Epigallocatechin-3-Gallate as a Novel Vaccine Adjuvant.

Vaccine adjuvants from natural resources have been utilized for enhancing vaccine efficacy against infectious diseases. This study examined the potential use of catechins, polyphenolic materials derived from green tea, as adjuvants for subunit and inactivated vaccines. Previously, catechins have been documented to have irreversible virucidal function, with the possible applicability in the inactivated viral vaccine platform. In a mouse model, the coadministration of epigallocatechin-3-gallate (EGCG) with influenza hemagglutinin (HA) antigens induced high levels of neutralizing antibodies, comparable to that induced by alum, providing complete protection against the lethal challenge. Adjuvant effects were observed for all types of HA antigens, including recombinant full-length HA and HA1 globular domain, and egg-derived inactivated split influenza vaccines. The combination of alum and EGCG further increased neutralizing (NT) antibody titers with the corresponding hemagglutination inhibition (HI) titers, demonstrating a dose-sparing effect. Remarkably, EGCG induced immunoglobulin isotype switching from IgG1 to IgG2a (approximately >64-700 fold increase), exerting a more balanced TH1/TH2 response compared to alum. The upregulation of IgG2a correlated with significant enhancement of antibody-dependent cellular cytotoxicity (ADCC) function (approximately 14 fold increase), providing a potent effector-mediated protection in addition to NT and HI. As the first report on a novel class of vaccine adjuvants with built-in virucidal activities, the results of this study will help improve the efficacy and safety of vaccines for pandemic preparedness.

RNA-dependent assembly of chimeric antigen nanoparticles as an efficient H5N1 pre-pandemic vaccine platform.

Highly pathogenic avian influenza viruses (HPAIVs) pose a significant threat to human health, with high mortality rates, and require effective vaccines. We showed that, harnessed with novel RNA-mediated chaperone function, hemagglutinin (HA) of H5N1 HPAIV could be displayed as an immunologically relevant conformation on self-assembled chimeric nanoparticles (cNP). A tri-partite monomeric antigen was designed including: i) an RNA-interaction domain (RID) as a docking tag for RNA to enable chaperna function (chaperna: chaperone + RNA), ii) globular head domain (gd) of HA as a target antigen, and iii) ferritin as a scaffold for 24 mer-assembly. The immunization of mice with the nanoparticles (~46 nm) induced a 25-30 fold higher neutralizing capacity of the antibody and provided cross-protection from homologous and heterologous lethal challenges. This study suggests that cNP assembly is conducive to eliciting antibodies against the conserved region in HA, providing potent and broad protective efficacy.

Built-in RNA-mediated chaperone (chaperna) for antigen folding tailored to immunized hosts.

High-quality antibody (Ab) production depends on the availability of immunologically relevant antigens. We present a potentially universal platform for generating soluble antigens from bacterial hosts, tailored to immunized animals for Ab production. A novel RNA-dependent chaperone, in which the target antigen is genetically fused with an RNA-interacting domain (RID) docking tag derived from the immunized host, promotes the solubility and robust folding of the target antigen. We selected the N-terminal tRNA-binding domain of lysyl-tRNA synthetase (LysRS) as the RID for fusion with viral proteins and demonstrated the expression of the RID fusion proteins in their soluble and native conformations; immunization predominantly elicited Ab responses to the target antigen, whereas the "self" RID tag remained nonimmunogenic. Differential immunogenicity of the fusion proteins greatly enriched and simplified the screening of hybridoma clones of monoclonal antibodies (mAbs), enabling specific and sensitive serodiagnosis of MERS-CoV infection. Moreover, mAbs against the consensus influenza hemagglutinin stalk domain enabled a novel assay for trivalent seasonal influenza vaccines. The Fc-mediated effector function was demonstrated, which could be harnessed for the design of next-generation "universal" influenza vaccines. The nonimmunogenic built-in antigen folding module tailored to a repertoire of immunized animal hosts will drive immunochemical diagnostics, therapeutics, and designer vaccines.

Hemagglutinin Quantitative ELISA-based Potency Assay for Trivalent Seasonal Influenza Vaccine Using Group-Specific Universal Monoclonal Antibodies.

