Induction of systemic and mucosal immunity and maintenance of its memory against influenza A virus by nasal vaccination using a new mucosal adjuvant SF-10 derived from pulmonary surfactant in young cynomolgus monkeys

Induction of systemic and mucosal immunity and maintenance of its memory was investigated in 12 young male cynomolgus monkeys after intranasal instillation of flu vaccine using a new mucosal adjuvant SF-10 derived from pulmonary surfactant constituents. Split-product of influenza virus A/California/7/2009(H1N1)pdm hemagglutinin vaccine (HAv) at 15 μg with or without SF-10 and the adjuvant alone were instilled intranasally three times every 2 weeks. SF-10-adjuvanted HAv (SF-10-HAv) elicited significantly higher HAv-specific IgG and hemagglutinin inhibition (HI) titers in serum and HAv-specific secretory IgA and its neutralizing activities in nasal washes compared with HAv antigen and SF-10 alone. Significant cross-neutralizing activities of nasal washes after the third vaccination to several other H1N1 and H3N2 strains were observed. HI titers in serum and neutralizing activities in nasal washes reached peak levels at 6 weeks after initial vaccination, then gradually decreased after 10 weeks and returned to the baseline levels at 36 weeks. A single intranasal revaccination of SF-10-HAv at 36 weeks rapidly and significantly increased both immunity in serum and nasal washes compared with naïve monkeys. Revaccination by one or two doses achieved almost maximal immunity at 2 or 4 weeks after instillation. Statistically significant adverse effects (e.g., body weight loss, elevated body temperature, nasal discharge, change in peripheral blood leukocyte and platelet counts) were not observed for 2 weeks after vaccination of SF-10-HAv, HAv or SF-10 and also during the experimental period. These results in young monkey model suggest the potential of clinical use SF-10 for intranasal flu vaccine.     

Retinaldehyde dehydrogenase 2 as a molecular adjuvant for enhancement of mucosal immunity during DNA vaccination

In order for vaccines to induce efficacious immune responses against mucosally transmitted pathogens, such as HIV-1, activated lymphocytes must efficiently migrate to and enter targeted mucosal sites. We have previously shown that all-trans retinoic acid (ATRA) can be used as a vaccine adjuvant to enhance mucosal CD8⁺ T cell responses during vaccination and improve protection against mucosal viral challenge. However, the ATRA formulation is incompatible with most recombinant vaccines, and the teratogenic potential of ATRA at high doses limits its usage in many clinical settings. We hypothesized that increasing in vivo production of retinoic acid (RA) during vaccination with a DNA vector expressing retinaldehyde dehydrogenase 2 (RALDH2), the rate-limiting enzyme in RA biosynthesis, could similarly provide enhanced programming of mucosal homing to T cell responses while avoiding teratogenic effects. Administration of a RALDH2- expressing plasmid during immunization with a HIVgag DNA vaccine resulted in increased systemic and mucosal CD8⁺ T cell numbers with an increase in both effector and central memory T cells. Moreover, mice that received RALDH2 plasmid during DNA vaccination were more resistant to intravaginal challenge with a recombinant vaccinia virus expressing the same HIVgag antigen (VACVgag). Thus, RALDH2 can be used as an alternative adjuvant to ATRA during DNA vaccination leading to an increase in both systemic and mucosal T cell immunity and better protection from viral infection at mucosal sites.     

Intranasal vaccination of mice against infection with Mycobacterium tuberculosis

The intranasal (i.n.) route of immunisation, has recently been of active interest in endeavours to improve the efficacy of vaccination against a number of respiratory infections. Here, we examined the outcome of tuberculous infection in BALB/c mice. I.n. application of the BCG-Pasteur strain was found to be highly protective against challenge infection with the pathogenic H37Rv strain given after a 4-week interval, reflected by the 100-fold reduction of CFUs in both lungs and spleens. Vaccination with the recombinant PstS-1 antigen and cholera toxin significantly protected against the challenge given 10 days later, but only marginally after 12 weeks. Histological examination showed, that i.n. vaccination abrogated the confluent infiltration of lungs with inflammatory cells, which surrounds the granulomas in H37Rv challenged control mice. In conclusion, the strong protection demonstrated by BCG suggests that the i.n. route of vaccine delivery deserves further attention toward improving vaccination against tuberculosis.     

Highly homogenous tri-acylated S-LPS acts as a novel clinically applicable vaccine against Shigella flexneri 2a infection

Shigellosis, a major cause of diarrhea worldwide, exhibits high morbidity and mortality in children. Specificity of Shigella immunity is determined by the structure of the main protective O-antigen polysaccharide component incorporated into the lipopolysaccharide (LPS) molecule. Endotoxicity, however, precludes LPS clinical use. Thus, there is still no vaccine against the most prevalent shigellosis species (serotype S. flexneri 2a), despite ongoing efforts focused on inducing serotype-specific immunity. As LPS is highly heterogenous, we hypothesized that more homogenous pools of LPS might be less toxic. We developed a method to generate a homogenous S. flexneri 2a LPS subfraction, Ac₃-S-LPS, containing long chain O-specific polysaccharide (S-LPS) and mainly tri-acylated lipid A, with no penta- and hexa-acylated, and rare tetra-acylated lipid A. Ac₃-S-LPS had dramatically reduced pyrogenicity and protected guinea pigs from shigellosis. In volunteers, 50?µg of injected Ac₃-S-LPS vaccine was safe, with low pyrogenicity, no severe and few minor adverse events, and did not induce pro-inflammatory cytokines. In spite of the profound lipid A modification, the vaccine induced a prevalence of IgG and IgA antibodies. Thus, we have developed the first safe immunogenic LPS-based vaccine candidate for human administration. Homogenous underacetylated LPSs may also be useful for treating other LPS-driven human diseases.Clinical trial registry: http://grls.rosminzdrav.ru/     

Development of a safe and effective novel synthetic mucosal adjuvant SF-10 derived from physiological metabolic pathways and function of human pulmonary surfactant

A safe and effective mucosal adjuvant is required for vaccination against influenza A virus (IAV) infection. Previously, we described that intranasally administration of surfacten®, a medicine derived from bovine pulmonary surfactant (PS), with IAV vaccine can induce IAV-specific IgA in the respiratory tract mucosa and IgG in serum. PS is secreted by alveolar type II cells and Clara cells and serves to reduce lung surface tension. PS finished its rules is incorporated by antigen presenting cells (APCs), such as alveolar macrophages and dendritic cells, and alveolar type II cells and rapidly metabolized. We focused on the metabolic pathways and rapid metabolic turnover of PS and developed a PS-based mucosal adjuvant. First, we determined the essential components of PS adjuvanticity and found that the complex of three PS lipids and surfactant protein-C can enhance to deliver the vaccine antigen and activate APCs. Later, we improved the safety, efficacy and ease of manufacture and finally succeeded in developing SF-10. The use of SF-10 with influenza split vaccine (HAv) (HAv-SF-10) enhances HAv incorporation into APCs both in vitro and in vivo, and intranasal instillation of HAv-SF-10 induced systemic and mucosal HAv-specific immunities in not only mice but also cynomolgus monkeys. The report that PS has physiological effects on the gastrointestinal mucosa prompted us develop a new SF-10-based vaccine that can be administered orally. In this review, We summarize our work on the development of clinically effective PS-based nasal and oral mucosal adjuvants for influenza vaccine.     

Nasal delivery of Protollin-adjuvanted H5N1 vaccine induces enhanced systemic as well as mucosal immunity in mice

Sporadic, yet frequent human infections with avian H5N1 influenza A viruses continue to pose a potential pandemic threat. Poor immunogenicity of unadjuvanted H5N1 vaccines warrants developing novel adjuvants and formulations as well as alternate delivery systems to improve their immunogenicity and efficacy. Here, we show that Protollin, a nasal adjuvant composed of Neisseria meningitides outer membrane proteins non-covalently linked to Shigella flexneri 2a lipopolysaccharide, is a potent nasal adjuvant for an inactivated split virion H5N1 clade 1 A/Viet Nam1203/2004 (A/VN/1203/04) vaccine in a mouse model. Protollin-adjuvanted vaccines elicited enhanced serum protective hemagglutination inhibition titers, mucosal IgA responses, and H5N1-specific cell-mediated immunity that resulted in complete protection against a lethal challenge with a homologous virus as well as a heterologous clade 2 virus A/Indonesia/05/2005 (A/IN/05/05). Detailed analysis of adaptive immunity revealed that Protollin increased the frequency of lymphoid- as well as local tissue-resident antibody-secreting cells, local germinal center reaction of B cells, broad-spectrum of CD4 T cell response. Our findings suggest that nasal delivery of H5N1 vaccine with Protollin adjuvant can overcome the poor immunogenicity of H5N1 vaccines, induce both cellular and humoral immune responses, enhance protection against challenge with clade 1 and clade 2 H5N1 viruses and achieve significant antigen dose-sparing.     

Co-administration of cholera toxin and apple polyphenol extract as a novel and safe mucosal adjuvant strategy

Although native cholera toxin (CT) is an extremely effective adjuvant, its toxicity prevents its use in humans. We report here that apple polyphenol extract (APE), obtained from unripe apples, reduces CT-induced morphological changes and cAMP accumulation. Based upon this finding, we have attempted to design a novel, effective and safe mucosal vaccine by using CT with several dosages of APE as nasal adjuvants. Mice nasally immunized with OVA plus CT and an optimal dosage of APE showed significantly reduced levels of inflammatory responses as well as total and OVA-specific IgE antibodies when compared with mice given without APE. However, levels of both mucosal and systemic OVA-specific antibody responses were maintained. Further, APE significantly down-regulated accumulation of CT in the olfactory nerves and epithelium. In summary, an optimal dosage of APE would take full advantage of mucosal adjuvanticity of native CT without any toxicity for application in humans.     

Nanoencapsulated retinoic acid as a safe tolerogenic adjuvant for intranasal vaccination against cutaneous leishmaniasis

Mucosal, but not peripheral, vaccination with whole Leishmania amazonensis antigen (LaAg) effectively protects mice against leishmaniasis, likely through a tolerogenic mechanism. Given the crucial role of retinoic acid (RA) in CD4⁺ Foxp3⁺ regulatory T cell (T_reg) differentiation and mucosal tolerance, here we evaluated the capacity of RA to improve intranasal (i.n.) vaccination with LaAg. To prevent degradation and possible mucosa irritation, RA was encapsulated in solid lipid nanoparticles (RA-SLN). Thus, BALB/c mice were given two i.n. doses of LaAg alone or in association with RA-SLN (LaAg/RA-SLN) prior to challenge with L. amazonensis. No histological sign of irritation or inflammation was produced in the nasal mucosa after RA-SLN administration. LaAg/RA-SLN vaccine was more effective in delaying lesion growth and reducing parasite burdens than LaAg alone (96% and 61% reduction, respectively). At two months after challenge, both vaccinated groups displayed similar T helper (Th) 1-skewed in situ cytokine responses, different from early infection where both Th1 and Th2 responses were suppressed, except for transforming growth factor (TGF)-β mRNA, that was higher in mice given RA-SLN. At the mucosa, RA-SLN promoted enhanced expression of interleukin (IL)-10 and CD4⁺ Foxp3⁺ T_reg population. In sum, these data show that RA-SLN is an effective and safe tolerogenic adjuvant for i.n. vaccination against leishmaniasis.     

Partial protective immunity against toxoplasmosis in mice elicited by recombinant Toxoplasma gondii malate dehydrogenase

Toxoplasma gondii can infect humans and wildlife, sometimes causing serious clinical presentations. Currently, no viable vaccine or effective drug strategies exist to prevent and control toxoplasmosis. T. gondii malate dehydrogenase (TgMDH) is a crucial enzyme in cellular redox reactions and has been shown to be an immunogenic compound that could be a potential vaccine candidate. Here, we investigate the protective efficacy of recombinant TgMDH (rTgMDH) against T. gondii infection in BALB/c mice. All mice were vaccinated via the nasal route. We determined the optimal vaccination dose by monitoring systemic and mucosal immune responses. The results showed that mice vaccinated with 30 μg of rTgMDH produced the highest antibody titers in serum, a strong lymphoproliferative response, marked increases in their levels of IL-2 and IFN-γ, and significantly greater levels of specific secretory IgA (sIgA) in mucosal washes. In addition, the vaccinated mice were orally challenged with tachyzoites of the virulent T. gondii RH strain 2 weeks after the final vaccination. Compared to the control group, we found that vaccination with rTgMDH increased the survival rate of infected mice by 47% and also significantly reduced the tachyzoite loads in their liver (by 58%) and brain (by 41%). Therefore, the rTgMDH protein triggers a strong systemic and mucosal immune response and provides partial protection against T. gondii infection.     

