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2.
Expert Rev Vaccines ; 18(9): 913-920, 2019 09.
Article in English | MEDLINE | ID: mdl-31373843

ABSTRACT

Introduction: Inovirus-associated vectors (IAVs) are derived from bacterial filamentous viruses (phages). As vaccine carriers, they have elicited both cellular and humoral responses against a variety of pathogens causing infectious diseases and other non-infectious diseases. By displaying specific antigen epitopes or proteins on their coat proteins, IAVs have merited much study, as their unique abilities are exploited for widespread vaccine development. Areas covered: The architectural traits of filamentous viruses and their derivatives, IAVs, facilitate the display of specific antigenic peptides which induce antibody production to prevent or curtail infection. Inoviruses provide a foundation for cost-efficient large-scale specific phage display. In this paper, the development of different applications of inovirus-based phage display vaccines across a broad range of pathogens and hosts is reviewed. The references cited in this review were selected from established databases based on the authors' knowledge of the study subject. Expert commentary: The importance of phage-display technology has been recently highlighted by the Nobel Prize in Chemistry 2018 awarded to George P. Smith and Sir Gregory P. Winter. Furthermore, the symbiotic nature of filamentous viruses infecting intestinal F+E. coli strains offers an attractive platform for the development of novel vaccines that stimulate mucosal immunity.


Subject(s)
Cell Surface Display Techniques/methods , Inovirus/immunology , Viral Vaccines/immunology , Animals , Drug Design , Epitopes/immunology , Escherichia coli , Humans , Immunity, Mucosal , Peptides
3.
J Cancer Res Ther ; 15(Supplement): S1-S10, 2019 Mar.
Article in English | MEDLINE | ID: mdl-30900613

ABSTRACT

A new modality of targeting therapeutic drugs based on the use of bacteriophage (virus), as an emerging tool for specific targeting and for vaccine development, has been an area of interest for genetic and cancer research. The approach is based on genetic manipulation and modification in the chemical structure of a filamentous bacteriophage that facilitates its application not only for in vivo imaging but also for therapeutic purpose, as a gene delivery vehicle, as drug carriers, and also as an immunomodulatory agent. Filamentous bacteriophage on account of its high surface holding ability with adaptable genetic engineering properties can effectively be used in loading of chemical and genetic drugs specifically on to the targeted lesion location. Moreover, the specific peptides/proteins exhibited on the phage surface can be applied directly as self-navigating drug delivery nanovehicles. The present review article has been framed with an objective to summarize the importance of bacteriophage in phage cancer therapy and to understand the possible future prospective of this approach in developing new tools for biotechnological and genetic research, especially in phage -mediated cancer therapy. Importantly, the peptides or proteins emerging from the surface of a nano carrier will make the expense of such peptides economically more effective as compared to other immunological tools, and this seems to be a potential approach for developing a new nanodrug carrier platform.


Subject(s)
Antineoplastic Agents/administration & dosage , Drug Carriers/chemistry , Genetic Vectors/genetics , Inovirus/genetics , Neoplasms/therapy , Animals , Genetic Engineering , Genetic Therapy/methods , Genetic Vectors/chemistry , Genetic Vectors/immunology , Humans , Inovirus/chemistry , Inovirus/immunology , Nanoparticles , Neoplasms/genetics , Neoplasms/immunology
4.
Cancer Immunol Immunother ; 67(2): 183-193, 2018 02.
Article in English | MEDLINE | ID: mdl-29026949

ABSTRACT

Colorectal cancer is a deadly disease, which is frequently diagnosed at advanced stages, where conventional treatments are no longer effective. Cancer immunotherapy has emerged as a new form to treat different malignancies by turning-on the immune system against tumors. However, tumors are able to evade antitumor immune responses by promoting an immunosuppressive microenvironment. Single-stranded DNA containing M13 bacteriophages are highly immunogenic and can be specifically targeted to the surface of tumor cells to trigger inflammation and infiltration of activated innate immune cells, overcoming tumor-associated immunosuppression and promoting antitumor immunity. Carcinoembryonic antigen (CEA) is highly expressed in colorectal cancers and has been shown to promote several malignant features of colorectal cancer cells. In this work, we targeted M13 bacteriophage to CEA, a tumor-associated antigen over-expressed in a high proportion of colorectal cancers but largely absent in normal cells. The CEA-targeted M13 bacteriophage was shown to specifically bind to purified CEA and CEA-expressing tumor cells in vitro. Both intratumoral and systemic administration of CEA-specific bacteriophages significantly reduced tumor growth of mouse models of colorectal cancer, as compared to PBS and control bacteriophage administration. CEA-specific bacteriophages promoted tumor infiltration of neutrophils and macrophages, as well as maturation dendritic cells in tumor-draining lymph nodes, suggesting that antitumor T-cell responses were elicited. Finally, we demonstrated that tumor protection provided by CEA-specific bacteriophage particles is mediated by CD8+ T cells, as depletion of circulating CD8+ T cells completely abrogated antitumor protection. In summary, we demonstrated that CEA-specific M13 bacteriophages represent a potential immunotherapy against colorectal cancer.


