with the decrease in global supply of fossil fuels is a mat... more The increase in energy demand coupled with the decrease in global supply of fossil fuels is a matter of great concern. In addition, there are omnipresent problems associated with the use of fossil fuel like pollution, increase in greenhouse gases and most important is its non-renewable nature. Hence there is an urgent need to address this crisis. The most promising solution is to use biofuels. Since Jatropha plant is the preferred choice of source of biofuel, the study aims to generate transgenic embryos of Jatropha curcas overexpressing DGAT2 (Diacyl Glycerol Acyl Transferase-2), a rate limiting enzyme of oil biosynthesis pathway. This strategy involves increase in metabolic flux towards oil production in Jatropha plant. In order to achieve the same we used plasmid pE172 with VfDGAT2 gene to transform Agrobacterium tumefaciens. We have successfully transformed A tumefaciens with pE172-VfDGAT using Triparental mating. Recombinant A tumefaciens was confirmed by plasmid isolation and ...
Indian Journal of Endocrinology and Metabolism, 2017
Fluorosis is controlled by the duration of fluoride exposure and calcium and Vitamin D nutrition ... more Fluorosis is controlled by the duration of fluoride exposure and calcium and Vitamin D nutrition status. To examine (a) prevalence of dental and skeletal fluorosis in adolescents from upper, middle, and lower socioeconomic strata (SES) and (b) association of fluorosis with calcium intake and Vitamin D status. A cross-sectional study conducted in 10-13.9 years apparently healthy adolescents (n = 90), from different SES of Patan (Gujarat, India). Dental fluorosis was graded as mild, moderate, and severe. Radiographs of the right hand and wrist were examined and graded. Serum 25 hydroxyvitamin D3 (25OHD) and parathyroid hormone concentrations were measured. Diet was recorded (24 h recall) and calcium intake was computed (C-diet V-2.1, 2013, Xenios Technologies Pvt. Ltd). Generalized linear model was used to analyze relationships between fluorosis, SES, serum 25OHD concentration, and calcium intake. Fluorosis was predominant in lower SES (17% had both dental and radiological features whereas 73% had dental fluorosis); no skeletal deformities were observed. Mean 25OHD concentrations and dietary calcium were 26.3 ± 4.9, 23.4 ± 4.7, and 18.6 ± 4 ng/ml and 441.2 ± 227.6, 484.3 ± 160.9, and 749.2 ± 245.4 mg/day, respectively, for lower, middle, and upper SES (P < 0.05). Fluorosis and SES showed a significant association (exponential β = 2.5, P = 0.01) as compared to upper SES, middle SES adolescents were at 1.3 times while lower SES adolescents were at 2.5 times higher risk. Serum 25OHD concentrations (P = 0.937) and dietary calcium intake (P = 0.825) did not show a significant association with fluorosis. Fluorosis was more common in lower SES adolescents, probably due to the lack of access to bottled water. Relatively adequate calcium intake and serum 25OHD concentrations may have increased the efficiency of dietary calcium absorption, thus preventing severe fluorosis.
Use of food grade Lactococcus lactis (L. lactis) is fast emerging as a safe alternative for deliv... more Use of food grade Lactococcus lactis (L. lactis) is fast emerging as a safe alternative for delivery of DNA vaccine. To attain efficient DNA delivery, L. lactis, a non-invasive bacterium is converted to invasive strain either by expressing proteins like Internalin A (InlA) or Fibronectin binding protein A (FnBPA) or through chemical treatments. However the safety status of invasive L. lactis is questionable. In the present report, we have shown that non-invasive L. lactis efficiently delivered the newly constructed reporter plasmid pPERDBY to mammalian cells without any chemical enhancers. The salient features of the vector are; I) Ability to replicate in two different hosts; Escherichia coli (E. coli) and Lactic Acid Bacteria (LAB), II) One of the smallest reporter plasmid for DNA vaccine, III) Enhanced Green Fluorescence Protein (EGFP) linked to Multiple Cloning Site (MCS), IV) Immunostimulatory CpG motifs functioning as an adjuvant. Expression of EGFP in pPERDBY transfected CHO-K...
Potential use of Lactococcus lactis (L. lactis) as a heterologous protein expression host as well... more Potential use of Lactococcus lactis (L. lactis) as a heterologous protein expression host as well as for delivery of multiple therapeutic proteins has been investigated extensively using Nisin Inducible Controlled Expression (NICE) system. Optimum inducible expression of heterologous protein by NICE system in L. lactis depends on multiple factors. To study the unexplored role of factors affecting heterologous protein expression in L. lactis using NICE, the present study outlines the optimization of various key parameters such as inducer concentration, host's proteases and precipitating agent using Outer membrane protein A (OmpA). For efficient expression and secretion of OmpA, pSEC:OmpA vector was successfully constructed. To circumvent the troubles encountered during detection of expressed OmpA, the precipitating agent was switched from TCA to methanol. Nevertheless, detection was achieved accompanied by degraded protein products. Speculating the accountability of observed degradation at higher inducer concentration, different nisin concentrations were evaluated. Lower nisin concentrations were found desirable for optimum expression of OmpA. Consistently observed degradation was eliminated by incorporation of protease inhibitor cocktail which inhibits intracellular proteases and expression in VEL1153 (NZ9000 ΔhtrA) strain which inhibits extracellular protease leading to optimum expression of OmpA. Versatility and complexity of NICE system in L. lactis requires fine-tuning of target protein specific parameters for optimum expression.
Vaccination with plasmid DNA offers several advantages over traditional vaccination methods. Rece... more Vaccination with plasmid DNA offers several advantages over traditional vaccination methods. Recent novel approach for genetic immunization includes use of attenuated pathogens such as Shigella, Yersinia, Listeria, Salmonella or Mycobaterium. However, reverting to wild type phenotype is of great threat. Hence, use of food grade Lactococcus lactis as a DNA delivery vehicle is an attractive alternative for genetic immunization. Various advantages like immunostimulatory property, extremely safe profile and great advance in L. lactis genetic manipulation tools played an important role in adopting L. lactis as a readily amenable delivery vector. Here, we have constructed a new E. coli-L. lactis shuttle vector, pPERDBY, harbouring eukaryotic expression cassette for genetic immunization. pPERDBY was constructed by fusion of two plasmids i.e. pSEC:Nuc and pEGFP-N1. Salient features of pPERDBY includes presence of I) a eukaryotic region, containing the CytoMegaloVirus promoter (pCMV), a mult...
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