Dataset Information

Structural Insight into CVB3-VLP Non-Adjuvanted Vaccine.

ABSTRACT: Coxsackievirus B (CVB) enteroviruses are common pathogens that can cause acute and chronic myocarditis, dilated cardiomyopathy, aseptic meningitis, and they are hypothesized to be a causal factor in type 1 diabetes. The licensed enterovirus vaccines and those currently in clinical development are traditional inactivated or live attenuated vaccines. Even though these vaccines work well in the prevention of enterovirus diseases, new vaccine technologies, like virus-like particles (VLPs), can offer important advantages in the manufacturing and epitope engineering. We have previously produced VLPs for CVB3 and CVB1 in insect cells. Here, we describe the production of CVB3-VLPs with enhanced production yield and purity using an improved purification method consisting of tangential flow filtration and ion exchange chromatography, which is compatible with industrial scale production. We also resolved the CVB3-VLP structure by Cryo-Electron Microscopy imaging and single particle reconstruction. The VLP diameter is 30.9 nm on average, and it is similar to Coxsackievirus A VLPs and the expanded enterovirus cell-entry intermediate (the 135s particle), which is ~2 nm larger than the mature virion. High neutralizing and total IgG antibody levels, the latter being a predominantly Th2 type (IgG1) phenotype, were detected in C57BL/6J mice immunized with non-adjuvanted CVB3-VLP vaccine. The structural and immunogenic data presented here indicate the potential of this improved methodology to produce highly immunogenic enterovirus VLP-vaccines in the future.

SUBMITTER: Hankaniemi MM

PROVIDER: S-EPMC7565060 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Structural Insight into CVB3-VLP Non-Adjuvanted Vaccine.

Hankaniemi Minna M MM Baikoghli Mo A MA Stone Virginia M VM Xing Li L Väätäinen Outi O Soppela Saana S Sioofy-Khojine Amirbabak A Saarinen Niila V V NVV Ou Tingwei T Anson Brandon B Hyöty Heikki H Marjomäki Varpu V Flodström-Tullberg Malin M Cheng R Holland RH Hytönen Vesa P VP Laitinen Olli H OH

Microorganisms 20200824 9

Coxsackievirus B (CVB) enteroviruses are common pathogens that can cause acute and chronic myocarditis, dilated cardiomyopathy, aseptic meningitis, and they are hypothesized to be a causal factor in type 1 diabetes. The licensed enterovirus vaccines and those currently in clinical development are traditional inactivated or live attenuated vaccines. Even though these vaccines work well in the prevention of enterovirus diseases, new vaccine technologies, like virus-like particles (VLPs), can offer ...[more]

PMID: 32846899

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Project description:BACKGROUND: Fundamental to vaccine development, manufacturing consistency, and product stability is an understanding of the vaccine structure-activity relationship. With the virus-like particle (VLP) approach for recombinant vaccines gaining popularity, there is growing demand for tools that define their key characteristics. We assessed a suite of non-intrusive VLP epitope structure and function characterization tools by application to the Hepatitis B surface antigen (rHBsAg) VLP-based vaccine. METHODOLOGY: The epitope-specific immune reactivity of rHBsAg epitopes to a given monoclonal antibody was monitored by surface plasmon resonance (SPR) and quantitatively analyzed on rHBsAg VLPs in-solution or bound to adjuvant with a competitive enzyme-linked immunosorbent assay (ELISA). The structure of recombinant rHBsAg particles was examined by cryo transmission electron microscopy (cryoTEM) and in-solution atomic force microscopy (AFM). PRINCIPAL FINDINGS: SPR and competitive ELISA determined relative antigenicity in solution, in real time, with rapid turn-around, and without the need of dissolving the particulate aluminum based adjuvant. These methods demonstrated the nature of the clinically relevant epitopes of HBsAg as being responsive to heat and/or redox treatment. In-solution AFM and cryoTEM determined vaccine particle size distribution, shape, and morphology. Redox-treated rHBsAg enabled 3D reconstruction from CryoTEM images--confirming the previously proposed octahedral structure and the established lipid-to-protein ratio of HBsAg particles. Results from these non-intrusive biophysical and immunochemical analyses coalesced into a comprehensive understanding of rHBsAg vaccine epitope structure and function that was important for assuring the desired epitope formation, determinants for vaccine potency, and particle stability during vaccine design, development, and manufacturing. SIGNIFICANCE: Together, the methods presented here comprise a novel suite of non-intrusive VLP structural and functional characterization tools for recombinant vaccines. Key VLP structural features were defined and epitope-specific antigenicity was quantified while preserving epitope integrity and particle morphology. These tools should facilitate the development of other VLP-based vaccines.

Project description:Ringworm is a worldwide distributed contagious disease infecting both man and animals that constitute an economic, zoonotic, and health problem concern all over the world. During the last decade, attention has been directed to vaccination as an ideal approach to the control of such diseases. In the present study, non-adjuvanted polyvalent vaccines were prepared from locally isolated hot and virulent dermatophyte species, namely Trichophyton verrucosum (T. verrucosum), Trichophyton mentagrophytes (T. mentagrophytes), and Microsporum canis (M. canis) were immunologically evaluated. The prepared vaccine evaluation was focused on the aspects of immunogenicity and protective efficacy using guinea pigs. Both in its living or inactivated forms, the vaccine-induced significant humoral and cell-mediated immune responses and achieve proper protection of guinea pigs against challenging infections with homologous and heterologous dermatophyte strains. On the other hand, investigations on dermatophyte exo-keratinases showed that it was better produced and more expressed in a mineral-based medium containing pure keratin (3 g/L) than in the same medium with human hair supplementation (2.6 g/L). The maximum dermatophyte productivity of exo-keratinases was found to be between 18 and 21 days post-incubation. Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two fractions with molecular weights of 40 kDa (fraction I) and 28 kDa (fraction II) have been identified in the culture filtrate of the three involved dermatophyte species. Both fractions demonstrated keratinolytic activity. The specific activity of the isolated keratinases (number of Keratinase units (KU)/mg protein) was stronger in fraction I, where it reached 18.75, 15.38, and 14 KU/mg protein as compared to 12.9, 8.74, and 12 KU/mg protein in fraction II of T. verrucosum, T. mentagrophytes, and M. canis, respectively. The dermatophyte exo-keratinases proved to be immunogenic as they stimulated high keratinase-specific antibody titers and induced strong delayed skin hypersensitivity reactions in vaccinated animals. Anti-keratinase-specific IgG was detected in sera of guinea pigs immunized with the inactivated or living polyvalent dermatophyte vaccines by a homemade enzyme-linked immunosorbent assay (ELISA) using dermatophyte exo-keratinases as coating antigen. The intradermal injection of dermatophyte exo-keratinases induced specific delayed skin reactions in guinea pigs immunized with the inactivated or the living polyvalent dermatophyte vaccines. The intradermal injection of dermatophyte exo-keratinases in the control non-sensitized guinea pigs was associated with itching, swelling, and bloody scar formation, however, no skin indurations were formed. The development of those post-exo-keratinases injection reactions in the control non-sensitized apparently healthy guinea pigs group, suggests an exo-keratinases possible role in the pathogenesis of dermatophytosis.

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Dataset Information

Structural Insight into CVB3-VLP Non-Adjuvanted Vaccine.

Publications

Structural Insight into CVB3-VLP Non-Adjuvanted Vaccine.

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