Subject(s)
Carcinoembryonic Antigen/immunology , Colorectal Neoplasms/immunology , Inovirus/immunology , Animals , Cell Line, Tumor , Disease Models, Animal , Humans , Mice , Mice, Inbred C57BL
5.
Curr Med Chem ; 24(35): 3907-3920, 2017 Nov 20.
Article in English | MEDLINE | ID: mdl-28901276

ABSTRACT

BACKGROUND: Population control of domestic, wild, invasive, and captive animal species is a global issue of importance to public health, animal welfare and the economy. There is pressing need for effective, safe, and inexpensive contraceptive technologies to address this problem. Contraceptive vaccines, designed to stimulate the immune system in order to block critical reproductive events and suppress fertility, may provide a solution. Filamentous bacteriophages can be used as platforms for development of such vaccines. OBJECTIVE: In this review authors highlight structural and immunogenic properties of filamentous phages, and discuss applications of phage-peptide vaccines for advancement of immunocontraception technology in animals. RESULTS: Phages can be engineered to display fusion (non-phage) peptides as coat proteins. Such modifications can be accomplished via genetic manipulation of phage DNA, or by chemical conjugation of synthetic peptides to phage surface proteins. Phage fusions with antigenic determinants induce humoral as well as cell-mediated immune responses in animals, making them attractive as vaccines. Additional advantages of the phage platform include environmental stability, low cost, and safety for immunized animals and those administering the vaccines. CONCLUSION: Filamentous phages are viable platforms for vaccine development that can be engineered with molecular and organismal specificity. Phage-based vaccines can be produced in abundance at low cost, are environmentally stable, and are immunogenic when administered via multiple routes. These features are essential for a contraceptive vaccine to be operationally practical in animal applications. Adaptability of the phage platform also makes it attractive for design of human immunocontraceptive agents.


Subject(s)
Contraception, Immunologic , Inovirus/metabolism , Vaccines, Contraceptive/immunology , Animals , Genetic Vectors/genetics , Genetic Vectors/metabolism , Inovirus/chemistry , Inovirus/immunology , Peptide Library , Vaccines, Subunit/immunology , Vaccines, Virus-Like Particle/immunology
6.
J Virol Methods ; 183(1): 63-8, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22575687

ABSTRACT

The focus of this study is on development of vaccines using filamentous phage as a delivery vector for immunogenic peptides. The use of phage as a carrier for immunogenic peptides provides significant benefits such as high immunogenicity, low production costs, and high stability of phage preparations. However, introduction of live recombinant phage into the environment might represent a potential ecological problem. This, for example, may occur when vaccines are used in oral or nasal formulations in field conditions for wild and feral animals. To address this issue, comparative studies of antigenic properties of live and inactivated (non-viable) phage were accomplished. Inactivated phage, if released, will not propagate and will degrade as any other protein. In these experiments, a model phage clone that was previously selected from a phage display library and shown to stimulate production of anti-sperm antibodies with contraceptive properties was used. Multiple methods of phage inactivation were tested, including drying, freezing, autoclaving, heating, and UV irradiation. Under studied conditions, heating at 76°C for 3h, UV irradiation, and autoclaving resulted in complete phage inactivation. Phage samples treated by heat and UV were characterized by spectrophotometry and electron microscopy. To test antigenicity, live and inactivated phage preparations were injected into mice and antibody responses assayed by ELISA. It was found that phage killed by heat causes little to no immune responses, probably due to destruction of phage particles. In contrast, UV-inactivated phage stimulated production of IgG serum antibodies at the levels comparable to live phage. Thus, vaccines formulated to include UV-inactivated filamentous phage might represent environmentally safe alternatives to live phage vaccines.


