Chitosan-based mucosal adjuvants: Sunrise on the ocean

Mucosal vaccination, which is shown to elicit systemic and mucosal immune responses, serves as a non-invasive and convenient alternative to parenteral administration, with stronger capability in combatting diseases at the site of entry. The exploration of potent mucosal adjuvants is emerging as a significant area, based on the continued necessity to amplify the immune responses to a wide array of antigens that are poorly immunogenic at the mucosal sites. As one of the inspirations from the ocean, chitosan-based mucosal adjuvants have been developed with unique advantages, such as, ability of mucosal adhesion, distinct trait of opening the junctions to allow the paracellular transport of antigen, good tolerability and biocompatibility, which guaranteed the great potential in capitalizing on their application in human clinical trials. In this review, the state of art of chitosan and its derivatives as mucosal adjuvants, including thermo-sensitive chitosan system as mucosal adjuvant that were newly developed by author's group, was described, as well as the clinical application perspective. After a brief introduction of mucosal adjuvants, chitosan and its derivatives as robust immune potentiator were discussed in detail and depth, in regard to the metabolism, safety profile, mode of actions and preclinical and clinical applications, which may shed light on the massive clinical application of chitosan as mucosal adjuvant.     

Efficacy of poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP) as mucosal adjuvant to induce protective immunity against respiratory pathogens

Polyphosphazene polyelectrolyte, a potent new mucosal adjuvant candidate, was tested for its ability to elicit protective immunity against several respiratory diseases. Groups of mice were intranasally (i.n.) vaccinated with poly[di(sodium carboxylatophenoxy)phosphazene] (PCPP) together with several vaccine antigens such as pertussis toxoid, pneumococcal surface protein A, and formalin-inactivated PR8 influenza virus. Results showed predominant levels of antigen-specific IgG and IgA antibodies in serum and bronchial alveolar lavage fluids after vaccination with PCPP plus antigen when compared to antigen alone. In addition, there were significantly higher levels of the secretory form of IgA antibody in the mucosal secretions (i.e., nasal wash, saliva, vaginal wash, and fecal extracts). Moreover, i.n. vaccination with PCPP resulted in brisk numbers of IgG and IgA antibody-forming cells in the nasal passage, lung, and sub-mandibular glands of vaccinated mice. Of note, PCPP administration resulted in mixed Th1 and Th2 type responses (i.e., high levels of IgG2a and IgG1 as well as IFN-γ and IL-4). Most interestingly, i.n. challenge with vaccine antigens together with PCPP elicited strong protective efficacy against respiratory infection with Bordetella pertussis, Streptococcus pneumoniae, and influenza virus. Taken together, these results suggest that PCPP may be a promising candidate for mucosal adjuvant to elicit protective immunity against respiratory infectious diseases.     

Bis-(3',5')-cyclic dimeric adenosine monophosphate: strong Th1/Th2/Th17 promoting mucosal adjuvant

New effective adjuvants are required to improve the performance of subunit vaccines. Here, we showed that bis-(3′,5′)-cyclic dimeric adenosine monophosphate (c-di-AMP), a second messenger molecule in bacteria and archaea, exerts strong adjuvant activities when delivered by mucosal route. In vitro studies showed that c-di-AMP was able to both stimulate pre-activated murine macrophages and promote the activation and maturation of dendritic cells of murine and human origin. Co-administration of c-di-AMP with β-galactosidase (β-Gal) by intranasal route to BALB/c mice resulted in the elicitation of significantly higher serum antigen-specific IgG titres than in controls. The induction of local immune responses was shown by the production of antigen-specific secretory IgA in different mucosal territories. In addition, strong cellular immune responses were observed against both the β-Gal protein and a peptide encompassing its MHC class I-restricted epitope. The ratio of β-Gal-specific antibodies and the secreted cytokine profiles by in vitro re-stimulated splenocytes suggested that a balanced Th1/Th2/Th17 response pattern is promoted by c-di-AMP. When C57BL/6 mice were immunized with OVA and c-di-AMP, vigorous in vivo CTL responses were also observed. These results indicated that c-di-AMP exhibits a high potential as adjuvant for the development of mucosal vaccines, in particular when cellular immunity is needed.     

