Recombinant proteins and in particular single domains or peptides are often poorly immunogenic unless conjugated to a carrier protein. Virus-like-particles are a very efficient means to confer high ...immunogenicity to antigens. We report here the development of virus-like-particles (VLPs) derived from the RNA bacteriophage AP205 for epitope-based vaccines.
Peptides of angiotensin II, S.typhi outer membrane protein (D2), CXCR4 receptor, HIV1 Nef, gonadotropin releasing hormone (GnRH), Influenza A M2-protein were fused to either N- or C-terminus of AP205 coat protein. The A205-peptide fusions assembled into VLPs, and peptides displayed on the VLP were highly immunogenic in mice. GnRH fused to the C-terminus of AP205 induced a strong antibody response that inhibited GnRH function in vivo. Exposure of the M2-protein peptide at the N-terminus of AP205 resulted in a strong M2-specific antibody response upon immunization, protecting 100% of mice from a lethal influenza infection.
AP205 VLPs are therefore a very efficient and new vaccine system, suitable for complex and long epitopes, of up to at least 55 amino acid residues in length. AP205 VLPs confer a high immunogenicity to displayed epitopes, as shown by inhibition of endogenous GnRH and protective immunity against influenza infection.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Advances in nanotechnology and nanomaterials have facilitated the development of silicon dioxide, or Silica, particles as a promising immunological adjuvant for the generation of novel prophylactic ...and therapeutic vaccines. In the present study, we have compared the adjuvanting potential of commercially available Silica nanoparticles (initial particles size of 10-20 nm) with that of aluminium hydroxide, or Alum, as well as that of complete and incomplete Freund's adjuvants for the immunisation of BALB/c mice with virus-like particles (VLPs) formed by recombinant full-length Hepatitis B virus core (HBc) protein. The induction of B-cell and T-cell responses was studied after immunisation. Silica nanoparticles were able to adsorb maximally 40% of the added HBc, whereas the adsorption capacity of Alum exceeded 90% at the same VLPs/adjuvant ratio. Both Silica and Alum formed large complexes with HBc VLPs that sedimented rapidly after formulation, as detected by dynamic light scattering, spectrophotometry, and electron microscopy. Both Silica and Alum augmented the humoral response against HBc VLPs to the high anti-HBc level in the case of intraperitoneal immunisation, whereas in subcutaneous immunisation, the Silica-adjuvanted anti-HBc level even exceeded the level adjuvanted by Alum. The adjuvanting of HBc VLPs by Silica resulted in the same typical IgG2a/IgG1 ratios as in the case of the adjuvanting by Alum. The combination of Silica with monophosphoryl lipid A (MPL) led to the same enhancement of the HBc-specific T-cell induction as in the case of the Alum and MPL combination. These findings demonstrate that Silica is not a weaker putative adjuvant than Alum for induction of B-cell and T-cell responses against recombinant HBc VLPs. This finding may have an essential impact on the development of the set of Silica-adjuvanted vaccines based on a long list of HBc-derived virus-like particles as the biological component.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Bacteriophage GA coat protein formed self-assembly competent virus-like particles (VLPs) have been expressed previously in bacterial and yeast cells. On the basis of our previous experiments on the ...yeast vector pESC-URA /
system containing two oppositely oriented promoters, new constructions were created with point-mutations in coat protein to mimic phage MS2-like RNA binding characteristics. Simultaneously, the MS2 operator sequence was added to mRNA desired for packaging. After the introduction of single-point mutations (S87N, K55N, R43K) and double-point mutations (S87N + K55N and S87N + R43K), the coat protein’s ability to form VLPs was retained, but yield from cells was decreased. Exchange of the 87
Ser to Asn in coat protein sequence in combination with bacteriophage MS2 translational operator provided specific packaging of the gene of interest (GFP). Although non-specific nucleic acid sequences were packaged, the remarkable specificity for packaging of the gene of interest can be achieved using the described approach.