Subject(s)
Drug Carriers , Genetic Vectors , Inovirus/genetics , Inovirus/immunology , Vaccines, Contraceptive/immunology , Animals , Antibodies/blood , Desiccation , Disinfection/methods , Enzyme-Linked Immunosorbent Assay , Freezing , Hot Temperature , Inovirus/radiation effects , Male , Mice , Spermatozoa/immunology , Ultraviolet Rays , Vaccines, Contraceptive/administration & dosage , Vaccines, Inactivated/administration & dosage , Vaccines, Inactivated/immunology , Vaccines, Synthetic/administration & dosage , Vaccines, Synthetic/immunology , Virus Inactivation/radiation effects
7.
Bioeng Bugs ; 2(5): 275-83, 2011.
Article in English | MEDLINE | ID: mdl-22008640

ABSTRACT

Filamentous bacteriophage are commonly used as immunogenic carriers for peptides and proteins displayed on the phage surface. Previously, we showed that immunization with phage to which peptides had been chemically conjugated can elicit a focused anti-peptide antibody response compared with traditional carrier molecules bearing the same peptide, perhaps due to the low surface complexity of the phage. The regularity of its surface also gives the phage other advantages as a carrier, including immunological simplicity and thousands of well-defined sites for chemical conjugation. More recently, we showed that focusing of antibody responses against 'target' peptides was enhanced when the phage's molecular surface was simplified by removal of immunodominant B-cell epitopes present on the minor coat protein, pIII. The pIII-truncated variant elicits an antibody response that is largely restricted to the exposed N-terminus of the major coat protein, pVIII, and to phage-associated bacterial lipopolysaccharide, and a significant fraction of this response cross-reacts with a 12-residue peptide covering the surface-exposed region of pVIII. This allows one to track antibody responses against the phage (and any associated haptens) as they develop over time, and characterize them using a combination of serological, flow cytometric, cellular and immunogenetic assays. The filamentous phage thus provides an excellent model system for studying various aspects of the antibody response, all with the goal of targeting antibody production against weakly immunogenic peptides, proteins and carbohydrates.


Subject(s)
Adjuvants, Immunologic/pharmacology , Antigens/immunology , Drug Delivery Systems/methods , Immunity, Humoral , Inovirus/immunology , Animals , Antibodies/immunology , Antigens/genetics , Capsid Proteins/genetics , Capsid Proteins/immunology , Drug Carriers/pharmacology , Humans , Inovirus/genetics , Mice , Models, Immunological
8.
Eur J Immunol ; 41(9): 2573-84, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21688262

ABSTRACT

The efficacy of a new vaccine-delivery vector, based on the filamentous bacteriophage fd displaying a single-chain antibody fragment known to bind the mouse DC surface molecule DEC-205, is reported. We demonstrate both in vitro and in vivo an enhanced receptor-mediated uptake of phage particles expressing the anti-DEC-205 fragment by DCs. We also report that DCs targeted by fd virions in the absence of other stimuli produce IFN-α and IL-6, and acquire a mature phenotype. Moreover, DC-targeting with fd particles double-displaying the anti-DEC-205 fragment on the pIII protein and the OVA(257-264) antigenic determinant on the pVIII protein induced potent inhibition of the growth of the B16-OVA tumor in vivo. This protection was much stronger than other immunization strategies and similar to that induced by adoptively transferred DCs. Since targeting DEC-205 in the absence of DC activation/maturation agents has previously been described to result in tolerance, the ability of fd bacteriophages to induce a strong tumor-specific immune response by targeting DCs through DEC-205 is unexpected, and further validates the potential employment of this safe, versatile and inexpensive delivery system for vaccine formulation.


Subject(s)
Cancer Vaccines , Dendritic Cells/metabolism , Inovirus/immunology , Single-Chain Antibodies/metabolism , Virion/metabolism , Animals , Antigens, CD/immunology , Capsid Proteins/genetics , Capsid Proteins/metabolism , Cell Differentiation , Dendritic Cells/immunology , Dendritic Cells/pathology , Dendritic Cells/virology , Enterobacteriaceae/virology , Inovirus/pathogenicity , Interferon-gamma/metabolism , Interleukin-6/metabolism , Lectins, C-Type/immunology , Melanoma, Experimental , Mice , Mice, Inbred C57BL , Minor Histocompatibility Antigens , Molecular Targeted Therapy , Ovalbumin/genetics , Ovalbumin/metabolism , Peptide Fragments/genetics , Peptide Fragments/metabolism , Receptors, Cell Surface/immunology , Single-Chain Antibodies/genetics , Transgenes/genetics , Tumor Burden , Vaccination , Virion/immunology , Virion/pathogenicity
9.
Immunol Lett ; 119(1-2): 62-70, 2008 Aug 15.
Article in English | MEDLINE | ID: mdl-18538862