Rotavirus enterotoxin NSP4 has mucosal adjuvant properties

Rotavirus nonstructural protein 4 (NSP4) is a protein with pleiotropic properties. It functions in rotavirus morphogenesis, pathogenesis, and is the first described viral enterotoxin. Since many bacterial toxins function as potent mucosal adjuvants, we evaluated whether baculovirus-expressed recombinant simian rotavirus SA11 NSP4 possesses adjuvant activity by co-administering NSP4 with keyhole limpet hemocyanin (KLH), tetanus toxoid (TT) or ovalbumin (OVA) as model antigens in mice. Following intranasal immunization, NSP4 significantly enhanced both systemic and mucosal immune responses to model immunogens, as compared to the control group, in an antigen-specific manner. Both full-length and a cleavage product of SA11 NSP4 had adjuvant activity, localizing this activity to the C-terminus of the protein. NSP4 forms from virulent and avirulent porcine rotavirus OSU strain, and SA11 NSP4 localized within a 2/6-virus-like particle (VLP) also exhibited adjuvant effects. These studies suggest that the rotavirus enterotoxin NSP4 can function as an adjuvant to enhance immune responses for a co-administered antigen.     

Immunomodulation of TH2 biased immunity with mucosal administration of nanoemulsion adjuvant

T_H2-biased immune responses are associated with inadequate protection against some pathogens and with cancer, colitis, asthma and allergy. Since most currently used vaccine adjuvants induce a T_H2-biased response, this has led to interest in developing adjuvants capable of activating T_H1 immunity and modulating existing T_H2 responses. Immunotherapies to shift immune responses from T_H2 to T_H1 have generally required prolonged immunization protocols and have not induced effective T_H1 responses. We have demonstrated that nanoscale emulsions (NE), a novel mucosal adjuvant, induce robust IgA and IgG antibody responses and T_H1/T_H17 cellular immunity resulting in protection against a variety of respiratory and mucosal infections. Because intranasal (i.n.) delivery of NE adjuvant consistently induces T_H1/T_H17 biased responses, we hypothesized that NE could be used as a therapeutic vaccine to redirect existing T_H2 polarized immunity towards a more balanced T_H1/T_H2 profile. To test this, a T_H2 immune response was established by intramuscular immunization of mice with alum-adjuvanted hepatitis B surface antigen (HBs), followed by a single subsequent i.n. immunization with NE-HBs. These animals exhibited increased T_H1 associated immune responses and IL-17, and decreased T_H2 cytokines (IL-4 and IL-5) and IgG1. NE immunization induced regulatory T cells and IL-10, and IL-10 was required for the suppression of T_H2 immunity. These data demonstrate that NE-based vaccines can modulate existing T_H2 immune responses to promote T_H1/T_H17 immunity and suggest the potential therapeutic use of NE vaccines for diseases associated with T_H2 immunity.     

A mucosal adjuvant for the inactivated poliovirus vaccine

The eradication of poliovirus from the majority of the world has been achieved through the use of two vaccines: the inactivated poliovirus vaccine (IPV) and the live-attenuated oral poliovirus vaccine (OPV). Both vaccines are effective at preventing paralytic poliomyelitis, however, they also have significant differences. Most importantly for this work is the risk of revertant virus from OPV, the greater cost of IPV, and the low mucosal immunity induced by IPV. We and others have previously described the use of an alphavirus-based adjuvant that can induce a mucosal immune response to a co-administered antigen even when delivered at a non-mucosal site. In this report, we describe the use of an alphavirus-based adjuvant (GVI3000) with IPV. The IPV-GVI3000 vaccine significantly increased systemic IgG, mucosal IgG and mucosal IgA antibody responses to all three poliovirus serotypes in mice even when administered intramuscularly. Furthermore, GVI3000 significantly increased the potency of IPV in rat potency tests as measured by poliovirus neutralizing antibodies in serum. Thus, an IPV-GVI3000 vaccine would reduce the dose of IPV needed and provide significantly improved mucosal immunity. This vaccine could be an effective tool to use in the poliovirus eradication campaign without risking the re-introduction of revertant poliovirus derived from OPV.     