The member of the cyclic di-nucleotide family bis-(3′, 5′)-cyclic dimeric inosine monophosphate exerts potent activity as mucosal adjuvant

Here we demonstrated that bis-(3′,5′)-cyclic dimeric inosine monophosphate (c-di-IMP) exhibits potent adjuvant properties. BALB/c or C57BL/6 mice were immunized with the model antigens beta-galactosidase (β-Gal) or Ovalbumin (OVA) alone or co-administered with c-di-IMP by the intranasal route. Animals receiving c-di-IMP showed significantly higher anti-β-Gal or OVA immunoglobulin G titres (IgG) in sera than those vaccinated with β-Gal or OVA alone. Furthermore, strong local immune responses were also detectable in different mucosal territories, as shown by the high levels of β-Gal-specific secretory IgA (sIgA). The analysis of the antigen-specific IgG isotypes in sera, together with the profiles of the cytokines and chemokines secreted by lymphocytes from vaccinated animals showed that the use of c-di-IMP resulted in stimulation of a mixed T_H1/T_H2/T_H17 response. Mucosal immunization of C57BL/6 mice with OVA using c-di-IMP as adjuvant also led to the stimulation of both humoral and cellular (i.e., 60% of antigen-specific lysis by in vivo CTL) responses. Our results demonstrated that the novel compound c-di-IMP exhibits strong adjuvant properties when co-administered with an antigen by the mucosal route, thereby representing a promising candidate adjuvant for the development of mucosal vaccination strategies.     

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.     

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.     

Cross-protection against influenza virus infection by intranasal administration of M1-based vaccine with chitosan as an adjuvant

The antigenic variation of influenza virus represents a major health problem, thus continuous efforts have been made to develop broad-spectrum vaccines against influenza virus. Matrix protein 1 (M1) protein is highly conserved in all influenza A strains. In this study, M1 protein was efficiently expressed in Escherichia coli (E. coli), then purified and used for immunization of BALB/c mice by intranasal drip using chitosan as adjuvant. The M1 protein was administered intranasally to mice in combination with chitosan adjuvant twice at an interval of 3 weeks. Three weeks after the second immunization, the mice were challenged with a lethal dose (5 × LD₅₀) of A/Chicken/Jiangsu/7/2002 (H9N2) virus, PR8 (H1N1) virus and A/Chicken/Henan/12/2004 (H5N1) virus. The protective immunity of the vaccine was evaluated by determining the survival rates, residual lung virus titers, bodyweight, and the serum antibody titers of the mice. The results showed that nasal administration of 100 μg M1 in combination with chitosan could not only completely protect the mice effectively against the challenge of the homologous virus but also protect 70% and 30% of the mice against the heterologous H1N1 and H5N1 viruses, respectively. The study indicated that the M1 protein was a candidate antigen for a broad-spectrum influenza virus vaccine and the adjuvant chitosan significantly improved the efficacy of the M1 vaccine.     

The induction of systemic and mucosal immune responses following the subcutaneous immunization of mature adult mice: characterization of the antibodies in mucosal secretions of animals immunized with antigen formulations containing a vitamin D3 adjuvant

Systemic and mucosal immune responses were effectively induced following the subcutaneous administration of Haemophilus influenzae type b oligosaccharide conjugated to diphtheria toxoid vaccine in a formulation containing the active form of vitamin D3. IgA and IgG antibodies with specificity for both the protein and oligosaccharide components of the vaccine were detectable in mucosal secretions following immunization. The IgA and IgG mucosal antibodies were produced locally, and were functional as demonstrated by their diphtheria toxin neutralizing activity. Our data suggests that subcutaneous tissues can effectively serve as effective antigen presenting sites for both mucosal and systemic immune responses to antigens administered in combination with vitamin D3.     

