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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the etiological agent of COVID-19, causes life-threatening disease. This novel coronavirus enters host cells via the respiratory tract, promoting the formation of severe pulmonary lesions and systemic disease. Few animal models can simulate the clinical signs and pathology of COVID-19 patients. Diverse preclinical studies using K18-hACE2 mice and Syrian golden hamsters, which are highly permissive to SARS-CoV-2 in the respiratory tract, are emerging; however, the systemic pathogenesis and cellular tropism of these models remain obscure. We intranasally infected K18-hACE2 mice and Syrian golden hamsters with SARS-CoV-2, and compared the clinical features, pathogenesis, cellular tropism and infiltrated immune-cell subsets. In K18-hACE2 mice, SARS-CoV-2 persistently replicated in alveolar cells and caused pulmonary and extrapulmonary disease, resulting in fatal outcomes. Conversely, in Syrian golden hamsters, transient SARS-CoV-2 infection in bronchial cells caused reversible pulmonary disease, without mortality. Our findings provide comprehensive insights into the pathogenic spectrum of COVID-19 using preclinical models.

The most widely used influenza vaccines are prepared by chemical inactivation. However, chemical, especially formalin, treatment-induced modifications of the antigenic structure of the virus are frequently associated with adverse effects including low efficacy of protection, unexpected immune responses, or exacerbation of disease. Gamma-irradiation was suggested as an alternative influenza virus inactivation method due to its great features of completely inactivating virus while not damaging the structures of protein antigens, and cross-protective ability against heterologous strains. However, immunological features of gamma radiation-inactivated influenza vaccine have not been fully understood. In this study, we aimed to investigate the humoral and cellular immune responses of gamma radiation-inactivated influenza vaccine. The gamma irradiation-inactivated influenza vaccine (RADVAX(FluA)) showed complete viral inactivation but retained normal viral structure with functional activities of viral protein antigens. Intranasal immunization of RADVAX(FluA) provided better protection against influenza virus infection than formalin-inactivated influenza virus (FIV) in mice. RADVAX(FluA) greatly enhanced the production of virus-specific serum IgG and alveolar mucosal IgA, which effectively neutralized HA (hemagglutinin) and NA (neuraminidase) activities, and blocked viral binding to the cells, respectively. Further analysis of IgG subclasses showed RADVAX(FluA)-immunized sera had higher levels of IgG1 and IgG2a than those of FIV-immunized sera. In addition, analysis of cellular immunity found RADVAX(FluA) induced strong dendritic cells (DC) activation resulting in higher DC-mediated activation of CD8(+) T cells than FIV. The results support improved immunogenicity by RADVAX(FluA).

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in significant morbidity and mortality worldwide. Despite a successful vaccination programme, the emergence of mutated variants that can escape current levels of immunity mean infections continue. Herein, we report the development of CT-P63, a broad-spectrum neutralizing monoclonal antibody. In vitro studies demonstrated potent neutralizing activity against the most prevalent variants, including Delta and the BA.1 and BA.2 sub-lineages of Omicron. In a transgenic mouse model, prophylactic CT-P63 significantly reduced wild-type viral titres in the respiratory tract and CT-P63 treatment proved efficacious against infection with Beta, Delta, and Omicron variants of SARS-CoV-2 with no detectable infectious virus in the lungs of treated animals. A randomized, double-blind, parallel-group, placebo-controlled, Phase I, single ascending dose study in healthy volunteers (NCT05017168) confirmed the safety, tolerability, and pharmacokinetics of CT-P63. Twenty-four participants were randomized and received the planned dose of CT-P63 or placebo. The safety and tolerability of CT-P63 were evaluated as primary objectives. Eight participants (33.3%) experienced a treatment-emergent adverse event (TEAE), including one grade ≥3 (blood creatine phosphokinase increased). There were no deaths, treatment-emergent serious adverse events, TEAEs of special interest, or TEAEs leading to study drug discontinuation in the CT-P63 groups. Serum CT-P63 concentrations rapidly peaked before declining in a biphasic manner and systemic exposure was dose proportional. Overall, CT-P63 was clinically safe and showed broad-spectrum neutralizing activity against SARS-CoV-2 variants in vitro and in vivo.

Highlights • RNA adjuvant was developed from the CrPV intergenic region IRES. • The RNA adjuvant functioned as an adjuvant with protein-based vaccines. • The RNA adjuvant increased vaccine efficacy and induced balanced Th1/Th2 response. • The RNA adjuvant enhanced APC chemotaxis., An ideal adjuvant should increase vaccine efficacy through balanced Th1/Th2 responses and be safe to use. Recombinant protein-based vaccines are usually formulated with aluminum (alum)-based adjuvants to ensure an adequate immune response. However, use of alum triggers a Th2-biased immune induction, and hence is not optimal. Although the adjuvanticity of RNA has been reported, a systematic and overall investigation on its efficacy is lacking. We found that single strand RNA (termed RNA adjuvant) derived from cricket paralysis virus intergenic region internal ribosome entry site induced the expression of various adjuvant-function-related genes, such as type 1 and 2 interferon (IFN) and toll-like receptor (TLR), T cell activation, and leukocyte chemotaxis in human peripheral blood mononuclear cells; furthermore, its innate and IFN transcriptome profile patterns were similar to those of a live-attenuated yellow fever vaccine. This suggests that protein-based vaccines formulated using RNA adjuvant function as live-attenuated vaccines. Application of the RNA adjuvant in mouse enhanced the efficacy of Middle East respiratory syndrome spike protein, a protein-subunit vaccine and human papillomavirus L1 protein, a virus-like particle vaccine, by activating innate immune response through TLR7 and enhancing pAPC chemotaxis, leading to a balanced Th1/Th2 responses. Moreover, the combination of alum and the RNA adjuvant synergistically induced humoral and cellular immune responses and endowed long-term immunity. Therefore, RNA adjuvants have broad applicability and can be used with all conventional vaccines to improve vaccine efficacy qualitatively and quantitively.