Self-adjuvanting modular virus-like particles for mucosal vaccination against group A streptococcus (GAS)

Group A streptococcus (GAS) causes a wide range of diseases, some of them related to autoimmune diseases triggered by repeated GAS infections. Despite the fact that GAS primarily colonizes the mucosal epithelium of the pharynx, the main mechanism of action of most vaccine candidates is based on development of systemic antibodies that do not cross-react with host tissues, neglecting the induction of mucosal immunity that could potentially block disease transmission. Peptide antigens from GAS M-surface protein can confer protection against infection; however, translation of such peptides into immunogenic mucosal vaccines that can be easily manufactured remains a challenge. In this work, a modular murine polyomavirus (MuPyV) virus-like particle (VLP) was engineered to display a GAS antigenic peptide, J8i. Heterologous modules containing one or two J8i antigen elements were integrated with the MuPyV VLP, and produced using microbial protein expression, standard purification techniques and in vitro VLP assembly. Both modular VLPs, when delivered intranasally to outbred mice without adjuvant, induced significant titers of J8i-specific IgG and IgA antibodies, indicating significant systemic and mucosal responses, respectively. GAS colonization in the throats of mice challenged intranasally was reduced in these immunized mice, and protection against lethal challenge was observed. This study shows that modular MuPyV VLPs prepared using microbial synthesis have potential to facilitate cost-effective vaccine delivery to remote communities through the use of mucosal immunization.     

Functional and structural characteristics of secretory IgA antibodies elicited by mucosal vaccines against influenza virus

Mucosal tissues are major targets for pathogens. The secretions covering mucosal surfaces contain several types of molecules that protect the host from infection. Among these, mucosal immunoglobulins, including secretory IgA (S-IgA) antibodies, are the major contributor to pathogen-specific immune responses. IgA is the primary antibody class found in many external secretions and has unique structural and functional features not observed in other antibody classes. Recently, extensive efforts have been made to develop novel vaccines that induce immunity via the mucosal route. S-IgA is a key molecule that underpins the mechanism of action of these mucosal vaccines. Thus, precise characterization of S-IgA induced by mucosal vaccines is important, if the latter are to be used successfully in a clinical setting. Intensive studies identified the fundamental characteristics of S-IgA, which was first discovered almost half a century ago. However, S-IgA itself has not gained much attention of late, despite its importance to mucosal immunity; therefore, some important questions remain. This review summarizes the current understanding of the molecular characteristics of S-IgA and its role in intranasal mucosal vaccines against influenza virus infection.     

Alphavirus replicon-based enhancement of mucosal and systemic immunity is linked to the innate response generated by primary immunization

Venezuelan equine encephalitis virus replicon particles (VRP) function as an effective systemic, cellular and mucosal adjuvant when codelivered with antigen, and show promise for use as a component in new and existing human vaccine formulations. We show here that VRP are effective at low dose and by intramuscular delivery, two useful features for implementation of VRP as a vaccine adjuvant. In mice receiving a prime and boost with antigen, we found that VRP are required in prime only to produce a full adjuvant effect. This outcome indicates that the events triggered during prime with VRP are sufficient to establish the nature and magnitude of the immune response to a second exposure to antigen. Events induced by VRP in the draining lymph node after prime include robust secretion of many inflammatory cytokines, upregulation of CD69 on leukocytes, and increased cellularity, with a disproportionate increase of a cell population expressing CD11c, CD11b, and F4/80. We show that antigen delivered 24 h after administration of VRP does not benefit from an adjuvant effect, indicating that the events which are critical to VRP-mediated adjuvant activity occur within the first 24 h. Further studies of the events induced by VRP will help elucidate the mechanism of VRP adjuvant activity and will advance the safe implementation of this adjuvant in human vaccines.     

Recombinant interleukin 6 with M cell-targeting moiety produced in Lactococcus lactis IL1403 as a potent mucosal adjuvant for peroral immunization

