Regina Renhofa

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.

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.

Silica Nanoparticles as the Adjuvant for the Immunisation of Mice Using Hepatitis B Core Virus-Like Particles

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 Freunds 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.

The True Story and Advantages of RNA Phage Capsids as Nanotools

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.

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.

The Hepatitis B Virus Core Variants that Expose Foreign C-Terminal Insertions on the Outer Surface of Virus-Like Particles

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.