The development of a vaccine to prevent Zika virus (ZIKV) infection has been one of the priorities in infectious disease research in recent years. There have been numerous attempts to develop an effective vaccine against ZIKV. It is imperative to choose the safest and the most effective ZIKV vaccine from all candidate vaccines to control this infection globally. We have employed a dual serotype of prime-boost recombinant vesicular stomatitis virus (VSV) vaccine strategy, to develop a ZIKV vaccine candidate, using a type 1 IFN-receptor knock-out (Ifnar −/−) mouse model for challenge studies. Prime vaccination with an attenuated recombinant VSV Indiana serotype (rVSVInd) carrying a genetically modified ZIKV envelope (E) protein gene followed by boost vaccination with attenuated recombinant VSV New Jersey serotype (rVSVNJ) carrying the same E gene induced robust adaptive immune responses. In particular, rVSV carrying the ZIKV E gene with the honeybee melittin signal peptide (msp) at the N terminus and VSV G protein transmembrane domain and cytoplasmic tail (Gtc) at the C terminus of the E gene induced strong protective immune responses. This vaccine regimen induced highly potent neutralizing antibodies and T cell responses in the absence of an adjuvant and protected Ifnar -/- mice from a lethal dose of the ZIKV challenge.

The development of safe and effective vaccines to prevent SARS-CoV-2 infections remains an urgent priority worldwide. We have used a recombinant vesicular stomatitis virus (rVSV)-based prime-boost immunization strategy to develop an effective COVID-19 vaccine candidate. We have constructed VSV genomes carrying exogenous genes resulting in the production of avirulent rVSV carrying the full-length spike protein (SF), the S1 subunit, or the receptor-binding domain (RBD) plus envelope (E) protein of SARS-CoV-2. Adding the honeybee melittin signal peptide (msp) to the N-terminus enhanced the protein expression, and adding the VSV G protein transmembrane domain and the cytoplasmic tail (Gtc) enhanced protein incorporation into pseudotype VSV. All rVSVs expressed three different forms of SARS-CoV-2 spike proteins, but chimeras with VSV-Gtc demonstrated the highest rVSV-associated expression. In immunized mice, rVSV with chimeric S protein-Gtc derivatives induced the highest level of potent neutralizing antibodies and T cell responses, and rVSV harboring the full-length msp-SF-Gtc proved to be the superior immunogen. More importantly, rVSV-msp-SF-Gtc vaccinated animals were completely protected from a subsequent SARS-CoV-2 challenge. Overall, we have developed an efficient strategy to induce a protective response in SARS-CoV-2 challenged immunized mice. Vaccination with our rVSV-based vector may be an effective solution in the global fight against COVID-19., Author summary The COVID-19 pandemic has had unprecedented global health, economic and societal impact globally. Vaccinating the majority of the world’s population is the best way to help prevent new infections. Many vaccines have been developed to prevent various viral diseases that are currently in use around the world. This has generated a high demand for these vaccines, putting a strain on production capacity and delivery. With new variants of concern starting to dominate the human pandemic, new derivatives of the current vaccines may be necessary for continued protection from SARS-CoV-2 infection. We have developed a vaccine that uses a safe vesicular stomatitis virus-based delivery vehicle to present a key SARS-CoV-2 protein to our immune system in order to train it to recognize and prevent SARS-CoV-2 infection. Our vaccine completely protected vaccinated animals from SARS-CoV-2 infection and significantly reduced lung damage, a major hallmark of COVID-19.

Coronavirus is an RNA virus with a higher mutation rate than DNA viruses. Therefore, a mutation in S-protein, which mediates viral infection by binding to a human cellular receptor, is expected to cause difficulties in vaccine development. Given that DNA-protein vaccines promote stronger cell-mediated immune responses than protein-only vaccination, we immunized mice with various combinations of DNA priming and protein boosting using the S-subunit sequences of the MERS-CoV EMC/2012 strain. We demonstrated a cross-protective effect against wild-type KOR/KNIH/002, a strain with two mutations in the S amino acids, including one in its RBD. The vaccine also provided cross-neutralization against 15 different S-pseudotyped viruses. These suggested that a vaccine targeting one variant of S can provide cross-protection against multiple viral strains with mutations in S. The regimen of DNA priming/Protein boosting can be applied to the development of other coronavirus vaccines., Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory illness and has a high mortality of ∼34%. However, since its discovery in 2012, an effective vaccine has not been developed for it. To develop a vaccine against multiple strains of MERS-CoV, we targeted spike glycoprotein (S) using prime-boost vaccination with DNA and insect cell-expressed recombinant proteins for the receptor-binding domain (RBD), S1, S2, SΔTM, or SΔER. Our S subunits were generated using an S sequence derived from the MERS-CoV EMC/2012 strain. We examined humoral and cellular immune responses of various combinations with DNA plasmids and recombinant proteins in mice. Mouse sera immunized with SΔER DNA priming/SΔTM protein boosting showed cross-neutralization against 15 variants of S-pseudovirions and the wild-type KOR/KNIH/002 strain. In addition, these immunizations provided full protection against the KOR/KNIH/002 strain challenge in human DPP4 knock-in mice. These findings suggest that vaccination with the S subunits derived from one viral strain can provide cross-protection against variant MERS-CoV strains with mutations in S. DNA priming/protein boosting increased gamma interferon production, while protein-alone immunization did not. The RBD subunit alone was insufficient to induce neutralizing antibodies, suggesting the importance of structural conformation. In conclusion, heterologous DNA priming with protein boosting is an effective way to induce both neutralizing antibodies and cell-mediated immune responses for MERS-CoV vaccine development. This study suggests a strategy for selecting a suitable platform for developing vaccines against MERS-CoV or other emerging coronaviruses. IMPORTANCE Coronavirus is an RNA virus with a higher mutation rate than DNA viruses. Therefore, a mutation in S-protein, which mediates viral infection by binding to a human cellular receptor, is expected to cause difficulties in vaccine development. Given that DNA-protein vaccines promote stronger cell-mediated immune responses than protein-only vaccination, we immunized mice with various combinations of DNA priming and protein boosting using the S-subunit sequences of the MERS-CoV EMC/2012 strain. We demonstrated a cross-protective effect against wild-type KOR/KNIH/002, a strain with two mutations in the S amino acids, including one in its RBD. The vaccine also provided cross-neutralization against 15 different S-pseudotyped viruses. These suggested that a vaccine targeting one variant of S can provide cross-protection against multiple viral strains with mutations in S. The regimen of DNA priming/Protein boosting can be applied to the development of other coronavirus vaccines.

Highlights • Identification of neutralizing mAbs using MERS-CoV spike-pseudotyped virus. • Transmembrane domain-deleted spike subunit protein induced neutralizing antibodies. • Neutralizing antibodies could bind to RBD of MERS-CoV spike, but not vice versa. • Mutation in residue 506–509 or 529 of S elicits neutralization escape of MERS-CoV. • Our mAbs can be utilized for identification of specific mutation of MERS-CoV., Middle East Respiratory Syndrome coronavirus (MERS-CoV) causes severe pulmonary infection, with ∼35 % mortality. Spike glycoprotein (S) of MERS-CoV is a key target for vaccines and therapeutics because S mediates viral entry and membrane-fusion to host cells. Here, four different S subunit proteins, receptor-binding domain (RBD; 358–606 aa), S1 (1–751 aa), S2 (752–1296 aa), and SΔTM (1–1296 aa), were generated using the baculoviral system and immunized in mice to develop neutralizing antibodies. We developed 77 hybridomas and selected five neutralizing mAbs by immunization with SΔTM against MERS-CoV EMC/2012 strain S-pseudotyped lentivirus. However, all five monoclonal antibodies (mAb) did not neutralize the pseudotyped V534A mutation. Additionally, one mAb RBD-14F8 did not show neutralizing activity against pseudoviruses with amino acid substitution of L506 F or D509 G (England1 strain, EMC/2012 L506 F, and EMC/2012 D509 G), and RBD-43E4 mAb could not neutralize the pseudotyped I529 T mutation, while three other neutralizing mAbs showed broad neutralizing activity. This implies that the mutation in residue 506–509, 529, and 534 of S is critical to generate neutralization escape variants of MERS-CoV. Interestingly, all five neutralizing mAbs have binding affinity to RBD, although most mAbs generated by RBD did not have neutralizing activity. Additionally, chimeric antibodies of RBD-14F8 and RBD-43E4 with human Fc and light chain showed neutralizing effect against wild type MERS-CoV KOR/KNIH/002, similar to the original mouse mAbs. Thus, our mAbs can be utilized for the identification of specific mutations of MERS-CoV.

There are several broadly neutralizing monoclonal antibodies that neutralize influenza viruses with different mechanisms from traditional polyclonal antibodies induced by vaccination. CT149, which is one of the broadly neutralizing antibodies, was also previously reported to neutralize group 2 and some of group 1 influenza viruses (13 out of 13 tested group 2 viruses and 5 out of 11 group 1 viruses). In this study, we developed another antibody with the aim of compensating partial coverage of CT149 against group 1 influenza viruses. CT120 was screened among different antibody candidates and mixed with CT149. Importantly, although the binding sites of CT120 and CT149 are close to each other, the two antibodies do not interfere. The mixture of CT120 and CT149, which we named as CT-P27, showed broad efficacy by neutralizing 37 viruses from 11 different subtypes, of both group 1 and 2 influenza A viruses. Moreover, CT-P27 showed in vivo therapeutic efficacy, long prophylactic potency, and synergistic effect with oseltamivir in influenza virus-challenged mouse models. Our findings provide a novel therapeutic opportunity for more efficient treatment of influenza.

Developing effective mucosal subunit vaccine for the Streptococcus pneumoniae has been unsuccessful mainly because of their poor immunogenicity with insufficient memory T and B cell responses. We thus address whether such limitation can be overcome by introducing effective adjuvants that can enhance immunity and show here that polysorbitol transporter (PST) serves as a mucosal adjuvant for a subunit vaccine against the Streptococcus pneumoniae. Pneumococcal surface protein A (PspA) with PST adjuvant induced protective immunity against S. pneumoniae challenge, especially long-term T and B cell immune responses. Moreover, we found that the PST preferentially induced T helper (Th) responses toward Th2 or T follicular helper (Tfh) cells and, importantly, that the responses were mediated through antigen-presenting cells via activating a peroxisome proliferator-activated receptor gamma (PPAR-γ) pathway. Thus, these data indicate that PST can be used as an effective and safe mucosal vaccine adjuvant against S. pneumoniae infection. STATE OF SIGNIFICANCE: In this study, we suggested the nanoparticle forming adjuvant, PST works as an effective adjuvant for the pneumococcal vaccine, PspA. The PspA subunit vaccine together with PST adjuvant efficiently induced protective immunity, even in the long-term memory responses, against Streptococcus pneumoniae lethal challenge. We found that PspA with PST adjuvant induced dendritic cell activation followed by follicular helper T cell responses through PPAR-γ pathway resulting long-term memory antibody-producing cells. Consequently, in this paper, we suggest the mechanism for safe nanoparticle forming subunit vaccine adjuvant against pneumococcal infection.

Background The pandemic strain of the influenza A virus (pH1N1) in 2009 caused many complications in patients. In this study, we introduce asthmatic symptoms as a complication of pH1N1 infection in children, not having a relationship with asthma history. The aim of this study was to quantify asthmatic symptoms in pH1N1-infected children and elucidate the underlying mechanisms of airway hyper-responsiveness (AHR) induced in a murine model of pH1N1 infection. Methods As a retrospective study, pH1N1-infected children who were hospitalized with moderate to severe acute asthmatic symptoms were enrolled and administered a methacholine challenge test (MCT) at 3 months post-discharge. Additionally, the induction of AHR by pH1N1 infection was measured by MCT in wild-type and Rag1−/− mice. The effect of the innate immune response on the development of AHR following pH1N1 infection was investigated. Results More than 70% of the pH1N1-infected children without a pre-infection diagnosis of asthma had a negative response on the MCT. None of these children had recurrent wheezing or asthma during the 3 years following pH1N1 infection. The development of AHR in pH1N1-infected mice was associated with an elevation in IL-33 and innate lymphoid cells 2 (ILC2). Conclusions This study demonstrates that pH1N1 infection directly induces transient asthmatic symptoms in patients regardless of their medical history. pH1N1 infection was shown to stimulate the rapid development of AHR and Th2-type cytokine secretion in mice via the activation of ILC2; it may be activated independently of adaptive immunity.

