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The Journal of Immunology, 2006, 176: 3697-3706.
Copyright © 2006 by The American Association of Immunologists

The Combined CTA1-DD/ISCOM Adjuvant Vector Promotes Priming of Mucosal and Systemic Immunity to Incorporated Antigens by Specific Targeting of B Cells1

Anja Helgeby*, Neil C. Robson{dagger}, Anne M. Donachie{dagger}, Helen Beackock-Sharp{dagger}, Karin Lövgren{ddagger}, Karin Schön*, Allan Mowat{dagger} and Nils Y. Lycke2,*

* Department of Clinical Immunology, University of Goteborg, Goteborg, Sweden; {dagger} Division of Immunology, Infection, and Inflammation, University of Glasgow, Glasgow, Scotland; and {ddagger} Isconova, Uppsala, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The cholera toxin A1 (CTA1)-DD/QuilA-containing, immune-stimulating complex (ISCOM) vector is a rationally designed mucosal adjuvant that greatly potentiates humoral and cellular immune responses. It was developed to incorporate the distinctive properties of either adjuvant alone in a combination that exerted additive enhancing effects on mucosal immune responses. In this study we demonstrate that CTA1-DD and an unrelated Ag can be incorporated together into the ISCOM, resulting in greatly augmented immunogenicity of the Ag. To demonstrate its relevance for protection against infectious diseases, we tested the vector incorporating PR8 Ag from the influenza virus. After intranasal immunization we found that the immunogenicity of the PR8 proteins were significantly augmented by a mechanism that was enzyme dependent, because the presence of the enzymatically inactive CTA1R7K-DD mutant largely failed to enhance the response over that seen with ISCOMs alone. The combined vector was a highly effective enhancer of a broad range of immune responses, including specific serum Abs and balanced Th1 and Th2 CD4+ T cell priming as well as a strong mucosal IgA response. Unlike unmodified ISCOMs, Ag incorporated into the combined vector could be presented by B cells in vitro and in vivo as well as by dendritic cells; it also accumulated in B cell follicles of draining lymph nodes when given s.c. and stimulated much enhanced germinal center reactions. Strikingly, the enhanced adjuvant activity of the combined vector was absent in B cell-deficient mice, supporting the idea that B cells are important for the adjuvant effects of the combined CTA1-DD/ISCOM vector.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
It has proved difficult to stimulate immune responses with Ags given at mucosal sites (1, 2). The natural response to mucosal Ag exposure is tolerance, and effective adjuvants are required to facilitate effective priming of mucosal as well as systemic immunity after oral or intranasal (i.n)3 immunization (3, 4, 5). Few adjuvants have been found to work when given mucosally, but the effects of QuilA-containing, immune-stimulating complexes (ISCOMs) have been well documented (6, 7, 8, 9, 10, 11). Perhaps the most potent mucosal adjuvants, though, are the closely related bacterial enteroxins, cholera toxin (CT) and Escherichia coli heat-labile toxin (LT) (4). These holotoxins are structurally AB5 complexes that bind to most mammalian cells through ganglioside receptors via their B subunits (12). The A1 subunit is an ADP-ribosylating enzyme that has been found to host strong adjuvant function, but is also responsible for the toxicity of the molecules (13, 14, 15). Because of their relative toxicity, the clinical use of CT or LT has been limited, and vaccine-induced diarrhea is an unwanted side effect when these holotoxins have been used as adjuvants in oral vaccines (16). Studies have also shown that the toxins may accumulate and affect the CNS after i.n. administration (17, 18, 19). An increased incidence of Bell’s palsy recently caused withdrawal from the market of an influenza vaccine containing the LT adjuvant, which was administered i.n. (20, 21).

To circumvent the toxicity problem, we have developed an alternative CT-based adjuvant, the CTA1-DD, a gene fusion protein that does not bind to ganglioside receptors (22). The CTA1-DD adjuvant is nontoxic in mice, but retained adjuvant function, comparable to that of CT, when given i.n. (22, 23). It consists of the enzymatically active CTA1 subunit fused in-frame with a gene encoding a dimer of the D domain from the Staphylococcus aureus protein A (24, 25), allowing it to specifically target B cells via binding to their Ig receptors. In contrast, ISCOMs are taken up by dendritic cells (DCs) preferentially (22, 23, 26, 27, 28, 29, 30) and are more potent than CTA1-DD when given orally. Therefore, we combined CTA1-DD and ISCOMs to create one of the first rationally designed adjuvant vectors (31). We found that the CTA1-DD/ISCOM vector was highly immunogenic by the i.n. as well as the oral route even with nanogram doses of Ag, inducing Ag-specific serum Abs, CD4 T cell priming, and IFN-{gamma} production (31).

Combinations of adjuvants have several potential advantages. In addition to targeting different APCs, they offer the possibility of improving the stability of pharmacologically active enzymes by incorporating in a stable vector that can deliver them linked to Ag. Micro- or nanoparticles especially have been found to be the most effective in these adjuvant combinations and, apart from ISCOMs, chitosan or virus-like particles have also been successfully tested in formulations together with mutant holotoxins or muramyl dipeptide (32, 33). Although our previous work with CTA1-DD and p323 peptides from OVA expressed as a gene fusion protein, CTA1-OVA-DD, supports the idea that greatly enhanced responses to CTA1-DD/ISCOM-linked Ag can be achieved, it will not be possible to create fusion proteins between CTA1 and any given Ag or peptide (31). Therefore, the aim of the present study was to extend the potential of CTA1-DD/ISCOMs as an effective mucosal vaccine delivery vehicle by incorporating CTA1-DD and the influenza virus PR8 Ag into the same ISCOM particles. In addition to the ability of the novel vector to stimulate local and systemic Ag-specific immunity, we determined to what extent the augmenting effects were CTA1 enzyme dependent and dissected the mechanisms involved in the presentation and immunogenicity of the vector.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Animals

BALB/c mice (H-2d) and, when indicated, C57BL/6 (H-2b) mice were obtained from B&K Universal or Harlan Olac. DO11.10 (H-2d) or OT-II (H-2b) mice, hosting TCR specific for the OVA323–339 peptide, were bred and maintained in the central research facility at University of Glasgow. The µMT mice on a BALB/c background were obtained from the animal facility at University of Goteborg. Mice were maintained under specific pathogen-free conditions, and sex- and age-matched animals were used in the experiments.

Immunizations

BALB/c mice were immunized i.n. with 20 µl containing 2 µg of CTA1-OVA-DD/ISCOMs or mutant CTA1R7K-OVA-DD/ISCOMs or with ISCOMs containing the PR8 Ag. When indicated, the immunizations were given as single s.c injections in the foot pad or complementing the i.n. immunizations of 10 µg of CTA1-DD or 2 µg of CT together with 2 µg of PR8 Ag. Alternatively, but only when indicated, C57BL/6 or µMT mice (on a BALB/c background) were used. Immunizations were given 10 days apart and were repeated once or twice. Animals were killed 7–9 days after the final immunization, and tissues, cells, serum, genital tract secretions, and bronchial lavage were recovered as described previously (34, 35). Specimens were freshly used (cells) or were stored at –80°C (tissues) or –20°C until analyzed.

Preparation of adjuvants and Ags

CTA1-OVA-DD and CTA1R7K-OVA-DD fusion proteins, containing a single copy of OVA323–339 peptide, were produced in Escherichia coli as previously described (26, 36). Protein analysis was performed with SDS-PAGE, and protein concentrations were determined using the Bio-Rad DC protein assay according to the manufacturer’s instructions. ADP-ribosyltransferase enzymatic activity was tested using the NAD:agmatine assay as described previously (12, 37). PR8 Ags were produced from the influenza virus envelope glycoproteins hemagglutinin and neuraminidase of human influenza A virus strain PR/8/34 (H1N1) as described. Briefly, the virus was propagated in 11-day-old embryonic hen eggs; after allantoic fluid was clarified by centrifugation, the virus was purified by sucrose density gradient centrifugation. PR8 Ag micelles, prepared from the envelope proteins, hemagglutinin and neuraminidase, were isolated by ultracentrifugation (30 min, 40,000 rpm, 20°C) from a sucrose layer (20% (w/v) sucrose containing 0.5% MEGA-10) and formed after removal of detergent by dialysis against PBS for 48 h at room temperature. Protein content in the preparations was assessed.

