Complement C3 Plays a Key Role in Inducing Humoral and Cellular Immune Responses to Influenza Virus Strain-Specific Hemagglutinin-Based or Cross-Protective M2 Extracellular Domain-Based Vaccination.

The complement pathway is involved in eliminating antigen immune complexes. However, the role of the C3 complement system remains largely unknown in influenza virus M2 extracellular (M2e) domain or hemagglutinin (HA) vaccine-mediated protection after vaccination. Using a C3 knockout (C3 KO) mouse model, we found that complement protein C3 was required for effective induction of immune responses to vaccination with M2e-based or HA-based vaccines, which include isotype class-switched antibodies and effector CD4 and CD8 T cell responses. C3 KO mice after active immunization with cross-protective nonneutralizing M2e-based vaccine were not protected against influenza virus, although low levels of M2e-specific antibodies were protective after passive coadministration with virus in wild-type mice. In contrast, C3 KO mice that were immunized with strain-specific neutralizing HA-based vaccine were protected against homologous virus challenge despite lower levels of HA antibody responses. C3 KO mice showed impaired maintenance of innate immune cells and a defect in innate immune responses upon exposure to antigens. The findings in this study suggest that C3 is required for effective induction of humoral and cellular adaptive immune responses as well as protective immunity after nonneutralizing influenza M2e vaccination.IMPORTANCE Complement is the well-known innate immune defense system involved in the opsonization and lysis of pathogens but is less studied in establishing adaptive immunity after vaccination. Influenza virus HA-based vaccination confers protection via strain-specific neutralizing antibodies, whereas M2e-based vaccination induces a broad spectrum of protection by immunity against the conserved M2e epitopes. This study revealed the critical roles of C3 complement in inducing humoral and cellular immune responses after immunization with M2e or HA vaccines. C3 was found to be required for protection by M2e-based but not by HA-based active vaccination as well as for maintaining innate antigen-presenting cells. Findings in this study have insight into better understanding the roles of C3 complement in inducing effective innate and adaptive immunity as well as in conferring protection by cross-protective conserved M2e vaccination.

Virus-Like Particle Vaccine Containing the F Protein of Respiratory Syncytial Virus Confers Protection without Pulmonary Disease by Modulating Specific Subsets of Dendritic Cells and Effector T Cells.

UNLABELLED: There is no licensed vaccine against respiratory syncytial virus (RSV) since the failure of formalin-inactivated RSV (FI-RSV) due to its vaccine-enhanced disease. We investigated immune correlates conferring protection without causing disease after intranasal immunization with virus-like particle vaccine containing the RSV fusion protein (F VLP) in comparison to FI-RSV and live RSV. Upon RSV challenge, FI-RSV immune mice showed severe weight loss, eosinophilia, and histopathology, and RSV reinfection also caused substantial RSV disease despite their viral clearance. In contrast, F VLP immune mice showed least weight loss and no sign of histopathology and eosinophilia. High levels of interleukin-4-positive (IL-4(+)) and tumor necrosis factor alpha-positive (TNF-α(+)) CD4(+) T cells were found in FI-RSV immune mice, whereas gamma interferon-positive (IFN-γ(+)) and TNF-α(+) CD4(+) T cells were predominantly detected in live RSV-infected mice. More importantly, in contrast to FI-RSV and live RSV that induced higher levels of CD11b(+) dendritic cells, F VLP immunization induced CD8α(+) and CD103(+) dendritic cells, as well as F-specific IFN-γ(+) and TNF-α(+) CD8(+) T cells. These results suggest that F VLP can induce protection without causing pulmonary RSV disease by inducing RSV neutralizing antibodies, as well as modulating specific subsets of dendritic cells and CD8 T cell immunity. IMPORTANCE: It has been a difficult challenge to develop an effective and safe vaccine against respiratory syncytial virus (RSV), a leading cause of respiratory disease. Immune correlates conferring protection but preventing vaccine-enhanced disease remain poorly understood. RSV F virus-like particle (VLP) would be an efficient vaccine platform conferring protection. Here, we investigated the protective immune correlates without causing disease after intranasal immunization with RSV F VLP in comparison to FI-RSV and live RSV. In addition to inducing RSV neutralizing antibodies responsible for clearing lung viral loads, we show that modulation of specific subsets of dendritic cells and CD8 T cells producing T helper type 1 cytokines are important immune correlates conferring protection but not causing vaccine-enhanced disease.