ABSTRACT

Several efforts have been invested in the identification of CTL and Th epitopes, as well as in the characterization of their immunodominance and MHC restriction, for the generation of a peptide-based HCMV vaccine. Small synthetic peptides are, however, poor antigens and carrier proteins are important for improving the efficacy of synthetic peptide vaccines. Recombinant bacteriophages appear as promising tools in the design of subunit vaccines. To investigate the antigenicity of peptides carried by recombinant bacteriophages we displayed different HCMV MHCII restricted peptides on the capsid of filamentous bacteriophage (fd) and found that hybrid bacteriophages are processed by human APC and activate HCMV-specific CD4 T-cells. Furthermore we constructed a reporter T-cell hybridoma expressing a chimeric TCR comprising murine alphabeta constant regions and human variable regions specific for the HLA-A2 restricted immunodominant NLV peptide of HCMV. Using the filamentous bacteriophage as an epitope carrier, we detected a more robust and long lasting response of the reporter T-cell hybridoma compared to peptide stimulation. Our results show a general enhancement of T-cell responses when antigenic peptides are carried by phages.


Subject(s)
Antigens, Viral/immunology , Cytomegalovirus Infections/prevention & control , Cytomegalovirus/immunology , Epitopes, T-Lymphocyte/genetics , Inovirus/immunology , Peptides/immunology , Vaccines, Synthetic/immunology , Animals , Antigen Presentation/immunology , Antigens, CD/genetics , Antigens, CD/immunology , Antigens, Differentiation, T-Lymphocyte/genetics , Antigens, Differentiation, T-Lymphocyte/immunology , Antigens, Viral/chemistry , Antigens, Viral/genetics , CD4-Positive T-Lymphocytes/immunology , Cell Line , Cell Proliferation , Cytokines/metabolism , Cytomegalovirus Infections/immunology , Epitopes, T-Lymphocyte/chemistry , Epitopes, T-Lymphocyte/immunology , Genetic Vectors , HLA-A2 Antigen/metabolism , Humans , Inovirus/genetics , Lectins, C-Type , Lymphocyte Activation/genetics , Lymphocyte Activation/immunology , Mice , Peptides/chemistry , Peptides/genetics , Receptors, Antigen, T-Cell/biosynthesis , Receptors, Antigen, T-Cell/genetics , Receptors, Antigen, T-Cell/immunology , Signal Transduction/genetics , Signal Transduction/immunology , T-Cell Antigen Receptor Specificity/genetics , Transfection , Transgenes/genetics , Vaccines, Subunit/immunology , Vaccines, Synthetic/chemistry
10.
Vaccine ; 24(19): 4188-200, 2006 May 08.
Article in English | MEDLINE | ID: mdl-16488517

ABSTRACT

Filamentous bacteriophage are widely used as immunogenic carriers for "phage-displayed" recombinant peptides. Here we report that they are an effective immunogenic carrier for synthetic peptides. The f1.K phage was engineered to have an additional Lys residue near the N-terminus of the major coat protein, pVIII, so as to enhance access to chemical cross-linking agents. The dimeric synthetic peptide, B2.1, was conjugated to f1.K (f1.K/B2.1) in high copy number and compared as an immunogen to B2.1 conjugated to ovalbumin (OVA/B2.1) and to phage-displayed, recombinant B2.1 peptide. All immunogens were administered without adjuvant. The serum antibody titers were measured against: the peptide, the carrier, and, if appropriate, the cross-linker. All immunogens elicited anti-peptide antibody titers, with those elicited by OVA/B2.1 exceeding those by f1.K/B2.1; both titers were greater than that elicited by recombinant B2.1 phage. Comparison of the anti-peptide and anti-carrier antibody responses showed that f1.K/B2.1 elicited a more focused anti-peptide antibody response than OVA/B2.1. The anti-peptide antibody response against f1.K/B2.1 was optimized for the injection route, dose and adjuvant. Dose and adjuvant did not have a significant effect on anti-peptide antibody titers, but a change in injection route from intraperitoneal (IP) to subcutaneous (SC) enhanced anti-peptide antibody titers after seven immunizations. The optimized anti-peptide antibody response exceeded the anti-carrier one by 21-fold, compared to 0.07-fold elicited by OVA/B2.1. This indicates that phage as a carrier can focus the antibody response against the peptide. The results are discussed with respect to the advantages of phage as an alternative to traditional carrier proteins for synthetic peptides, carbohydrates and haptens, and to further improvements in phage as immunogenic carriers.


