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1.
ACS Omega ; 8(31): 27953-27968, 2023 Aug 08.
Article in English | MEDLINE | ID: mdl-37576639

ABSTRACT

Adjuvants are the important part of vaccine manufacturing as they elicit the vaccination effect and enhance the durability of the immune response through controlled release. In light of this, nanoadjuvants have shown unique broad spectrum advantages. As nanoparticles (NPs) based vaccines are fast-acting and better in terms of safety and usability parameters as compared to traditional vaccines, they have attracted the attention of researchers. A vaccine nanocarrier is another interesting and promising area for the development of next-generation vaccines for prophylaxis. This review looks at the various nanoadjuvants and their structure-function relationships. It compiles the state-of-art literature on numerous nanoadjuvants to help domain researchers orient their understanding and extend their endeavors in vaccines research and development.

2.
Vaccines (Basel) ; 11(1)2023 Jan 02.
Article in English | MEDLINE | ID: mdl-36679955

ABSTRACT

BACKGROUND: The global pharma sector is fast shifting from generics to biologics and biosimilars with the first approval in Europe in 2006 followed by US approval in 2015. In the form of Hepatitis B vaccine, India saw its first recombinant biologics approval in 2000. Around 20% of generic medications and 62% of vaccines are now supplied by the Indian pharmaceutical industry. It is this good position in biologics and biosimilars production that could potentially improve healthcare via decreased treatment cost. India has witnessed large investments in biosimilars over the years. Numerous India-bred new players, e.g., Enzene Biosciences Ltd., are keen on biosimilars and have joined the race alongside the emerging giants, e.g., Biocon and Dr. Reddy's. A very positive sign was the remarkable disposition during the COVID-19 pandemic by Bharat Biotech and the Serum Institute of India. India's biopharmaceutical industry has been instrumental in producing and supplying preventives and therapeutics to fight COVID-19. Despite a weak supply chain and workforce pressure, the production was augmented to provide reasonably priced high-quality medications to more than 133 nations. Biosimilars could cost-effectively treat chronic diseases involving expensive conventional therapies, including diabetes, respiratory ailments, cancer, and connective tissue diseases. Biologics and biosimilars have been and are being tested to treat and manage COVID-19 symptoms characterized by inflammation and respiratory distress. PURPOSE OF REVIEW: Although India boasts many universities, research centers, and a relatively skilled workforce, its global University-Industry collaboration ranking is 24, IPR ranking remains 47 and innovation ranking 39. This reveals a wide industry-academia gap to bridge. There are gaps in effective translational research in India that must be promptly and appropriately addressed. Innovation demands strong and effective collaborations among universities, techno-incubators, and industries. METHODOLOGY: Many successful research findings in academia do not get translation opportunities supposedly due to low industrial collaboration, low IP knowledge, and publication pressure with stringent timelines. In light of this, a detailed review of literature, including policy papers, government initiatives, and corporate reviews, was carried out, and the compilation and synthesis of the secondary data were meticulously summarized for the easy comprehension of the facts and roadmap ahead. For easy comprehension, charts, figures, and compiled tables are presented. RESULTS: This review assesses India's situation in the biosimilar space, the gaps and areas to improve for Indian investment strategies, development, and innovation, addressing need for a more skilled workforce, industrial collaboration, and business models. CONCLUSIONS: This review also proposes forward an approach to empowering technopreneurs to develop MSMEs for large-scale operations to support India in taking innovative thoughts to the global level to ultimately realize a self-reliant India. The limitations of the compilation are also highlighted towards the end.