Evaluation of adenovirus 19a as a novel vector for mucosal vaccination against influenza A viruses

Since preexisting immunity and enhanced infection rates in a clinical trial of an HIV vaccine have raised some concerns on adenovirus (Ad) serotype 5-based vaccines, we evaluated the subgroup D adenovirus serotype Ad19a for its suitability as novel viral vector vaccine against mucosal infections. In BALB/c mice, we compared the immunogenicity and efficacy of E1/E3-deleted Ad19a vectors encoding the influenza A virus (IAV)-derived antigens hemagglutinin (HA) and nucleoprotein (NP) to the most commonly used Ad5 vectors. The adenoviral vectors were applied intranasally and induced detectable antigen-specific T cell responses in the lung and in the spleen as well as robust antibody responses. A prior DNA immunization significantly improved the immunogenicity of both vectors and resulted in full protection against a lethal infection with a heterologous H3N2 virus. Nevertheless, the Ad5-based vectors were slightly superior in reducing viral replication in the lung which corresponded to higher NP-specific T cell responses measured in the lungs.     

Contrasting effects of type I interferon as a mucosal adjuvant for influenza vaccine in mice and humans

To identify an adjuvant that enhances antibody responses in respiratory secretions to inactivated influenza virus vaccine (IVV), a comparison was made of responses to intranasal vaccinations of mice with IVV containing monophosphoryl lipid A (MPL), type I interferon (IFN) or cholera toxin B (CTB). Antibody in nasal secretions and lung wash fluids from mice was increased after vaccination and lung virus was significantly reduced after challenge to a similar level in each adjuvant group. Interferon was selected for a trial in humans. Trivalent inactivated influenza vaccine was given intranasally to healthy adult volunteers alone or with 1 million units (Mu) or 10 Mu of alpha interferon. Vaccinations were well tolerated but neither serum hemagglutination-inhibiting nor neutralizing antibody responses among the vaccine groups were significantly different. Similarly, neither neutralizing nor IgA antibody responses in nasal secretions were significantly different. Thus, despite exhibiting a significant adjuvant effect in mice, interferon did not exhibit an adjuvant effect for induction of antibody in respiratory secretions of humans to inactivated influenza virus vaccine given intranasally.     

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.     

The mucosal adjuvant effect of plant polysaccharides for induction of protective immunity against Helicobacter pylori infection

Some plant polysaccharides (PPSs) had been used as the adjuvants for systemic vaccination. In this study, we investigated whether PPSs could exhibit adjuvant effect at the mucosa. Groups of mice were intranasally immunized with Epimedium Polysaccharide (EPS), Trollius chinensis polysaccharide (TCPS), Siberian solomonseal rhizome polysaccharide (SSRPS) and Astragalus polysaccharides (APS) together with ovalbumin (OVA). Significantly higher levels of OVA-specific IgG in serum and secretory IgA in saliva, vaginal wash and intestinal lavage fluid were induced after immunization with OVA plus one of the four PPSs compared to OVA alone. Antigen absorption and TLR2 (Toll-like receptor 2) activation may be related to their mucosal adjuvant effect. Of note, when APS used as an adjuvant, intranasally vaccination with recombination UreB (rUreB, Urease subunit B) conferred more robust protection against Helicobacter pylori (H. pylori). Immunized with rUreB in combination APS resulted in mixed specific Th1 and Th17 immune response, which may contribute to the inhibition of H. pylori colonization. Though specific Th2-dominant responses were elicited when the other three PPS intranasally immunized with rUreB, no significant difference in the protective effect were found between those groups and rUreb alone group. Taken together, the four PPSs may be promising candidates for mucosal adjuvant, and APS could enhance rUreB-specific protective immunity against H. pylori infection.     

Prospects for use of interleukin-12 as a mucosal adjuvant for vaccination of humans to protect against respiratory pneumococcal infection

Mucosal vaccination against pneumococcal disease offers potential protection against otitis media, pneumonia and invasive disease, including providing herd benefit by reducing pathogen carriage. The major obstacle, however, remains the lack of a suitable adjuvant for use in humans. Animal models have demonstrated success of interleukin-12 (IL-12) as an adjuvant for mucosal vaccines using recombinant pneumococcal protein antigens. This review examines the biology of the IL-12 cytokine family, the toxicity of IL-12 in human studies and suggests approaches by which IL-12 could be developed as a mucosal adjuvant with pneumococcal protein based vaccines, for use in humans.     

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.