Preparation of ISCOMs

Briefly, ISCOMs were prepared by mixing 1.0 mg of Ag (PR8 Ags alone or mixed with CTA1-OVA-DD or CTA1R7K-OVA-DD) with 1.0 mg of cholesterol (C-8503; Sigma-Aldrich), 1.0 mg of phosphatidylcholine of egg origin (Lipoid), and 5.0 mg of Quillaja saponins (Spikoside; Isconova) in a total volume of 1.0 ml and a final concentration of 2% Mega-10 (Bachem). To determine the optimum conditions for efficient incorporation of the different proteins with retained biological activities, several buffers were used to remove the detergent during the first overnight dialysis at room temperature (0.1 M acetate (pH 4.5), 0.2 M acetate (pH 6.0), PBS, and 0.1 M phosphate (pH 8.0)). Thereafter, dialysis buffer was changed to PBS, and dialysis was continued for another 24 h at room temperature. All references in the text to Ag or adjuvant concentrations refer to the protein content of incorporated protein with a ratio of protein content to Quillaja saponins of 1:2 in the ISCOMs. The formation of ISCOMs was confirmed by electron microscopy, and the various components were analyzed for comigration of protein and Quillaja saponins into fractions isolated from an analytical 10–50% (w/w) sucrose gradient after centrifugation (18 h at 200,000 x g, 10°C). The contents of protein and saponins in the different fractions were determined as described, using ELISA and spectrophotometric analysis (at A214 nm), respectively. The amino acid content in each preparation was assessed. In some experiments we labeled the ISCOMs with CFSE. Briefly, 2 µl of solution, freshly prepared from a stock solution (100 mM in DMSO) of CFSE (Molecular Probes) was added per milligram of cholesterol as the ISCOMs were prepared by mixing the lipids. Endotoxin contaminations were determined in the ISCOM preparations using the Limulus amebocyte lysate test (LAL Endochrome; Charles River Endostestafe). The endotoxin levels were <110 endotoxin units/mg protein in all ISCOM preparations, whereas CTA1-OVA-DD, CTA1R7K-OVA-DD, and CT had endotoxin levels < 50 endotoxin units/mg protein.

Quality assessment of ISCOMs

We routinely analyzed the protein content in all ISCOMs using specific ELISA. When sucrose-derived fractions were used, we incubated 50 µl of each fraction in the first row of a 96-well microtiter Maxisorp plate (Nunc) at a 1/3 dilution of 50 mM carbonate buffer (pH 9.6). Three-fold dilutions in subsequent subwells were performed, and the plates were incubated at 4°C overnight. After washing, each well plate was blocked for 1 h at room temperature with PBS-Tween 20 containing 2% fat-free dried milk powder. For detection of PR8, a biotinylated chicken anti-PR8 polyclonal serum (importantly, chicken Abs do not bind protein A and, thus, do not bind DD), followed by HRP-conjugated avidin (DakoCytomation). For detection of the native or mutant CTA1-OVAp-DD, we used an HRP-conjugated rabbit anti-mouse antiserum (DakoCytomation). All incubations were performed with gentle agitation for 1 h at room temperature. Plates were washed three times in PBS-Tween 20 between all steps and before the incubation in tetramethylbenzidine substrate (Svanova), and the enzymatic reaction was read at A450 nm using a spectrophotometer.

