Development of vaccine preventing HIV-1 infection is hindered by high variability of envelope glycoprotein (Env), known target for neutralizing antibodies. Identification of several monoclonal antibodies neutralizing broad range of HIV-1 variants (bn-mAbs) recognizing Env glycans as a part of their epitopes provides the new opportunity for vaccine design. As the composite glycans-containing epitopes are less effective immunogens than protein epitopes, we propose to develop a set of peptides mimicking epitopes recognized by above bn-mAbs using high-affinity binders approach developed by the co-applicant. Binders will be selected based on specific interaction with respective bn-mAbs, fused to albumin, and used for immunization of animals in form of proteoliposomes or DNA vaccines. Sera will be characterized concerning to their specificity, affinity, and neutralization activity. This approach provides the possibility to identify the candidate antigen for future HIV-1 vaccine and provide a platform for development of glycan-mimicking vaccines eliciting good immunological memory.

Vývoj vakcíny proti HIV-1 infekci je brzděn enormní variabilitou obalového glykoproteinu (Env), cíle neutralizačních protilátek. Identifikace sady monoklonálních protilátek neutralizujících široké spektrum variant HIV-1 (bn-mAb), reagujících s Env glykany, jako součástí jejich epitopu, nabízí novou cestu vývoje vakcíny. Jelikož kompozitní epitopy obsahující glykany jsou méně efektivní imunogeny než proteinové epitopy navrhujeme vyvinout sadu peptidů napodobujících epitopy rozlišované výše popsanými bn-mAb pomocí metody vysoce afinitních ligandů (binderů) vyvinutých spolunavrhovatelem projektu. Bindery budou selektovány na základě jejich specifické vazby na jednotlivé bn-mAb, budou fúzovány s albuminem a kotvami a ve formě proteoliposomů nebo DNA vakcín užity k imunizaci experimentálních zvířat. Poté bude charakterizována specificita, afinita a neutralizační aktivita sérových protilátek. Přístup skýtá možnost identifikovat kandidátní antigen pro vakcínu proti HIV-1 a představuje platformu pro vývoj vakcín napodobujících glykanové epitopy navozujících dobrou imunologickou paměť.

• MeSH
• epitopy terapeutické užití   MeSH
• experimenty na zvířatech MeSH
• HIV infekce terapie   MeSH
• HIV obalový protein gp120 MeSH
• HIV-1 MeSH
• imunizace MeSH
• látky proti HIV MeSH
• ligandy MeSH
• neutralizující protilátky MeSH
• rekombinantní proteiny terapeutické užití   MeSH
• vakcíny proti AIDS MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• dermatovenerologie
• infekční lékařství
• alergologie a imunologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

New synthetic aminooxy lipid was designed and synthesized as a building block for the formulation of functionalised nanoliposomes (presenting onto the outer surface of aminooxy groups) by microfluidic mixing. Orthogonal binding of cellular mannan (Candida glabrata (CCY 26-20-1) onto the outer surface of functionalised nanoliposomes was modified by orthogonal binding of reducing termini of mannans to oxime lipids via a click chemistry reaction based on aminooxy coupling (oxime ligation). The aminooxy lipid was proved as a suitable active component for preparation of functionalised nanoliposomes by the microfluidic mixing method performed with the instrument NanoAssemblrTM. This "on-chip technology" can be easily scaled-up. The structure of mannan-liposomes was visualized by transmission and scanning electron microscopy, including immunogold staining of recombinant mannan receptor bound onto mannosylated-liposomes. The observed structures are in a good correlation with data obtained by DLS, NTA, and TPRS methods. In vitro experiments on human and mouse dendritic cells demonstrate selective internalisation of fluorochrome-labelled mannan-liposomes and their ability to stimulate DC comparable to lipopolysaccharide. We describe a potentially new drug delivery platform for mannan receptor-targeted antimicrobial drugs as well as for immunotherapeutics. Furthermore, the platform based on mannans bound orthogonally onto the surface of nanoliposomes represents a self-adjuvanted carrier for construction of liposome-based recombinant vaccines for both systemic and mucosal routes of administration.

• MeSH
• adjuvancia imunologická farmakologie   MeSH
• antigeny povrchové metabolismus   MeSH
• Candida glabrata chemie   MeSH
• click chemie MeSH
• dendritické buňky imunologie   MeSH
• hydroxylaminy chemická syntéza   chemie   MeSH
• lektiny typu C imunologie   MeSH
• lektiny vázající mannosu imunologie   MeSH
• lidé MeSH
• lipidy chemická syntéza   chemie   MeSH
• liposomy chemie   imunologie   farmakologie   MeSH
• mannany chemie   imunologie   farmakologie   MeSH
• mikrofluidika metody   MeSH
• myši inbrední BALB C MeSH
• nanočástice chemie   MeSH
• receptory buněčného povrchu imunologie   MeSH
• velikost částic MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

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.

