A molecular mucosal adjuvant to enhance immunity against pneumococcal infection in the elderly.

Streptococcus pneumoniae (the pneumococcus) causes a major upper respiratory tract infection often leading to severe illness and death in the elderly. Thus, it is important to induce safe and effective mucosal immunity against this pathogen in order to prevent pnuemocaccal infection. However, this is a very difficult task to elicit protective mucosal IgA antibody responses in older individuals. A combind nasal adjuvant consisting of a plasmid encoding the Flt3 ligand cDNA (pFL) and CpG oligonucleotide (CpG ODN) successfully enhanced S. pneumoniae-specific mucosal immunity in aged mice. In particular, a pneumococcal surface protein A-based nasal vaccine given with pFL and CpG ODN induced complete protection from S. pneumoniae infection. These results show that nasal delivery of a combined DNA adjuvant offers an attractive potential for protection against the pneumococcus in the elderly.

Potential roles of CCR5(+) CCR6(+) dendritic cells induced by nasal ovalbumin plus Flt3 ligand expressing adenovirus for mucosal IgA responses.

We assessed the role of CCR5(+)/CCR6(+)/CD11b(+)/CD11c(+) dendritic cells (DCs) for induction of ovalbumin (OVA)-specific antibody (Ab) responses following mucosal immunization. Mice given nasal OVA plus an adenovirus expressing Flt3 ligand (Ad-FL) showed early expansion of CCR5(+)/CCR6(+)/CD11b(+)/CD11c(+) DCs in nasopharyngeal-associated lymphoid tissue (NALT) and cervical lymph nodes (CLNs). Subsequently, this DC subset became resident in submandibular glands (SMGs) and nasal passages (NPs) in response to high levels of CCR-ligands produced in these tissues. CD11b(+)/CD11c(+) DCs were markedly decreased in both CCR5(-/-) and CCR6(-/-) mice. Chimera mice reconstituted with bone marrow cells from CD11c-diphtheria toxin receptor (CD11c-DTR) and CCR5(-/-) or CD11c-DTR and CCR6(-/-) mice given nasal OVA plus Ad-FL had elevated plasma IgG, but reduced IgA as well as low anti-OVA secretory IgA (SIgA )Ab responses in saliva and nasal washes. These results suggest that CCR5(+)CCR6(+) DCs play an important role in the induction of Ag-specific SIgA Ab responses.

Notch-ligand expression by NALT dendritic cells regulates mucosal Th1- and Th2-type responses.

Our previous studies showed that an adenovirus (Ad) serotype 5 vector expressing Flt3 ligand (Ad-FL) as nasal adjuvant activates CD11c(+) dendritic cells (DCs) for the enhancement of antigen (Ag)-specific IgA antibody (Ab) responses. In this study, we examined the molecular mechanism for activation of CD11c(+) DCs and their roles in induction of Ag-specific Th1- and Th2-cell responses. Ad-FL activated CD11c(+) DCs expressed increased levels of the Notch ligand (L)-expression and specific mRNA. When CD11c(+) DCs from various mucosal and systemic lymphoid tissues of mice given nasal OVA plus Ad-FL were cultured with CD4(+) T cells isolated from non-immunized OVA TCR-transgenic (OT II) mice, significantly increased levels of T cell proliferative responses were noted. Furthermore, Ad-FL activated DCs induced IFN-γ, IL-2 and IL-4 producing CD4(+) T cells. Of importance, these APC functions by Ad-FL activated DCs were down-regulated by blocking Notch-Notch-L pathway. These results show that Ad-FL induces CD11c(+) DCs to the express Notch-ligands and these activated DCs regulate the induction of Ag-specific Th1- and Th2-type cytokine responses.

A novel combined adjuvant for nasal delivery elicits mucosal immunity to influenza in aging.

Since a combination of flt3 ligand plasmid (pFL) and CpG-oligodeoxynucleotides (ODN)(3) as a dendritic cell (DC)-targeting double mucosal adjuvant elicited ovalbumin-specific secretory IgA (S-IgA) antibody (Ab) responses, we examined whether this double adjuvant could induce influenza-specific protective immunity in aged mice. A double adjuvant plus A/Puerto Rico/8/34 (PR8) hemagglutinin (HA) induced increased numbers of CD11b(+) CD11c(+) DCs and both CD4(+) Th1- and Th2-type responses in the nasopharyngeal-associated lymphoreticular tissue, nasal passages and cervical lymph nodes. Further, increased levels of PR8 HA-specific S-IgA Ab responses were detected in the upper respiratory tact (URT) of aged and young adult mice given nasal PR8 HA with this double adjuvant. Thus, when mice were challenged with PR8 virus via the nasal route, both aged and young adult mice given nasal vaccine exhibited complete protection. Further, IgA-deficient mice nasally immunized with a double adjuvant influenza vaccine failed to provide protection against PR8 challenge. These results indicate that a nasal double adjuvant successfully induces PR8 HA-specific IgA Ab responses in both young adult and aged mice, which are essential for the prevention of influenza infection in the murine URT.

Oral-nasopharyngeal dendritic cells mediate T cell-independent IgA class switching on B-1 B cells.

Native cholera toxin (nCT) as a nasal adjuvant was shown to elicit increased levels of T-independent S-IgA antibody (Ab) responses through IL-5- IL-5 receptor interactions between CD4+ T cells and IgA+ B-1 B cells in murine submandibular glands (SMGs) and nasal passages (NPs). Here, we further investigate whether oral-nasopharyngeal dendritic cells (DCs) play a central role in the induction of B-1 B cell IgA class switch recombination (CSR) for the enhancement of T cell-independent (TI) mucosal S-IgA Ab responses. High expression levels of activation-induced cytidine deaminase, Iα-Cµ circulation transcripts and Iµ-Cα transcripts were seen on B-1 B cells purified from SMGs and NPs of both TCRß⁻/⁻ mice and wild-type mice given nasal trinitrophenyl (TNP)-LPS plus nCT, than in the same tissues of mice given nCT or TNP-LPS alone. Further, DCs from SMGs, NPs and NALT of mice given nasal TNP-LPS plus nCT expressed significantly higher levels of a proliferation-inducing ligand (APRIL) than those in mice given TNP-LPS or nCT alone, whereas the B-1 B cells in SMGs and NPs showed elevated levels of transmembrane activator and calcium modulator cyclophilin ligand interactor (TACI) expression. Interestingly, high frequencies of IgA+ B-1 B cells were induced when peritoneal IgA⁻ IgM+ B cells were stimulated with mucosal DCs from mice given nasal TNP-LPS plus nCT. Taken together, these findings show that nasal nCT plays a key role in the enhancement of mucosal DC-mediated TI IgA CSR by B-1 B cells through their interactions with APRIL and TACI.

A novel adenovirus expressing Flt3 ligand enhances mucosal immunity by inducing mature nasopharyngeal-associated lymphoreticular tissue dendritic cell migration.

Previously, we showed that nasal administration of a naked cDNA plasmid expressing Flt3 ligand (FL) cDNA (pFL) enhanced CD4(+) Th2-type, cytokine-mediated mucosal immunity and increased lymphoid-type dendritic cell (DC) numbers. In this study, we investigated whether targeting nasopharyngeal-associated lymphoreticular tissue (NALT) DCs by a different delivery mode of FL, i.e., an adenovirus (Ad) serotype 5 vector expressing FL (Ad-FL), would provide Ag-specific humoral and cell-mediated mucosal immunity. Nasal immunization of mice with OVA plus Ad-FL as mucosal adjuvant elicited high levels of OVA-specific Ab responses in external secretions and plasma as well as significant levels of OVA-specific CD4(+) T cell proliferative responses and OVA-induced IFN-gamma and IL-4 production in NALT, cervical lymph nodes, and spleen. We also observed higher levels of OVA-specific CTL responses in the spleen and cervical lymph nodes of mice given nasal OVA plus Ad-FL than in mice receiving OVA plus control Ad. Notably, the number of CD11b(+)CD11c(+) DCs expressing high levels of costimulatory molecules was preferentially increased. These DCs migrated from the NALT to mucosal effector lymphoid tissues. Taken together, these results suggest that the use of Ad-FL as a nasal adjuvant preferentially induces mature-type NALT CD11b(+)CD11c(+) DCs that migrate to effector sites for subsequent CD4(+) Th1- and Th2-type cytokine-mediated, Ag-specific Ab and CTL responses.

Ovalbumin-protein sigma 1 M-cell targeting facilitates oral tolerance with reduction of antigen-specific CD4+ T cells.

BACKGROUND & AIMS: The follicle-associated epithelium (FAE) plays key roles in antigen uptake and subsequent induction of mucosal immunity. In this study, we examined whether M-cell targeting using a protein antigen (Ag) delivery system would induce oral tolerance instead of enhancement of Ag-specific mucosal antibody (Ab) responses. METHODS: Mice were fed different doses of a recombinant protein sigma 1 of reovirus genetically conjugated to ovalbumin (OVA-psigma1), psigma1 only, or phosphate-buffered saline (PBS) before oral challenge with OVA plus cholera toxin as mucosal adjuvant. OVA-specific Ab and CD4-positive (CD4(+)) T-cell responses were determined. RESULTS: A low dose of OVA-psigma1 reduced anti-OVA Ab and CD4(+) T-cell responses in both mucosal and systemic lymphoid tissues. OVA/MHC I-A(d) tetramer staining showed that the numbers of OVA-specific CD4(+) T cells were significantly reduced in lamina propria of mice fed OVA-psigma1 than those fed psigma1 only or PBS only. In fact, Foxp3 expressing CD25(+) CD4(+) T cells were markedly increased in this tissue. Nonetheless, CD25(+) CD4(+) T cells from the spleen, mesenteric lymph nodes, and Peyer's patches of orally tolerized mice showed increased transforming growth factor beta1 (TGF-beta1) and interleukin-10 (IL-10) production compared with nontolerized mice. CONCLUSIONS: These results show that an FAE M-cell targeting protein Ag delivery system facilitates oral tolerance induction because of a reduction in Ag-specific CD4(+) T cells and increased levels of TGF-beta1 and IL-10 producing, CD25(+) CD4(+) regulatory T cells in both systemic and mucosal lymphoid tissues.

Nasal cholera toxin elicits IL-5 and IL-5 receptor alpha-chain expressing B-1a B cells for innate mucosal IgA antibody responses.

In this study, we examine whether native cholera toxin (nCT) as a mucosal adjuvant can support trinitrophenyl (TNP)-LPS-specific mucosal immune responses. C57BL/6 mice were given nasal TNP-LPS in the presence or absence of nCT. Five days later, significantly higher levels of TNP-specific mucosal IgA Ab responses were induced in the nasal washes, saliva, and plasma of mice given nCT plus TNP-LPS than in those given TNP-LPS alone. High numbers of TNP-specific IgA Ab-forming cells were also detected in mucosal tissues such as the nasal passages (NPs), the submandibular glands (SMGs), and nasopharyngeal-associated lymphoreticular tissue of mice given nCT. Flow cytometric analysis showed that higher numbers of surface IgA+, CD5+ B cells (B-1a B cells) in SMGs and NPs of mice given nasal TNP-LPS plus nCT than in those given TNP-LPS alone. Furthermore, increased levels of IL-5R alpha-chain were expressed by B-1a B cells in SMGs and NPs of mice given nasal TNP-LPS plus nCT. Thus, CD4+ T cells from these mucosal effector lymphoid tissues produce high levels of IL-5 at both protein and mRNA levels. When mice were treated with anti-IL-5 mAb, significant reductions in TNP-specific mucosal IgA Ab responses were noted in external secretions. These findings show that nasal nCT as an adjuvant enhances mucosal immune responses to a T cell-independent Ag due to the cross-talk between IL-5Ralpha+ B-1a B cells and IL-5-producing CD4+ T cells in the mucosal effector lymphoid tissues.

Nasal Flt3 ligand cDNA elicits CD11c+CD8+ dendritic cells for enhanced mucosal immunity.