A novel avian-origin H3N2 canine influenza A virus (CIV) that showed high sequence similarities in hemagglutinin and neuraminidase genes with those of non-pathogenic avian influenza viruses was isolated in our routine surveillance program in South Korea. We previously reported that the pathogenicity of this strain could be reproduced in dogs and cats. In the present study, the host tropism of H3N2 CIV was examined by experimental inoculation into several host species, including chickens, pigs, mice, guinea pigs, and ferrets. The CIV infection resulted in no overt symptoms of disease in these host species. However, sero-conversion, virus shedding, and gross and histopathologic lung lesions were observed in guinea pig and ferrets but not in pigs, or mice. Based on the genetic similarity of our H3N2 CIV with currently circulating avian influenza viruses and the presence of α-2,3-linked rather than α-2,6-linked sialic acid receptors in the respiratory tract of dogs, we believed that this strain of CIV would have avian virus-like receptor specificity, but that seems to be contrary to our findings in the present study. Further studies are needed to determine the co-receptors of hemagglutinin or post-attachment factors related to virus internalization or pathogenesis in other animals.

Adjuvants are essential vaccine components used to enhance, accelerate, and/or prolong adaptive immunity against specific vaccine antigens. In this study, we compared the adjuvanticity of two adjuvant formulations containing de-O-acylated lipooligosaccharide (dLOS), a toll-like receptor 4 agonist, on the Japanese encephalitis (JE) vaccine in mice. Mice were immunized once or twice at a two-week interval with inactivated JE vaccine in the absence or presence of adjuvant. We found that both the alum- and the liposome-based formulation induced significantly faster and higher serum IgG antibody responses as compared with the non-adjuvanted vaccine after either one or two immunizations. The antibody titers of the mouse immune sera correlated with 50% plaque reduction neutralization test (PRNT50) antibody titers. In addition, the dLOS/liposome formulation was more effective in inducing a Th1-type immune response than the dLOS/alum formulation, as suggested by a strong antigen-specific interferon (IFN)-γ response. Based on these results, we suggest that both alum- and liposome-based adjuvant formulations containing dLOS may be used for the development of JE vaccines with improved immunogenicity.

Potential use of cholera toxin (CT) as a mucosal vaccine adjuvant has been documented in a variety of animal models. However, native CT is highly toxic to be used as a mucosal adjuvant in humans. Here, we demonstrate a new approach to generate a mucosal adjuvant by replacing the B subunit of CT with HIV-1 Tat protein transduction domain (PTD), which efficiently delivers fusion proteins into the cell cytoplasm by unspecific binding to cell surface. We compared the adjuvanticity and toxicity of Tat PTD-CTA1-Tat PTD (TCTA1T) with those of CT. Our results indicate that intranasal (i.n.) delivery of ovalbumin (OVA) with TCTA1T significantly augments the OVA-specific systemic and mucosal antibody responses to levels comparable to those seen with CT adjuvant. Moreover,in vivocytotoxic T lymphocyte activity elicited by TCTA1T was significantly higher than that elicited by a mutant TCTA1T (TmCTA1T) lacking ADP-ribosyltransferase function. In addition, coadministration of influenza M2 protein with TCTA1T conferred near complete protection against lethal influenza virus challenge. Importantly, TCTA1T, in contrast to CT, did not induce serum IgG antibody responses to itself and was shown to be nontoxic. These results suggest that TCTA1T may be a safe and effective adjuvant when given by mucosal routes.

Respiratory syncytial virus (RSV) is a major viral pathogen that causes severe lower respiratory tract infections in infants and the elderly worldwide. Infants with severe RSV bronchiolitis tend to experience more wheezing and asthma in later childhood. Because invariant natural killer T (iNKT) cells are associated with the asthma pathology, we investigated whether neonatal iNKT cells are involved in the aggravation of pulmonary diseases following RSV infection in mice. Intranasal exposure to the iNKT cell ligand alpha-galactosylceramide (alpha-GC) with RSV primary infection in neonatal mice elicited neither cytokine production (except for a slight increase of IL-5) nor pulmonary eosinophilia, despite the presence of both CD1d(+) cells and NKT cells. Interestingly, in adult mice re-infected with RSV, neonatal iNKT cell sensitization by alpha-GC during RSV primary infection resulted in much higher levels of pulmonary Th2 cytokines and elevated eosinophilia with airway hyperresponsiveness, whereas this was not observed in cd1d knockout mice. In contrast, alpha-GC priming of adults during RSV re-infection did not induce more severe airway symptoms than RSV re-infection in the absence of alpha-GC. alpha-GC co-administration during RSV primary infection facilitated RSV clearance regardless of age, but viral clearance following re-infection was not iNKT cell-dependent. This study clearly demonstrates that RSV-induced immune responses can be altered by iNKT cells, suggesting that neonatal iNKT cell sensitization during RSV primary infection is associated with exacerbation of pulmonary diseases following RSV re-infection in adulthood.

Oral vaccine responsiveness is often lower in children from less developed countries. Childhood malnutrition may be associated with poor immune response to oral vaccines. The present study was designed to investigate whether protein energy malnutrition (PEM) impairs B cell immunity and ultimately reduces oral vaccine efficacy in a mouse model. Purified isocaloric diets containing low protein (1/10 the protein of the control diet) were used to determine the effect of PEM. PEM increased both nonspecific total IgA and oral antigen-specific IgA in serum without alteration of gut permeability. However, PEM decreased oral antigen-specific IgA in feces, which is consistent with decreased expression of polymeric Immunoglobulin receptor (pIgR) in the small intestine. Of note, polymeric IgA was predominant in serum under PEM. In addition, PEM altered B cell development status in the bone marrow and increased the frequency of IgA-secreting B cells, as well as IgA secretion by long-lived plasma cells in the small intestinal lamina propria. Moreover, PEM reduced the protective efficacy of the mucosally administered cholera vaccine and recombinant attenuated Salmonella enterica serovar Typhimurium vaccine in a mouse model. Our results suggest that PEM can impair mucosal immunity where IgA plays an important role in host protection and may partly explain the reduced efficacy of oral vaccines in malnourished subjects.

