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.
- Klíčová slova
- Albumin-binding domain scaffold, Antibody paratope mimetics, Combinatorial protein library, HIV-1 vaccine, Neutralizing antibody, Protein docking,
- MeSH
- antigeny virové chemie imunologie MeSH
- epitopy chemie imunologie MeSH
- HIV infekce imunologie virologie MeSH
- HIV obalový protein gp120 imunologie MeSH
- HIV protilátky krev imunologie MeSH
- HIV-1 imunologie MeSH
- imunoglobulin G krev imunologie MeSH
- konformace proteinů MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- molekulární modely MeSH
- myši MeSH
- neutralizující protilátky krev imunologie MeSH
- sekvence aminokyselin MeSH
- vakcíny proti AIDS imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antigeny virové MeSH
- epitopy MeSH
- HIV obalový protein gp120 MeSH
- HIV protilátky MeSH
- imunoglobulin G MeSH
- neutralizující protilátky MeSH
- vakcíny proti AIDS MeSH
BACKGROUND/AIMS: IgA nephropathy is associated with aberrant O-glycosylation of IgA1, which is recognized by autoantibodies leading to the formation of circulating immune complexes. Some of them, after deposition into kidney mesangium, trigger glomerular injury. In patients with active disease nonresponding to angiotensin-converting enzyme inhibitors or angiotensin II blockers, corticosteroids are recommended. METHODS: The relationship between the corticosteroid therapy and serum levels of IgA, aberrantly O-glycosylated IgA1, IgA-containing immune complexes and their mesangioproliferative activity was analyzed in IgA nephropathy patients and disease and healthy controls. RESULTS: Prednisone therapy significantly reduced proteinuria and levels of serum IgA, galactose-deficient IgA1, and IgA-IgG immune complexes in IgA nephropathy patients and thus reduced differences in all of the above parameters between IgAN patients and control groups. A moderate but not significant reduction of mesangioproliferative potential of IgA-IgG immune complexes and IgA sialylation was detected. CONCLUSION: The prednisone therapy reduces overall aberrancy in IgA1 O-glycosylation in IgA nephropathy patients, but the measurement of IgA1 parameters does not allow us to predict the prednisone therapy outcome in individual patients.
- Klíčová slova
- Circulating immune complexes, Galactose deficiency, Glycan-specific antibodies, IgA1 hinge region, Iga nephropathy, O-glycosylation,
- MeSH
- glukokortikoidy farmakologie terapeutické užití MeSH
- glykosylace účinky léků MeSH
- IgA nefropatie diagnóza farmakoterapie MeSH
- imunoglobulin A krev imunologie metabolismus MeSH
- imunokomplex krev MeSH
- lidé MeSH
- prednison terapeutické užití MeSH
- prognóza MeSH
- protilátky krev MeSH
- studie případů a kontrol MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- glukokortikoidy MeSH
- imunoglobulin A MeSH
- imunokomplex MeSH
- prednison MeSH
- protilátky MeSH
Lasioglossins are a group of peptides with identified antimicrobial activity. The inhibitory effects of two synthetic lasioglossin derivatives, LLIII and D-isomeric variant LLIII-D, on morphological changes in Candida albicans in vitro and the effect of local administration of LLIII during experimental murine candidiasis were investigated. C. albicans blastoconidia were grown in the presence of lasioglossin LLIII or LLIII-D at concentrations of 11.5 μM and 21 μM, respectively, for 1, 2 and 3 days and their viability determined by flow cytometry using eosin Y staining. Morphological changes were examined by light and fluorescent microscopy. The Candida-inhibitory effect of daily intravaginal administration of 0.7 or 1.4 μg of LLIII was assessed in mice with experimentally-induced vaginal candidiasis. LLIII and LLIII-D lasioglossins exhibited candidacidal activity in vitro (>76% after 24 hr and >84% after 48 hr of incubation). After 72 hr incubation of Candida with low concentration of lasioglossins, an increase in viability was detected, probably due to a Candida antimicrobial peptides evasion strategy. Furthermore, lasioglossins inhibited temperature-induced morphotype changes toward hyphae and pseudohyphae with sporadic occurrence of atypical cells with two or enlarged nuclei, suggesting interference with mitosis or cytokinesis. Local application of LLIII reduced the duration of experimental candidiasis with no evidence of adverse effects. Lasioglossin LLIII is a promising candidate for development as an antimicrobial drug for treating the vaginal candidiasis.
