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

Induction of Broadly Cross-Reactive Antibodies by Displaying Receptor Binding Domains of SARS-CoV-2 on Virus-like Particles.

The impact of the COVID-19 pandemic has been reduced since the application of vaccination programs, mostly shown in the reduction of hospitalized patients. However, the emerging variants, in particular Omicron, have caused a steep increase in the number of infections; this increase is, nevertheless, not matched by an increase in hospitalization. Therefore, a vaccine that induces cross-reactive antibodies against most or all variants is a potential solution for the issue of emerging new variants. Here, we present a vaccine candidate which displays receptor-binding domain (RBD) of SARS-CoV-2 on virus-like particles (VLP) that, in mice, not only induce strong antibody responses against RBD but also bind RBDs from other variants of concern (VOCs). The antibodies induced by wild-type (wt) RBD displayed on immunologically optimized Cucumber mosaic virus incorporated tetanus toxin (CuMVTT) VLPs bind to wt as well as RBDs of VOCs with high avidities, indicating induction of strongly cross-reactive IgG antibodies. Interestingly, similar cross-reactive IgA antibodies were induced in immunized mice. Furthermore, these cross-reactive antibodies demonstrated efficacy in neutralizing wt (Wuhan) as well as SARS-CoV-2 VOCs (Beta, Delta, and Gamma). In summary, RBDs displayed on VLPs are capable of inducing protective cross-reactive IgG and IgA antibodies in mice, indicating that it may be possible to cover emerging VOCs with a single vaccine based on wt RBD

Virus-Like Particles Are Efficient Tools for Boosting mRNA-Induced Antibodies.

mRNA based vaccines against COVID-19 have proven most successful at keeping SARS-CoV-2 pandemic at bay in many countries. Recently, there is an increased interest in heterologous prime-boost vaccination strategies for COVID-19 to maintain antibody responses for the control of continuously emerging SARS-CoV-2 variants of concern (VoCs) and to overcome other obstacles such as supply shortage, costs and reduced safety issues or inadequatly induced immune-responses. In this study, we investigated the antibody responses induced by heterologous prime-boost with vaccines based on mRNA and virus-like particles (VLPs). The VLP-based mCuMVTT-RBM vaccine candidate and the approved mRNA-1273 vaccine were used for this purpose. We find that homologous prime boost regimens with either mRNA or VLP induced high levels of high avidity antibodies. Optimal antibody responses were, however, induced by heterologous regimens both for priming with mRNA and boosting with VLP and vice versa, priming with VLP and boosting with mRNA. Thus, heterologous prime boost strategies may be able to optimize efficacy and economics of novel vaccine strategies

Influence of antigen density and TLR ligands on preclinical efficacy of a VLP-based vaccine against peanut allergy.

BACKGROUND Virus-like particle (VLP) Peanut is a novel immunotherapeutic vaccine candidate for the treatment of peanut allergy. The active pharmaceutical ingredient represents cucumber mosaic VLPs (CuMVTT -VLPs) that are genetically fused with one of the major peanut allergens, Ara h 2 (CuMVTT -Ara h 2). We previously demonstrated the immunogenicity and the protective capacity of VLP Peanut-based immunization in a murine model for peanut allergy. Moreover, a Phase I clinical trial has been initiated using VLP Peanut material manufactured following a GMP-compliant manufacturing process. Key product characterization studies were undertaken here to understand the role and contribution of critical quality attributes that translate as predictive markers of immunogenicity and protective efficacy for clinical vaccine development. METHOD The role of prokaryotic RNA encapsulated within VLP Peanut on vaccine immunogenicity was assessed by producing a VLP Peanut batch with a reduced RNA content (VLP Peanut low RNA). Immunogenicity and peanut allergen challenge studies were conducted with VLP Peanut low RNA, as well as with VLP Peanut in WT and TLR 7 KO mice. Furthermore, mass spectrometry and SDS-PAGE based methods were used to determine Ara h 2 antigen density on the surface of VLP Peanut particles. This methodology was subsequently applied to investigate the relationship between Ara h 2 antigen density and immunogenicity of VLP Peanut. RESULTS A TLR 7 dependent formation of Ara h 2 specific high-avidity IgG antibodies, as well as a TLR 7 dependent change in the dominant IgG subclass, was observed following VLP Peanut vaccination, while total allergen-specific IgG remained relatively unaffected. Consistently, a missing TLR 7 signal caused only a weak decrease in allergen tolerability after vaccination. In contrast, a reduced RNA content for VLP Peanut resulted in diminished total Ara h 2 specific IgG responses, followed by a significant impairment in peanut allergen tolerability. The discrepant effect on allergen tolerance caused by an absent TLR 7 signal versus a reduced RNA content is explained by the observation that VLP Peanut-derived RNA not only stimulates TLR 7 but also TLR 3. Additionally, a strong correlation was observed between the number of Ara h 2 antigens displayed on the surface of VLP Peanut particles and the vaccine's immunogenicity and protective capacity. CONCLUSIONS Our findings demonstrate that prokaryotic RNA encapsulated within VLP Peanut, including antigen density of Ara h 2 on viral particles, are key contributors to the immunogenicity and protective capacity of the vaccine. Thus, antigenicity and RNA content are two critical quality attributes that need to be determined at the stage of manufacturing, providing robust information regarding the immunogenicity and protective capacity of VLP Peanut in the mouse which has translational relevance to the human setting