• 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

New synthetic aminoxy lipids are designed and synthesized as building blocks for the formulation of functionalized nanoliposomes by microfluidization using a NanoAssemblr. Orthogonal binding of hyaluronic acid onto the outer surface of functionalized nanoliposomes via aminoxy coupling ( N-oxy ligation) is achieved at hemiacetal function of hyaluronic acid and the structure of hyaluronic acid-liposomes is visualized by transmission electron microscopy and cryotransmission electron microscopy. Observed structures are in a good correlation with data obtained by dynamic light scattering (size and ζ-potential). In vitro experiments on cell lines expressing CD44 receptors demonstrate selective internalization of fluorochrome-labeled hyaluronic acid-liposomes, while cells with down regulated CD44 receptor levels exhibit very low internalization of hyaluronic acid-liposomes. A method based on microfluidization mixing was developed for preparation of monodispersive unilamellar liposomes containing aminoxy lipids and orthogonal binding of hyaluronic acid onto the liposomal surface was demonstrated. These hyaluronic acid-liposomes represent a potentially new drug delivery platform for CD44-targeted anticancer drugs as well as for immunotherapeutics and vaccines.

• MeSH
• antigeny CD44 analýza   metabolismus   MeSH
• buněčné linie MeSH
• endocytóza MeSH
• fluorescenční barviva MeSH
• kyselina hyaluronová chemie   metabolismus   MeSH
• lékové transportní systémy metody   MeSH
• lidé MeSH
• lipidy chemická syntéza   MeSH
• liposomy chemie   terapeutické užití   MeSH
• mikrofluidika MeSH
• nádory farmakoterapie   MeSH
• protinádorové látky aplikace a dávkování   MeSH
• transmisní elektronová mikroskopie MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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).

• MeSH
• acetylmuramyl-alanyl-isoglutamin analogy a deriváty   chemie   imunologie   farmakologie   MeSH
• adjuvancia imunologická chemie   farmakologie   MeSH
• antigeny povrchové chemie   imunologie   farmakologie   MeSH
• bakteriální vakcíny chemie   imunologie   farmakologie   MeSH
• Borrelia burgdorferi imunologie   MeSH
• HEK293 buňky MeSH
• imunizace MeSH
• lidé MeSH
• lipoproteiny chemie   imunologie   farmakologie   MeSH
• lymeská nemoc imunologie   mikrobiologie   MeSH
• myši inbrední BALB C MeSH
• myši MeSH
• protein NLRP3 agonisté   imunologie   MeSH
• proteiny vnější bakteriální membrány chemie   imunologie   farmakologie   MeSH
• RAW 264.7 buňky MeSH
• tvorba protilátek MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Nanofibre-based mucoadhesive films were invented for oromucosal administration of nanocarriers used for delivery of drugs and vaccines. The mucoadhesive film consists of an electrospun nanofibrous reservoir layer, a mucoadhesive film layer and a protective backing layer. The mucoadhesive layer is responsible for tight adhesion of the whole system to the oral mucosa after application. The electrospun nanofibrous reservoir layer is intended to act as a reservoir for polymeric and lipid-based nanoparticles, liposomes, virosomes, virus-like particles, dendrimers and the like, plus macromolecular drugs, antigens and/or allergens. The extremely large surface area of nanofibrous reservoir layers allows high levels of nanoparticle loading. Nanoparticles can either be reversibly adsorbed to the surface of nanofibres or they can be deposited in the pores between the nanofibres. After mucosal application, nanofibrous reservoir layers are intended to promote prolonged release of nanoparticles into the submucosal tissue. Reversible adsorption of model nanoparticles as well as sufficient mucoadhesive properties were demonstrated. This novel system appears appropriate for the use in oral mucosa, especially for sublingual and buccal tissues. To prove this concept, trans-/intramucosal and lymph-node delivery of PLGA-PEG nanoparticles was demonstrated in a porcine model. This system can mainly be used for sublingual immunization and the development of "printed vaccine technology".

• MeSH
• adheziva chemie   MeSH
• aplikace bukální MeSH
• aplikace sublinguální MeSH
• léčivé přípravky aplikace a dávkování   MeSH
• lékové transportní systémy metody   MeSH
• liposomy chemie   MeSH
• lymfatické uzliny metabolismus   MeSH
• myši MeSH
• nanočástice chemie   MeSH
• nanovlákna chemie   MeSH
• polyethylenglykoly chemie   MeSH
• polyglactin 910 chemie   MeSH
• prasata MeSH
• ústní sliznice metabolismus   MeSH
• vakcinace metody   MeSH
• vakcíny aplikace a dávkování   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH