Efficacy of genotype-matched vaccine against re-emerging genotype V Japanese encephalitis virus.

Japanese encephalitis (JE), caused by the Japanese encephalitis virus (JEV), is a highly threatening disease with no specific treatment. Fortunately, the development of vaccines has enabled effective defense against JE. However, re-emerging genotype V (GV) JEV poses a challenge as current vaccines are genotype III (GIII)-based and provide suboptimal protection. Given the isolation of GV JEVs from Malaysia, China, and the Republic of Korea, there is a concern about the potential for a broader outbreak. Under the hypothesis that a GV-based vaccine is necessary for effective defense against GV JEV, we developed a pentameric recombinant antigen using cholera toxin B as a scaffold and mucosal adjuvant, which was conjugated with the E protein domain III of GV by genetic fusion. This GV-based vaccine antigen induced a more effective immune response in mice against GV JEV isolates compared to GIII-based antigen and efficiently protected animals from lethal challenges. Furthermore, a bivalent vaccine approach, inoculating simultaneously with GIII- and GV-based antigens, showed protective efficacy against both GIII and GV JEVs. This strategy presents a promising avenue for comprehensive protection in regions facing the threat of diverse JEV genotypes, including both prevalent GIII and GI as well as emerging GV strains.

Egg microneedles for transdermal vaccination of inactivated influenza virus.

The use of dissolving microneedles (DMNs) is a drug delivery technique in which drug dissolution occurs once it is administered into the skin. The skin is a remarkable site for vaccination due to its significant immunologic properties. Compared to the traditional hypodermic intramuscular (IM) injection, vaccination via DMN does not require cold chains and allows for minimal invasive drug delivery. On account of the significance of skin vaccination, preceding studies have been conducted to elucidate the importance of the DMN technology in vaccination. Most of these studies focused on formulations that maintain the activity of the vaccine, so formulations designed to be specific to the mechanical properties of the microneedle could not be used together independently. In this study, we have developed influenza vaccine loaded egg microneedles (EMN) and characterized the specificity of layer-specific functions of EMN by distinguishing between formulations that can maintain the activity of the vaccine and have the mechanical strength. By the use of in vitro tests such as ELISA and SRID assays, we quantitively evaluated the antigen activity of the formulation candidates to be 87% and 91%, respectively. In vivo tests were also conducted as mouse groups were inoculated with the formulation constructed into egg microneedles (FLU-EMN) to determine the protective efficacy against infection. The results demonstrated that FLU-EMN with functionalized formulations successfully enabled protective immune response even with a fractional dose compared to IM injection.

Comparative Evaluation of Recombinant and Acellular Pertussis Vaccines in a Murine Model.

Since the 2000s, sporadic outbreaks of whooping cough have been reported in advanced countries, where the acellular pertussis vaccination rate is relatively high, and in developing countries. Small-scale whooping cough has also continued in many countries, due in part to the waning of immune protection after childhood vaccination, necessitating the development of an improved pertussis vaccine and vaccination program. Currently, two different production platforms are being actively pursued in Korea; one is based on the aP (acellular pertussis) vaccine purified from B. pertussis containing pertussis toxoid (PT), filamentous hemagglutin (FHA) and pertactin (PRN), and the other is based on the recombinant aP (raP), containing genetically detoxified pertussis toxin ADP-ribosyltransferase subunit 1 (PtxS1), FHA, and PRN domain, expressed and purified from recombinant E. coli. aP components were further combined with diphtheria and tetanus vaccine components as a prototype DTaP vaccine by GC Pharma (GC DTaP vaccine). We evaluated and compared the immunogenicity and the protective efficacy of aP and raP vaccines in an experimental murine challenge model: humoral immunity in serum, IgA secretion in nasal lavage, bacterial clearance after challenge, PTx (pertussis toxin) CHO cell neutralization titer, cytokine secretion in spleen single cell, and tissue resident memory CD4+ T cell (CD4+ TRM cell) in lung tissues. In humoral immunogenicity, GC DTaP vaccines showed high titers for PT and PRN and showed similar patterns in nasal lavage and IL-5 cytokine secretions. The GC DTaP vaccine and the control vaccine showed equivalent results in bacterial clearance after challenge, PTx CHO cell neutralization assay, and CD4+ TRM cell. In contrast, the recombinant raP vaccine exhibited strong antibody responses for FHA and PRN, albeit with low antibody level of PT and low titer in PTx CHO neutralization assay, as compared to control and GC DTaP vaccines. The raP vaccine provided a sterile lung bacterial clearance comparable to a commercial control vaccine after the experimental challenge in murine model. Moreover, raP exhibited a strong cytokine response and CD4+ TRM cell in lung tissue, comparable or superior to the experimental and commercial DTaP vaccinated groups. Contingent on improving the biophysical stability and humoral response to PT, the raP vaccine warrants further development as an effective alternative to aP vaccines for the control of a pertussis outbreak.

Harnessing Pentameric Scaffold of Cholera Toxin B (CTB) for Design of Subvirion Recombinant Dengue Virus Vaccine.

Dengue virus is an enveloped virus with an icosahedral assembly of envelope proteins (E). The E proteins are arranged as a head-to-tail homodimer, and domain III (EDIII) is placed at the edge of the dimer, converging to a pentamer interface. For a structure-based approach, cholera toxin B (CTB) was harnessed as a structural scaffold for the five-fold symmetry of EDIII. Pivoted by an RNA-mediated chaperone for the protein folding and assembly, CTB-EDIII of dengue serotype 1 (DV1) was successfully produced as soluble pentamers in an E. coli host with a high yield of about 28 mg/L. Immunization of mice with CTB-DV1EDIII elicited increased levels of neutralizing antibodies against infectious viruses compared to the control group immunized with DV1EDIII without CTB fusion. IgG isotype switching into a balanced Th1/Th2 response was also observed, probably triggered by the intrinsic adjuvant activity of CTB. Confirming the immune-enhancing potential of CTB in stabilizing the pentamer assembly of EDIII, this study introduces a low-cost bacterial production platform designed to augment the soluble production of subunit vaccine candidates, particularly those targeting flaviviruses.

AB5-Type Toxin as a Pentameric Scaffold in Recombinant Vaccines against the Japanese Encephalitis Virus.

Japanese encephalitis virus (JEV) is an enveloped icosahedral capsid virus with a prime neutralizing epitope present in E protein domain III (EDIII). E dimers are rearranged into a five-fold symmetry of icosahedrons. Cholera toxin B (CTB) and heat-labile enterotoxin B (LTB) of AB5-type toxin was used as the structural scaffold for emulating the pentameric axis of EDIII. We produced homo-pentameric EDIII through the genetic fusion of LTB or CTB in E. coli without recourse to additional refolding steps. Harnessing an RNA-mediated chaperone further enhanced the soluble expression and pentameric assembly of the chimeric antigen. The pentameric assembly was validated by size exclusion chromatography (SEC), non-reduced gel analysis, and a GM1 binding assay. CTB/LTB-EDIII chimeric antigen triggered high neutralizing antibodies against the JEV Nakayama strain after immunization in mice. Altogether, our proof-of-principle study creating a JEV-protective antigen via fusion with an AB5-type toxin as both a pentameric scaffold and a built-in adjuvant posits the bacterially produced recombinant chimeric antigen as a cost-effective alternative to conventional inactivated vaccines against JEV.

Highly chromophoric dual-terminus labeling of an intrinsically disordered native eukaryotic protein of interest at nanoscale.

Chemical conjugation of purified proteins of interest (POIs) in Escherichia coli cells is effective for high expression but has limitations for highly chromogenic dual labeling of intrinsically disordered native proteins (IDNPs). Our probes can tag IDNPs using chemical conjugation during protein synthesis and folding while preserving biologically active structures in mammalian cells. We fluorescently labeled IDNPs in mammalian cells using pure fluorescent methionine and ATTO 565-biotin at the N-or C-terminus, respectively. The dual-labeled Tat protein was used as a model for IDNPs in HeLa cells and detected using Ni-NTA beads to estimate its highly chromogenic concentration. We also demonstrated highly chromogenic double labeling of genetically encoded fluorescent-Tat expression in eukaryotic cells using a single fluorescent dye pair with Förster resonance energy transfer (FRET) ratio and two-color correlation analysis. This study aims to solve native POI processing and achieve ultra-sensitive protein folding for biological and ecological applications at the nanoscale.

Impact of hemagglutination activity and M2e immunity on conferring protection against influenza viruses.