- Klíčová slova
- Antimicrobial peptides, Candida albicans, lasioglossins, vaginal candidiasis,
- MeSH
- antifungální látky aplikace a dávkování MeSH
- aplikace intravaginální MeSH
- Candida albicans účinky léků růst a vývoj MeSH
- kandidóza vulvovaginální farmakoterapie mikrobiologie MeSH
- kationické antimikrobiální peptidy aplikace a dávkování MeSH
- lidé MeSH
- myši inbrední DBA MeSH
- myši MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- antifungální látky MeSH
- kationické antimikrobiální peptidy MeSH
BACKGROUND: Galactose-deficient O-glycans in the hinge region (HR) of immunoglobulin A1 (IgA1) play a key role in the pathogenesis of IgA nephropathy (IgAN). O-Glycans of circulatory IgA1 consist of N-acetylgalactosamine (GalNAc) with a β1,3-linked galactose; both sugars may be sialylated. In patients with IgAN, α2,6-sialylated GalNAc is a frequent form of the galactose-deficient O-glycans. Prior analyses of IgA1-producing cells had indicated that α2,6-sialyltransferase II (ST6GalNAc-II) is likely responsible for sialylation of GalNAc of galactose-deficient IgA1, but direct evidence is missing. METHODS: We produced a secreted variant of recombinant human ST6GalNAc-II and an IgA1 fragment comprised of Cα1-HR-Cα2. This IgA1 fragment and a synthetic HR peptide with enzymatically attached GalNAc residues served as acceptors. ST6GalNAc-II activity was assessed in vitro and the attachment of sialic acid to these acceptors was detected by lectin blot and mass spectrometry. RESULTS: ST6GalNAc-II was active with both acceptors. High-resolution mass spectrometry analysis revealed that up to three sialic acid residues were added to the GalNAc residues of the HR glycopeptide. CONCLUSIONS: Our data provide direct evidence that ST6GalNAc-II can sialylate GalNAc of galactose-deficient IgA1. As serum levels of galactose-deficient IgA1 with sialylated glycoforms are increased in IgAN patients, our data explain the corresponding part of the biosynthetic pathway.
- Klíčová slova
- IgA nephropathy, aberrant O-glycosylation, galactose-deficient IgA1, immunoglobulin A1, α2,6 sialyltransferase ST6GalNAc-II,
- MeSH
- autoantigeny imunologie MeSH
- galaktosa nedostatek MeSH
- glykosylace MeSH
- hmotnostní spektrometrie MeSH
- IgA nefropatie enzymologie imunologie patologie MeSH
- imunoglobulin A metabolismus MeSH
- kultivované buňky MeSH
- kyselina N-acetylneuraminová metabolismus MeSH
- lidé MeSH
- rekombinantní proteiny imunologie metabolismus MeSH
- sialyltransferasy metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- autoantigeny MeSH
- galactosyl-1-3-N-acetylgalactosaminyl-specific 2,6-sialyltransferase MeSH Prohlížeč
- galaktosa MeSH
- imunoglobulin A MeSH
- kyselina N-acetylneuraminová MeSH
- rekombinantní proteiny MeSH
- sialyltransferasy MeSH
BACKGROUND: HIV-1 entry into host cells is mediated by interactions between the virus envelope glycoprotein (gp120/gp41) and host-cell receptors. N-glycans represent approximately 50% of the molecular mass of gp120 and serve as potential antigenic determinants and/or as a shield against immune recognition. We previously reported that N-glycosylation of recombinant gp120 varied, depending on the producer cells, and the glycosylation variability affected gp120 recognition by serum antibodies from persons infected with HIV-1 subtype B. However, the impact of gp120 differential glycosylation on recognition by broadly neutralizing monoclonal antibodies or by polyclonal antibodies of individuals infected with other HIV-1 subtypes is unknown. METHODS: Recombinant multimerizing gp120 antigens were expressed in different cells, HEK 293T, T-cell, rhabdomyosarcoma, hepatocellular carcinoma, and Chinese hamster ovary cell lines. Binding of broadly neutralizing monoclonal antibodies and polyclonal antibodies from sera of subtype A/C HIV-1-infected subjects with individual gp120 glycoforms was assessed by ELISA. In addition, immunodetection was performed using Western and dot blot assays. Recombinant gp120 glycoforms were tested for inhibition of infection of reporter cells by SF162 and YU.2 Env-pseudotyped R5 viruses. RESULTS: We demonstrated, using ELISA, that gp120 glycans sterically adjacent to the V3 loop only moderately contribute to differential recognition of a short apex motif GPGRA and GPGR by monoclonal antibodies F425 B4e8 and 447-52D, respectively. The binding of antibodies recognizing longer peptide motifs overlapping with GPGR epitope (268 D4, 257 D4, 19b) was significantly altered. Recognition