Our previous research showed that the best yield of virus-like particles (VLPs) formed by RNA bacteriophage GA coat protein was obtained by expression in yeast Pichia pastoris, while other used ...expression systems in Saccharomyces cerevisiae gave much lower amounts of capsids. The main reasons to attempt further studies in Saccharomyces cerevisiae were to improve the yield of GA-based VLPs using constructs with optimised nucleotide triplets in coding sequences, and to exploit the possibilities of the two-promoter Gal1/Gal10 system of expression vector pESC-URA for production of the desired mosaic VLPs and for packaging of mRNAs into VLPs in vivo
9 9 Mūsu iepriekšējie pētījumi parādījuši, ka lielākais RNS bakteriofāga GA apvalka proteīna veidoto vīrusiem līdzīgo daļiņu (VLD) ieguvums ir ekspresējot tās raugos Pichia pastoris; savukārt, izmantojot Saccharomyces cerevisiae ekspresijas sistēmu, kapsīdu ieguvums ir daudz mazāks. Divi galvenie mērķi turpmākajām studijām raugos Saccharomyces cerevisiae bija paaugstināt GA apvalka proteīna veidoto VLD ieguvumu, izmantojot konstrukcijas ar optimizētu nukleotīdu tripletu sekvencēm, un izpētīt iespējas, ko varētu sniegt divu promoteru Gall/GallO saturošs ekspresijas vektors pESC-URA vēlamo mozaīkveida VLD iegūšanai un mRNS iepakošanai VLD in vivo
Previously, we have demonstrated that hepatitis B virus (HBV) core particles tolerate the insertion of the amino-terminal 120 amino acids (aa) of the Puumala hantavirus nucleocapsid (N) protein. ...Here, we demonstrate that the insertion of 120 amino-terminal aa of N proteins from highly virulent Dobrava and Hantaan hantaviruses allows the formation of chimeric core particles. These particles expose the inserted foreign protein segments, at least in part, on their surface. Analysis by electron cryomicroscopy of chimeric particles harbouring the Puumala virus (PUUV) N segment revealed 90%
T = 3 and 10%
T = 4 shells. A map computed from
T = 3 shells shows additional density splaying out from the tips of the spikes producing the effect of an extra shell of density at an outer radius compared with wild-type shells. The inserted Puumala virus N protein segment is flexibly linked to the core spikes and only partially icosahedrally ordered. Immunisation of mice of two different haplotypes (BALB/c and C57BL/6) with chimeric core particles induces a high-titered and highly cross-reactive N-specific antibody response in both mice strains.
Hepatitis B virus (HBV) core (HBc) virus-like particles (VLPs) are one of the most powerful protein engineering tools utilised to expose immunological epitopes and/or cell-targeting signals and for ...the packaging of genetic material and immune stimulatory sequences. Although HBc VLPs and their numerous derivatives are produced in highly efficient bacterial and yeast expression systems, the existing purification and packaging protocols are not sufficiently optimised and standardised. Here, a simple alkaline treatment method was employed for the complete removal of internal RNA from bacteria- and yeast-produced HBc VLPs and for the conversion of these VLPs into empty particles, without any damage to the VLP structure. The empty HBc VLPs were able to effectively package the added DNA and RNA sequences. Furthermore, the alkaline hydrolysis technology appeared efficient for the purification and packaging of four different HBc variants carrying lysine residues on the HBc VLP spikes. Utilising the introduced lysine residues and the intrinsic aspartic and glutamic acid residues exposed on the tips of the HBc spikes for chemical coupling of the chosen peptide and/or nucleic acid sequences ensured a standard and easy protocol for the further development of versatile HBc VLP-based vaccine and gene therapy applications.
Chemokines and their receptors are now under intensive investigation from different points of view: theoretically, to understand "how they work", and practically, to discover methods to affect or to ...force cellular processes into organisms in the intended direction. Evidence gathered during recent years suggests an important role for chemokines in normal cell proliferation, migration, intracellular communication, but what is of greater interest is their number of pathophysiological processes, such as chronic and acute inflammation, infection diseases, modulation of angiogenesis, tumour growth and spread. Chemokines have been also in several brain areas, where ligand-receptor systems can seriously alter the action of neuronally active pharmacological agents. Our interest is focused on the receptor/ligand pair CXCR4 / CXCL12 (SDF-1). We attempted the overexpression of this receptor into several eukaryotic cell lines for detailed interaction studies of receptor CXCR4 with ligands that were virus-like particles-based analogs of its only known natural ligand SDF-1 or stromal cell derived factor. This study evaluates the expression of CXCR4 into cells CHO, HEK293 and BHK21 with the idea of developing a handy "instrument" for further investigations.
