Comparison of Bacterial Expression Systems Based on Potato Virus Y-like Particles for Vaccine Generation.

Plant-based virus-like particle (VLP) vaccines have been studied for years, demonstrating their potential as antigen-presenting platforms. In this paper, we describe the development of, and compare between, simple Escherichia coli-based antigen display platforms for the generation of potato virus Y (PVY) VLP-derived vaccines, thus allowing the production of vaccines from a single bacterial cell culture. We constructed four systems with the major cat allergen Fel d 1; namely, direct fusion with plant virus PVY coat protein (CP), mosaic PVY VLPs, and two coexpression variants of conjugates (SpyTag/SpyCatcher) allowing coexpression and conjugation directly in E. coli cells. For control experiments, we included PVY VLPs chemically coupled with Fel d 1. All constructed PVY-Fel d 1 variants were well expressed and soluble, formed PVY-like filamentous particles, and were recognized by monoclonal Fel d 1 antibodies. Our results indicate that all vaccine variants induced high titers of anti-Fel d 1 antibodies in murine models. Mice that were immunized with the chemically coupled Fel d 1 antigen exhibited the highest antibody titers and antibody-antigen interaction specificity, as detected by binding avidity and recognition of native Fel d 1. IgG1 subclass antibodies were found to be the dominant IgG class against PVY-Fel d 1. PVY CP-derived VLPs represent an efficient platform for the comparison of various antigen presentation systems to help evaluate different vaccine designs

Bacterial expression systems based on Tymovirus-like particles for the presentation of vaccine antigens.

Virus-like particles (VLPs) are virus-derived artificial nanostructures that resemble a native virus-stimulating immune system through highly repetitive surface structures. Improved safety profiles, flexibility in vaccine construction, and the ease of VLP production and purification have highlighted VLPs as attractive candidates for universal vaccine platform generation, although exploration of different types of expression systems for their development is needed. Here, we demonstrate the construction of several simple Escherichia coli expression systems for the generation of eggplant mosaic virus (EMV) VLP-derived vaccines. We used different principles of antigen incorporation, including direct fusion of EMV coat protein (CP) with major cat allergen Feld1, coexpression of antigen containing and unmodified (mosaic) EMV CPs, and two coexpression variants of EMV VLPs and antigen using synthetic zipper pair 18/17 (SYNZIP 18/17), and coiled-coil forming peptides E and K (Ecoil/Kcoil). Recombinant Fel d 1 chemically coupled to EMV VLPs was included as control experiments. All EMV-Feld1 variants were expressed in E. coli, formed Tymovirus-like VLPs, and were used for immunological evaluation in healthy mice. The immunogenicity of these newly developed vaccine candidates demonstrated high titers of Feld1-specific Ab production; however, a comparably high immune response against carrier EMV was also observed. Antibody avidity tests revealed very specific Ab production (more than 50% specificity) for four out of the five vaccine candidates. Native Feld1 recognition and subclass-specific antibody tests suggested that the EMV-SZ18/17-Feld1 complex and chemically coupled EMV-Feld1 vaccines may possess characteristics for further development

Synthesis and Immunological Evaluation of Virus-Like Particle-Milbemycin A3/A4 Conjugates

Milbemycins are macrolide antibiotics with a broad spectrum of nematocidal, insecticidal, and acaricidal activity. To obtain milbemycin A3/A4 derivatives suitable for chemical conjugation to protein carriers (milbemycin haptens), succinate linker and a novel 17-atom-long linker containing a terminal carboxylic acid group were attached to the milbemycin core in a protecting group-free synthesis. The obtained milbemycin A3/A4 derivatives were coupled to Potato virus Y-like nanoparticles by the activated ester method. The reaction products were characterized and used in mice immunization experiments. It was found that the mice developed weak specific immune responses toward all tested milbemycin haptens

Identification and Full Genome Analysis of the First Putative Virus of Sea Buckthorn (Hippophae rhamnoides L.)

The agricultural importance of sea buckthorn (SBT; Hippophae rhamnoides L.) is rapidly increasing. Several bacterial and fungal pathogens infecting SBT have been identified and characterized; however, the viral pathogens are not yet known. In this study, we identified, isolated, and sequenced a virus from a wild plantation of SBT for the first time. Sequence analysis of the obtained viral genome revealed high similarity with several viruses belonging to the genus Marafivirus. The genome of the new virus is 6989 nucleotides (nt) in length according to 5′, 3′ RACE (without polyA-tail), with 5′ and 3′ 133 and 109 nt long untranslated regions, respectively. The viral genome encoded two open reading frames (ORFs). ORF1 encoded a polyprotein of 1954 amino acids with the characteristic marafivirus non-structural protein domains—methyltransferase, Salyut domain, papain-like cysteine protease, helicase, and RNA-dependent RNA polymerase. ORF1 was separated from ORF2 by 6 nt, encoding the coat protein (CP) with typical signatures of minor and major forms. Both CP forms were cloned and expressed in a bacterial expression system. Only the major CP was able to self-assemble into 30 nm virus-like particles that resembled the native virus, thus demonstrating that minor CP is not essential for virion assembly