Indulis Cielens

Versatile Virus-Like Particle Carrier for Epitope Based Vaccines

Background 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. Methodology/Principal Findings 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. Conclusions/Significance 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.

Mosaic Qβ coats as a new presentation model

The new protein carrier was developed on the basis of recombinant RNA phage Qβ capsid. C‐terminal UGA extension of the short form of Qβ coat, so‐called A1 extension, served as a target for presentation of foreign peptides on the outer surface of mosaic Qβ particles. In conditions of enhanced UGA suppression, the proportion of A1‐extended to short coats in mosaic particles dropped from 48% to 14%, with an increase of the length of A1 extension. A model insertion, short preS1 epitope 31‐DPAFR‐35 of hepatitis B surface antigen, demonstrated superficial location on the mosaic Qβ particles and ensured specific antigenicity and immunogenicity.

RNA Phage Qβ Coat Protein as a Carrier for Foreign Epitopes

The Qβ gene C has been proposed as a new carrier for the exposure of foreign peptide sequences. Contrary to well-known ‘display vectors’ on the basis of coat proteins of RNA phage group I, group III p

Efficient homologous prime-boost strategies for T cell vaccination based on virus-like particles.

Induction of high frequencies of specific T cells by vaccination requires prime‐boost regimens. To reach optimal immune responses, it is necessary to use different vectors for priming and boosting as e.g. DNA vaccination followed by boosting with a recombinant viral vector. Here, we show that vaccines based on virus‐like particles (VLP) displaying peptide epitopes are equally effective to induce CTL responses if used in a homologous or heterologous prime‐boost setting. Strikingly, high frequencies (>20% of CD8+ cells) of protective CTL could be induced and maintained by weekly injection of VLP. Thus, the use of VLP may avoid the requirement for complicated heterologous prime‐boost regimens, facilitating the development of effective T cell‐based vaccines.

Mutilation of RNA phage Qβ virus-like particles: from icosahedrons to rods

Icosahedral virus‐like particles (VLPs) of RNA phage Qβ are stabilized by four disulfide bonds of cysteine residues 74 and 80 within the loop between β‐strands F and G (FG loop) of the monomeric subunits, which determine the five‐fold and quasi‐six‐fold symmetry contacts of the VLPs. In order to reduce the stability of Qβ VLPs, we mutationally converted the amino acid stretch 76‐ANGSCD‐81 within the FG loop into the 76‐VGGVEL‐81 sequence. It led to production in Escherichia coli cells of aberrant rod‐like Qβ VLPs, along with normal icosahedral capsids. The length of the rod‐like particles exceeded 4–30 times the diameter of icosahedral Qβ VLPs.

Preparation by alkaline treatment and detailed characterisation of empty hepatitis B virus core particles for vaccine and gene therapy applications

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.

Assembly of mixed rod-like and spherical particles from group I and II RNA bacteriophage coat proteins

The capsids of single-stranded RNA bacteriophages show remarkable structural similarity. In an attempt to test whether the coat protein (CP) from one bacteriophage could substitute for the CP of another and form mixed particles, we reassembled capsids in vitro from a mixture of different RNA phage CP dimers together with E. coli ribosomal RNA. Surprisingly, mixing CPs from phages belonging to groups I and II led to appearance of rod-like particles along with icosahedral spherical capsids, both containing a mixture of the two CPs. Rods and mixed spherical capsids containing host RNA were also obtained in vivo in bacteria expressing simultaneously fr and GA CPs. In a co-infection of the two phages, however, only authentic fr and GA virions were formed. Coat protein mutants in the FG loop were unable to assemble into rods, suggesting that these loops are involved in the formation of the aberrant particles.

EXPRESSION OF GA COAT PROTEIN-DERIVED MOSAIC VIRUS-LIKE PARTICLES IN Saccharomyces cerevisiae AND PACKAGING in vivo OF mRNAs INTO PARTICLES

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