3.
Cureus ; 15(12): e50424, 2023 Dec.
Article in English | MEDLINE | ID: mdl-38222131

ABSTRACT

In the wake of rising rabies cases worldwide, especially after the COVID-19 pandemic, it is time to understand the scenario better and suggest technically sound and plausible countermeasures. This article is an attempt at this perspective. Although a critical zoonotic viral disease, rabies is preventable. Medico-legally, the ailment is classified as furious rabies and paralytic rabies. The four world bodies, namely, the World Health Organisation (WHO), the Food and Agriculture Organisation (FAO), the World Organisation for Animal Health (OIE), and the Global Alliance for Rabies Control (GARC) endorsed framing a global support system to eradicate human death from dog-mediated rabies under the 'Zero by 30' framework. The framework calls for extending the vaccination of dogs to reduce the risk of human rabies. Stray dogs became aggressive primarily due to their food shortage during the pandemic lockdown. As many adopted stray dogs were disowned post-pandemic, decreased human-dog interactions increased the aggressiveness among dogs. As a result, 'dog-bite' cases rose, with a sudden spike in rabies cases and dog-bite-induced deaths in India and elsewhere. Jeopardising the 'Zero by 30' plan is certainly a public health concern. Stray dog sterilisation through the irreversible ductal occlusion technique and reversible inhibition of sperm under guidance (RISUG) are other suggested interventions to control rabies. Importantly, wildlife like foxes, raccoons, skunks, and bats could also be rabid. Three out of the four WHO-pre-qualified human vaccines against rabies are intradermally administered as post-exposure prophylaxis, while the intramuscular one is more popular. Even though 'Zero by 30' may not be achieved within the set timeframe, it is time for a concerted and planned strategy by global agencies to curb the globally rising rabies cases and manage the disease better. The 'One Health' model seems to be a plausible guideline and the ultimate countermeasure to achieve this.

4.
Biotechnol Adv ; 34(1): 1-13, 2016.
Article in English | MEDLINE | ID: mdl-26657051

ABSTRACT

Vaccines are derived from a variety of sources including tissue extracts, bacterial cells, virus particles, recombinant mammalian, yeast and insect cell produced proteins and nucleic acids. The most common method of vaccine production is based on an initial fermentation process followed by purification. Production of vaccines is a complex process involving many different steps and processes. Selection of the appropriate purification method is critical to achieving desired purity of the final product. Clarification of vaccines is a critical step that strongly impacts product recovery and subsequent downstream purification. There are several technologies that can be applied for vaccine clarification. Selection of a harvesting method and equipment depends on the type of cells, product being harvested, and properties of the process fluids. These techniques include membrane filtration (microfiltration, tangential-flow filtration), centrifugation, and depth filtration (normal flow filtration). Historically vaccine harvest clarification was usually achieved by centrifugation followed by depth filtration. Recently membrane based technologies have gained prominence in vaccine clarification. The increasing use of single-use technologies in upstream processes necessitated a shift in harvest strategies. This review offers a comprehensive view on different membrane based technologies and their application in vaccine clarification, outlines the challenges involved and presents the current state of best practices in the clarification of vaccines.


Subject(s)
Biotechnology/methods , Vaccines , Animals , Bacterial Vaccines/isolation & purification , Centrifugation/methods , Chick Embryo/virology , Fermentation , Filtration/methods , Pneumococcal Vaccines/isolation & purification , Tetanus Toxin/isolation & purification , Toxoids , Vaccines, DNA/isolation & purification , Viral Vaccines/isolation & purification
5.
Expert Rev Vaccines ; 13(12): 1537-51, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25308708

ABSTRACT

The demand for plasmid DNA (pDNA) has vastly increased over the past decade in response to significant advances that have been made in its application for gene therapy and vaccine development. Plasmid DNA-based vaccines are experiencing a resurgence due to success with prime-boost immunization strategies. The challenge has always been poor productivity and delivery of pDNA. Plasmid DNA-based vaccines have traditionally required milligram scale of GMP-grade product for vaccination due to the relatively low efficacy and duration of gene expression. However, efforts to increase pDNA vaccine effectiveness are evolving in genetic manipulations of bacterial host, improvements in product recovery and innovative delivery methods. This review summarizes recent advances in large-scale pDNA vaccine manufacturing, ranging from upstream processing, downstream processing and formulation, as such information is usually not available to the scientific community. The article will highlight technology gaps and offer insight on further scope of innovation.


Subject(s)
Drug Discovery/methods , Drug Discovery/trends , Plasmids/isolation & purification , Vaccines, DNA/genetics , Vaccines, DNA/isolation & purification , Chemistry, Pharmaceutical , Humans , Vaccination/methods , Vaccines, DNA/administration & dosage
6.
Biotechnol Prog ; 30(6): 1319-25, 2014.
Article in English | MEDLINE | ID: mdl-25080096

ABSTRACT

Rigid chromatography resins, such as controlled pore glass based adsorbents, offer the advantage of high permeability and a linear pressure-flow relationship irrespective of column diameter which improves process time and maximizes productivity. However, the rigidity and irregularly shaped nature of these resins often present challenges in achieving consistent and uniform packed beds as formation of bridges between resin particles can hinder bed consolidation. The standard flow-pack method when applied to irregularly shaped particles does not yield well-consolidated packed beds, resulting in formation of a head space and increased band broadening during operation. Vibration packing methods requiring the use of pneumatically driven vibrators are recommended to achieve full packed bed consolidation but limitations in manufacturing facilities and equipment may prevent the implementation of such devices. The stop-flow packing method was developed as an improvement over the flow-pack method to overcome these limitations and to improve bed consolidation without the use of vibrating devices. Transition analysis of large-scale columns packed using the stop-flow method over multiple cycles has shown a two- to three-fold reduction of change in bed integrity values as compared to a flow-packed bed demonstrating an improvement in packed bed stability in terms of the height equivalent to a theoretical plate (HETP) and peak asymmetry (As ).


Subject(s)
Chromatography, Liquid/instrumentation , Glass/chemistry , Models, Theoretical , Laboratory Chemicals , Rheology
7.
Protein Pept Lett ; 19(5): 509-19, 2012 May.
Article in English | MEDLINE | ID: mdl-22486646

ABSTRACT

Dengue hemorrhagic fever and dengue shock syndrome are the severe manifestations of dengue infection. The quest for reliable dengue diagnostics and a dengue vaccine remained elusive for decades. Domain III of dengue virus envelope contains multiple conformation dependant neutralizing epitopes, thus making it an attractive diagnostic and vaccine candidate. In this report we show the expression of dengue virus type 3 envelope domain III protein (D3EDIII) and demonstrate its potential as a diagnostic and vaccine candidate. Accordingly, D3EDIII was expressed to high levels in Escherichia coli and purified by Ni-NTA affinity chromatography. The purified protein was used to develop an in-house plate ELISA and was further tested with a panel of 40 dengue infected serum samples previously characterized by commercially available serological tests. The in-house results were in excellent agreement with the commercial kits. D3EDIII was refolded by rapid dilution method and the refolded monomer protein was purified by Ion exchange chromatography. Further, the recombinant protein was biologically functional and found to inhibit dengue virus type 3 plaque formation on LLC-MK2 cells demonstrating its function of receptor interaction. Furthermore, D3EDIII in combination with Freund's complete adjuvant induced high antibody titers in BALB/c mice and these antibodies efficiently neutralized dengue 3 virus. Additionally, D3EDIII induced expression of Th1 cytokines that can inhibit the intracellular viral infections. Thus, our results demonstrate that D3EDIII protein has tremendous potential both in diagnosis of dengue infections and in vaccine development.


Subject(s)
Dengue Virus/chemistry , Dengue Virus/immunology , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Viral Envelope Proteins/immunology , Viral Envelope Proteins/isolation & purification , Animals , Antibodies, Neutralizing/blood , Antibodies, Viral/blood , Dengue Vaccines/biosynthesis , Dengue Vaccines/chemistry , Dengue Vaccines/immunology , Dengue Vaccines/isolation & purification , Enzyme-Linked Immunosorbent Assay , Escherichia coli/chemistry , Escherichia coli/genetics , Escherichia coli/metabolism , Immunoglobulin G/blood , Immunoglobulin M/blood , Mice , Mice, Inbred BALB C , Neutralization Tests , Protein Structure, Tertiary , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Vaccines, Synthetic/biosynthesis , Vaccines, Synthetic/chemistry , Vaccines, Synthetic/immunology , Vaccines, Synthetic/isolation & purification , Viral Envelope Proteins/biosynthesis , Viral Envelope Proteins/chemistry
8.
Protein Pept Lett ; 16(11): 1334-41, 2009.
Article in English | MEDLINE | ID: mdl-20001924