Determination of immunogenicity

T cell-dependent immunity was assessed in cervical lymph nodes (CLNs) or spleens from immunized or control mice as previously described (38). Briefly, single-cell suspensions were prepared by passing the tissue through a nylon mesh. RBCs were lysed with a hypotonic ammonium chloride-Tris solution and washed in HBSS (Invitrogen Life Technologies). The cell suspensions were resuspended at a final concentration of 2 x 106 cells/ml and cultured in 200-µl aliquots in 96-well microtiter plates (Nunc) in Iscove’s medium (Biochrom) supplemented with 10% heat-inactivated FCS (Biochrom), 50 µM 2-ME (Sigma-Aldrich), 1 mM L-glutamine (Biochrom), and 50 µg/ml gentamicin (Sigma-Aldrich; Iscove’s complete medium) and cultured for 72 h at 37°C in 5% CO2 either alone or with 1 µM p323 (KJ Ross-Petersen) or 1 µg/ml PR8 micelles (Isconova). Proliferation was assessed after addition of 1 µCi/well [3H]thymidine (Amersham Biosciences) for the last 6 h of culturing. [3H]Thymidine uptake was determined using a beta scintillation counter (Beckman Coulter). After 96 h of culture, in vitro unstimulated and PR8 micell-restimulated cell supernatants were stored at –70°C until assayed. Cytokine responses to recall Ag in vitro were analyzed by multicytokine analysis (Luminex), which involved incubation with Ab-conjugated beads directed against mouse IL-5, IL-10, and IFN-{gamma} (Bio-Rad) according to the manufacturer’s instructions. The assay was read on a Luminex 100 and analyzed using Bio-Plex Manager software; the concentrations of cytokines were determined against a panel of cytokine preparations of known concentrations.

Specific Ab responses in serum, genital tract secretions, or bronchial lavage were determined using ELISA as described previously (26). PR8-specific total IgG, IgG1, IgG2a, and IgA concentrations were determined using polystyrene, 96-well microtiter plates (Nunc) coated with PR8 micelles (1 µg/ml). Total IgE in serum was assessed using rat anti-mouse IgE (Serotec)-coated, soft, 96-well ELISA plates (Dynatech Laboratories). After blocking with 0.1% BSA/PBS, serum samples were diluted 1/500 for specific responses or 1/20 for total IgE levels, followed by serial dilutions in 0.1% BSA/PBS in subsequent subwells. Bronchial lavage or vaginal secretions were diluted 1/10 and serially diluted. Alkaline phosphatase-conjugated, isotype-specific, goat anti-mouse Abs at 1/500 (Southern Biotechnology Associates) or 0.125 µg/ml biotin-conjugated anti-mouse IgE (Serotec) were then added, followed by 2.1 µg/ml Extravidin peroxidase (Sigma-Aldrich). Nitrophenyl phosphatase (1 mg/ml; Sigma-Aldrich) in ethanolamine buffer (pH 9.8) or o-phenylenediamine substrates (1 mg/ml; Sigma-Aldrich) in citrate buffer (pH 4.5) containing 0.04% H2O2 were used, and enzymatic reactions were read in a Titer-Tek Multiscan spectrophotometer (Labsystems). IgE concentrations were calculated in micrograms per milliliter from a standard curve generated by serial dilutions of purified IgE of known concentration (BD Pharmingen). PR8-specific log10 Ab titers (means ± SD) were defined as the interpolated reading giving rise to an absorbance of 0.4 above background, which consistently gave readings on the linear part of the curve.

In vivo distribution of ISCOMs

CFSE-labeled ISCOMs containing 5 µg of either CTA1-OVA-DD or OVA were injected s.c. into the footpad of BALB/c mice. Two, 4, 24, and 48 h later, the draining popliteal lymph nodes were harvested; they were either frozen for immunohistochemistry, or single-cell suspensions were prepared by forcing through a fine nylon mesh. The cells were washed in Iscove’s complete medium, followed by incubation for 5–15 min at 4°C with 1 µl of the 2.4G2-FcR-blocking Ab (BD Pharmingen) in cold PBS containing 0.1% BSA (BSA/PBS), and were added to cells aliquoted in 100 µl. The cells were labeled with 1/100 PE-conjugated anti-mouse CD19 or CD11c (BD Pharmingen) by incubation for 30 min at 4°C. After washing twice, the cells were analyzed on a FACScan flow cytometer (BD Biosciences) using CellQuest software. Cells were gated on the lymphocyte population with the gate set on either the CD19 or CD11c population, and 10,000 cells were collected, which were positive for CD19 or CD11c positive for CFSE (detected in the FL-1 channel).

