Eliciting unnatural immune responses by activating cryptic epitopes in viral antigens.

Antigenic variation in viral surface antigens is a strategy for escaping the host's adaptive immunity, whereas regions with pivotal functions for infection are less subject to antigenic variability. We hypothesized that genetically invariable and immunologically dormant regions of a viral surface antigen could be exposed to the host immune system and activated by rendering them susceptible to antigen-processing machinery in professional antigen-presenting cells (APCs). Considering the frequent antigen drift and shift in influenza viruses, we identified and used structural modeling to evaluate the conserved regions on the influenza hemagglutinin (HA) surface as potential epitopes. Mutant viruses containing the cleavage motifs of cathepsin S within HA were generated. Immunization of mice showed that the mutant, but not the wild-type virus, elicited specific antibodies against the cryptic epitope. Those antibodies were purified, and specific binding to HA was confirmed. These results suggest that an unnatural immune response can be elicited through the processing of target antigens in APCs, followed by presentation via the major histocompatibility complex, if not subjected to regulatory pathways. By harnessing the antigen-processing machinery, our study shows a proof-of-principle for designer vaccines with increased efficacy and safety by either activating cryptic, or inactivating naturally occurring, epitopes of viral antigens.-Lee, Y. J., Yu, J. E., Kim, P., Lee, J.-Y., Cheong, Y. C., Lee, Y. J., Chang, J., Seong, B. L. Eliciting unnatural immune responses by activating cryptic epitopes in viral antigens.

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.

Development of a diagnostic system for detection of specific antibodies and antigens against Middle East respiratory syndrome coronavirus.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a single-stranded RNA virus that causes severe respiratory disease in humans with a high fatality rate. Binding of the receptor binding domain (RBD) of the spike (S) glycoprotein to dipeptidyl peptidase 4 is the critical step in MERS-CoV infection of a host cell. No vaccines or clinically applicable treatments are currently available for MERS-CoV. Therefore, rapid diagnosis is important for improving patient outcomes through prompt treatment and protection against viral outbreaks. In this study, the aim was to establish two ELISA systems for detecting antigens and antibodies against MERS-CoV. Using a recombinant full-length S protein, an indirect ELISA was developed and found to detect MERS-CoV-specific antibodies in animal sera and sera of patient with MERS. Moreover, MAbs were induced with the recombinant S protein and RBD and used for sandwich ELISA to detect the MERS-CoV S protein. Neither ELISA system exhibited significant intra-assay or inter-assay variation, indicating good reproducibility. Moreover, the inter-day precision and sensitivity were adequate for use as a diagnostic kit. Thus, these ELISAs can be used clinically to diagnose MERS-CoV.

Recombinant adenylate kinase 3 from liver fluke Clonorchis sinensis for histochemical analysis and serodiagnosis of clonorchiasis.

Due to the lack of an effective prophylactic intervention and diagnosis, human liver fluke Clonorchis sinensis continues to afflict a large human population, causing a chronic inflammatory bile duct disease. With an aim to identify target antigens for sensitive serodiagnosis, adenylate kinase 3 of C. sinensis (CsAK3) was successfully expressed in soluble form in Escherichia coli by fusion to an RNA-interacting domain derived from human Lys-tRNA synthetase and purified by Ni2+-affinity chromatography. Anti-CsAK3 serum was raised by immunization of mice, and Western blotting confirmed that CsAK3 was expressed in adult-stage C. sinensis. Histochemical analysis showed that CsAK3 was localized to the subtegumental tissue of C. sinensis and was excreted into the bile duct of the host. When tested against sera from various parasite-infected patients by enzyme-linked immunosorbent assay, the recombinant CsAK3 elicited a specific response to C. sinensis-infected sera. The results suggest that CsAK3, either alone or in combination with other antigens, could be used for improving the clinical diagnosis of clonorchiasis.

The folding competence of HIV-1 Tat mediated by interaction with TAR RNA.

The trans-activator Tat protein of HIV-1 belongs to the large family of intrinsically disordered proteins (IDPs), and is known to recruit various host proteins for the transactivation of viral RNA synthesis. Tat protein interacts with the transactivator response RNA (TAR RNA), exhibiting RNA chaperone activities for structural rearrangement of interacting RNAs. Here, considering that Tat-TAR RNA interaction is mutually cooperative, we examined the potential role of TAR RNA as Chaperna - RNA that provides chaperone function to proteins - for the folding of HIV-1 Tat. Using EGFP fusion as an indirect indicator for folding status, we monitored Tat-EGFP folding in HeLa cells via time-lapse fluorescence microscopy. The live cell imaging showed that the rate and the extent of folding of Tat-EGFP were stimulated by TAR RNA. The purified Tat-EGFP was denatured and the fluorescence was monitored in vitro under renaturation condition. The fluorescence was significantly increased by TAR RNA, and the mutations in TAR RNA that affected the interaction with Tat protein failed to promote Tat refolding. The results suggest that TAR RNA stabilizes Tat as unfolded, but prevents it from misfolding, and maintaining its folding competence for interaction with multiple host factors toward its transactivation. The Chaperna function of virally encoded RNA in establishing proteome link at the viral-host interface provides new insights to as yet largely unexplored RNA mediated protein folding in normal and dysregulated cellular metabolism.