Hemokīni un to receptori tiek intensīvi pētīti dažādos aspektos - teorētiski, lai saprastu to darbības principus, un praktiski, lai atklātu metodes, kas ļautu ietekmēt organismā notiekošos šūnu procesus vai pat vadīt tos vēlamā virzienā. Pēdējo gadu pētījumi liecina par hemokīnu nozīmīgu lomu normālā šūnu proliferācijā, migrācijā un starpšūnu komunikācijā, bet interesi rada hemokīnu loma daudzos patofiziologiskos procesos - hroniskos un akūtos iekaisumos, infekcijas slimībās, angiogenēzes modulācijā un ļaundabīgo audzēju attīstībā. Hemokīni ir atrasti arī atsevišķos smadzeņu rajonos, kur ligandu-receptoru sistēma spēj nopietni ietekmēt neironāli aktīvu farmakologisko agentu darbību. Mūsu interese ir vērsta uz receptora-liganda pāri CXCR4 / CXCL12 (SDF-1). Mēs mēginājām pārekspresēt šo receptoru vairākās eikariotisko šūnu līnijās, lai detalizētāk noskaidrotu receptora CXCR4 mijiedarbību ar ligandu - uz vīrusiem līdzīgām daļiņām balstītu analogu, kas satur receptora vienīgā dabiskā liganda SDF-1 (stromālo šūnu faktora) fragmentus. Šis pētījums ir veltīts CXCR4 ekspresēšanai CHO, HEK293 un BHK21 šūnās cerībā izveidot noderīgu "instrumentu" turpmākajām studijām.
RNA phages are often used as prototypes for modern recombinant virus-like particle (VLP) technologies. Icosahedral RNA phage VLPs can be formed from coat proteins (CPs) and are efficiently produced ...in bacteria and yeast. Both genetic fusion and chemical coupling have been successfully used for the production of numerous chimeras based on RNA phage VLPs. In this review, we describe advances in RNA phage VLP technology along with the history of the Leviviridae family, including its taxonomical organization, genomic structure, and important role in the development of molecular biology. Comparative 3D structures of different RNA phage VLPs are used to explain the level of VLP tolerance to foreign elements displayed on VLP surfaces. We also summarize data that demonstrate the ability of CPs to tolerate different organic (peptides, oligonucleotides, and carbohydrates) and inorganic (metal ions) compounds either chemically coupled or noncovalently added to the outer and/or inner surfaces of VLPs. Finally, we present lists of nanotechnological RNA phage VLP applications, such as experimental vaccines constructed by genetic fusion and chemical coupling methodologies, nanocontainers for targeted drug delivery, and bioimaging tools.
The major immunodominant region (MIR) and N-terminus of the hepatitis B virus (HBV) core (HBc) protein were used to expose foreign insertions on the outer surface of HBc virus-like particles (VLPs). ...The additions to the HBc positively charged arginine-rich C-terminal (CT) domain are usually not exposed on the VLP surface. Here, we constructed a set of recombinant HBcG vectors in which CT arginine stretches were substituted by glycine residues. In contrast to natural HBc VLPs and recombinant HBc VLP variants carrying native CT domain, the HBcG VLPs demonstrated a lowered capability to pack bacterial RNA during expression in Escherichia coli cells. The C-terminal addition of a model foreign epitope from the HBV preS1 sequence to the HBcG vectors resulted in the exposure of the inserted epitope on the VLP surface, whereas the same preS1 sequences added to the native CT of the natural HBc protein remained buried within the HBc VLPs. Based on the immunisation of mice, the preS1 epitope added to the HBcG vectors as a part of preS1(20–47) and preS1phil sequences demonstrated remarkable immunogenicity. The same epitope added to the original C-terminus of the HBc protein did not induce a notable level of anti-preS1 antibodies. HBcG vectors may contribute to the further development of versatile HBc VLP-based vaccine and gene therapy applications.
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