To improve cross-protection of influenza vaccination, we tested conjugation of conserved M2e epitopes to the surface of inactivated influenza virus (iPR8-M2e*). Treatment of virus with chemical cross-linker led to diminished hemagglutination activity and failure to induce hemagglutination inhibiting antibodies. Conjugated iPR8-M2e* vaccine was less protective against homologous and heterosubtypic viruses, despite the induction of virus-specific binding IgG antibodies. In alternative approaches to enhance cross-protection, we developed a genetically linked chimeric protein (M2e-B stalk) vaccine with M2e of influenza A and hemagglutinin (HA) stalk of influenza B virus. Vaccination of mice with inactivated influenza A virus supplemented with M2e-B stalk effectively induced hemagglutination inhibiting antibodies, humoral and cellular M2e immune responses, and enhanced heterosubtypic protection. This study demonstrates the importance of HA functional integrity in influenza vaccine efficacy and that supplementation of influenza vaccines with M2e-B stalk protein could be a feasible strategy of improving cross-protection against influenza viruses.

Prospects on Repurposing a Live Attenuated Vaccine for the Control of Unrelated Infections.

Live vaccines use attenuated microbes to acquire immunity against pathogens in a safe way. As live attenuated vaccines (LAVs) still maintain infectivity, the vaccination stimulates diverse immune responses by mimicking natural infection. Induction of pathogen-specific antibodies or cell-mediated cytotoxicity provides means of specific protection, but LAV can also elicit unintended off-target effects, termed non-specific effects. Such mechanisms as short-lived genetic interference and non-specific innate immune response or long-lasting trained immunity and heterologous immunity allow LAVs to develop resistance to subsequent microbial infections. Based on their safety and potential for interference, LAVs may be considered as an alternative for immediate mitigation and control of unexpected pandemic outbreaks before pathogen-specific therapeutic and prophylactic measures are deployed.

Thermostable H1 hemagglutinin stem with M2e epitopes provides broad cross-protection against group1 and 2 influenza A viruses.

Hemagglutinin (HA) stem-based vaccines have limitations in providing broad and effective protection against cross-group influenza viruses, despite being a promising universal vaccine target. To overcome the limited cross-protection and low efficacy by HA stem vaccination, we genetically engineered a chimeric conjugate of thermostable H1 HA stem and highly conserved M2e repeat (M2e-H1stem), which was expressed at high yields in Escherichia coli. M2e-H1stem protein presented native-like epitopes reactive to antisera of live virus infection. M2e-H1stem protein vaccination of mice induced strong M2e- and HA stem-specific immune responses, conferring broadly effective cross-protection against both antigenically distinct group 1 (H1N1, H5N1, and H9N2 subtypes) and group 2 (H3N2 and H7N9 subtypes) seasonal and pandemic potential influenza viruses. M2e-H1stem vaccination generated CD4+ and CD8+ T cell responses and antibody-dependent cytotoxic cellular and humoral immunity, which contributed to enhancing cross-protection. Furthermore, comparable broad cross-group protection was observed in older aged mice after M2e-H1stem vaccination. This study provides evidence warranting further development of chimeric M2e-stem proteins as a promising universal influenza vaccine candidate in adult and aged populations.

A chimeric thermostable M2e and H3 stalk-based universal influenza A virus vaccine.

We developed a new chimeric M2e and H3 hemagglutinin (HA) stalk protein vaccine (M2e-H3 stalk) by genetic engineering of modified H3 stalk domain conjugated with conserved M2e epitopes to overcome the drawbacks of low efficacy by monomeric domain-based universal vaccines. M2e-H3 stalk protein expressed and purified from Escherichia coli was thermostable, displaying native-like antigenic epitopes recognized by antisera of different HA subtype proteins and influenza A virus infections. Adjuvanted M2e-H3 stalk vaccination induced M2e and stalk-specific IgG antibodies recognizing viral antigens on virus particles and on the infected cell surface, CD4+ and CD8+ T-cell responses, and antibody-dependent cytotoxic cell surrogate activity in mice. M2e-H3 stalk was found to confer protection against heterologous and heterosubtypic cross-group subtype viruses (H1N1, H5N1, H9N2, H3N2, H7N9) at similar levels in adult and aged mice. These results provide evidence that M2e-H3 stalk chimeric proteins can be developed as a universal influenza A virus vaccine candidate for young and aged populations.

Enhanced cross protection by hetero prime-boost vaccination with recombinant influenza viruses containing chimeric hemagglutinin-M2e epitopes.