Subject(s)
Inovirus/immunology , Peptides/administration & dosage , Peptides/immunology , Vaccines, Subunit/administration & dosage , Adjuvants, Immunologic/administration & dosage , Amino Acid Sequence , Animals , Antibody Formation , Base Sequence , Cross-Linking Reagents , DNA Primers/genetics , Dimerization , Drug Carriers , Genetic Engineering , Inovirus/genetics , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Ovalbumin/administration & dosage , Ovalbumin/immunology , Peptides/chemistry , Vaccines, Subunit/chemistry
11.
Trends Microbiol ; 14(3): 141-7, 2006 Mar.
Article in English | MEDLINE | ID: mdl-16460941

ABSTRACT

Microbial infections are dependent on the panoply of interactions between pathogen and host and identifying the molecular basis of such interactions is necessary to understand and control infection. Phage display is a simple functional genomic methodology for screening and identifying protein-ligand interactions and is widely used in epitope mapping, antibody engineering and screening for receptor agonists or antagonists. Phage display is also used widely in various forms, including the use of fragment libraries of whole microbial genomes, to identify peptide-ligand and protein-ligand interactions that are of importance in infection. In particular, this technique has proved successful in identifying microbial adhesins that are vital for colonization.


Subject(s)
Communicable Diseases , Epitope Mapping/methods , Peptide Library , Adhesins, Bacterial/isolation & purification , Humans , Inovirus/genetics , Inovirus/immunology , Inovirus/metabolism
12.
Vaccine ; 23(31): 4088-96, 2005 Jul 01.
Article in English | MEDLINE | ID: mdl-15963364

ABSTRACT

Epitope LKVIRK on 47 kDa of heat shock protein (Hsp) 90 of Candida albicans, corresponding to residues 386-391 of the Hsp90, is recognized by patients recovering from invasive candidiasis. The efficacy of hybrid phage displaying epitope LKVIRK in the N-terminal region of the major coat protein (pVIII) in inducing anti-invasive candidiasis immune response was studied in C57BL/6 mice. Indirect phage-ELISA results demonstrated that the mice immunized with hybrid phage had significantly higher titers of epitope LKVIRK-specific serum IgG as compared to those immunized with heat-killed C. albicans (HK-CA). C57BL/6 mice immunized either with hybrid phage or with wild-type phage also developed significant levels of delayed-type hypersensitivity (DTH) response and splenocyte proliferation, as well as with HK-CA. In addition, high levels of IFN-gamma in the CD4(+) splenocytes from phage-immunized mice were detected as well during 1 week post-inoculation. Furthermore, mice immunized with hybrid phage acquired a resistance to systemic C. albicans infection as confirmed by fewer C. albicans cells in the kidneys, and had a longer lifespan compared to control groups following intravenous challenge with C. albicans. These results indicate that hybrid phage displaying epitope LKVIRK may serve as a potential vaccine conferring a resistance to systemic candidiasis.


Subject(s)
Candida albicans/immunology , Candidiasis/prevention & control , Epitopes, B-Lymphocyte/immunology , HSP90 Heat-Shock Proteins/immunology , Heat-Shock Proteins/immunology , Inovirus/immunology , Animals , Antibodies, Fungal/blood , CD4-Positive T-Lymphocytes/immunology , Candidiasis/pathology , Hypersensitivity, Delayed , Inovirus/genetics , Interferon-gamma/analysis , Mice , Mice, Inbred C57BL , Peptide Library
13.
Expert Opin Biol Ther ; 5(6): 773-81, 2005 Jun.
Article in English | MEDLINE | ID: mdl-15952908

ABSTRACT

Cocaine addiction continues to be a major health and social problem in spite of governmental efforts devoted towards educating the public in the dangers of illicit drug use. A variety of pharmacotherapies and psychosocial programmes have been proposed in an effort to provide a method for alleviating the physical and psychological symptoms of cocaine abuse. Unfortunately, these methods have been met with limited success, illustrating a critical need for new effective approaches for the treatment of cocaine addiction. The authors have recently disclosed an alternative cocaine abuse treatment strategy using intranasal administration of an engineered filamentous bacteriophage displaying cocaine-sequestering antibodies on its surface. These phage particles are an effective vector for central nervous system penetration and are capable of binding cocaine, thereby blocking its behavioural effects in a rodent model.


Subject(s)
Antibodies/administration & dosage , Central Nervous System/metabolism , Cocaine-Related Disorders/therapy , Drug Delivery Systems , Inovirus/immunology , Proteins/metabolism , Administration, Intranasal , Animals , Antibodies/genetics , Antibodies/immunology , Cocaine/immunology , Cocaine/metabolism , Cocaine-Related Disorders/prevention & control , Disease Models, Animal , Inovirus/genetics , Proteins/administration & dosage , Proteins/genetics , Rats
14.
Eur J Immunol ; 35(7): 2041-50, 2005 Jul.
Article in English | MEDLINE | ID: mdl-15940671

ABSTRACT

It has been shown that exogenous antigens can access the MHC class I pathway of professional antigen-processing cells. However, details as to how the MHC class I-peptide complex forms in the presentation pathway are still poorly understood. Here we used MHC class I-peptide-specific antibodies to investigate the formation and intracellular location of class I-peptide complexes in macrophages. We observed that the formation of class I-peptide complexes occurs within a few hours and lasts for another few hours on the cell surface of macrophages following loading with filamentous phage particles. The class I-peptide complexes in the process were co-localized with MHC class II molecules and endocytic system markers. Moreover, endosomal compartments containing class I-peptide complexes were found within intracellular organelles stained by DiOC6 and calnexin. In addition, the cross-presentation of phage particles was transporter associated with antigen processing (TAP)-dependent and sensitive to proteasome inhibitors and NH(4)Cl. These data suggest that endocytosed phage particles may be processed and cross-presented in organelles positive for phagosome and endoplasmic reticulum (ER) markers via a classical ER MHC class I loading mechanism.


Subject(s)
Antigens, Viral/immunology , Cross-Priming/immunology , Endoplasmic Reticulum/immunology , Histocompatibility Antigens Class II/immunology , Inovirus/immunology , Virion/immunology , Ammonium Chloride/pharmacology , Animals , Biomarkers/metabolism , Carrier Proteins/metabolism , Endoplasmic Reticulum/metabolism , Histocompatibility Antigens Class I/immunology , Histocompatibility Antigens Class II/metabolism , Hydrogen-Ion Concentration , Kinetics , Lipoproteins/metabolism , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mice, Knockout , Phagocytosis/immunology , Proteasome Inhibitors , Trans-Activators/metabolism , Transport Vesicles/immunology
15.
Immunol Lett ; 101(1): 87-94, 2005 Oct 15.
Article in English | MEDLINE | ID: mdl-15939481

ABSTRACT

Elevated levels of B-cell-activating factor of the TNF family (BAFF) have been implicated in the pathogenesis of autoimmune diseases in human. We now report the isolation by phage display of human single-chain antibody fragment (scFv) anti-BAFF. After four rounds of panning against BAFF, thirty-two out of 92 phage clones displayed BAFF binding activity. One of the positive clones, designated F8, bound to BAFF with relatively high affinity and neutralized BAFF bioactivity in vitro. F8 clone was expressed in soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography (IMAC). The purified scFv recognized BAFF with the affinity constant (K(aff)) of 2.5 x 10(7)M(-1) without cross-reaction to APRIL. In addition to binding, the purified scFv could does-dependently inhibit BAFF-induced mouse spleen B lymphocyte proliferation. Together with its fully human mature, F8 scFv may have therapeutic implications in therapy of autoimmune disorders mediated by BAFF.


Subject(s)
Antibody Specificity/immunology , B-Cell Activating Factor/immunology , Immunoglobulin Fragments/physiology , Immunoglobulin Variable Region/physiology , Peptide Library , Amino Acid Sequence , Animals , Cloning, Molecular , Humans , Immunoglobulin Fragments/genetics , Immunoglobulin Fragments/isolation & purification , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/isolation & purification , Inovirus/genetics , Inovirus/immunology , Inovirus/metabolism , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Protein Binding/genetics , Protein Binding/immunology
16.
J Immunoassay Immunochem ; 26(2): 89-95, 2005.
Article in English | MEDLINE | ID: mdl-15794119

ABSTRACT

Filamentous bacteriophage (Ff) displayed random peptide and antibody libraries are widely used to identify specific, high affinity, binding ligands. A critical element in the identification of target-specific phages is to determine phage titers, not only at every round of selection, but also for normalizing phage titers of a set of individual clones for their comparative binding analysis. The conventional ELISA-based Ff titration methods require a minimum of 4-5 hr assay time and their lowest reported detection limit is approximately 50,000 particles/well. In this report, we present a sandwich ELISA that allows detection of approximately 1000 Ff particles/well in less than 2.5 hr. The values of correlation of coefficient (r2) for the curves at low phage concentrations (up to 106 TU/well) were about 0.999 in our ELISA. Experiments conducted at different temperatures suggest using 40 degrees C incubations when titering low phage concentration samples. Experiments were also conducted with conventional ELISA for comparison. Our ELISA method derives an advantage from using a chemiluminescence substrate that gives much larger signals and wide linear range of measurement, thus allowing discrimination between background and low Ff phage concentrations. In conclusion, the Ff titration method presented here is highly sensitive, rapid, and amenable to high throughput analysis.


Subject(s)
Enzyme-Linked Immunosorbent Assay/methods , Inovirus/isolation & purification , Luminescent Measurements/methods , Antibody Affinity , Antigen-Antibody Reactions , Automation , Binding Sites , Inovirus/immunology , Peptide Library , Peptides/immunology , Sensitivity and Specificity , Temperature
17.
Curr Drug Targets ; 5(1): 1-15, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14738215

ABSTRACT

Antigenic epitopes are the part (contact points) of an antigen involved in specific interaction with the antigen-binding site (the paratope) of an antibody or a T-cell receptor. Detailed analysis of epitopes is important both for the understanding of immunological events and for the development of more effective vaccine and diagnostic tools for various diseases. Identification and characterization of epitopes is a complex process. Although various methods have been developed in this area, there still lacks a simple common approach which can be applied to all epitopes. Since its first introduction more than a decade ago, phage display technology has made a major impact in this area of research. With the exponential growth in this area, it is impractical to review the entire literature detailing all possible applications. Instead, this review aims to focus on specific applications related to the discovery and identification of epitopes which have potential as vaccine candidates or can be used in disease diagnosis.


Subject(s)
Epitopes , Peptide Library , Vaccines , Animals , Bacteriophages/genetics , Bacteriophages/immunology , Biomarkers , Carbohydrates/chemistry , Carbohydrates/immunology , Epitope Mapping , Epitopes/genetics , Epitopes/immunology , Gene Library , Humans , Inovirus/genetics , Inovirus/immunology , Molecular Mimicry , Peptides/chemistry , Peptides/genetics , Peptides/immunology
18.
Biochem Biophys Res Commun ; 298(4): 566-73, 2002 Nov 08.
Article in English | MEDLINE | ID: mdl-12408989

ABSTRACT

Functional expressions of proteins often depend on the presence of host specific factors. Frequently recombinant expression strategies of proteins in foreign hosts, such as bacteria, have been associated with poor yields or significant loss of functionality. Improvements in the performance of heterologous expression systems will benefit present-day quests in structural and functional genomics where high amounts of active protein are required. One example, which has been the subject of considerable interest, is recombinant antibodies or fragments thereof as expressions of these in bacteria constitute an easy and inexpensive method compared to hybridoma cultures. Such approaches have, however, often suffered from low yields and poor functionality. A general method is described here which enables expressions of functional antibody fragments when fused to the amino-terminal domain(s) of the filamentous phage coat protein III. Furthermore, it will be shown that the observed effect is neither due to improved stability nor increased avidity.


Subject(s)
Capsid Proteins/immunology , Immunoglobulin Fragments/immunology , Inovirus/immunology , Base Sequence , Capsid Proteins/chemistry , Chromatography, Gel , DNA Primers , Enzyme-Linked Immunosorbent Assay , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/isolation & purification
19.
J Immunol ; 166(6): 4170-6, 2001 Mar 15.
Article in English | MEDLINE | ID: mdl-11238668

ABSTRACT

Celiac disease (CD) is an intestinal malabsorption characterized by intolerance to cereal proteins accompanied by immunological responses to dietary gliadins and tissue transglutaminase, an autoantigen located in the endomysium. Tissue transglutaminase belongs to the family of enzymes that catalyze protein cross-linking reactions and is constitutively expressed in many tissues as well as being activated during apoptosis. The role of gliadins in eliciting the immune response in CD and how transglutaminase is linked to the primary reaction are still unclear. In this work, we report the production and analysis of six phage Ab libraries from the peripheral and intestinal lymphocytes of three CD patients. We were able to isolate Abs to transglutaminase from all intestinal lymphocytes libraries but not from those obtained from peripheral lymphocytes. This is in contrast to Abs against gliadin, which could be obtained from all libraries, indicating that the humoral response against transglutaminase occurs at the local level, whereas that against gliadin occurs both peripherally and centrally. Abs from all three patients recognized the same transglutaminase epitopes with a bias toward the use of the V(H)5 Ab variable region family. The possible role of these anti-transglutaminase Abs in the onset of CD and associated autoimmune pathologies is discussed.