Development and application of safe and effective mucosal adjuvants are important to improve immunization efficiency in oral vaccine. Here, we report a novel mucosal adjuvant, IL-6-CKS9, a recombinant cytokine generated by conjugating an M cell-targeting peptide (CKS9) with c-terminus of the murine interleukin 6 (IL-6), which facilitated enhancement of mucosal immune response. Lactococcus lactis IL1403, a food-grade strain of lactic acid bacteria (LAB) which is widely used in dairy industry, was used as a host cell to express and secrete the IL-6-CKS9 for a mucosal vaccine adjuvant. The recombinant L. lactis IL1403 secreting IL-6-CKS9 was orally administered with a model antigen protein, M-BmpB (Brachyspira membrane protein B conjugated with CKS9), to BALB/c mice for mucosal immunization. ELISA analyses showed consistent enhancement tendencies in induction of anti-M-BmpB antibody levels both with mucosal (IgA) and systemic (IgG) immune responses in IL-6-CKS9-LAB treated group compared with other groups tested by conducting two separated mice immunization assays. In addition, we characterized that the oral administration of model protein antigen with live LAB producing IL-6-CKS9 could induce both Th1 and Th2 type immune responses by analysis of the specific anti-BmpB IgG1 and IgG2a isotypes in the sera and also investigated possible oral tolerance in our vaccine strategy. Collectively, our results showed successful production and secretion of recombinant murine IL-6 with M cell-targeting moiety (IL-6-CKS9) from L. lactis IL1403 and demonstrated the live recombinant LAB producing IL-6-CKS9 could have a potential to be used as an efficient adjuvant for peroral vaccination.     

Intranasal and sublingual delivery of inactivated polio vaccine

Polio is on the brink of eradication. Improved inactivated polio vaccines (IPV) are needed towards complete eradication and for the use in the period thereafter. Vaccination via mucosal surfaces has important potential advantages over intramuscular injection using conventional needle and syringe, the currently used delivery method for IPV. One of them is the ability to induce both serum and mucosal immune responses: the latter may provide protection at the port of virus entry.The current study evaluated the possibilities of polio vaccination via mucosal surfaces using IPV based on attenuated Sabin strains. Mice received three immunizations with trivalent sIPV via intramuscular injection, or via the intranasal or sublingual route. The need of an adjuvant for the mucosal routes was investigated as well, by testing sIPV in combination with the mucosal adjuvant cholera toxin.Both intranasal and sublingual sIPV immunization induced systemic polio-specific serum IgG in mice that were functional as measured by poliovirus neutralization. Intranasal administration of sIPV plus adjuvant induced significant higher systemic poliovirus type 3 neutralizing antibody titers than sIPV delivered via the intramuscular route. Moreover, mucosal sIPV delivery elicited polio-specific IgA titers at different mucosal sites (IgA in saliva, fecal extracts and intestinal tissue) and IgA-producing B-cells in the spleen, where conventional intramuscular vaccination was unable to do so. However, it is likely that a mucosal adjuvant is required for sublingual vaccination. Further research on polio vaccination via sublingual mucosal route should include the search for safe and effective adjuvants, and the development of novel oral dosage forms that improve antigen uptake by oral mucosa, thereby increasing vaccine immunogenicity. This study indicates that both the intranasal and sublingual routes might be valuable approaches for use in routine vaccination or outbreak control in the period after complete OPV cessation and post-polio eradication.     

Onjisaponins, from the root of Polygala tenuifolia Willdenow, as effective adjuvants for nasal influenza and diphtheria-pertussis-tetanus vaccines

Active substances from hot water extracts from 267 different Chinese and Japanese medicinal herbs were screened for mucosal adjuvant activity with influenza HA vaccine in mice. The extract from the root of Polygala tenuifolia was found to contain potent mucosal adjuvant activity. The active substances were purified and identified as onjisaponins A, E, F, and G. When each onjisaponin (10 μg) was intranasally (i.n.) inoculated with influenza vaccine (10 μg) in mice, serum hemagglutination-inhibiting (HI) antibody titers increased 3–14 times over control mice administered vaccine alone after 4 weeks. When each onjisaponin (10 μg) was i.n. inoculated with the vaccine (10 μg) followed by i.n. vaccination of the vaccine alone after 3 weeks, serum HI antibody titers increased 27–50 fold over those mice given i.n. vaccinations without onjisaponins. These same conditions also significantly increased nasal anti-influenza virus IgA antibody titers. Two inoculations with onjisaponin F (1 μg) and influenza HA vaccine (1 μg) at 3 weeks intervals, significantly increased serum HI antibody and nasal anti-influenza virus IgA and IgG antibody titers after only 1 week over mice given HA vaccine alone after the secondary vaccination. Intranasal vaccination with onjisaponin F inhibited proliferation of mouse adapted influenza virus A/PR/8/34 in bronchoalveolar lavages of infected mice. Separate intranasal vaccinations with onjisaponins A, E, F, and G (10 μg) each and diphtheria–pertussis–tetanus (DPT) vaccine (10 μg) of mice followed by i.n. vaccination with DPT vaccine alone after 4 weeks showed significant increases in serum IgG and nasal IgA antibody titers after 2 weeks following secondary vaccination over mice vaccinated with DPT vaccine alone. All onjisaponins showed little hemolytic activity at concentrations up to 100 μg/ml. The results of this study suggest that onjisaponins may provide safe and potent adjuvants for intranasal inoculation of influenza HA and DPT vaccines.     