Identification of a Small Benzamide Inhibitor of Influenza Virus Using a Cell-Based Screening.

BACKGROUND: The zoonotic transmission of highly pathogenic avian influenza viruses and the global pandemic of H1N1 influenza in 2009 signified the need for a wider coverage of therapeutic options for the control of influenza. METHODS: An in-house compound library was screened using a cytopathic effect inhibition assay. Selected hits were then tested in vivo and used as a core skeleton for derivative synthesis. RESULTS: The hit compound (BMD-2601505) was effective [50% effective concentration (EC50) of 60-70 µM] in reducing the death rate of cells infected with human influenza A and B viruses as well as avian influenza A virus. Furthermore, BMD-2601505 reduced the weight loss and increased the survival after lethal infection. The compound was further modified to enhance its antiviral potency. Results show that one derivative with bromobenzene moiety was most effective (EC50 of 22-37 µM) against the influenza viruses tested. CONCLUSION: We identified a small benzamide compound exhibiting antiviral activity against influenza viruses. The results warrant further evaluation of antiviral activities against drug-resistant influenza isolates.

Intranasal adenovirus-vectored vaccine for induction of long-lasting humoral immunity-mediated broad protection against influenza in mice.

UNLABELLED: Influenza vaccines aimed at inducing antibody (Ab) responses against viral surface hemagglutinin (HA) and neuraminidase (NA) provide sterile immunity to infection with the same subtypes. Vaccines targeting viral conserved determinants shared by the influenza A viruses (IAV) offer heterosubtypic immunity (HSI), a broad protection against different subtypes. We proposed that vaccines targeting both HA and the conserved ectodomain of matrix protein 2 (M2e) would provide protection against infection with the same subtype and also HSI against other subtypes. We report here that single intranasal immunization with a recombinant adenovirus (rAd) vector encoding both HA of H5 virus and M2e (rAdH5/M2e) induced significant HA- and M2e-specific Ab responses, along with protection against heterosubtypic challenge in mice. The protection is superior compared to that induced by rAd vector encoding either HA (rAdH5), or M2e (rAdM2e). While protection against homotypic H5 virus is primarily mediated by virus-neutralizing Abs, the cross-protection is associated with Abs directed to conserved stalk HA and M2e that seem to have an additive effect. Consistently, adoptive transfer of antisera induced by rAdH5/M2e provided the best protection against heterosubtypic challenge compared to that provided by antisera derived from mice immunized with rAdH5 or rAdM2e. These results support the development of rAd-vectored vaccines encoding both H5 and M2e as universal vaccines against different IAV subtypes. IMPORTANCE: Current licensed influenza vaccines provide protection limited to the infection with same virus strains; therefore, the composition of influenza vaccines has to be revised every year. We have developed a new universal influenza vaccine that is highly efficient in induction of long-lasting cross-protection against different influenza virus strains. The cross-protection is associated with a high level of vaccine-induced antibodies against the conserved stalk domain of influenza virus hemagglutinin and the ectodomain of matrix protein. The vaccine could be used to stimulate cross-protective antibodies for the prevention and treatment of influenza with immediate effect for individuals who fail to respond to or receive the vaccine in due time. The vaccine offers a new tool to control influenza outbreaks, including pandemics.

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.

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.

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.

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.

Cold-adapted pandemic 2009 H1N1 influenza virus live vaccine elicits cross-reactive immune responses against seasonal and H5 influenza A viruses.

The rapid transmission of the pandemic 2009 H1N1 influenza virus (pH1N1) among humans has raised the concern of a potential emergence of reassortment between pH1N1 and highly pathogenic influenza strains, especially the avian H5N1 influenza virus. Here, we report that the cold-adapted pH1N1 live attenuated vaccine (CApH1N1) elicits cross-reactive immunity to seasonal and H5 influenza A viruses in the mouse model. Immunization with CApH1N1 induced both systemic and mucosal antibodies with broad reactivity to seasonal and H5 strains, including HAPI H5N1 and the avian H5N2 virus, providing complete protection against heterologous and heterosubtypic lethal challenges. Our results not only accentuate the merit of using live attenuated influenza virus vaccines in view of cross-reactivity but also represent the potential of CApH1N1 live vaccine for mitigating the clinical severity of infections that arise from reassortments between pH1N1 and highly pathogenic H5 subtype viruses.

Type I interferon signaling regulates Ly6C(hi) monocytes and neutrophils during acute viral pneumonia in mice.

Type I interferon (IFN-I) plays a critical role in the homeostasis of hematopoietic stem cells and influences neutrophil influx to the site of inflammation. IFN-I receptor knockout (Ifnar1⁻/⁻) mice develop significant defects in the infiltration of Ly6C(hi) monocytes in the lung after influenza infection (A/PR/8/34, H1N1). Ly6C(hi) monocytes of wild-type (WT) mice are the main producers of MCP-1 while the alternatively generated Ly6C(int) monocytes of Ifnar1⁻/⁻ mice mainly produce KC for neutrophil influx. As a consequence, Ifnar1⁻/⁻ mice recruit more neutrophils after influenza infection than do WT mice. Treatment of IFNAR1 blocking antibody on the WT bone marrow (BM) cells in vitro failed to differentiate into Ly6C(hi) monocytes. By using BM chimeric mice (WT BM into Ifnar1⁻/⁻ and vice versa), we confirmed that IFN-I signaling in hematopoietic cells is required for the generation of Ly6C(hi) monocytes. Of note, WT BM reconstituted Ifnar1⁻/⁻ chimeric mice with increased numbers of Ly6C(hi) monocytes survived longer than influenza-infected Ifnar1⁻/⁻ mice. In contrast, WT mice that received Ifnar1⁻/⁻ BM cells with alternative Ly6C(int) monocytes and increased numbers of neutrophils exhibited higher mortality rates than WT mice given WT BM cells. Collectively, these data suggest that IFN-I contributes to resistance of influenza infection by control of monocytes and neutrophils in the lung.

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.

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.

Evaluation of the antiviral activity of a green tea solution as a hand-wash disinfectant.

Based on the broad-spectrum antiviral effect of green tea catechins, we established an experimental skin contact model for influenza virus transmission and evaluated the use of a green tea solution as a first-hand disinfectant. The infectivity of the virus on the skin cell layer became obsolete when washed with the green tea solution. The skin contact model could be applied to develop non-pharmaceutical intervention measures for reducing human transmission of the influenza virus.

Risk factors affecting seroconversion after influenza A/H1N1 vaccination in hemodialysis patients.

BACKGROUND: Hemodialysis (HD) patients have multiple causes of immune dysfunction and poor immune response to influenza vaccination. We investigated the antibody response rate to a pandemic H1N1/2009 influenza vaccination and clinical parameters influencing the induction of antibody responses in HD patients. METHODS: A total of 114 HD patients were vaccinated with a monovalent adjuvanted H1N1 inactivated influenza vaccine. Titers of neutralizing antibodies were evaluated by hemagglutination inhibition (HI) assay at pre- and 4 weeks after vaccination. Seroconversion was defined as either a pre-vaccination HI titer < 1:10 and a post vaccination HI titer > 1:40 or a pre-vaccination HI titer ≥ 1:10 and a minimum four-fold rise in post-vaccination HI antibody titer. Seventeen out of 114 HD patients (14.9%) tested positive for antibodies against influenza A/H1N1/2009 before vaccination. The remaining 97 baseline sero-negative patients were included in the analysis. RESULTS: Only 30 (30.9%) HD patients had seroconversion 4 weeks after vaccination. The elderly patients, those over 65 years of age, showed significantly lower seroconversion rate compared to younger HD patients (20.5% vs. 39.6%, p = 0.042). Furthermore, patients with hemoglobin values less than 10 g/dL had a significantly lower seroconversion rate compared to those with higher hemoglobin values (20.0 vs. 38.6%, p = 0.049). By multivariate logistic regression analysis, only age ≥65 years (OR = 0.336, 95% confidence interval (CI) 0.116-0.971, p = 0.044) and hemoglobin levels <10 g/dL (OR = 0.315, 95% CI 0.106-0.932, p = 0.037) were independently associated with seroconversion after vaccination. CONCLUSIONS: Our data show that HD patients, especially who are elderly with low hemoglobin levels, are at increased risk for lower seroconversion rate after influenza A/H1N1 vaccination. Further studies are needed to improve the efficacy of vaccination in these high risk patients.

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.

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 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.