Polyethyleneimine is a potent mucosal adjuvant for viral glycoprotein antigens.
ABSTRACT: Protection against mucosally transmitted infections probably requires immunity at the site of pathogen entry, yet there are no mucosal adjuvant formulations licensed for human use. Polyethyleneimine (PEI) represents a family of organic polycations used as nucleic acid transfection reagents in vitro and DNA vaccine delivery vehicles in vivo. Here we show that diverse PEI forms have potent mucosal adjuvant activity for viral subunit glycoprotein antigens. A single intranasal administration of influenza hemagglutinin or herpes simplex virus type-2 (HSV-2) glycoprotein D with PEI elicited robust antibody-mediated protection from an otherwise lethal infection, and was superior to existing experimental mucosal adjuvants. PEI formed nanoscale complexes with antigen, which were taken up by antigen-presenting cells in vitro and in vivo, promoted dendritic cell trafficking to draining lymph nodes and induced non-proinflammatory cytokine responses. PEI adjuvanticity required release of host double-stranded DNA that triggered Irf3-dependent signaling. PEI therefore merits further investigation as a mucosal adjuvant for human use.
Project description:Increasing evidence suggests that mucosally targeted vaccines will enhance local humoral and cellular responses whilst still eliciting systemic immunity. We therefore investigated the capacity of nasal, sublingual or vaginal delivery of DNA-PEI polyplexes to prime immune responses prior to mucosal protein boost vaccination. Using a plasmid expressing the model antigen HIV CN54gp140 we show that each of these mucosal surfaces were permissive for DNA priming and production of antigen-specific antibody responses. The elicitation of systemic immune responses using nasally delivered polyplexed DNA followed by recombinant protein boost vaccination was equivalent to a systemic prime-boost regimen, but the mucosally applied modality had the advantage in that significant levels of antigen-specific IgA were detected in vaginal mucosal secretions. Moreover, mucosal vaccination elicited both local and systemic antigen-specific IgG(+) and IgA(+) antibody secreting cells. Finally, using an Influenza challenge model we found that a nasal or sublingual, but not vaginal, DNA prime/protein boost regimen protected against infectious challenge. These data demonstrate that mucosally applied plasmid DNA complexed to PEI followed by a mucosal protein boost generates sufficient antigen-specific humoral antibody production to protect from mucosal viral challenge.
Project description: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.
Project description:Vaccine-induced memory T cells localized at mucosal sites can provide rapid protection from viral infection. All-trans-retinoic acid (ATRA) has been shown to act physiologically to induce the expression of gut-homing receptors on lymphocytes. We tested whether the administration of exogenous ATRA during a systemic vaccination of mice could enhance the generation of mucosal CD8(+) T cell immunity, which might represent a strategy for establishing better protection from viral infection via mucosal routes. ATRA induced the expression of CCR9 and α4β7 on both mouse and human CD8(+) T cells activated in vitro. The administration of ATRA to mice during in vivo priming with a replication-defective recombinant adenovirus vector expressing the lymphocytic choriomeningitis virus glycoprotein (LCMVgp) (Ad5gp) increased numbers of both effector and memory T cells in intestinal mucosal tissues and showed higher frequencies of systemic central memory-like T cells that exhibited enhanced proliferation during boosting immunization with recombinant modified vaccinia virus Ankara expressing LCMVgp (MVAgp). Mice that received ATRA during Ad5gp vaccination were more resistant to intravaginal challenge by recombinant vaccinia virus expressing LCMVgp (VVgp), reflecting in part stronger T cell recall responses in situ. Thus, ATRA appears to be useful as an adjuvant during vaccination to increase memory T cell responses and protection from viral infection at mucosal sites and may facilitate the development of more effective vaccines against mucosally transmitted pathogens such as HIV.
Project description:Consecutive cases of human infection with H7N9 influenza viruses since 2013 in China have prompted efforts to develop an effective treatment. Subunit vaccines introduced by intranasal administration can block an infection at its primary site; flagellin (fliC) and polyethyleneimine (PEI) have been shown to be potent adjuvants. We previously generated the hemagglutinin (HA)1-2-fliC fusion protein consisting of the globular head domain (HA1-2; amino acids 62-284) of HA fused with <i>Salmonella typhimurium</i> fliC. In the present study, we investigated its effectiveness of both flagellin and PEI as mucosal adjuvants for the H7N9 influenza subunit vaccine. Mice immunized intranasally with HA1-2-fliC and HA1-2-PEI showed higher HA1-2-specific immunoglobulin (Ig)G and IgA titers in serum, nasal wash, and bronchial alveolar lavage fluid. Moreover, splenocyte activation and proliferation and the number of HA1-2-specific interferon (IFN)-?- and interleukin (IL)-4-producing splenocytes were markedly increased in the fliC and PEI groups; in the latter, there were more cells secreting IL-4 than IFN-?, suggesting that fliC induced T helper type (Th)1 and Th2 immune responses, and PEI induced Th2-biased responses, consistent with the serum antibody isotype pattern (IgG1/IgG2a ratio). Furthermore, virus challenge was performed in a chicken model. The results showed that chickens receiving fliC and PEI adjuvant vaccine exhibited robust immune responses leading to a significant reduction in viral loads of throat and cloaca compared to chickens receiving only HA1-2. These findings provide a basis for the development of H7N9 influenza HA1-2 mucosal subunit vaccines.
Project description:Cholera toxin (CT) is a potent adjuvant for inducing mucosal immune responses. However, the mechanism by which CT induces adjuvant activity remains unclear. Here we show that the microbiota is critical for inducing antigen-specific IgG production after intranasal immunization. After mucosal vaccination with CT, both antibiotic-treated and germ-free (GF) mice had reduced amounts of antigen-specific IgG, smaller recall-stimulated cytokine responses, impaired follicular helper T (TFH) cell responses and reduced numbers of plasma cells. Recognition of symbiotic bacteria via the nucleotide-binding oligomerization domain containing 2 (Nod2) sensor in cells that express the integrin CD11c (encoded by Itgax) was required for the adjuvanticity of CT. Reconstitution of GF mice with a Nod2 agonist or monocolonization with Staphylococcus sciuri, which has high Nod2-stimulatory activity, was sufficient to promote robust CT adjuvant activity, whereas bacteria with low Nod2-stimulatory activity did not. Mechanistically, CT enhanced Nod2-mediated cytokine production in dendritic cells via intracellular cyclic AMP. These results show a role for the microbiota and the intracellular receptor Nod2 in promoting the mucosal adjuvant activity of CT.
Project description:Nasal vaccination has the potential to elicit systemic and mucosal immunity against pathogens. However, split and subunit vaccines lack potency at stimulating mucosal immunity, and an adjuvant is indispensable for eliciting potent mucosal immune response to nasal vaccines. Endocine, a lipid-based mucosal adjuvant, potentiates both systemic and mucosal immune responses. Although Endocine has shown efficacy and tolerability in animal and clinical studies, its mechanism of action remains unknown. It has been reported recently that endogenous danger signals are essential for the effects of some adjuvants such as alum or MF59. However, the contribution of danger signals to the adjuvanticity of Endocine has not been explored. Here, we show that RNA is likely to be an important mediator for the adjuvanticity of Endocine. Administration of Endocine generated nucleic acids release, and activated dendritic cells (DCs) in draining lymph nodes in vivo. These results suggest the possibility that Endocine indirectly activates DCs via damage-associated molecular patterns. Moreover, the adjuvanticity of Endocine disappeared in mice lacking TANK-binding kinase 1 (Tbk1), which is a downstream molecule of nucleic acid sensing signal pathway. Furthermore, co-administration of RNase A reduced the adjuvanticity of Endocine. These data suggest that RNA is important for the adjuvanticity of Endocine.
Project description:Human infections with highly pathogenic avian influenza H5N1 viruses persist as a major global health concern. Vaccination remains the primary protective strategy against H5N1 and other novel avian influenza virus infections. We investigated the use of <i>E. coli</i> type IIb heat labile enterotoxin B subunit (LTIIb-B5) as a mucosal adjuvant for intranasal immunizations with recombinant HA proteins against H5N1 avian influenza viruses. Use of LTIIb-B5 adjuvant elicited more potent IgG, IgA, and neutralizing antibody titers in both sera and bronchoalveolar lavage fluids, thus increasing protection against lethal virus challenges. LTIIb-B5 mucosal adjuvanticity was found to trigger stronger Th17 cellular response in spleen lymphocytes and cervical lymph nodes. Studies of anti-IL-17A monoclonal antibody depletion and IL-17A knockout mice also suggest the contribution from Th17 cellular response to anti-H5N1 protective immunity. Our results indicate a link between improved protection against H5N1 live virus challenges and increased Th17 response due to the use of LTIIb-B5 mucosal adjuvant with HA subunit proteins.