Salmonella flagellins are potent adjuvants for intranasally administered whole inactivated influenza vaccine

Bacterial flagellins are potent inducers of innate immune responses in the mouse lung because they bind to TLR5 expressed on the apical surfaces of airway epithelial cells. TLR engagement leads to the initiation of a signaling cascade that results in a pro-inflammatory response with subsequent up-regulation of several cytokines and leads to adaptive immune responses. We examined the ability of two soluble flagellins, a monomeric flagellin expressed in Escherichia coli and a highly purified polymeric flagellin directly isolated from Salmonella, to enhance the efficacy of influenza vaccines in mice. Here we demonstrate that both flagellins co-administered intranasally with inactivated A/PR/8/34 (PR8) virus induced robust increases of systemic influenza-specific IgA and IgG titers and resulted in a more comprehensive humoral response as indicated by the increase of IgG2a and IgG2b subclass responses. Groups immunized with the adjuvanted vaccines were fully protected against high dose lethal challenge by homologous virus whereas inactivated PR8 alone conferred only partial protection. Finally we show that shortly after immunization the adjuvanted vaccines induced a dramatic increase in pro-inflammatory cytokines in the lung, resulting in extensive lung infiltration by granulocytes and monocytes/macrophages. Our results reveal a promising perspective for the use of both soluble monomeric and polymeric flagellin as mucosal vaccine adjuvants to improve protection against influenza epidemics as well as a range of other infectious diseases.     

A lipidic delivery system of a triple vaccine adjuvant enhances mucosal immunity following nasal administration in mice

We previously developed an highly efficacious combination adjuvant comprised of innate defense regulator (IDR)-1002 peptide, poly(I:C) and polyphosphazene (TriAdj). Here we aimed to design and test the in vivo efficacy of a mucoadhesive nasal formulation of this adjuvant. To determine the physical properties of the formulation, the effect of addition of each individual component was characterised by gel electrophoresis and fluorescence quenching using rhodamine-poly(I:C). Cationic liposomes comprised of didodecyl dimethylammonium bromide (DDAB), dioleoyl phosphatidylethanolamine (DOPE) (50:50 or 75:25?mol:mol) and DDAB, L-α-phosphatidylcholine (egg PC) and DOPE (40:50:10?mol:mol:mol) were prepared by the thin-film extrusion method. The liposomes and TriAdj were combined by simple mixing. The formed complex (L-TriAdj) was characterized by dynamic light scattering, zeta potential, and mucin interactions. We found that IDR-1002 peptide, polyphosphazene and poly(I:C) self-assembled in solution forming an anionic complex. Exposure of RAW267.4 mouse macrophage cells to TriAdj alone vs. L-TriAdj indicated that DDAB/DOPE (50:50) and DDAB/EPC/cholesterol (40:50:10) complexation reduced TriAdj toxicity. Next, TriAdj-containing cationic liposomes were prepared at several molar ratios to determine optimal size, stability and desired positive charge. Transmission electron microscopy showed rearrangement of lipid structures on binding of liposomes to TriAdj and to mucin. Stable particles (<200?nm over 24?h) showed mucin binding of DDAB/DOPE?+?TriAdj was greater than DDAB/EPC/DOPE?+?TriAdj. To verify in vivo efficacy, mice were administered the DDAB/DOPE?+?TriAdj complex intranasally with ovalbumin as the antigen, and the immunogenic response was measured by ELISA (serum IgG1, IgG2a, IgA) and ELISpot assays (splenocyte IL-5, IFN-γ). Mice administered adjuvant showed a significantly greater immune response with L-TriAdj than TriAdj alone, with a dose-response proportionate to the triple adjuvant content, and an overall balanced Th1/Th2 immune response representing both systemic and mucosal immunity.     