The assurance of vaccine potency is important for the timely release and distribution of influenza vaccines. As an alternative to Single Radial Immunodiffusion (SRID), we report a new quantitative enzyme-linked immunosorbent assay (ELISA) for seasonal trivalent influenza vaccine (TIV). The consensus hemagglutinin (cHA) stalks for group 1 influenza A virus (IAV), group 2 IAV, and influenza B virus (IBV) were designed and produced in bacterial recombinant host in a soluble form, and monoclonal antibodies (mAbs) were generated. The group-specific 'universal' mAbs (uAbs) bound to various subtypes of HAs in the same group from recombinant hosts, embryonated eggs, and commercial vaccine lots. The calibration curves were generated to assess the sensitivity, specificity, accuracy, and linear dynamic range. The quantitative ELISA was validated for the potency assay of individual components of TIV- H1, H3, and IBV- with good correlation with the SRID method. This new assay could be extended to pandemic or pre-pandemic mock-up vaccines of H5 of group 1 and H7 virus of group 2, and novel HA stalk-based universal vaccines.

Universal monoclonal antibody-based influenza hemagglutinin quantitative enzyme-linked immunosorbent assay.

Seasonal and pandemic influenza infections remain a serious public health concern. Many health authorities recommend annual vaccination as the most effective way to control influenza infection. Accordingly, regulatory guidelines ask vaccine manufacturers to determine vaccine potency at the time of release and throughout shelf-life to ensure vaccine quality. The potency of inactivated influenza vaccine is related to the quantity of hemagglutinin (HA). Since 1970s, single radial immunodiffusion (SRID) assay has been standardly used for the quantitation of HA in influenza vaccine. However, SRID is labor-intensive, inaccurate, and requires standard reference reagents that should be updated annually. Therefore, there have been extensive efforts to develop alternative potency assays. In this study, we developed and tested a new HA quantitative enzyme-linked immunosorbent assay (ELISA) using a universal monoclonal antibody that can bind to HAs from various subtypes in group 1 influenza A virus (IAV). We analyzed the conserved stalk domain of HA via a library approach to design a consensus HA antigen for group 1 IAV. The antigens were expressed as a soluble form in E. coli and were purified by Ni-affinity chromatography. When tested with variety of HAs from IAVs or influenza B viruses (IBVs), the mAbs exhibited specific binding to group 1 HAs, with potential exception to H9 subtype. Among various conditions of pH, urea, and reducing agents, pretreatment of HA at low pH exposing the conserved stalk domain was crucially important for optimal ELISA performance. Calibration curves for various HAs were generated to determine accuracy, specificity, sensitivity, and linear dynamic range. The ELISA method shows high sensitivity and accuracy compared with the SRID assay. The HA group specific universal mAbs against the consensus stalk domain of HA are conducive to establishing an ELISA-based standard procedure for the quantitation of HA antigens for annual vaccination against influenza infection.

Harnessing an RNA-mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation.

A novel protein-folding function of RNA has been recognized, which can outperform previously known molecular chaperone proteins. The RNA as a molecular chaperone (chaperna) activity is intrinsic to some ribozymes and is operational during viral infections. Our purpose was to test whether influenza hemagglutinin (HA) can be assembled in a soluble, trimeric, and immunologically activating conformation by means of an RNA molecular chaperone (chaperna) activity. An RNA-interacting domain (RID) from the host being immunized was selected as a docking tag for RNA binding, which served as a transducer for the chaperna function for de novo folding and trimeric assembly of RID-HA1. Mutations that affect tRNA binding greatly increased the soluble aggregation defective in trimer assembly, suggesting that RNA interaction critically controls the kinetic network in the folding/assembly pathway. Immunization of mice resulted in strong hemagglutination inhibition and high titers of a neutralizing antibody, providing sterile protection against a lethal challenge and confirming the immunologically relevant HA conformation. The results may be translated into a rapid response to a new influenza pandemic. The harnessing of the novel chaperna described herein with immunologically tailored antigen-folding functions should serve as a robust prophylactic and diagnostic tool for viral infections.-Yang, S. W., Jang, Y. H., Kwon, S. B., Lee, Y. J., Chae, W., Byun, Y. H., Kim, P., Park, C., Lee, Y. J., Kim, C. K., Kim, Y. S., Choi, S. I., Seong, B. L. Harnessing an RNA-mediated chaperone for the assembly of influenza hemagglutinin in an immunologically relevant conformation.