In developing countries,Shigellais a primary cause of diarrhea in infants and young children. Although antibiotic therapy is an effective treatment for shigellosis, therapeutic options are narrowing due to the emergence of antibiotic resistance. Thus, preventive vaccination could become the most efficacious approach for controlling shigellosis. We have identified several conserved protein antigens that are shared by multipleShigellaserotypes and species. Among these, one antigen induced cross-protection against experimental shigellosis, and we have named it pan-Shigellasurface protein 1 (PSSP-1). PSSP-1-induced protection requires a mucosal administration route and coadministration of an adjuvant. When PSSP-1 was administered intranasally, it induced cross-protection againstShigella flexneriserotypes 2a, 5a, and 6,Shigella boydii,Shigella sonnei, andShigella dysenteriaeserotype 1. Intradermally administered PSSP-1 induced strong serum antibody responses but failed to induce protection in the mouse lung pneumonia model. In contrast, intranasal administration elicited efficient local and systemic antibody responses and production of interleukin 17A and gamma interferon. Interestingly, blood samples from patients with recent-onset shigellosis showed variable but significant mucosal antibody responses to other conservedShigellaprotein antigens but not to PSSP-1. We suggest that PSSP-1 is a promising antigen for a broadly protective vaccine againstShigella.

Current influenza vaccines are produced in embryonated chicken eggs. However, egg-based vaccines have various problems. To address these problems, recombinant protein vaccines have been developed as new vaccine candidates. Unfortunately, recombinant proteins frequently encounter aggregation and low stability during their biogenesis. It has been previously demonstrated that recombinantly expressed proteins can be greatly stabilized with high solubility by fusing stabilizing peptide (SP) derived from the C-terminal acidic tail of human synuclein (ATS). To investigate whether SP fusion proteins can induce protective immunity in mice, we produced influenza HA and SP fusion protein using a baculovirus expression system. In in vitro tests, SP-fused recombinant HA1 (SP-rHA1) was shown to be more stable than recombinant HA1 (rHA1). Mice were immunized intramuscularly with baculovirus-expressed rHA1 protein or SP-rHA1 protein (2 μg/mouse) formulated with aluminum hydroxide. Antibody responses were determined by ELISA and hemagglutination inhibition assay. We observed that SP-rHA1 immunization elicited HA-specific antibody responses that were comparable to rHA1 immunization. These results indicate that fusion of SP to rHA1 does not negatively affect the immunogenicity of the vaccine candidate. Therefore, it is possible to apply SP fusion technology to develop stable recombinant protein vaccines with high solubility.

To develop a safe and effective mucosal vaccine against pathogenic influenza viruses, we constructed recombinant Lactobacillus casei strains that express conserved matrix protein 2 with (pgsA-CTA1-sM2/L. casei) or without (pgsA-sM2/L. casei) cholera toxin subunit A1 (CTA1) on the surface. The surface localization of the fusion protein was verified by cellular fractionation analyses, flow cytometry and immunofluorescence microscopy. Oral and nasal inoculations of recombinant L. casei into mice resulted in high levels of serum immunoglobulin G (IgG) and mucosal IgA. However, the conjugation of cholera toxin subunit A1 induced more potent mucosal, humoral and cell-mediated immune responses. In a challenge test with 10 MLD50 of A/EM/Korea/W149/06(H5N1), A/Puerto Rico/8/34(H1N1), A/Aquatic bird /Korea/W81/2005(H5N2), A/ Aquatic bird/Korea/W44/2005(H7N3), and A/Chicken/Korea/116/2004(H9N2) viruses, the recombinant pgsA-CTA1-sM2/L. casei provided better protection against lethal challenges than pgsA-sM2/L. casei, pgsA/L. casei and PBS in mice. These results indicate that mucosal immunization with recombinant L. casei expressing CTA1-conjugated sM2 protein on its surface is an effective means of eliciting protective immune responses against diverse influenza subtypes.

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infection in infants and young children worldwide, but currently no safe and effective vaccine is available. The RSV G glycoprotein (RSVG), a major attachment protein, is an important target for the induction of protective immune responses during RSV infection. However, it has been thought that a CD4(+)T cell epitope (a. a. 183-195) within RSVG is associated with pathogenic pulmonary eosinophilia. To develop safe and effective RSV vaccine using RSV G protein core fragment (Gcf), several Gcf variants resulting from modification to CD4(+)T cell epitope were constructed. Mice were immunized with each variant Gcf, and the levels of RSV-specific serum IgG were measured. At day 4 post-challenge with RSV subtype A or B, lung viral titers and pulmonary eosinophilia were determined and changes in body weight were monitored. With wild type Gcf derived from RSV A2 (wtAGcf), although RSV A subtype-specific immune responses were induced, vaccine-enhanced disease characterized by excessive pulmonary eosinophil recruitment and body weight loss were evident, whereas wtGcf from RSV B1 (wtBGcf) induced RSV B subtype-specific immune responses without the signs of vaccine-enhanced disease. Mice immunized with Th-mGcf, a fusion protein consisting CD4(+)T cell epitope from RSV F (F51-66) conjugated to mGcf that contains alanine substitutions at a. a. position 185 and 188, showed higher levels of RSV-specific IgG response than mice immunized with mGcf. Both wtAGcf and Th-mGcf provided complete protection against RSV A2 and partial protection against RSV B. Importantly, mice immunized with Th-mGcf did not develop vaccine-enhanced disease following RSV challenge. Immunization of Th-mGcf provided protection against RSV infection without the symptom of vaccine-enhanced disease. Our study provides a novel strategy to develop a safe and effective mucosal RSV vaccine by manipulating the CD4(+)T cell epitope within RSV G prot