of gp120 glycoforms by monoclonal antibodies specific for other than V3-loop epitopes was significantly affected by cell types used for gp120 expression. These epitopes included CD4-binding site (VRC03, VRC01, b12), discontinuous epitope involving V1/V2 loop with the associated glycans (PG9, PG16), and an epitope including V3-base-, N332 oligomannose-, and surrounding glycans-containing epitope (PGT 121). Moreover, the different gp120 glycoforms variably inhibited HIV-1 infection of reporter cells. CONCLUSION: Our data support the hypothesis that the glycosylation machinery of different cells shapes gp120 glycosylation and, consequently, impacts envelope recognition by specific antibodies as well as the interaction of HIV-1 gp120 with cellular receptors. These findings underscore the importance of selection of appropriately glycosylated HIV-1 envelope as a vaccine antigen.
- Klíčová slova
- Deglycosylation resistance, Glycan-specific antibody, Neutralization inhibition, gp120 glycosylation,
- Publikační typ
- časopisecké články MeSH
Lyme disease caused by spirochete Borrelia burgdorferi sensu lato, is a tick-born illness. If the infection is not eliminated by the host immune system and/or antibiotics, it may further disseminate and cause severe chronic complications. The immune response to Borrelia is mediated by phagocytic cells and by Borrelia-specific complement-activating antibodies associated with Th1 cell activation. A new experimental vaccine was constructed using non-lipidized form of recombinant B. burgdorferi s.s. OspC protein was anchored by metallochelating bond onto the surface of nanoliposomes containing novel nonpyrogenic lipophilized norAbuMDP analogues denoted MT05 and MT06. After i.d. immunization, the experimental vaccines surpassed Alum with respect to OspC-specific titers of IgG2a, IgG2b isotypes when MT06 was used and IgG3, IgM isotypes when MT05 was used. Both adjuvants exerted a high adjuvant effect comparable or better than MDP and proved themselves as nonpyrogenic.
- MeSH
- acetylmuramyl-alanyl-isoglutamin chemie toxicita MeSH
- antigeny bakteriální imunologie MeSH
- Borrelia burgdorferi imunologie MeSH
- chelátory chemie toxicita MeSH
- diferenciální skenovací kalorimetrie MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- ELISA MeSH
- liposomy MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- nanočástice chemie toxicita MeSH
- nosiče léků chemie toxicita MeSH
- proteiny vnější bakteriální membrány imunologie MeSH
- protilátky bakteriální krev MeSH
- radiační rozptyl MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- světlo MeSH
- transmisní elektronová mikroskopie MeSH
- vakcína proti lymeské nemoci aplikace a dávkování imunologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- acetylmuramyl-alanyl-isoglutamin MeSH
- antigeny bakteriální MeSH
- chelátory MeSH
- liposomy MeSH
- nosiče léků MeSH
- OspC protein MeSH Prohlížeč
- proteiny vnější bakteriální membrány MeSH
- protilátky bakteriální MeSH
- vakcína proti lymeské nemoci MeSH
Hsp90-CA is present in cell wall of Candida pseudohyphae or hyphae-typical pathogenic morphotype for both systemic and mucosal Candida infections. Heat shock protein from Candida albicans (hsp90-CA) is an important target for protective antibodies during disseminated candidiasis of experimental mice and human. His-tagged protein rHsp90 was prepared and used as the antigen for preparation of experimental recombinant liposomal vaccine. Nickel-chelating liposomes (the size around 100nm, PDI≤0.1) were prepared from the mixture of egg phosphatidyl choline and nickel-chelating lipid DOGS-NTA-Ni (molar ratio 95:5%) by hydration of lipid film and extrusion methods. New non-pyrogenic hydrophobised derivative of MDP (C18-O-6-norAbuMDP) was incorporated into liposomes as adjuvans. rHsp90 was attached onto the surface of metallochelating liposomes by metallochelating bond and the structure of these proteoliposomes was studied by dynamic light scattering, AF microscopy, TEM and GPC. The liposomes with surface-exposed C18-O-6-norAbuMDP were well recognised and phagocyted by human dendritic cells in vitro. In vivo the immune response towards this experimental vaccine applied in mice (i.d.) demonstrated both TH1 and TH2 response comparable to FCA, but without any side effects. Metallochelating liposomes with lipophilic derivatives of muramyl dipeptide represent a new biocompatible platform for construction of experimental recombinant vaccines and drug-targeting systems.