Virus-Like Particles Are Efficient Tools for Boosting mRNA-Induced Antibodies

mRNA based vaccines against COVID-19 have proven most successful at keeping SARS-CoV-2 pandemic at bay in many countries. Recently, there is an increased interest in heterologous prime-boost vaccination strategies for COVID-19 to maintain antibody responses for the control of continuously emerging SARS-CoV-2 variants of concern (VoCs) and to overcome other obstacles such as supply shortage, costs and reduced safety issues or inadequatly induced immune-responses. In this study, we investigated the antibody responses induced by heterologous prime-boost with vaccines based on mRNA and virus-like particles (VLPs). The VLP-based mCuMVTT-RBM vaccine candidate and the approved mRNA-1273 vaccine were used for this purpose. We find that homologous prime boost regimens with either mRNA or VLP induced high levels of high avidity antibodies. Optimal antibody responses were, however, induced by heterologous regimens both for priming with mRNA and boosting with VLP and vice versa, priming with VLP and boosting with mRNA. Thus, heterologous prime boost strategies may be able to optimize efficacy and economics of novel vaccine strategies

The three-dimensional structure of cocksfoot mottle virus at 2.7 å resolution

AbstractCocksfoot mottle virus is a plant virus that belongs to the genus Sobemovirus. The structure of the virus has been determined at 2.7 Å resolution. The icosahedral capsid has T = 3 quasisymmetry and 180 copies of the coat protein. Except for a couple of stacked bases, the viral RNA is not visible in the electron density map. The coat protein has a jelly-roll β-sandwich fold and its conformation is very similar to that of other sobemoviruses and tobacco necrosis virus. The N-terminal arm of one of the three quasiequivalent subunits is partly ordered and follows the same path in the capsid as the arm in rice yellow mottle virus, another sobemovirus. In other sobemoviruses, the ordered arm follows a different path, but in both cases the arms from three subunits meet and form a similar structure at a threefold axis. A comparison of the structures and sequences of viruses in this family shows that the only conserved parts of the protein–protein interfaces are those that form binding sites for calcium ions. Still, the relative orientations and position of the subunits are mantained

Rigid Polyurethane Foam Thermal Insulation Protected with Mineral Intumescent Mat

One of the biggest disadvantages of rigid polyurethane (PU) foams is its low thermal resistance, high flammability and high smoke production. Greatest advantage of this thermal insulation material is its low thermal conductivity (λ), which at 18-28 mW/(m•K) is superior to other materials. To lower the flammability of PU foams, different flame retardants (FR) are used. Usually, industrially viable are halogenated liquid FRs but recent trends in EU regulations show that they are not desirable any more. Main concern is toxicity of smoke and health hazard form volatiles in PU foam materials. Development of intumescent passive fire protection for foam materials would answer problems with flammability without using halogenated FRs. It is possible to add expandable graphite (EG) into PU foam structure but this increases the thermal conductivity greatly. Thus, the main advantage of PU foam is lost. To decrease the flammability of PU foams, three different contents 3%; 9% and 15% of EG were added to PU foam formulation. Sample with 15% of EG increased λ of PU foam from 24.0 to 30.0 mW/(m•K). This paper describes the study where PU foam developed from renewable resources is protected with thermally expandable intumescent mat from Technical Fibre Products Ltd. (TFP) as an alternative to EG added into PU material. TFP produces range of mineral fibre mats with EG that produce passive fire barrier. Two type mats were used to develop sandwich-type PU foams. Also, synergy effect of non-halogenated FR, dimethyl propyl phosphate and EG was studied. Flammability of developed materials was assessed using Cone Calorimeter equipment. Density, thermal conductivity, compression strength and modulus of elasticity were tested for developed PU foams. PU foam morphology was assessed from scanning electron microscopy images