ABSTRACT

Japanese encephalitis virus (JEV) is a mosquito-borne viral zoonosis of public health importance. Global efforts have been made towards development of vaccine for prevention of Japanese encephalitis. The envelope protein of JEV is associated with viral binding to cellular receptors, membrane fusion, and the induction of protective neutralizing antibody response in hosts. Here we report that the antibodies raised against refolded domain III of envelope protein of JEV neutralize the JE virus and inhibit the JEV infection to Porcine Stable Kidney (PS) cells. A reverse transcriptase-PCR amplified gene encoding domain III of JEV envelope protein was cloned into pET28a+ vector and over expressed in E. coli. The recombinant JEV-DIII protein was purified by affinity chromatography under denaturing conditions. The rJEV-DIII was refolded by oxido-redux shuffle and purified to homogeneity by ion-exchange chromatography. Refolded rJEV-DIII was characterized using biochemical and biophysical methods. The polyclonal antibodies were raised against in vitro refolded rJEV-DIII protein in BALB/c mice with Freunds adjuvant. Ninety percent JEV is neutralized when the serum against refolded rJEV-DIII is used at a dilution of 1:80 as against 60.5% neutralization capacity with the same dilution of serum raised against denatured rJEV-DIII. The method of expression and purification of biologically functional rJEV-DIII protein described in this study may help in better understanding the biology of JE virus and the development of better vaccine candidate. Since the expression system uses E. coli as the heterologous host, the process is easy and amenable to inexpensive scale-up.


Subject(s)
Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Encephalitis Virus, Japanese/immunology , Encephalitis, Japanese/immunology , Viral Envelope Proteins/immunology , Animals , Antibodies, Neutralizing/metabolism , Antibodies, Viral/metabolism , Cells, Cultured , Chromatography, Gel , Chromatography, Ion Exchange , Circular Dichroism , Escherichia coli/genetics , Mice , Mice, Inbred BALB C , Neutralization Tests , Protein Folding , Protein Structure, Tertiary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Sulfhydryl Compounds/analysis , Swine , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics , Viral Envelope Proteins/isolation & purification
9.
Article in English | MEDLINE | ID: mdl-19008160

ABSTRACT

The antibody fragments generated from hyperimmune equine IgG is widely used as anti-snake venom, anti-scorpion venom, anti-diphtheria, anti-tetanus, anti-gangrene and anti-rabies agents. Antibody fragments, F(ab)(2), because of their specificity and absence of undesired reactivity are preferred over complete IgG. This paper discusses a novel purification technique for chromatographic purification of anti-rabies immunoglobulin G (IgG) fragment F(ab)(2) from horse serum. F(ab)(2) was purified by two successive chromatography steps using Cellufine A-200 and ProSep-vA Ultra media. The purified F(ab)(2) was characterized using biochemical and biophysical methods and shown to be pure and homogeneous. The purified F(ab)(2) was reactive to rabies antigen in immuno-electrophoresis and diffusion tests. The purified F(ab)(2) was biologically functional and was found to show a potency of 1500 IU ml(-1). Comparative analysis of the purity with commercially available F(ab)(2) by HPLC analysis and SDS-PAGE indicated that the present product is better in purity. To our knowledge, this is the first report providing evidence on purification of equine antibody fragment using controlled pore glass based protein A chromatography media.


Subject(s)
Chromatography, Affinity/methods , Chromatography, Ion Exchange/methods , Horses/blood , Immunoglobulin Fab Fragments/isolation & purification , Immunoglobulin G/isolation & purification , Animals , Rabies Vaccines/immunology , Staphylococcal Protein A/immunology
10.
Vaccine ; 26(36): 4655-63, 2008 Aug 26.
Article in English | MEDLINE | ID: mdl-18640168

ABSTRACT

Dengue fever, a mosquito borne viral disease, has become a major public health problem with dramatic expansion in recent decades. Several dengue vaccines are at developing stage, none are yet available for humans. There is no vaccine or antiviral therapy available for dengue fever till date. Domain III of envelope protein is involved in binding to host receptors and it contains type and subtype-specific epitopes that elicit virus neutralizing antibodies. Hence domain III is an attractive vaccine candidate. In the present study we report the immunomodulatory potential of refolded D4EIII protein in combination with various adjuvants (Freunds Complete adjuvant, Montanide ISA720, Alum). Mice were tested for humoral immune responses by ELISA, immunofluorescence assay and plaque reduction neutralization test. Cell mediated immune response was tested by lymphocyte proliferation assay and cytokine profiling. All the formulations resulted in high antibody titers that neutralized the virus entry in vitro. D4EIII in combination with montanide ISA720 and Feuds complete adjuvant gave highest antibody endpoint titers followed by alum. The level of antigen-stimulated splenocyte proliferation and cytokine production was comparable to that obtained from Con A stimulation and cytokine profiling of stimulated splenocyte culture supernatants indicated that all the adjuvant formulations have induced cell mediated immune response as well. These findings suggest that D4EIII in combination with compatible adjuvants is highly immunogenic and can elicit high titer neutralizing antibodies and cell mediated immune response which plays an important role in intracellular infections, which proves that refolded D4EIII can be a potential vaccine candidate.