- MeSH
- antigeny fungální imunologie metabolismus MeSH
- biokompatibilní potahované materiály metabolismus MeSH
- buněčná imunita * MeSH
- Candida imunologie MeSH
- chelátory chemie metabolismus MeSH
- dendritické buňky imunologie metabolismus MeSH
- kultivované buňky MeSH
- lidé MeSH
- liposomy MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- nikl imunologie metabolismus MeSH
- proteiny tepelného šoku HSP90 imunologie metabolismus MeSH
- syntetické vakcíny imunologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- antigeny fungální MeSH
- biokompatibilní potahované materiály MeSH
- chelátory MeSH
- liposomy MeSH
- nikl MeSH
- proteiny tepelného šoku HSP90 MeSH
- syntetické vakcíny MeSH
Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the interaction between a variably glycosylated envelope glycoprotein (gp120) and host-cell receptors. Approximately half of the molecular mass of gp120 is contributed by N-glycans, which serve as potential epitopes and may shield gp120 from immune recognition. The role of gp120 glycans in the host immune response to HIV-1 has not been comprehensively studied at the molecular level. We developed a new approach to characterize cell-specific gp120 glycosylation, the regulation of glycosylation, and the effect of variable glycosylation on antibody reactivity. A model oligomeric gp120 was expressed in different cell types, including cell lines that represent host-infected cells or cells used to produce gp120 for vaccination purposes. N-Glycosylation of gp120 varied, depending on the cell type used for its expression and the metabolic manipulation during expression. The resultant glycosylation included changes in the ratio of high-mannose to complex N-glycans, terminal decoration, and branching. Differential glycosylation of gp120 affected envelope recognition by polyclonal antibodies from the sera of HIV-1-infected subjects. These results indicate that gp120 glycans contribute to antibody reactivity and should be considered in HIV-1 vaccine design.
- MeSH
- AIDS imunologie MeSH
- buněčné linie MeSH
- buňky Hep G2 metabolismus MeSH
- ELISA metody MeSH
- glykosidhydrolasy metabolismus MeSH
- glykosylace MeSH
- HIV obalový protein gp120 genetika imunologie izolace a purifikace metabolismus MeSH
- HIV séropozitivita imunologie metabolismus MeSH
- HIV-1 imunologie metabolismus MeSH
- hmotnostní spektrometrie MeSH
- Jurkat buňky metabolismus MeSH
- komplementární DNA genetika MeSH
- lektin vázající mannosu genetika MeSH
- lidé MeSH
- mannosa metabolismus MeSH
- nádorové buněčné linie MeSH
- oligosacharidy chemie izolace a purifikace MeSH
- plazmidy MeSH
- polysacharidy chemie izolace a purifikace MeSH
- protilátky imunologie MeSH
- specificita protilátek MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- glykosidhydrolasy MeSH
- gp120 protein, Human immunodeficiency virus 1 MeSH Prohlížeč
- HIV obalový protein gp120 MeSH
- komplementární DNA MeSH
- lektin vázající mannosu MeSH
- mannosa MeSH
- oligosacharidy MeSH
- polysacharidy MeSH
- protilátky MeSH
