5 results on '"Barkocziova L"'
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2. Preparation and properties of recombinant Clostridium ramosum IgA proteinase. Isolation of Fc-SC and Fab fragments of human secretory IgA.
- Author
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Krupka M, Raskova Kafkova L, Barkocziova L, Sloupenska K, Brokesova D, Sebela M, and Raska M
- Subjects
- Bacterial Proteins genetics, Firmicutes genetics, Humans, Peptide Hydrolases genetics, Recombinant Proteins chemistry, Recombinant Proteins genetics, Bacterial Proteins chemistry, Firmicutes enzymology, Immunoglobulin A, Secretory chemistry, Immunoglobulin Fab Fragments chemistry, Immunoglobulin Fab Fragments isolation & purification, Immunoglobulin Fc Fragments chemistry, Immunoglobulin Fc Fragments isolation & purification, Peptide Hydrolases chemistry
- Abstract
Immunoglobulin A (IgA) proteinase from Clostridium ramosum is the enzyme which cleaves IgA of both subclasses; in contrast, the other bacterial proteinases cleave only IgA1 proteins. Previous reports characterized the activity of proteinase naturally secreted by C. ramosum specific for the normal human serum IgA of IgA1 and IgA2m(1) subclasses and also for secretory IgA (SIgA). Its amino acid sequence was determined, and the recombinant proteinase which cleaved IgA of both subclasses was prepared. Here we report the optimized expression, purification, storage conditions and activity testing against purified human milk SIgA. The recombinant C. ramosum IgA proteinase isolated in the high degree of purity exhibited almost complete cleavage of SIgA of both subclasses. The proteinase remained active upon storage for more than 10 month at -20 °C without substantial loss of enzymatic activity. Purified SIgA fragments are suitable for studies of all antigen-binding and Fc-dependent functions of SIgA involved in the protection against infections with mucosal pathogens., (Copyright © 2021 Elsevier Inc. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
3. Proteins mimicking epitope of HIV-1 virus neutralizing antibody induce virus-neutralizing sera in mice.
- Author
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Kosztyu P, Kuchar M, Cerny J, Barkocziova L, Maly M, Petrokova H, Czernekova L, Liskova V, Raskova Kafkova L, Knotigova P, Masek J, Turanek J, Maly P, and Raska M
- Subjects
- AIDS Vaccines immunology, Amino Acid Sequence, Animals, Antibodies, Neutralizing blood, Antigens, Viral chemistry, Disease Models, Animal, Epitopes chemistry, HIV Antibodies blood, HIV Envelope Protein gp120 immunology, HIV Infections virology, Humans, Immunoglobulin G blood, Immunoglobulin G immunology, Mice, Models, Molecular, Protein Conformation, Antibodies, Neutralizing immunology, Antigens, Viral immunology, Epitopes immunology, HIV Antibodies immunology, HIV Infections immunology, HIV-1 immunology
- Abstract
Background: The development of an effective vaccine preventing HIV-1 infection is hindered by the enormous antigenic variability and unique biochemical and immunological properties of HIV-1 Env glycoprotein, the most promising target for HIV-1 neutralizing antibody. Functional studies of rare elite neutralizers led to the discovery of broadly neutralizing antibodies., Methods: We employed a highly complex combinatorial protein library derived from a 5 kDa albumin-binding domain scaffold, fused with support protein of total 38 kDa, to screen for binders of broadly neutralizing antibody VRC01 paratope. The most specific binders were used for immunization of experimental mice to elicit Env-specific antibodies and to test their neutralization activity using a panel of HIV-1 clade C and B pseudoviruses., Findings: Three most specific binders designated as VRA017, VRA019, and VRA177 exhibited high specificity to VRC01 antibody. Immunized mice produced Env-binding antibodies which neutralize eight of twelve HIV-1 Tier 2 pseudoviruses. Molecular modelling revealed a shape complementarity between VRA proteins and a part of VRC01 gp120 interacting surface., Interpretation: This strategy based on the identification of protein replicas of broadly neutralizing antibody paratope represents a novel approach in HIV-1 vaccine development. This approach is not affected by low immunogenicity of neutralization-sensitive epitopes, variability, and unique biochemical properties of HIV-1 Env used as a crucial antigen in the majority of contemporary tested vaccines. FUND: Czech Health Research Council 15-32198A, Ministry of Health, Czech Republic., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2019
- Full Text
- View/download PDF
4. Nonpyrogenic Molecular Adjuvants Based on norAbu-Muramyldipeptide and norAbu-Glucosaminyl Muramyldipeptide: Synthesis, Molecular Mechanisms of Action, and Biological Activities in Vitro and in Vivo.