Subject(s)
Adjuvants, Immunologic/administration & dosage , Dengue Vaccines/immunology , Viral Envelope Proteins/immunology , Alum Compounds/administration & dosage , Animals , Antibodies, Viral/blood , Cell Proliferation , Cytokines/metabolism , Enzyme-Linked Immunosorbent Assay , Freund's Adjuvant/administration & dosage , Humans , Lymphocytes/immunology , Mannitol/administration & dosage , Mannitol/analogs & derivatives , Mice , Mice, Inbred BALB C , Neutralization Tests , Oleic Acids/administration & dosage , Spleen/immunology , Vaccines, Synthetic/immunology , Viral Plaque Assay
11.
Biotechnol Prog ; 23(2): 488-93, 2007.
Article in English | MEDLINE | ID: mdl-17256968

ABSTRACT

Dengue virus infections have recently undergone dramatic expansion in range, affecting several tropical and subtropical regions of the world. Early detection of dengue infection based on the identification of antibodies has emerged as a practical and reliable means of diagnosis of dengue fever. The recombinant dengue multiepitope (rDME-M) protein specific to IgM in E. coli was produced in a 5-L fermentor for use in diagnostic purpose. After fermentation, dry cell weight was approximately 11.8 g/L of the culture. The rDME-M protein was purified under denaturing conditions using single-step nickel nitrilotriacetate (Ni-NTA) affinity chromatography. The final yield of purified rDME-M protein from this method was approximately 68.5 mg/L of the culture. The purity of rDME-M protein was checked by SDS-PAGE analysis, and the reactivity of this protein was further checked by Western blotting and enzyme-linked immunosorbent assay (ELISA). The purified protein was used as an antigen in the development of an in-house dipstick ELISA and evaluated with a panel of 80 patient sera, characterized using commercially available tests for detection of dengue antibody. The results were in excellent agreement with those of IgM capture ELISA (Pan-Bio) and rapid immunochromatography (IC) test (Pan-Bio). These results show that the in-house dipstick ELISA using rDME-M protein can be used as a promising kit because of its comparable sensitivity, specificity, field applicability, and low cost.


Subject(s)
Dengue Virus/immunology , Dengue/diagnosis , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin M/immunology , Recombinant Proteins/immunology , Viral Proteins/immunology , Viral Proteins/metabolism , Dengue/immunology , Dengue Virus/genetics , Epitopes/genetics , Epitopes/immunology , Escherichia coli/genetics , Escherichia coli/metabolism , Humans , Immunoglobulin M/genetics , Immunoglobulin M/metabolism , Protein Engineering/methods , Recombinant Proteins/biosynthesis , Viral Proteins/isolation & purification
12.
Biotechnol Appl Biochem ; 46(Pt 2): 105-13, 2007 Feb.
Article in English | MEDLINE | ID: mdl-16999685

ABSTRACT

Dengue is an acute mosquito-borne viral disease of humankind. Dengue fever, dengue haemorrhagic fever and dengue shock syndrome have become global public health problems in recent years. rDME-G (recombinant dengue multiepitope protein that can specifically detect IgG) was produced in a 5-litre fermenter in Escherichia coli for use in diagnosis. The culture was induced with 1 mM isopropyl beta-D-thiogalactoside and cells were further grown for 4 h before harvesting. After fermentation, dry cell weight resulted in approx. 16.2 g/l. The rDME-G protein was purified from inclusion bodies using affinity chromatography. The final yield of purified rDME-G protein from fermentation resulted in approx. 168 mg/l of pure biologically active rDME-G protein. The purity of rDME-G protein was checked by SDS/PAGE analysis and the reactivity of this protein was further determined by Western blotting. The purified protein was used to develop an in-house dipstick ELISA and tested using a panel of 60 patient sera characterized using the commercially available tests for detection of dengue antibody. We compared our results with IgG-capture ELISA (Pan-Bio, Windsor, QLD, Australia) and rapid IC (immuno-chromatography) test (Pan-Bio). By using rDME-G protein as an antigen, in the dipstick ELISA, the results were in excellent agreement with commercial rapid IC test and IgG capture ELISA. These results show that the product has a promising potential to be used for diagnosis of dengue in both laboratory- and field-based detection systems with minimum cost and a high degree of sensitivity and specificity.


Subject(s)
Dengue Virus/metabolism , Epitopes/immunology , Epitopes/isolation & purification , Escherichia coli/metabolism , Protein Engineering/methods , Viral Proteins/immunology , Viral Proteins/isolation & purification , Dengue Virus/genetics , Epitopes/chemistry , Epitopes/genetics , Escherichia coli/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Recombinant Proteins/isolation & purification , Viral Proteins/chemistry , Viral Proteins/genetics
13.
Vaccine ; 25(5): 806-13, 2007 Jan 15.
Article in English | MEDLINE | ID: mdl-17045706

ABSTRACT

Malaria parasites require specific receptor-ligand interactions to invade host erythrocytes. The 175 kDa Plasmodium falciparum erythrocyte binding antigen (EBA-175) binds sialic acid residues on glycophorin A to mediate erythrocyte invasion. The amino-terminal, conserved, cysteine-rich region of EBA-175, referred to as F2, contains receptor-binding sites. We propose to develop a recombinant malaria vaccine based on region F2. Recombinant P. falciparum region F2 (PfF2) was expressed in Escherichia coli, purified from inclusion bodies under denaturing conditions by metal affinity chromatography, renatured by oxidative refolding and purified further by ion-exchange and gel filtration chromatography. Recombinant PfF2 was characterized and shown to be pure, homogeneous and functionally active in that it binds human erythrocytes with specificity. The immunogenicity of recombinant PfF2 formulated with three human compatible adjuvants, namely, Montanide ISA720, AS02A and alum was tested in mice. All the formulations tested elicited high titer antibodies that block erythrocyte invasion in vitro. The AS02 formulation yielded sera with the highest end-point ELISA titers followed by Montanide ISA720 and alum. Analysis of cellular immune responses indicated that all formulations resulted in significant splenocyte proliferation. Analysis of cytokines secreted by proliferating splenocytes indicated that all the adjuvant formulations tested induced Th1 type responses. These results suggest that recombinant PfF2 formulated with human compatible adjuvants is immunogenic and can elicit high titer invasion inhibitory antibodies providing support for further clinical development of this promising vaccine candidate.


Subject(s)
Antigens, Protozoan/immunology , Malaria Vaccines/immunology , Protozoan Proteins/immunology , Vaccines, Synthetic/immunology , Adjuvants, Immunologic/administration & dosage , Animals , Antigens, Protozoan/chemistry , Binding Sites , Cytokines/biosynthesis , Malaria Vaccines/administration & dosage , Mice , Mice, Inbred BALB C , Protein Folding , Protozoan Proteins/chemistry , Vaccination , Vaccines, Synthetic/administration & dosage
14.
J Chromatogr B Analyt Technol Biomed Life Sci ; 846(1-2): 184-94, 2007 Feb 01.
Article in English | MEDLINE | ID: mdl-17011249

ABSTRACT

An arboviral infection like dengue fever/dengue hemorrhagic fever (DHF) with high morbidity and mortality rate are extensively prevalent in several parts of the world. Global efforts have been directed towards development of vaccine for prevention of dengue. However, lack of thorough understanding about biology and pathogenesis of dengue virus restricts us from development of an effective vaccine. Here we report molecular interaction of domain III of envelope protein of dengue virus type-4 with heparan sulfate. A codon optimized synthetic gene encoding domain III of dengue virus type-4 envelope protein was expressed in Escherichia coli and purified under denaturing conditions, refolded and purified to homogeneity. Refolded Den4-DIII was characterized using biochemical and biophysical methods and shown to be pure and homogeneous. The purified protein was recognized in Western analyses by monoclonal antibody specific for the 6x His tag as well as the H241 monoclonal antibody. The in vitro refolded recombinant protein preparation was biologically functional and found to bind cell free heparan sulfate. This is the first report providing molecular evidence on binding of dengue-4 envelope protein to heparan sulfate. We developed a homology model of dengue-4 envelope protein (domain III) and mapped the possible amino acid residues critical for binding to heparan sulfate. Domain III envelope protein of dengue virus is a lead vaccine candidate. Our findings further the understanding on biology of dengue virus and will help in development of bioassay for the proposed vaccine candidate.