The induction of strong cytotoxic T-lymphocyte (CTL) and humoral responses appear to be essential for the elimination of persistently infecting viruses, such as hepatitis C virus (HCV). Here, we tested several vaccine regimens and demonstrate that a combined vaccine regimen, consisting of HCV E2 DNA priming and boosting with recombinant E2 protein, induces the strongest immune responses to HCV E2 protein. This combined vaccine regimen augments E2-specific immunoglobulin G2a (IgG2a) and CD8+CTL responses to a greater extent than immunizations with recombinant E2 protein and E2 DNA alone, respectively. In addition, the data showed that a protein boost following one DNA priming was also effective, but much less so than those following two DNA primings. These data indicate that sufficient DNA priming is essential for the enhancement of DNA encoded antigen-specific immunity by a booster immunization with recombinant E2 protein. Furthermore, the enhanced CD8+CTL and IgG2a responses induced by our combined vaccine regimens are closely associated with the protection of BALB/c mice from challenge with modified CT26 tumor cells expressing HCV E2 protein. Together, our results provide important implications for vaccine development for many pathogens, including HCV, which require strong antibody and CTL responses.

Background Influenza vaccines are prepared annually based on global epidemiological surveillance data. However, since there is no method by which to predict the influenza strain that will cause the next pandemic, the demand to develop new vaccination strategies with broad cross-reactivity against influenza viruses are clearly important. The ectodomain of the influenza M2 protein (M2e) is an attractive target for developing a vaccine with broad cross-reactivity. For these reasons, we investigated the efficacy of an inactivated H9N2 virus vaccine (a-H9N2) mixed with M2e (1xM2e or 4xM2e) proteins expressed in Escherichia coli, which contains the consensus of sequence the extracellular domain of matrix 2 (M2e) of A/chicken/Vietnam/27262/09 (H5N1) avian influenza virus, and investigated its humoral immune response and cross-protection against influenza A viruses. Results Mice were intramuscularly immunized with a-H9N2, 1xM2e alone, 4xM2e alone, a-H9N2/1xM2e, or a-H9N2/4xM2e. Three weeks post-vaccination, mice were challenged with lethal homologous (A/ chicken /Korea/ma163/04, H9N2) or heterosubtypic virus (A/Philippines/2/82, H3N2 and A/aquatic bird/Korea/maW81/05, H5N2). Our studies demonstrate that the survival of mice immunized with a-H9N2/1xM2e or with a-H9N2/4xM2e (100% survival) was significantly higher than that of mouse-adapted H9N2 virus-infected mice vaccinated with 1xM2e alone or with 4xM2e alone (0% survival). We also evaluated the protective efficacy of the M2e + vaccine against infection with mouse-adapted H5N2 influenza virus. Protection from death in the control group (0% survival) was similar to that of the 1×M2e alone and 4xM2e alone-vaccinated groups (0% survival). Only 40% of mice vaccinated with vaccine alone survived challenge with H5N2, while the a-H9N2/1×M2e and a-H9N2/4×M2e groups showed 80% and 100% survival following mouse-adapted H5N2 challenge, respectively. We also examined cross-protection against human H3N2 virus and found that the a-H9N2/1×M2e group displayed partial cross-protection against H3N2 (40% survival), whereas vaccine alone, 1×M2e alone, 4×M2e alone, or H9N2/1×M2e groups showed incomplete protection (0% survival) in response to challenge with a lethal dose of human H3N2 virus. Conclusions Taken together, these results suggest that prokaryote-expressed M2e protein improved inactivated H9N2 virus vaccine efficacy and achieved cross-protection against lethal influenza A virus infection in mice.

Background Sublingual (s.l.) administration of soluble protein antigens, inactivated viruses, or virus-like particles has been shown to induce broad immune responses in mucosal and extra-mucosal tissues. Recombinant replication-defective adenovirus vectors (rADVs) infect mucosa surface and therefore can serve as a mucosal antigen delivery vehicle. In this study we examined whether s.l. immunization with rADV encoding spike protein (S) (rADV-S) of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) induces protective immunity against SARS-CoV and could serve as a safe mucosal route for delivery of rADV. Results Here, we show that s.l. administration of rADV-S induced serum SARS-CoV neutralizing and airway IgA antibodies in mice. These antibody responses are comparable to those induced by intranasal (i.n.) administration. In addition, s.l. immunization induced antigen-specific CD8+ T cell responses in the lungs that are superior to those induced by intramuscular immunization. Importantly, unlike i.n. administration, s.l. immunization with rADV did not redirect the rADV vector to the olfactory bulb. Conclusion Our study indicates that s.l. immunization with rADV-S is safe and effective in induction of a broad spectrum of immune responses and presumably protection against infection with SARS-CoV.

Influenza viruses are respiratory pathogens that continue to pose a significantly high risk of morbidity and mortality of humans worldwide. Vaccination is one of the most effective strategies for minimizing damages by influenza outbreaks. In addition, rapid development and production of efficient vaccine with convenient administration is required in case of influenza pandemic. In this study, we generated recombinant influenza virus hemagglutinin protein 1 (sHA1) of 2009 pandemic influenza virus as a vaccine candidate using a well-established bacterial expression system and administered it into mice via sublingual (s.l.) route. We found that s.l. immunization with the recombinant sHA1 plus cholera toxin (CT) induced mucosal antibodies as well as systemic antibodies including neutralizing Abs and provided complete protection against infection with pandemic influenza virus A/CA/04/09 (H1N1) in mice. Indeed, the protection efficacy was comparable with that induced by intramuscular (i.m.) immunization route utilized as general administration route of influenza vaccine. These results suggest that s.l. vaccination with the recombinant non-glycosylated HA1 protein offers an alternative strategy to control influenza outbreaks including pandemics.