- Author
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Effenberg R, Turánek Knötigová P, Zyka D, Čelechovská H, Mašek J, Bartheldyová E, Hubatka F, Koudelka Š, Lukáč R, Kovalová A, Šaman D, Křupka M, Barkocziova L, Kosztyu P, Šebela M, Drož L, Hučko M, Kanásová M, Miller AD, Raška M, Ledvina M, and Turánek J
- Subjects
- Acetylmuramyl-Alanyl-Isoglutamine chemistry, Acetylmuramyl-Alanyl-Isoglutamine immunology, Adjuvants, Immunologic chemistry, Animals, Antibody Formation, Antigens, Surface chemistry, Antigens, Surface immunology, Bacterial Outer Membrane Proteins chemistry, Bacterial Outer Membrane Proteins immunology, Bacterial Vaccines chemistry, Bacterial Vaccines immunology, Female, HEK293 Cells, Humans, Immunization, Lipoproteins chemistry, Lipoproteins immunology, Lyme Disease immunology, Lyme Disease microbiology, Mice, Mice, Inbred BALB C, NLR Family, Pyrin Domain-Containing 3 Protein agonists, NLR Family, Pyrin Domain-Containing 3 Protein immunology, RAW 264.7 Cells, Acetylmuramyl-Alanyl-Isoglutamine analogs & derivatives, Acetylmuramyl-Alanyl-Isoglutamine pharmacology, Adjuvants, Immunologic pharmacology, Antigens, Surface pharmacology, Bacterial Outer Membrane Proteins pharmacology, Bacterial Vaccines pharmacology, Borrelia burgdorferi immunology, Lipoproteins pharmacology
- Abstract
Fatty acyl analogues of muramyldipeptide (MDP) (abbreviated N-L18 norAbuGMDP, N-B30 norAbuGMDP, norAbuMDP-Lys(L18), norAbuMDP-Lys(B30), norAbuGMDP-Lys(L18), norAbuGMDP-Lys(B30), B30 norAbuMDP, L18 norAbuMDP) are designed and synthesized comprising the normuramyl-l-α-aminobutanoyl (norAbu) structural moiety. All new analogues show depressed pyrogenicity in both free (micellar) state and in liposomal formulations when tested in rabbits in vivo (sc and iv application). New analogues are also shown to be selective activators of NOD2 and NLRP3 (inflammasome) in vitro but not NOD1. Potencies of NOD2 and NLRP3 stimulation are found comparable with free MDP and other positive controls. Analogues are also demonstrated to be effective in stimulating cellular proliferation when the sera from mice are injected sc with individual liposome-loaded analogues, causing proliferation of bone marrow-derived GM-progenitors cells. Importantly, vaccination nanoparticles prepared from metallochelation liposomes, His-tagged antigen rOspA from Borrelia burgdorferi, and lipophilic analogue norAbuMDP-Lys(B30) as adjuvant, are shown to provoke OspA-specific antibody responses with a strong Th1-bias (dominance of IgG2a response). In contrast, the adjuvant effects of Alum or parent MDP show a strong Th2-bias (dominance of IgG1 response).
- Published
- 2017
- Full Text
- View/download PDF
5. The Position of His-Tag in Recombinant OspC and Application of Various Adjuvants Affects the Intensity and Quality of Specific Antibody Response after Immunization of Experimental Mice.
- Author
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Krupka M, Masek J, Barkocziova L, Turanek Knotigova P, Kulich P, Plockova J, Lukac R, Bartheldyova E, Koudelka S, Chaloupkova R, Sebela M, Zyka D, Droz L, Effenberg R, Ledvina M, Miller AD, Turanek J, and Raska M
- Subjects
- Animals, Antigens, Bacterial administration & dosage, Antigens, Bacterial chemistry, Bacterial Outer Membrane Proteins administration & dosage, Bacterial Outer Membrane Proteins chemistry, Enzyme-Linked Immunosorbent Assay, Immunization, Lyme Disease immunology, Mice, Models, Animal, Protein Stability, Protein Structure, Secondary, Proteolipids, Recombinant Fusion Proteins administration & dosage, Recombinant Fusion Proteins chemistry, Recombinant Fusion Proteins isolation & purification, Adjuvants, Immunologic, Antibodies, Bacterial immunology, Antibody Formation immunology, Antibody Specificity immunology, Antigens, Bacterial immunology, Bacterial Outer Membrane Proteins immunology, Borrelia burgdorferi immunology, Recombinant Fusion Proteins immunology
- Abstract
Lyme disease, Borrelia burgdorferi-caused infection, if not recognized and appropriately treated by antibiotics, may lead to chronic complications, thus stressing the need for protective vaccine development. The immune protection is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies, associated with the Th1 immune response. Surface antigen OspC is involved in Borrelia spreading through the host body. Previously we reported that recombinant histidine tagged (His-tag) OspC (rOspC) could be attached onto liposome surfaces by metallochelation. Here we report that levels of OspC-specific antibodies vary substantially depending upon whether rOspC possesses an N' or C' terminal His-tag. This is the case in mice immunized: (a) with rOspC proteoliposomes containing adjuvants MPLA or non-pyrogenic MDP analogue MT06; (b) with free rOspC and Montanide PET GEL A; (c) with free rOspC and alum; or (d) with adjuvant-free rOspC. Stronger responses are noted with all N'-terminal His-tag rOspC formulations. OspC-specific Th1-type antibodies predominate post-immunization with rOspC proteoliposomes formulated with MPLA or MT06 adjuvants. Further analyses confirmed that the structural features of soluble N' and C' terminal His-tag rOspC and respective rOspC proteoliposomes are similar including their thermal stabilities at physiological temperatures. On the other hand, a change in the position of the rOspC His-tag from N' to C' terminal appears to affect substantially the immunogenicity of rOspC arguably due to steric hindrance of OspC epitopes by the C' terminal His-tag itself and not due to differences in overall conformations induced by changes in the His-tag position in rOspC variants.
- Published
- 2016
- Full Text
- View/download PDF
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