Project description:The generation of effective levels of antigen-specific immunity at the mucosal sites of pathogen entry is a key goal for vaccinologists. We explored topical vaginal application as an approach to initiate local antigen-specific immunity, enhance previously existing systemic immunity or re-target responses to the mucosae. To deliver a protein vaccine formulation to the vaginal mucosal surface, we used a novel vaginal ring device comprising a silicone elastomer body into which three freeze-dried, rod-shaped, hydroxypropylmethylcellulose inserts were incorporated. Each rod contained recombinant HIV-1 CN54gp140 protein (167?g)±R848 (167?g) adjuvant. The inserts were loaded into cavities within each ring such that only the ends of the inserts were initially exposed. Sheep received a prime-boost vaccination regime comprising intramuscular injection of 100?g CN54gp140+200?g R848 followed by three successive ring applications of one week duration and separated by one month intervals. Other sheep received only the ring devices without intramuscular priming. Serum and vaginal mucosal fluids were sampled every two weeks and analysed by CN54gp140 ELISA and antigen-specific B cells were measured by flow cytometry at necropsy. Vaccine antigen-specific serum antibody responses were detected in both the intramuscularly-primed and vaginal mucosally-primed groups. Those animals that received only vaginal vaccinations had identical IgG but superior IgA responses. Analysis revealed that all animals exhibited mucosal antigen-specific IgG and IgA with the IgA responses 30-fold greater than systemic levels. Importantly, very high numbers of antigen-specific B cells were detected in local genital draining lymph nodes. We have elicited local genital antigen-specific immune responses after topical application of an adjuvanted antigen formulation within a novel vaginal ring vaccine release device. This regimen and delivery method elicited high levels of antigen-specific mucosal IgA and large numbers of local antigen-reactive B cells, both likely essential for effective mucosal protection.
Project description:Prophylactic vaccination against HIV-1 sexual transmission will probably require antibody elicitation at genital mucosal surfaces. However, HIV-1 envelope glycoprotein (Env)-based antigens are weakly immunogenic, particularly when applied mucosally. The polyanion PRO 2000 is safe for human vaginal application, and thus may represent a potential formulating agent for vaginal delivery of experimental vaccine immunogens. Based upon its biochemical properties, we hypothesized that PRO 2000 might enhance mucosal immunogenicity of HIV-1 envelope glycoprotein (Env)-based antigens, promoting local and systemic immune responses. Vaginal immunization with Env-PRO 2000 resulted in significantly increased titres of Env-specific mucosal IgA and IgG in mice and rabbits, respectively, compared to Env alone, revealing modest but significant mucosal adjuvant activity for PRO 2000. In vitro, PRO 2000 associated with Env, protecting the glycoprotein from proteolytic degradation in human vaginal lavage. Unexpectedly, PRO 2000 antagonized TLR4 activation, suppressing local production of inflammatory cytokines. Since inflammation-mediated recruitment of viral target cells is a major risk factor in HIV-1 transmission, the immune modulatory and anti-inflammatory activities of PRO 2000 combined with its intravaginal safety profile suggests promise as an HIV-1 mucosal vaccine formulating agent.
Project description:The immunogenicity of bacterial flagellin has been reported in different studies. By its close interaction with the immune system, the flagellin represents an interesting adjuvant and vaccine candidate. Salmonella Typhimurium flagellin has already been tested as adjuvant to stimulate mucosal immunity. Here, we assessed the ability of Clostridium difficile flagellin FliC to act as a mucosal adjuvant, first combined with ovalbumin as antigen and second with a C. difficile surface protein, the precursor of the S-layer proteins SlpA. Using ovalbumin as antigen, we compared the gut mucosal adjuvanticity of FliC to Salmonella Typhimurium flagellin and cholera toxin. Two routes of immunization were tested in a mouse model: intra-rectal and intra-peritoneal, following which, gut mucosal and systemic antibody responses against ovalbumin (Immunoglobulins G and Immunoglobulins A) were analyzed by Enzyme-Linked Immuno Assay in intestinal contents and in sera. In addition, ovalbumin-specific immunoglobulin producing cells were detected in the intestinal lamina propria by Enzyme-Linked Immunospot. Results showed that FliC as adjuvant for immunization targeting ovalbumin was able to stimulate a gut mucosal and systemic antibody response independently of the immunization route. In order to develop a mucosal vaccine to prevent C. difficile intestinal colonization, we assessed in a mouse model the efficacy of FliC as adjuvant compared with cholera toxin co-administrated with the C. difficile S-layer precursor SlpA as antigen. After challenge, a significant decrease of C. difficile intestinal colonization was observed in immunized groups compared to the control group. Our results showed that C. difficile FliC could be used as adjuvant in mucosal vaccination strategy against C. difficile infections.