The nonstructural protein 1 (NS1) of influenza A virus (IAV) enables the virus to disarm the host cell type 1 IFN defense system. Mutation or deletion of the NS1 gene leads to attenuation of the virus and enhances host antiviral response making such live-attenuated influenza viruses attractive vaccine candidates. Sublingual (SL) immunization with live influenza virus has been found to be safe and effective for inducing protective immune responses in mucosal and systemic compartments. Here we demonstrate that SL immunization with NS1 deleted IAV (DeltaNS1 H1N1 or DeltaNS1 H5N1) induced protection against challenge with homologous as well as heterosubtypic influenza viruses. Protection was comparable with that induced by intranasal (IN) immunization and was associated with high levels of virus-specific antibodies (Abs). SL immunization with DeltaNS1 virus induced broad Ab responses in mucosal and systemic compartments and stimulated immune cells in mucosa-associated and systemic lymphoid organs. Thus, SL immunization with DeltaNS1 offers a novel potential vaccination strategy for the control of influenza outbreaks including pandemics.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine to prevent RSV infection. The G glycoprotein of RSV, a major attachment protein, is a potentially important target for protective antiviral immune responses and has been shown to exhibit chemotactic activity through CX3C mimicry. Here, we show that sublingual or intranasal immunization of a purified G protein fragment of amino acids from 131 to 230, designated Gcf, induces strong serum IgG and mucosal IgA responses. Interestingly, these antibody responses could be elicited by Gcf even in the absence of any adjuvant, indicating a novel self-adjuvanting property of our vaccine candidate. Gcf exhibited potent chemotactic activity in in vitro cell migration assay and cysteine residues are necessary for chemotactic activity and self-adjuvanticity of Gcf in vivo. Mucosal immunization with Gcf also provides protection against RSV challenge without any significant lung eosinophilia or vaccine-induced weight loss. Together, our data demonstrate that mucosal administration of Gcf vaccine elicits beneficial protective immunity and represents a promising vaccine regimen preventing RSV infection.

Background: The ectodomain of matrix protein 2 (M2e) of influenza A virus is a rationale target antigen candidate for the development of a universal vaccine against influenza as M2e undergoes little sequence variation amongst human influenza A strains. Vaccine-induced M2e-specific antibodies (Abs) have been shown to display significant cross-protective activity in animal models. M2e-based vaccine constructs have been shown to be more protective when administered by the intranasal (i.n.) route than after parenteral injection. However, i.n. administration of vaccines poses rare but serious safety issues associated with retrograde passage of inhaled antigens and adjuvants through the olfactory epithelium. In this study, we examined whether the sublingual (s.l.) route could serve as a safe and effective alternative mucosal delivery route for administering a prototype M2e-based vaccine. The mechanism whereby s.l. immunization with M2e vaccine candidate induces broad protection against infection with different influenza virus subtypes was explored. Methods and Results: A recombinant M2 protein with three tandem copies of the M2e (3M2eC) was expressed in Escherichia coli. Parenteral immunizations of mice with 3M2eC induced high levels of M2e-specific serum Abs but failed to provide complete protection against lethal challenge with influenza virus. In contrast, s.l. immunization with 3M2eC was superior for inducing protection in mice. In the latter animals, protection was associated with specific Ab responses in the lungs. Conclusions: The results demonstrate that s.l. immunization with 3M2eC vaccine induced airway mucosal immune responses along with broad cross-protective immunity to influenza. These findings may contribute to the understanding of the M2-based vaccine approach to control epidemic and pandemic influenza infections. Citation: Shim B-S, Choi YK, Yun C-H, Lee E-G, Jeon YS, et al. (2011) Sublingual Immunization with M2-Based Vaccine Induces Broad Protective Immunity against

a b s t r a c t Heat killed spores of the Gram-positive bacterium Bacillus subtilis have been evaluated as a vaccine deliv- ery system with mucosal adjuvant properties for influenza. Killed spores were able to bind H5N1 virions (NIBRG-14; clade 1) and, when intra-nasally administered to mice, resulting immune responses, both humoral and cell mediated, were enhanced compared to immunization with the virion alone. Levels of both systemic IgG and mucosal sIgA specific to the virion were elevated. Levels of IgG2a (a Th1 antibody type) were strongly enhanced when the virion was co-administered with killed spores. Cytokine induc- tion in stimulated splenocytes was also apparent indicating balanced Th1 and Th2 responses. Evidence of cross-neutralization of clade 2.2 viruses was shown. In a challenge experiment mice dosed two times with spores adsorbed with just 20 ng HA (hemagglutinin) of inactivated NIBRG-14 were fully protected against challenge with 20 LD50 of H5N2 virus. Interestingly, partial protection (60%) was observed in ani- mals dosed only with killed spores. Mice dosed only with killed spores were shown to be fully protected against H5N2 (5 LD50) infection indicating that innate immunity and its stimulation by spores may play an important role in protection. Supporting this killed spores were (i) shown to stimulate TLR-mediated expression of NF-B, and (ii) able to recruit NK cells into lungs and induce maturation of DCs. This work demonstrates the potential and underlying mechanism for the use of bacterial spores as an adjuvant for H5N1 vaccination.

Background Immunization with the spike protein (S) of severe acute respiratory syndrome (SARS)-coronavirus (CoV) in mice is known to produce neutralizing antibodies and to prevent the infection caused by SARS-CoV. Polyethylenimine 25K (PEI) is a cationic polymer which effectively delivers the plasmid DNA. Results In the present study, the immune responses of BALB/c mice immunized via intranasal (i.n.) route with SARS DNA vaccine (pci-S) in a PEI/pci-S complex form have been examined. The size of the PEI/pci-S nanoparticles appeared to be around 194.7 ± 99.3 nm, and the expression of the S mRNA and protein was confirmed in vitro. The mice immunized with i.n. PEI/pci-S nanoparticles produced significantly (P < 0.05) higher S-specific IgG1 in the sera and mucosal secretory IgA in the lung wash than those in mice treated with pci-S alone. Compared to those in mice challenged with pci-S alone, the number of B220+ cells found in PEI/pci-S vaccinated mice was elevated. Co-stimulatory molecules (CD80 and CD86) and class II major histocompatibility complex molecules (I-Ad) were increased on CD11c+ dendritic cells in cervical lymph node from the mice after PEI/pci-S vaccination. The percentage of IFN-γ-, TNF-α- and IL-2-producing cells were higher in PEI/pci-S vaccinated mice than in control mice. Conclusion These results showed that intranasal immunization with PEI/pci-S nanoparticles induce antigen specific humoral and cellular immune responses.