Advances and challenges in mucosal adjuvant technology

Adjuvants play attractive roles in enhancement of immune response during vaccination; however, due to several challenges, only a limited number of adjuvants are licensed by health authorities. The lack of an effective mucosal adjuvant is even more significant as none of the licensed adjuvants revealed a strong enhancement in immune system after mucosal administration. Over the past two decades, several mucosal adjuvants have been developed to deliver antigens to the target cells in the mucosal immune system and increase specific immune responses. However, the safety and efficacy of these adjuvants for testing in human trials is still an important issue, requiring further study. In this article, we briefly review the challenges associated with most common mucosal adjuvants and discuss potential strategies for targeting the mucosal immune system.     

The novel adjuvant dmLT promotes dose sparing,mucosal immunity and longevity of antibody responses to the inactivated polio vaccine in a murine model

One option for achieving global polio eradication is to replace the oral poliovirus vaccine (OPV), which has the risk of reversion to wild-type virulence, with the inactivated poliovirus vaccine (IPV) vaccine. Adjuvants and alternate routes of immunization are promising options that may reduce antigen dose in IPV vaccinations, potentially allowing dose sparing and cost savings. Use of adjuvants and alternate routes of immunization could also help promote mucosal immunity, potentially mimicking the protection against intestinal virus shedding seen with OPV. In the current study, we examined the impact of combining the novel adjuvant dmLT with trivalent IPV for dose sparing, induction of mucosal immunity and increasing longevity of anti-poliovirus (PV) responses in a mouse model following either intradermal (ID) or intramuscular (IM) delivery.We found that non-adjuvanted ID delivery was not superior to IM delivery for fractional dose sparing, but was associated with development of mucosal immunity. Vaccination with IPV + dmLT promoted serum anti-PV neutralizing antibodies with fractional IPV doses by either IM or ID delivery, achieving at least five-fold dose sparing above non-adjuvanted fractional doses. These responses were most noticeable with the PV1 component of the trivalent vaccine. dmLT also promoted germinal center formation and longevity of serum anti-PV neutralizing titers. Lastly, dmLT enhanced mucosal immunity, as defined by fecal and intestinal anti-PV IgA secretion, when included in IPV immunization by ID or IM delivery. These studies demonstrate that dmLT is an effective adjuvant for either IM or ID delivery of IPV. Inclusion of dmLT in IPV immunizations allows antigen dose sparing and enhances mucosal immunity and longevity of anti-PV responses.     

Onjisaponins, from the root of Polygala tenuifolia Willdenow, as effective adjuvants for nasal influenza and diphtheria–pertussis–tetanus vaccines

Active substances from hot water extracts from 267 different Chinese and Japanese medicinal herbs were screened for mucosal adjuvant activity with influenza HA vaccine in mice. The extract from the root of Polygala tenuifolia was found to contain potent mucosal adjuvant activity. The active substances were purified and identified as onjisaponins A, E, F, and G. When each onjisaponin (10 μg) was intranasally (i.n.) inoculated with influenza vaccine (10 μg) in mice, serum hemagglutination-inhibiting (HI) antibody titers increased 3–14 times over control mice administered vaccine alone after 4 weeks. When each onjisaponin (10 μg) was i.n. inoculated with the vaccine (10 μg) followed by i.n. vaccination of the vaccine alone after 3 weeks, serum HI antibody titers increased 27–50 fold over those mice given i.n. vaccinations without onjisaponins. These same conditions also significantly increased nasal anti-influenza virus IgA antibody titers. Two inoculations with onjisaponin F (1 μg) and influenza HA vaccine (1 μg) at 3 weeks intervals, significantly increased serum HI antibody and nasal anti-influenza virus IgA and IgG antibody titers after only 1 week over mice given HA vaccine alone after the secondary vaccination. Intranasal vaccination with onjisaponin F inhibited proliferation of mouse adapted influenza virus A/PR/8/34 in bronchoalveolar lavages of infected mice. Separate intranasal vaccinations with onjisaponins A, E, F, and G (10 μg) each and diphtheria–pertussis–tetanus (DPT) vaccine (10 μg) of mice followed by i.n. vaccination with DPT vaccine alone after 4 weeks showed significant increases in serum IgG and nasal IgA antibody titers after 2 weeks following secondary vaccination over mice vaccinated with DPT vaccine alone. All onjisaponins showed little hemolytic activity at concentrations up to 100 μg/ml. The results of this study suggest that onjisaponins may provide safe and potent adjuvants for intranasal inoculation of influenza HA and DPT vaccines.     

