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.

A Host-Restricted Self-Attenuated Influenza Virus Provides Broad Pan-Influenza A Protection in a Mouse Model.

Influenza virus infections can cause a broad range of symptoms, form mild respiratory problems to severe and fatal complications. While influenza virus poses a global health threat, the frequent antigenic change often significantly compromises the protective efficacy of seasonal vaccines, further increasing the vulnerability to viral infection. Therefore, it is in great need to employ strategies for the development of universal influenza vaccines (UIVs) which can elicit broad protection against diverse influenza viruses. Using a mouse infection model, we examined the breadth of protection of the caspase-triggered live attenuated influenza vaccine (ctLAIV), which was self-attenuated by the host caspase-dependent cleavage of internal viral proteins. A single vaccination in mice induced a broad reactive antibody response against four different influenza viruses, H1 and rH5 (HA group 1) and H3 and rH7 subtypes (HA group 2). Notably, despite the lack of detectable neutralizing antibodies, the vaccination provided heterosubtypic protection against the lethal challenge with the viruses. Sterile protection was confirmed by the complete absence of viral titers in the lungs and nasal turbinates after the challenge. Antibody-dependent cellular cytotoxicity (ADCC) activities of non-neutralizing antibodies contributed to cross-protection. The cross-protection remained robust even after in vivo depletion of T cells or NK cells, reflecting the strength and breadth of the antibody-dependent effector function. The robust mucosal secretion of sIgA reflects an additional level of cross-protection. Our data show that the host-restricted designer vaccine serves an option for developing a UIV, providing pan-influenza A protection against both group 1 and 2 influenza viruses. The present results of potency and breadth of protection from wild type and reassortant viruses addressed in the mouse model by single immunization merits further confirmation and validation, preferably in clinically relevant ferret models with wild type challenges.

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.

RNA-assisted self-assembly of monomeric antigens into virus-like particles as a recombinant vaccine platform.

Representing highly ordered repetitive structures of antigen macromolecular assemblies, virus-like particles (VLPs) serve as a high-priority vaccine platform against emerging viral infections, as alternatives to traditional cell culture-based vaccines. RNAs can function as chaperones (Chaperna) and are extremely effective in promoting protein folding. Beyond their canonical function as translational adaptors, tRNAs may moonlight as chaperones for the kinetic control of macromolecular antigen assembly. Capitalizing on genomic RNA co-assembly in infectious virions, we present the first report of a biomimetic assembly of viral capsids that was assisted by non-viral host RNAs into genome-free, non-infectious empty particles. Here, we demonstrate the assembly of bacterially-produced soluble norovirus VP1 forming VLPs (n = 180) in vitro. A tRNA-interacting domain (tRID) was genetically fused with the VP1 capsid protein, as a tRNA docking tag, in the bacterial host to transduce chaperna function for de novo viral antigen folding. tRID/tRNA removal prompted the in vitro assembly of monomeric antigens into highly ordered repetitive structures that elicited robust protective immune responses after immunization. The chaperna-based assembly of monomeric antigens will impact the development and deployment of VLP vaccines for emerging and re-emerging viral infections.

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.

Brazilin Isolated from Caesalpinia sappan suppresses nuclear envelope reassembly by inhibiting barrier-to-autointegration factor phosphorylation.

To date, many anticancer drugs have been developed by directly or indirectly targeting microtubules, which are involved in cell division. Although this approach has yielded many anticancer drugs, these drugs produce undesirable side effects. An alternative strategy is needed, and targeting mitotic exit may be one alternative approach. Localization of phosphorylated barrier-to-autointegration factor (BAF) to the chromosomal core region is essential for nuclear envelope compartment relocalization. In this study, we isolated brazilin from Caesalpinia sappan Leguminosae and demonstrated that it inhibited BAF phosphorylation in vitro and in vivo. Moreover, we demonstrated direct binding between brazilin and BAF. The inhibition of BAF phosphorylation induced abnormal nuclear envelope reassembly and cell death, indicating that perturbation of nuclear envelope reassembly could be a novel approach to anticancer therapy. We propose that brazilin isolated from C. sappan may be a new anticancer drug candidate that induces cell death by inhibiting vaccinia-related kinase 1-mediated BAF phosphorylation.

Luteolin suppresses cancer cell proliferation by targeting vaccinia-related kinase 1.

Uncontrolled proliferation, a major feature of cancer cells, is often triggered by the malfunction of cell cycle regulators such as protein kinases. Recently, cell cycle-related protein kinases have become attractive targets for anti-cancer therapy, because they play fundamental roles in cellular proliferation. However, the protein kinase-targeted drugs that have been developed so far do not show impressive clinical results and also display severe side effects; therefore, there is undoubtedly a need to investigate new drugs targeting other protein kinases that are critical in cell cycle progression. Vaccinia-related kinase 1 (VRK1) is a mitotic kinase that functions in cell cycle regulation by phosphorylating cell cycle-related substrates such as barrier-to-autointegration factor (BAF), histone H3, and the cAMP response element (CRE)-binding protein (CREB). In our study, we identified luteolin as the inhibitor of VRK1 by screening a small-molecule natural compound library. Here, we evaluated the efficacy of luteolin as a VRK1-targeted inhibitor for developing an effective anti-cancer strategy. We confirmed that luteolin significantly reduces VRK1-mediated phosphorylation of the cell cycle-related substrates BAF and histone H3, and directly interacts with the catalytic domain of VRK1. In addition, luteolin regulates cell cycle progression by modulating VRK1 activity, leading to the suppression of cancer cell proliferation and the induction of apoptosis. Therefore, our study suggests that luteolin-induced VRK1 inhibition may contribute to establish a novel cell cycle-targeted strategy for anti-cancer therapy.

HnRNP A1 phosphorylated by VRK1 stimulates telomerase and its binding to telomeric DNA sequence.

The telomere integrity is maintained via replication machinery, telomere associated proteins and telomerase. Many telomere associated proteins are regulated in a cell cycle-dependent manner. Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), a single-stranded oligonucleotide binding protein, is thought to play a pivotal role in telomere maintenance. Here, we identified hnRNP A1 as a novel substrate for vaccinia-related kinase 1 (VRK1), a cell cycle regulating kinase. Phosphorylation by VRK1 potentiates the binding of hnRNP A1 to telomeric ssDNA and telomerase RNA in vitro and enhances its function for telomerase reaction. VRK1 deficiency induces a shortening of telomeres with an abnormal telomere arrangement and activation of DNA-damage signaling in mouse male germ cells. Together, our data suggest that VRK1 is required for telomere maintenance via phosphorylation of hnRNP A1, which regulates proteins associated with the telomere and telomerase RNA.