Subject(s)
Autoantibodies/biosynthesis , Autoantibodies/genetics , Celiac Disease/enzymology , Celiac Disease/immunology , GTP-Binding Proteins/immunology , Transglutaminases/immunology , Adult , Amino Acid Sequence , Antibody Affinity , Autoantibodies/metabolism , Autoantigens/immunology , Autoantigens/metabolism , Binding, Competitive/immunology , Clone Cells , Epitope Mapping , Fetal Blood/immunology , Fluorescent Antibody Technique, Direct , GTP-Binding Proteins/metabolism , Gene Library , Gene Rearrangement, B-Lymphocyte, Heavy Chain , Gene Rearrangement, B-Lymphocyte, Light Chain , Genetic Vectors/immunology , Humans , Immune Sera/metabolism , Immunoglobulin Heavy Chains/biosynthesis , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/metabolism , Immunoglobulin Light Chains/biosynthesis , Immunoglobulin Light Chains/genetics , Immunoglobulin Light Chains/metabolism , Immunoglobulin Variable Region/biosynthesis , Immunoglobulin Variable Region/genetics , Immunoglobulin Variable Region/metabolism , Inovirus/genetics , Inovirus/immunology , Molecular Sequence Data , Muscle Fibers, Skeletal/immunology , Mutation , Protein Glutamine gamma Glutamyltransferase 2 , Solubility , Transglutaminases/metabolism
20.
Int Immunol ; 13(2): 223-32, 2001 Feb.
Article in English | MEDLINE | ID: mdl-11157855

ABSTRACT

Autoantibodies to double-stranded (ds) DNA are an important diagnostic marker and pathogenic factor for systemic lupus erythematosus (SLE). Identifying dsDNA mimotopes is a way to discover diagnostic and therapeutic candidates for SLE. 'Mono-specific' SLE anti-dsDNA antibodies were obtained by affinity purification using dsDNA-coupled Sepharose column. Using the anti-dsDNA antibodies to screen a phage peptide library, we were able to identify a mimotope that has a motif peptide sequence of RLTSSLRYNP. This chemically synthesized peptide could be recognized by 88% (37 out of 42) of anti-dsDNA antibody-positive SLE sera with a cut-off point at mean + 3 SD of the negative control sera at OD(492). The reaction of the peptide with SLE sera in ELISA was highly correlated with that of dsDNA (r = 0.809, P < 0.0001). Of particular interest, not only dsDNA but also single-stranded (ss) DNA and native RNA could inhibit the binding of the peptide with SLE sera, suggesting that the mimotope is shared by ds and ssDNAs as well as native RNA, whereas denatured RNA was not observed to inhibit the binding. The peptide was also able to elicit an immune response in rabbits and the anti-peptide rabbit serum was observed to cross-react with the peptide, ss and dsDNAs, and ss and dsDNAs could inhibit the binding of the anti-peptide serum and the peptide. However, the inhibition was not obtained with RNA. Our findings demonstrate the potential of the peptide mimic in diagnostic tests of SLE, and in the investigation of anti-DNA antibody origin and of DNA-anti-DNA antibody interaction.


Subject(s)
DNA/immunology , Immunodominant Epitopes/immunology , Lupus Erythematosus, Systemic/immunology , Molecular Mimicry/immunology , Peptides/immunology , Adolescent , Adult , Aged , Amino Acid Motifs , Amino Acid Sequence , Antibodies, Antinuclear/metabolism , Binding Sites, Antibody , Binding, Competitive/immunology , Cloning, Molecular , DNA/blood , DNA/metabolism , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Immune Sera/metabolism , Immunodominant Epitopes/metabolism , Immunoglobulin G/metabolism , Inovirus/genetics , Inovirus/immunology , Lupus Erythematosus, Systemic/blood , Middle Aged , Molecular Sequence Data , Peptide Library , Peptides/metabolism , RNA/metabolism
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