Cholera toxin (CT) is a potent vaccine adjuvant, which promotes mucosal immunity to protein antigen given by nasal route. It has been suggested that CT promotes T helper type 2 (Th2) response and suppresses Th1 response. We here report the induction of Th17-dominated responses in mice by intranasal delivery of CT. This dramatic Th17-driving effect of CT, which was dependent on the B subunit, was observed even in Th1 or Th2-favored conditions of respiratory virus infection. These dominating Th17 responses resulted in the significant neutrophil accumulation in the lungs of mice given CT. Both in vitro and in vivo treatment of CT induced strongly augmented IL-6 production, and Th17-driving ability of CT was completely abolished in IL-6 knockout mice, indicating a role of this cytokine in the Th17-dominated T-cell responses by CT. These data demonstrate a novel Th17-driving activity of CT, and help understand the mechanisms of CT adjuvanticity to demarcate T helper responses.

The prevention of Mycobacterium tuberculosis (M. tuberculosis) reactivation would greatly reduce the incidence of the disease, particularly among the elderly. Here, we evaluated the efficacy of DNA vaccine in combination with a conventional TB chemotherapy on the prevention of M. tuberculosis reactivation. Mice were treated with isoniazid and pyrazinamide for 3 months from 4 weeks after aerosol infection with M. tuberculosis H37Rv. During this period of chemotherapy, DNA immunization was performed three times monthly with an antigen 85A (Ag85A) DNA or an IL-12 mutant (IL-12N220L) DNA, which is known to lead to a reduction in the secretion of the p40 subunit, but not of a bioactive IL-12p70. The reactivation of M. tuberculosis was dramatically reduced in mice treated with either Ag85A DNA (P0.01) or IL-12N220L DNA (P0.05) in combination with chemotherapy, compared with control mice receiving only chemotherapy. Ag85A DNA vaccine showed higher IFN-gamma responses to Ag85A protein, but a lower response to culture filtrate than IL-12N220L DNA vaccine. In addition, Ag85A DNA vaccine prevented the reactivation of M. tuberculosis more efficiently than IL-12N220L DNA vaccine, indicating that Ag85A-specific IFN-gamma response might correlate with M. tuberculosis control. This study suggests that immunotherapy using Ag85A or IL-12N220L DNA vaccine combined with conventional chemotherapy might be effective clinically for the prevention of tuberculosis reactivation and may offer a more effective cure for humans than chemotherapy alone.

CpG oligodeoxynucleotides (ODNs) are promising immunomodulatory agents for treating human diseases and vaccine development. Phosphodiester CpG ODNs were demonstrated to have poor immunostimulatory potentials for cytokine production. However, the conjugation of consecutive deoxyriboguanosine residues, called a dG run, at the 3′ terminus of phosphodiester CpG ODNs significantly enhanced TNF-α and IL-12 production from mouse splenic dendritic cells (DCs). The optimal induction of cytokine production was achieved by the addition of a hexameric dG (dG6) run. In contrast, the existence of a dG6 run either at the 5′ terminus of phosphodiester CpG ODNs or at the 3′ terminus of phosphorothioate CpG ODNs diminished CpG-mediated cytokine induction, suggesting that the effects of a dG run depend on its location and the chemical property of the ODN backbone, respectively. In addition, we provided the evidence that the conjugation of a dG6 run caused the structural transformation of CpG ODNs, which facilitates their targeting into mouse APCs such as splenic DCs, B cells, and peritoneal macrophages with a scavenger receptor type A ligand specificity. Among primary APCs, DCs were the most potent for CpG ODN-mediated IL-12 production. Furthermore, we demonstrated that the conjugation of a dG6 run into the 3′ terminus of phosphodiester CpG ODNs was crucial for their ability to generate Th1 immunity in vivo. Thus, the conjugation of a dG6 run into phosphodiester CpG ODNs would be an alternative way to optimize their immunostimulatory potentials in vitro and in vivo.

The nonstructural protein NS3 of the hepatitis C virus (HCV) is indispensable for virus replication and a multifunctional enzyme that contains three catalytic activities such as serine protease, helicase, and NTPase. Here, we demonstrated that the internal cleavage of the HCV NS3 protein occurs in various mammalian cells such as HepG2, COS-7, and NIH3T3. As is observed for the internal cleavage mechanism of the NS3 protein of dengue virus 2, the internal processing of HCV NS3 protein was catalyzed by the active NS3 serine protease and NS4A, but not NS3 alone. From the data acquired from extensive site-directed mutagenesis, we observed that the NS3 protein was internally cleaved at two different sites, FCH(1395) ||S(1396)KK and IPT(1428) ||S(1429)GD, within RNA helicase domain. The internal cleavage of NS3 protein by NS34A protease was also confirmed in a different isolate of HCV-1b strain. In addition, in vitro transforming assays demonstrated that the internal cleavage product of NS3, NS3a-1, appeared to have higher oncogenic potential than does intact NS3. Taken together, our results suggest that the internal cleavage of NS3 may be associated with the replication and oncogenesis of HCV.

Influenza vaccines that target the highly variable surface glycoproteins hemagglutinin and neuraminidase cause inconvenience of having vaccination every year. For this reason, development of universal vaccines targeting conserved viral components is needed. In this study, we generated recombinant adenovirus (rAd) vaccine encoding nucleoprotein (NP) of A/PR/8/34 influenza virus, designated rAd/NP. BALB/c mice were immunized intranasally or sublingually with rAd/NP vaccine and subsequently challenged with lethal doses of heterologous as well as homologous influenza viruses. We found that intranasal immunization of rAd/NP elicited strong mucosal IgA responses as well as stronger CD8 T-cell responses toward immunodominant K(d)-restricted NP147-155 epitope than sublingual immunization. Importantly, only single intranasal but not sublingual immunization of rAd/NP provides potent protection against both homologous and heterologous influenza virus challenges. These results suggest that recombinant rAd/NP could be a universal vaccine candidate for mucosal administration against influenza virus.