Subject(s)
Dengue Virus/metabolism , Escherichia coli/genetics , Viral Envelope Proteins/metabolism , Blotting, Western , Chromatography, Affinity , Chromatography, Gel , Chromatography, High Pressure Liquid , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Heparitin Sulfate , Models, Molecular , Protein Binding , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Spectrometry, Mass, Electrospray Ionization , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/genetics
15.
J Postgrad Med ; 52(3): 174-8, 2006.
Article in English | MEDLINE | ID: mdl-16855316

ABSTRACT

BACKGROUND: Dengue, Japanese encephalitis, West Nile encephalitis, yellow fever are the common flaviviral diseases associated with high morbidity and mortality. The initial symptoms of most of the flaviviral infections are similar to each other as well as to some other viral diseases. Making clinical diagnosis, therefore, becomes a challenging task for the clinician. Several studies have been reported on using detection of serum antibodies against flavivirus for the diagnosis of specific flaviviral disease; no field-based pan-flavi virus detection system is available, which can be used in low-endemicity areas for differentiation of flaviviral disease from other viral diseases. AIM: To identify a conserved amino acid sequence among all flaviviruses and evaluate the antibody formed against the conserved peptide to develop pan-flavivirus detection system. MATERIALS AND METHODS: In the present study we have compared amino acid sequences of several flaviviruses and identified a conserved amino acid sequence lying in domain II of envelope protein. RESULTS: A peptide having the conserved amino acid sequence was used to generate polyclonal antibodies and these antibodies were used to detect several flaviviruses. Anti-peptide polyclonal antibodies selectively recognized flaviviruses and did not detect non-flaviviruses. Anti-peptide antibodies detected presence of virus in serum spiked with pure virus preparations. CONCLUSION: The study offers a rationale for development of pan-flavivirus capture assay suitable for low endemic areas.


Subject(s)
Flavivirus Infections/diagnosis , Flavivirus Infections/virology , Flavivirus/isolation & purification , Peptides/immunology , Amino Acid Sequence , Animals , Antibodies, Viral/blood , Biomarkers , Early Diagnosis , Enzyme-Linked Immunosorbent Assay , Flavivirus/chemistry , Mice , Peptides/chemistry , Protein Structure, Tertiary , Viral Envelope Proteins/chemistry
16.
Biotechnol Lett ; 28(14): 1109-14, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16794771

ABSTRACT

A recombinant blood-stage vaccine for Plasmodium vivax malaria based on the functional receptor-binding domain of PvDBP (PvRII) has been developed. A synthetic gene coding for PvRII was expressed in Escherichia coli using codon optimization. Expression level of recombinant PvRII was 10% of the total cellular proteins. Truncated PvRII products, seen when the native PvRII gene was expressed, were absent in case of synthetic gene.


Subject(s)
Antigens, Protozoan/analysis , Antigens, Protozoan/metabolism , Genetic Enhancement/methods , Malaria Vaccines/analysis , Malaria Vaccines/metabolism , Plasmodium vivax/metabolism , Protein Engineering/methods , Protozoan Proteins/analysis , Protozoan Proteins/metabolism , Receptors, Cell Surface/analysis , Receptors, Cell Surface/metabolism , Animals , Antigens, Protozoan/genetics , Codon/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Malaria Vaccines/genetics , Plasmodium vivax/genetics , Protein Structure, Tertiary , Protozoan Proteins/genetics , Receptors, Cell Surface/genetics , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Transfection/methods
17.
Rev Med Virol ; 16(3): 151-65, 2006.
Article in English | MEDLINE | ID: mdl-16710839