Systemic immunization with CCL27/CTACK modulates immune responses at mucosal sites in mice and macaques

Plasmid DNA is a promising vaccine platform that has been shown to be safe and able to be administered repeatedly without vector interference. Enhancing the potency of DNA vaccination through co-delivery of molecular adjuvants is one strategy currently under investigation. Here we describe the use of the novel chemokine adjuvant CCL27/CTACK to enhance immune responses to an HIV-1 or SIV antigen in mice and rhesus macaques. CCL27 has been shown to play a role in inflammatory responses through chemotaxis of CCR10+ cells, and we hypothesized that CCL27 may modulate adaptive immune responses.     

Intranasal administration of a flagellin-adjuvanted inactivated influenza vaccine enhances mucosal immune responses to protect mice against lethal infection

The influenza virus, a mucosal pathogen that infects the respiratory tract, is a major global health issue. There have been attempts to mucosally administer inactivated influenza vaccines to induce both mucosal and systemic immune responses. However, mucosally administered inactivated influenza vaccine has low immunogenicity, which is partially due to the lack of an effective mucosal adjuvant. The development of a safe and effective mucosal adjuvant is a prerequisite to the practical use of a mucosal inactivated influenza vaccine. We have previously demonstrated that a bacterial flagellin, Vibrio vulnificus FlaB, when mixed with antigen and administered intranasally, exerts a strong mucosal adjuvant activity by stimulating the Toll-like receptor 5 (TLR5). In this study, we tested whether the FlaB protein could serve as an effective mucosal adjuvant for an inactivated trivalent influenza vaccine (TIV) manufactured for humans; in a murine vaccination model, this vaccine consists of A/Brisbane/59/07 (H1N1 subtype), A/Uruguay/716/07 (H3N2 subtype), and B/Florida/4/06 (B type). Intranasal co-administration of the TIV with FlaB induced prominent humoral responses as demonstrated by high influenza-specific IgA levels in both the mucosal secretions and serum and significant specific IgG induction in the systemic compartment. The FlaB protein significantly potentiated influenza-specific cytokine production by draining lymph node cells and splenocytes. The FlaB mucosal adjuvant conferred excellent protection against a lethal challenge with a live virulent virus with high hemagglutination inhibition (HAI) antibody (Ab) titers. The FlaB did not accumulate in the olfactory nerve and epithelium, guaranteeing against a retrograde uptake into the central nervous system. These results suggest that FlaB can be used as a promising mucosal adjuvant for nasal inactivated influenza vaccine development.     

Development of a respiratory syncytial virus vaccine using human hepatitis B core-based virus-like particles to induce mucosal immunity

BackgroundRespiratory syncytial virus (RSV) is an important viral pathogen responsible for severe infection of the lower respiratory tract in children under the age of 5 years. No vaccines against RSV are currently in clinical use. Vaccine-associated enhanced respiratory disease (ERD) caused by excess Th2 type responses was observed in a clinical trial of formalin-inactivated RSV (FI-RSV) in antigen-naïve infants. Thus, inducing a balanced immune response is a crucial issue in the development of an RSV vaccine.MethodsIn this study, we constructed, expressed, and purified a recombinant RSV vaccine candidate (i.e., HRØ24) containing the two heptad repeat regions and the antigenic sites Ø, II, and IV of the RSV F protein. The RSV vaccine candidate was intranasally administrated to BALB/c and C57BL/6 mice in combination with virus-like particles (VLPs) derived from the core protein of the hepatitis B virus (HBc). Mucosal immunity to HRØ24 was then assessed.ResultsIntranasal administration of HBc VLPs in combination with HRØ24 induced serum IgGs against HRØ24 as well as lung HRØ24-specific sIgAs in both C57BL/6 and BALB/c mouse models. The secretion of IFN-γ from splenocyte re-stimulation and an elevated ratio of serum IgG2a to IgG1 indicated that the immune response induced by the HBc VLPs/HRØ24 mixture was Th1-biased. Weight loss of <5% and no to low eosinophil infiltration was observed in histological analysis of the lung following a challenge with the RSV A2 strain. These results suggest that the HBc VLPs/HRØ24 combination conferred substantial partial protection against RSV-induced illness in mice.ConclusionsLong-term immunity to RSV-induced illness was achieved via intranasal vaccination using a mixture of HBc VLPs and HRØ24 in mouse models.