Annual repeat influenza vaccination raises concerns about protective efficacy against mismatched viruses. We investigated the impact of heterologous prime-boost vaccination on inducing cross protection by designing recombinant influenza viruses with chimeric hemagglutinin (HA) carrying M2 extracellular domains (M2e-HA). Heterologous prime-boost vaccination of C57BL/6 mice with M2e-HA chimeric virus more effectively induced M2e and HA stalk specific IgG antibodies correlating with cross protection than homologous prime-boost vaccination. Induction of M2e and HA stalk specific IgG antibodies was compromised in 1-year old mice, indicating significant aging effects on priming subdominant M2e and HA stalk IgG antibody responses. This study demonstrates that a heterologous prime-boost strategy with recombinant influenza virus expressing extra M2e epitopes provides more effective cross protection than homologous vaccination.

Comparison of the effects of different potent adjuvants on enhancing the immunogenicity and cross-protection by influenza virus vaccination in young and aged mice.

Vaccination against influenza viruses suffers from low efficacy in conferring homologous and cross-protection, particularly in older adults. Here, we compared the effects of three different adjuvant types (QS-21+MPL, CpG+MPL and bacterial cell wall CWS) on enhancing the immunogenicity and homologous and heterosubtypic protection of influenza vaccination in young adult and aged mouse models. A combination of saponin QS-21 and monophosphoryl lipid A (QS-21+MPL) was most effective in inducing T helper type 1 (Th1) T cell and cross-reactive IgG as well as hemagglutination inhibiting antibody responses to influenza vaccination. Both combination adjuvants (QS-21+MPL and CpG+MPL) exhibited high potency by preventing weight loss and reducing viral loads and enhanced homologous and cross-protection by influenza vaccination in adult and aged mouse models. Bacillus Calmette-Guerin cell-wall skeleton (CWS) displayed substantial adjuvant effects on immune responses to influenza vaccination but lower adjuvant efficacy in inducing Th1 IgG responses, cross-protection in adult mice, and in conferring homologous protection in aged mice. This study has significance in comparing the effects of potent adjuvants on enhancing humoral and cellular immune responses to influenza virus vaccination, inducing homologous and cross-protection in adult and aged populations.

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.

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.

Filamentous anti-influenza agents wrapping around viruses.

Owing to the emerging resistance to current anti-influenza therapies, strategies for blocking virus-cell interaction with agents that mimic interactions with host cell receptors are garnering interest. In this context, a multivalent presentation of sialyl groups on various types of scaffold materials such as dendrimers, liposomes, nanoparticles, and natural/synthetic polymers has been investigated for the inhibition of influenza A virus infection. However, the development of versatile antiviral agents based on monodisperse scaffolds capable of precise molecular design remains challenging. Whether an anisotropically extended filamentous nanostructure can serve as an effective scaffold for maximum inhibition of viral cell attachment has not been investigated. In this study, the preparation of a series of sialyllactose-conjugated filamentous bacteriophages (SLPhages), with controlled loading levels, ligand valencies, and two types of sialyllactose (α2,3' and α2,6'), is demonstrated. With optimal ligand loading and valency, SLPhages showed inhibitory activity (in vitro) against influenza A viruses at concentrations of tens of picomolar. This remarkable inhibition is due to the strong interaction between the SLPhage and the virus; this interaction is adequately potent to compensate for the cost of the bending and wrapping of the SLPhage around the influenza virus. Our study may open new avenues for the development of filamentous anti-viral agents, in which virus-wrapping or aggregation is the primary feature responsible for the blocking of cell entry.

Solubility, Stability, and Avidity of Recombinant Antibody Fragments Expressed in Microorganisms.

Solubility of recombinant proteins (i.e., the extent of soluble versus insoluble expression in heterogeneous hosts) is the first checkpoint criterion for determining recombinant protein quality. However, even soluble proteins often fail to represent functional activity because of the involvement of non-functional, misfolded, soluble aggregates, which compromise recombinant protein quality. Therefore, screening of solubility and folding competence is crucial for improving the quality of recombinant proteins, especially for therapeutic applications. The issue is often highlighted especially in bacterial recombinant hosts, since bacterial cytoplasm does not provide an optimal environment for the folding of target proteins of mammalian origin. Antibody fragments, such as single-chain variable fragment (scFv), single-chain antibody (scAb), and fragment antigen binding (Fab), have been utilized for numerous applications such as diagnostics, research reagents, or therapeutics. Antibody fragments can be efficiently expressed in microorganisms so that they offer several advantages for diagnostic applications such as low cost and high yield. However, scFv and scAb fragments have generally lower stability to thermal stress than full-length antibodies, necessitating a judicious combination of designer antibodies, and bacterial hosts harnessed with robust chaperone function. In this review, we discuss efforts on not only the production of antibodies or antibody fragments in microorganisms but also scFv stabilization via (i) directed evolution of variants with increased stability using display systems, (ii) stabilization of the interface between variable regions of heavy (V H ) and light (V L ) chains through the introduction of a non-native covalent bond between the two chains, (iii) rational engineering of V H -V L pair, based on the structure, and (iv) computational approaches. We also review recent advances in stability design, increase in avidity by multimerization, and maintaining the functional competence of chimeric proteins prompted by various types of chaperones.