Roles of major histocompatibility complex class II in inducing protective immune responses to influenza vaccination.

Major histocompatibility complex class II-deficient (MHC-II KO; Aß(-/-)) mice were used to assess the roles of MHC-II molecules in inducing protective immune responses to vaccination. After vaccination with influenza A/PR8 virus-like particle (VLP) vaccine, in vivo and in vitro vaccine antigen-specific IgG isotype antibodies were not detected in MHC-II KO mice, which is quite different from CD4 T cell-deficient mice that induced vaccine-specific IgG antibodies. The deficiency in MHC-II did not significantly affect the induction of antigen-specific IgM antibody in sera. MHC-II KO mice that were vaccinated with influenza VLP, whole inactivated influenza virus, or live attenuated influenza virus vaccines were not protected against lethal infection with influenza A/PR8 virus. Adoptive transfer of fractionated spleen cells from wild-type mice to MHC-II KO mice indicated that CD43(+) cell populations with MHC-II contributed more significantly to producing vaccine-specific IgG antibodies than CD43(-) B220(+) conventional B cell or CD4 T cell populations, as well as conferring protection against lethal infection. Bone marrow-derived dendritic cells from MHC-II KO mice showed a significant defect in producing interleukin-6 and tumor necrosis factor alpha cytokines. Thus, results indicate that MHC-II molecules play multiple roles in inducing protective immunity to influenza vaccination. Importance: Major histocompatibility complex class II (MHC-II) has been known to activate CD4 T helper immune cells. A deficiency in MHC-II was considered to be equivalent to the lack of CD4 T cells in developing host immune responses to pathogens. However, the roles of MHC-II in inducing protective immune responses to vaccination have not been well understood. In the present study, we demonstrate that MHC-II-deficient mice showed much more significant defects in inducing protective antibody responses to influenza vaccination than CD4 T cell-deficient mice. Further analysis showed that CD43 marker-positive immune cells with MHC-II, as well as an innate immunity-simulating adjuvant, could rescue some defects in inducing protective immune responses in MHC-II-deficient mice. These results have important implications for our understanding of host immunity-inducing mechanisms to vaccination, as well as in developing effective vaccines and adjuvants.

Supplementation of influenza split vaccines with conserved M2 ectodomains overcomes strain specificity and provides long-term cross protection.

Current influenza vaccines do not provide good protection against antigenically different influenza A viruses. As an approach to overcome strain specificity of protection, this study demonstrates significantly improved long-term cross protection by supplementing split vaccines with a conserved molecular target, a repeat of the influenza M2 ectodomain (M2e) expressed on virus-like particles (M2e5x VLPs) in a membrane-anchored form. Intramuscular immunization with H1N1 split vaccine (A/California/07/2009) supplemented with M2e5x VLPs induced M2e-specific humoral and cellular immune responses, and shaped the host responses to the vaccine in the direction of T-helper type 1 responses inducing dominant IgG2a isotype antibodies as well as interferon-γ (IFN-γ) producing cells in systemic and mucosal sites. Upon lethal challenge, M2e5x VLP-supplemented vaccination lowered lung viral loads and induced long-term cross protection against H3N2 or H5N1 subtype influenza viruses over 12 months. M2e antibodies, CD4 T cells, and CD8 T cells were found to contribute to improving heterosubtypic cross protection. In addition, improved cross protection by supplemented vaccination with M2e5x VLPs was mediated via Fc receptors. The results support evidence that supplementation with M2e5x VLPs is a promising approach for overcoming the limitation of strain-specific protection by current influenza vaccination.

Virus-like nanoparticle and DNA vaccination confers protection against respiratory syncytial virus by modulating innate and adaptive immune cells.

Respiratory syncytial virus (RSV) is an important human pathogen. Expression of virus structural proteins produces self-assembled virus-like nanoparticles (VLP). We investigated immune phenotypes after RSV challenge of immunized mice with VLP containing RSV F and G glycoproteins mixed with F-DNA (FdFG VLP). In contrast to formalin-inactivated RSV (FI-RSV) causing vaccination-associated eosinophilia, FdFG VLP immunization induced low bronchoalveolar cellularity, higher ratios of CD11c(+) versus CD11b(+) phenotypic cells and CD8(+) T versus CD4(+) T cells secreting interferon (IFN)-γ, T helper type-1 immune responses, and no sign of eosinophilia upon RSV challenge. Furthermore, RSV neutralizing activity, lung viral clearance, and histology results suggest that FdFG VLP can be comparable to live RSV in conferring protection against RSV and in preventing RSV disease. This study provides evidence that a combination of recombinant RSV VLP and plasmid DNA may have a potential anti-RSV prophylactic vaccine inducing balanced innate and adaptive immune responses.

Distinct B-cell populations contribute to vaccine antigen-specific antibody production in a transgenic mouse model.

The generation of memory B cells by vaccination plays a critical role in maintaining antigen-specific antibodies and producing antibody responses upon re-exposure to a pathogen. B-cell populations contributing to antibody production and protection by vaccination remain poorly defined. We used influenza virus-like particle (VLP) vaccine in a transgenic mouse model that would identify germinal centre-derived memory B cells with the expression of yellow fluorescent protein (YFP(+) cells). Immunization with influenza VLP vaccine did not induce significant increases in YFP(+) cells although vaccine antigen-specific antibodies in sera were found to confer protection against a lethal dose of influenza A virus (A/PR8). In addition, CD43(+)  B220(-) populations with low YFP(+) cells mainly contributed to the production of vaccine antigen-specific IgG isotype-switched antibodies whereas CD43(-)  B220(+) populations with high YFP(+) cells were able to produce vaccine antigen-specific IgM antibodies. Challenge infection of immunized transgenic mice with live influenza A virus resulted in significant increases in YFP(+) cells in the B220(-) populations of spleen and bone marrow cells. These results suggest that CD43(+)  B220(-) B cells generated by vaccination are important for producing influenza vaccine antigen-specific antibodies and conferring protection.

Virus-like particles containing multiple M2 extracellular domains confer improved cross-protection against various subtypes of influenza virus.

The extracellular domain of M2 (M2e), a small ion channel membrane protein, is well conserved among different human influenza A virus strains. To improve the protective efficacy of M2e vaccines, we genetically engineered a tandem repeat of M2e epitope sequences (M2e5x) of human, swine, and avian origin influenza A viruses, which was expressed in a membrane-anchored form and incorporated in virus-like particles (VLPs). The M2e5x protein with the transmembrane domain of hemagglutinin (HA) was effectively incorporated into VLPs at a several 100-fold higher level than that on influenza virions. Intramuscular immunization with M2e5x VLP vaccines was highly effective in inducing M2e-specific antibodies reactive to different influenza viruses, mucosal and systemic immune responses, and cross-protection regardless of influenza virus subtypes in the absence of adjuvant. Importantly, immune sera were found to be sufficient for conferring protection in naive mice, which was long-lived and cross-protective. Thus, molecular designing and presenting M2e immunogens on VLPs provide a promising platform for developing universal influenza vaccines without using adjuvants.

Implementation of the remote measuring system for addiction patients in rehabilitation applying vital sensor.

Recently, with the rapid development of related ubiquitous industries, ubiquitous-Zone (u-Zone) development is being promoted to build a ubiquitous environment within a specific area. From a health care system perspective, in particular, u-Zone is expected to contribute to reducing cost and effort to manage patients' condition such as in-patients, addiction patients and mental patients. In contrast, the current health care system only targets specific persons or continues to expand the internal system of hospitals. As addiction patients are on the rise in terms of drug addiction, including alcohol and narcotics, behavioural addiction attributable to the exposure to games, gambling, Internet and mobile communications and shopping is also becoming a problem. That is why it is difficult to collect data for the daily addiction status, which causes difficulties in systematic management and accurate diagnosis. Therefore, this paper suggests a remote measuring system to collect continuous condition data, which monitors the addiction patients via the vital sign measuring sensor within u-Zone. That is, the system collects their condition information from the sensors measuring heart rate, body temperature and acceleration, based on which the specialists determine the patient's emotional state. These data are expected to become the basis of diagnosing and managing addiction patients.

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.

The bacterial fermentation product butyrate influences epithelial signaling via reactive oxygen species-mediated changes in cullin-1 neddylation.

The human enteric flora plays a significant role in intestinal health and disease. Populations of enteric bacteria can inhibit the NF-kappaB pathway by blockade of IkappaB-alpha ubiquitination, a process catalyzed by the E3-SCF(beta-TrCP) ubiquitin ligase. The activity of this ubiquitin ligase is regulated via covalent modification of the Cullin-1 subunit by the ubiquitin-like protein NEDD8. We previously reported that interaction of viable commensal bacteria with mammalian intestinal epithelial cells resulted in a rapid and reversible generation of reactive oxygen species (ROS) that modulated neddylation of Cullin-1 and resulted in suppressive effects on the NF-kappaB pathway. Herein, we demonstrate that butyrate and other short chain fatty acids supplemented to model human intestinal epithelia in vitro and human tissue ex vivo results in loss of neddylated Cul-1 and show that physiological concentrations of butyrate modulate the ubiquitination and degradation of a target of the E3- SCF(beta-TrCP) ubiquitin ligase, the NF-kappaB inhibitor IkappaB-alpha. Mechanistically, we show that physiological concentrations of butyrate induces reactive oxygen species that transiently alters the intracellular redox balance and results in inactivation of the NEDD8-conjugating enzyme Ubc12 in a manner similar to effects mediated by viable bacteria. Because the normal flora produces significant amounts of butyrate and other short chain fatty acids, these data provide a functional link between a natural product of the intestinal normal flora and important epithelial inflammatory and proliferative signaling pathways.

Broad cross protection by recombinant live attenuated influenza H3N2 seasonal virus expressing conserved M2 extracellular domain in a chimeric hemagglutinin.

Hemagglutinin (HA)-based current vaccines provide suboptimum cross protection. Influenza A virus contains an ion channel protein M2 conserved extracellular domain (M2e), a target for developing universal vaccines. Here we generated reassortant influenza virus rgH3N2 4xM2e virus (HA and NA from A/Switzerland/9715293/2013/(H3N2)) expressing chimeric 4xM2e-HA fusion proteins with 4xM2e epitopes inserted into the H3 HA N-terminus. Recombinant rgH3N2 4xM2e virus was found to retain equivalent growth kinetics as rgH3N2 in egg substrates. Intranasal single inoculation of mice with live rgH3N2 4xM2e virus was effective in priming the induction of M2e specific IgG antibody responses in mucosal and systemic sites as well as T cell responses. The rgH3N2 4xM2e primed mice were protected against a broad range of different influenza A virus subtypes including H1N1, H3N2, H5N1, H7N9, and H9N2. The findings support a new approach to improve the efficacy of current vaccine platforms by recombinant influenza virus inducing immunity to HA and cross protective M2e antigens.

Production of 3-nitrosoindole derivatives by Escherichia coli during anaerobic growth.

When Escherichia coli K-12 is grown anaerobically in medium containing tryptophan and sodium nitrate, it produces red compounds. The reaction requires functional genes for trytophanase (tnaA), a tryptophan permease (tnaB), and a nitrate reductase (narG), as well as a natural drop in the pH of the culture. Mass spectrometry revealed that the purified chromophores had mass/charge ratios that closely match those for indole red, indoxyl red, and an indole trimer. These compounds are known products of chemical reactions between indole and nitrous acid. They are derived from an initial reaction of 3-nitrosoindole with indole. Apparently, nitrite that is produced from the metabolic reduction of nitrate is converted in the acid medium to nitrous acid, which leads to the nitrosation of the indole that is generated by tryptophanase. An nfi (endonuclease V) mutant and a recA mutant were selectively killed during the period of chromophore production, and a uvrA strain displayed reduced growth. These effects depended on the addition of nitrate to the medium and on tryptophanase activity in the cells. Unexpectedly, the killing of a tnaA(+) nfi mutant was not accompanied by marked increases in mutation frequencies for several traits tested. The vulnerability of three DNA repair mutants indicates that a nitrosoindole or a derivative of a nitrosoindole produces lethal DNA damage.

Respiratory Syncytial Virus Fusion Protein-encoding DNA Vaccine Is Less Effective in Conferring Protection against Inflammatory Disease than a Virus-like Particle Platform.

Formalin-inactivated respiratory syncytial virus (RSV) vaccination causes vaccine-enhanced disease (VED) after RSV infection. It is considered that vaccine platforms enabling endogenous synthesis of RSV immunogens would induce favorable immune responses than non-replicating subunit vaccines in avoiding VED. Here, we investigated the immunogenicity, protection, and disease in mice after vaccination with RSV fusion protein (F) encoding plasmid DNA (F-DNA) or virus-like particles presenting RSV F (F-VLP). F-DNA vaccination induced CD8 T cells and RSV neutralizing Abs, whereas F-VLP elicited higher levels of IgG2a isotype and neutralizing Abs, and germinal center B cells, contributing to protection by controlling lung viral loads after RSV challenge. However, mice that were immunized with F-DNA displayed weight loss and pulmonary histopathology, and induced F specific CD8 T cell responses and recruitment of monocytes and plasmacytoid dendritic cells into the lungs. These innate immune parameters, RSV disease, and pulmonary histopathology were lower in mice that were immunized with F-VLP after challenge. This study provides important insight into developing effective and safe RSV vaccines.

Antigenicity and immunogenicity of unique prefusion-mimic F proteins presented on enveloped virus-like particles.

Pre-fusion stabilizing mutations (DS-Cav1) in soluble fusion (F) proteins of human respiratory syncytial virus (RSV) were previously reported. Here we investigated the antigenic and immunogenic properties of pre-fusion like RSV F proteins on enveloped virus-like particles (VLP). Additional mutations were introduced to DS-Cav1 (F-dcmTM VLP); fusion peptide deletion and cleavage mutation site 1 (F1d-dcmTM VLP) or both sites (F12d-dcmTM VLP). F1d-dcmTM VLP and F12d-dcmTM VLP displayed higher reactivity against pre-fusion specific site Ø and antigenic site I and II specific monoclonal antibodies, compared to F-dcmTM VLP with DS-Cav1 only. Mice immunized with F1d-dcmTM VLP and F12d-dcmTM VLP induced higher levels of DS-Cav1 pre-fusion specific IgG antibodies, RSV neutralizing activity titers, and effective lung viral clearance after challenge. These results suggest that cleavage site mutations and fusion peptide deletion in addition to DS-Cav1 mutations have contributed to structural stabilization of pre-fusion like F conformation on enveloped VLP, capable of inducing high levels of pre-fusion F specific and RSV neutralizing antibodies.

A unique combination adjuvant modulates immune responses preventing vaccine-enhanced pulmonary histopathology after a single dose vaccination with fusion protein and challenge with respiratory syncytial virus.

Alum adjuvanted formalin-inactivated respiratory syncytial virus (RSV) vaccination resulted in enhanced respiratory disease in young children upon natural infection. Here, we investigated the adjuvant effects of monophosphoryl lipid A (MPL) and oligodeoxynucleotide CpG (CpG) on vaccine-enhanced respiratory disease after fusion (F) protein prime vaccination and RSV challenge in infant and adult mouse models. Combination CpG + MPL adjuvant in RSV F protein single dose priming of infant and adult age mice was found to promote the induction of IgG2a isotype antibodies and neutralizing activity, and lung viral clearance after challenge. CpG + MPL adjuvanted F protein (Fp) priming of infant and adult age mice was effective in avoiding lung histopathology, in reducing interleukin-4+ CD4 T cells and cellular infiltration of monocytes and neutrophils after RSV challenge. This study suggests that combination CpG and MPL adjuvant in RSV subunit vaccination might contribute to priming protective immune responses and preventing inflammatory RSV disease after infection.

The efficacy of inactivated split respiratory syncytial virus as a vaccine candidate and the effects of novel combination adjuvants.

Clinical trials with alum-adjuvanted formalin-inactivated human respiratory syncytial virus (FI-RSV) vaccine failed in children due to vaccine-enhanced disease upon RSV infection. In this study, we found that inactivated, detergent-split RSV vaccine (Split) displayed higher reactivity against neutralizing antibodies in vitro and less histopathology in primed adult mice after challenge, compared to FI-RSV. The immunogenicity and efficacy of FI-RSV and Split RSV vaccine were further determined in 2 weeks old mice after a single dose in the absence or presence of monophosphoryl lipid A (MPL) + CpG combination adjuvant. Split RSV with MPL + CpG adjuvant was effective in increasing T helper type 1 (Th1) immune responses and IgG2a isotype antibodies, neutralizing activity, and lung viral clearance as well as modulating immune responses to prevent pulmonary histopathology after RSV vaccination and challenge. This study demonstrates the efficacy of Split RSV as an effective vaccine candidate.

Small RNA ArrF regulates the expression of sodB and feSII genes in Azotobacter vinelandii.

Azotobacter vinelandii contains a prrF-like sequence in a noncoding region of the chromosome. Like the Pseudomonas aeruginosa PrrF small RNA-encoding genes, the expression of the sequence, herein named arrF (Azotobacter regulatory RNA involving Fe), was increased 100-fold in wild-type cells in response to iron depletion. The level of ArrF was also increased to the same degree in the iron-replete fur mutant, but down back to a wild-type level when this fur mutant was complemented with the wild-type fur gene. These results, with the location of arrF gene in a noncoding region, suggest that this gene encodes an iron-responsive small RNA whose expression is negatively regulated by the Fur-Fe(2+) complex. Disruption of this arrF gene upregulated the expression of iron-containing superoxide dismutase and FeSII protein, whereas fur mutation or iron depletion decreased the level of their transcript. A short region in the 5'-untranslated region of each transcript was predicted to be quite complementary to the core sequence of ArrF, assuming that ArrF represses the expression of the genes under Fur control by an antisense RNA mechanism. However, unlike the P. aeruginosa PrrF that has extensive targets in the tricarboxylic acid cycle and glyoxylate cycle, ArrF had little effect on those genes. The findings that there is a poor overlap between ArrF and PrrF targets and that the FeSII gene, which is present only in the chromosome of nitrogen-fixing bacterial species, is controlled by ArrF suggest that ArrF might have unique targets, some of which are involved in N(2) fixation.

Age, Period, and Cohort Effects on Suicide Mortality in South Korea, 1992⁻2015.

Although the effects of age, period, and cohort (APC) on suicide are important, previous work in this area may have been invalid because of an identification problem. We analyzed these effects under three different scenarios to identify vulnerable groups and thus overcame the identification problem. We extracted the annual numbers of suicides from the National Death Register of Korea (1992⁻2015) and estimated the APC effects. The annual average suicide rates in 1992⁻2015 were 31.5 and 14.7 per 100,000 males and females, respectively. The APC effects on suicide were similar in both sexes. The age effect was clearly higher in older subjects, in contrast to the minimal changes apparent during earlier adulthood. The birth cohort effect showed an inverted U shape; a higher cohort effect was evident in females born in the early 1980s when period drift was larger than 3.7%/year. Period effect increased sharply during the early 1990s and 2000s. We found that elderly and young females may be at a particularly high risk of suicide in Korea.

Virus-Like Particles Are a Superior Platform for Presenting M2e Epitopes to Prime Humoral and Cellular Immunity against Influenza Virus.

Influenza virus M2 protein has a highly conserved ectodomain (M2e) as a cross-protective antigenic target. We investigated the antigenic and immunogenic properties of tandem repeat M2e (5xM2e) proteins and virus-like particles (5xM2e VLP) to better understand how VLP and protein platform vaccines induce innate and protective adaptive immune responses. Despite the high antigenic properties of 5xM2e proteins, the 5xM2e VLP was superior to 5xM2e proteins in inducing IgG2a isotype antibodies, T cell responses, plasma cells and germinal center B cells as well as in conferring cross protection. Mice primed with 5xM2e VLP were found to be highly responsive to 5xM2e protein boost, overcoming the low immunogenicity and protective efficacy of 5xM2e proteins. Immunogenic differences between VLPs and proteins in priming immune responses might be due to an intrinsic ability of 5xM2e VLP to stimulate dendritic cells secreting T helper type 1 (Th1) cytokines. We also found that 5xM2e VLP was effective in inducing inflammatory cytokines and chemokines, and in recruiting macrophages, monocytes, neutrophils, and CD11b⁺ dendritic cells at the injection site. Therefore, this study provides evidence that 5xM2e VLP is an effective vaccine platform, inducing cross-protection by stimulating innate and adaptive immune responses.

Virus-like particles presenting flagellin exhibit unique adjuvant effects on eliciting T helper type 1 humoral and cellular immune responses to poor immunogenic influenza virus M2e protein vaccine.

Current licensed adjuvants including aluminum hydroxide (alum) bias immune responses toward T helper type 2 (Th2) immune responses. We tested whether virus-like particles presenting flagellin (Flag VLP) exhibit adjuvant effects on eliciting Th1 type immune responses and improving the efficacy of poor immunogenic tandem repeat M2e (M2e5x) protein vaccine against influenza virus. Co-immunization of mice with Flag VLP and M2e5x protein vaccine induced significantly higher levels of IgG2a isotype (Th1) antibodies in sera and mucosal sites, effector CD4+ T cells secreting IFN-γ and granzyme B, and more effective lung viral clearance and protection compared to alum adjuvant. Flag VLP stimulated primary macrophages and dendritic cells to secrete inflammatory cytokines, which is partially dependent on the Toll-like receptor 5. This study provides insight into developing effective vaccine adjuvants.