Nasal immunization is an effective way to induce both mucosal and systemic immune responses. In this study, we assessed a cDNA vector for Flt3 ligand (FL) for its potential to enhance mucosal immunity or tolerance. Interestingly, tolerance was avoided and elevated levels of OVA-specific Ab responses were induced in nasal washes, fecal extracts, and saliva as well as in plasma when compared with mice given nasal OVA plus DNA plasmid without the FL gene. In addition, significant levels of OVA-specific CD4+ T cell proliferative responses and OVA-induced IL-4 and IL-2 production were noted in spleen and cervical lymph nodes. Further, marked increases in FL protein occurred in the nasal lamina propria and submandibular glands and the frequencies of CD11c+CD8+ dendritic cells (DCs) significantly increased in the mucosal tissues. Moreover, these DCs expressed high levels of CD40, CD80, CD86, and MHC class II molecules. Nasal delivery of plasmid FL with OVA resulted in FL expression in both mucosal inductive and effector sites and resulted in expanded activated lymphoid DCs. Thus, nasal plasmid FL prevents mucosal tolerance and enhances active immunity when given by a mucosal route.

Lack of oral tolerance in aging is due to sequential loss of Peyer's patch cell interactions.

Our past studies showed that Peyer's patches were required for the induction of oral tolerance to the protein antigen ovalbumin (OVA), but not to the hapten 2,4,6-trinitrobenzene sulfonic acid (TNBS). In the present study, the effects of immunosenescence on oral tolerance induction were assessed with these two toleragens. Significant reductions in OVA-specific serum IgG antibody and CD4(+) T cell responses to subsequent challenge were observed in OVA-fed, young adult mice. Importantly, these reduced anti-OVA antibody responses were associated with delayed-type hypersensitivity, and antigen-induced CD4(+) T(h)1- and T(h)2-type cytokine responses. On the other hand, aged mice fed OVA failed to develop oral tolerance. Thus, CD4(+) T cells from Peyer's patches produced selected T(h)2- but no T(h)1-type cytokines. The TNP-specific serum IgG antibody and T cell responses were significantly diminished by prior TNBS feeding in young adult, 6- to 8-month-old and 12- to 14-month-old, but not in senescent, 2-year-old mice. Finally, we have directly assessed dendritic cell subsets and T cell responses in Peyer's patches, and their function in tolerance induction was impaired at an earlier stage of life. These results suggest that lack of oral tolerance to the protein OVA during aging is the result of dysfunctional Peyer's patches.

Protective mucosal immunity in aging is associated with functional CD4+ T cells in nasopharyngeal-associated lymphoreticular tissue.

Our previous studies showed that mucosal immunity was impaired in 1-year-old mice that had been orally immunized with OVA and native cholera toxin (nCT) as mucosal adjuvant. In this study, we queried whether similar immune dysregulation was also present in mucosal compartments of mice immunized by the nasal route. Both 1-year-old and young adult mice were immunized weekly with three nasal doses of OVA and nCT or with a nontoxic chimeric enterotoxin (mutant cholera toxin-A E112K/B subunit of native labile toxin) from Brevibacillus choshinensis. Elevated levels of OVA-specific IgG Abs in plasma and secretory IgA Abs in mucosal secretions (nasal washes, saliva, and fecal extracts) were noted in both young adult and 1-year-old mice given nCT or chimeric enterotoxin as mucosal adjuvants. Significant levels of OVA-specific CD4(+) T cell proliferative and OVA-induced Th1- and Th2-type cytokine responses were noted in cervical lymph nodes and spleen of 1-year-old mice. In this regard, CD4(+), CD45RB(+) T cells were detected in greater numbers in the nasopharyngeal-associated lymphoreticular tissues of 1-year-old mice than of young adult mice, but the same did not hold true for Peyer's patches or spleen. One-year-old mice given nasal tetanus toxoid plus the chimeric toxin as adjuvant were protected from lethal challenge with tetanus toxin. This result reinforced our findings that age-associated immune alterations occur first in gut-associated lymphoreticular tissues, and thus nasal delivery of vaccines for nasopharyngeal-associated lymphoreticular tissue-based mucosal immunity offers an attractive possibility to protect the elderly.