Causality Assessment Guidelines for Adverse Events Following Immunization with a Focus on Guillain-Barré Syndrome.

South Korea operates a National Vaccine Injury Compensation Program (VICP) for people who experience adverse events following immunization (AEFI). To run this program rationally, it is a prerequisite to confirm whether adverse events were caused by immunization. Guillain-Barré syndrome (GBS), a severe neurological disease with limb pain and muscle weakness as cardinal symptoms, is attracting attention as an AEFI. However, algorithm or guidelines for assessing the causality between vaccination and the incidence of GBS are lacking. We aimed to develop guidelines for causality assessment of GBS as an AEFI and suggest using these guidelines in alignment with the VICP. We systematically searched for other previously published algorithms or guidelines and found a WHO-AEFI guideline used worldwide; however, it only provides general instructions and is not tailored to specific adverse events. We translated and locally adapted the structure of this guideline and then added contents related to GBS. The GBS-specific guideline consists of four steps: case ascertainment of GBS, checklist (including (1) order of incidence, (2) temporal proximity, (3) evidence for other causes and (4) published evidence), an algorithm, and final classification. We listed key information on confirming GBS and whether any other causes of GBS were present. For real world application of the guideline along with the VICP, we collaborated with a panel of neurologists, epidemiologic investigators, and committee members from the VICP. To ensure transparency and a scientific approach, regular updates and collaboration with neurologists are essential. We expect that this guideline will contribute to logical causality assessment and compensation decisions for GBS and will provide the basic structure for causality assessment of other AEFIs.

Bead based facile assay for sensitive quantification of native state green fluorescent protein.

A facile method for the quantification of native state protein is strongly required to accurately determine the amount of expressed protein of interest. Here we report a simple bead-based assay, which can sensitively quantify the amount of native state green fluorescent protein using Ni-NTA (nickel-nitrilotriacetic acid)-modified microbead particles. The bead-based method is simple and straightforward to perform and it showed a highly sensitive capability to detect the expressed fluorescent protein because of the enriched fluorescent protein on the beads.

The Quest for a Truly Universal Influenza Vaccine.

There is an unmet public health need for a universal influenza vaccine (UIV) to provide broad and durable protection from influenza virus infections. The identification of broadly protective antibodies and cross-reactive T cells directed to influenza viral targets present a promising prospect for the development of a UIV. Multiple targets for cross-protection have been identified in the stalk and head of hemagglutinin (HA) to develop a UIV. Recently, neuraminidase (NA) has received significant attention as a critical component for increasing the breadth of protection. The HA stalk-based approaches have shown promising results of broader protection in animal studies, and their feasibility in humans are being evaluated in clinical trials. Mucosal immune responses and cross-reactive T cell immunity across influenza A and B viruses intrinsic to live attenuated influenza vaccine (LAIV) have emerged as essential features to be incorporated into a UIV. Complementing the weakness of the stand-alone approaches, prime-boost vaccination combining HA stalk, and LAIV is under clinical evaluation, with the aim to increase the efficacy and broaden the spectrum of protection. Preexisting immunity in humans established by prior exposure to influenza viruses may affect the hierarchy and magnitude of immune responses elicited by an influenza vaccine, limiting the interpretation of preclinical data based on naive animals, necessitating human challenge studies. A consensus is yet to be achieved on the spectrum of protection, efficacy, target population, and duration of protection to define a "universal" vaccine. This review discusses the recent advancements in the development of UIVs, rationales behind cross-protection and vaccine designs, and challenges faced in obtaining balanced protection potency, a wide spectrum of protection, and safety relevant to UIVs.