ABSTRACT

Kyasanur forest disease (KFD) was first recognised as a febrile illness in the Shimoga district of Karnataka state of India. The causative agent, KFD virus (KFDV), is a highly pathogenic member in the family Flaviviridae, producing a haemorrhagic disease in infected human beings. KFD is a zoonotic disease and has so far been localised only in a southern part of India. The exact cause of its emergence in the mid 1950s is not known. A variant of KFDV, characterised serologically and genetically as Alkhurma haemorrhagic fever virus (AHFV), has been recently identified in Saudi Arabia. KFDV and AHFV share 89% sequence homology, suggesting common ancestral origin. Homology modelling of KFDV envelope (E) protein exhibited a structure similar to those of other flaviviruses, suggesting a common mechanism of virus-cell fusion. The possible mechanism of receptor-ligand interaction involved in infection by KFDV may resemble that of other flavivirses. Present understanding is that KFDV may be persisting silently in several regions of India and that antigenic and structural differences from other tick borne viruses may be related to the unique host specificity and pathogenicity of KFDV. From January 1999 through January 2005, an increasing number of KFD cases have been detected in Karnataka state of Indian subcontinent despite routine vaccination, suggesting insufficient efficacy of the current vaccine protocol. However, the exact cause of the increase of KFD cases needs further investigation. Considering the requirement of safer and more effective vaccines in general, there is clearly a need for developing an alternative vaccine as well as a rapid diagnostic system for KFD. The changing ecology of the prime focus of the KFD also warrants attention, as it may lead to establishment of the disease in newer localities, never reported before.


Subject(s)
Encephalitis Viruses, Tick-Borne/growth & development , Kyasanur Forest Disease/epidemiology , Kyasanur Forest Disease/veterinary , Monkey Diseases/epidemiology , Monkey Diseases/virology , Zoonoses/epidemiology , Zoonoses/virology , Animals , Encephalitis Viruses, Tick-Borne/genetics , Encephalitis Viruses, Tick-Borne/immunology , Female , Haplorhini , Humans , India/epidemiology , Kyasanur Forest Disease/immunology , Kyasanur Forest Disease/virology , Male , Models, Molecular , Monkey Diseases/immunology , Phylogeny
19.
Appl Biochem Biotechnol ; 126(2): 79-92, 2005 Aug.
Article in English | MEDLINE | ID: mdl-16118464

ABSTRACT

During last decade there has been significant progress in the development of analytical techniques for evaluation of receptor-ligand interaction. Surface plasmon resonance (SPR)-based optical biosensors are now being used extensively to define the kinetics of wide variety of macromolecular interactions and high- and low-affinity small molecule interactions. The experimental design data analysis methods are evolving along with widespread applications in ligand fishing, microbiology, virology, host-pathogen interaction, epitope mapping, and protein-, cell-, membrane-, nucleic acid-protein interactions. SPR-based biosensors have strong impact on basic and applied research significantly. This brief review describes the SPR technology and few of its applications in relation to receptor-ligand interaction that has brought significant change in the methodology, analysis, interpretation, and application of the SPR technology.


Subject(s)
Biosensing Techniques , Surface Plasmon Resonance , Kinetics , Ligands , Protein Binding , Surface Plasmon Resonance/instrumentation , Surface Plasmon Resonance/methods
20.
Microbiol Res ; 160(2): 213-8, 2005.
Article in English | MEDLINE | ID: mdl-15881839

ABSTRACT

Effect of environmental factors on production of lichenin, a bacteriocin-like compound produced by Bacillus licheniformis 26L-10/3RA isolated from buffalo rumen was studied. Lichenin represents the first anaerobiosis-specific expression of broad-spectrum antibacterial compound effective only under anaerobic conditions. Production of lichenin by B. licheniformis 26L-10/3RA was found to be very high at 39 degrees C in L-10 medium supplemented with 0.5% glucose and 20% (w/v) inert thermocol beads. Lichenin production was highest at pH 6.8 after 72-96h of incubation. Our study also indicated that Lichenin is not a plasmid-linked characteristic and is encoded by chromosomal DNA. Results obtained can be used in large-scale production of Lichenin for potential application in manipulating rumen function intended for improving productivity of the ruminants.


Subject(s)
Bacillus/growth & development , Chromosomes, Bacterial/genetics , Glucans/biosynthesis , Anaerobiosis , Animals , Bacillus/genetics , Bacillus/metabolism , Buffaloes , Culture Media , DNA, Bacterial/genetics , Glucans/genetics , Hydrogen-Ion Concentration , Plasmids , Rumen/microbiology , Temperature
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