Uptake of ISCOMs in vitro

Bone marrow (BM)-derived DCs or purified spleen B cells were incubated with CFSE-labeled CTA1-OVA-DD/ISCOMs or OVA/ISCOMs in RPMI 1640/10% FCS medium for 1 h at 37°C in 24-well, low adhesion plates, then washed three times, and the amount of uptake was assessed by FACS.

Immunohistochemistry

Frozen sections (6 µm) were prepared on microslides using a cryostat, fixed in 100% acetone for 10 min at room temperature, and dried before washing in PBS. The slides were then treated with 5% horse serum in PBS for 15 min in a humidified chamber. To identify germinal centers (GCs), sections were double labeled with FITC-conjugated GL-7 (BD Pharmingen) mAb and a Texas Red-conjugated anti-IgM Ab (Southern Biotechnology Associates) at a 1/100 dilution. GCs were counted in each lymph node, and the total GL-7-positive area in the B cell follicles was measured in four CLNs per mouse and five mice per group. To assess the distribution of CFSE-labeled ISCOMs in vivo, the sections were stained with biotinylated anti-CD45R/B220 or CD4 (or 7-amino-4-methylcoumarin-3-acetic acid-labeled anti-CD4; BD Pharmingen), and Texas Red-labeled streptavidin (Vector Laboratories), diluted 1/100, was used as a secondary Ab. The slides were mounted with a fluorescence mounting medium (DakoCytomation) and evaluated using a Leica LSC microscope, with digital storing of photographs.

FACS analysis

The frequency of GL-7 B lymphocytes in the CLNs was analyzed by FACS. Briefly, single-cell suspensions of CLN cells were labeled with anti-GL7-FITC and anti-B220-PE (BD Pharmingen) and analyzed by FACS. The frequency of double-positive CLN cells was calculated for each group of five mice, and the mean percentage was calculated. The background labeling of CLN cells was 1% from naive mice lacking GCs in frozen sections.

Enrichment of B cells and BM-derived DCs

Naive B cells were purified from the spleens or lymph nodes of BALB/c mice by negative selection using MACS. Briefly, after preparation of single-cell suspensions, RBCs were lysed by addition of 5 ml of NH4Cl (0.14 M) for 5 min at room temperature, and the remaining cells were washed in MACS medium (PBS and 2% FCS), before being resuspended in MACS-medium and counted. Following centrifugation, the cells were resuspended at a concentration of 1 x 107 cells/100 µl MACS medium, and 10 µl/107 cells anti-CD43-coated microbeads (Miltenyi Biotec) were added for 15 min at 4°C. The labeled cells were then passed over a CS MACS column according to the manufacturer’s instructions (Miltenyi Biotec). B cells from Ag-immunized mice were positively selected by MACS using anti-CD19-coated microbeads (Miltenyi Biotec) according to the manufacturer’s instructions. Eluted cells were >96% B220+ as assessed by flow cytometry.

To obtain DCs, BM cells were washed out of the femurs of adult mice in RPMI 1640 using a syringe and a 21-gauge needle. Aliquots of 3 x 106 BM cells were seeded in 90-cm petri dishes (Bibby Sterilin) and cultured in RPMI 1640 containing 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1.25 µg/ml fungizone (all from Invitrogen Life Technologies), and 10% FCS (Harlan Sera Laboratories; complete medium) at 37°C in 5% CO2. On days 0, 3, 6, and 8 of culture, the medium was supplemented with 10% supernatant from the X-63 fibroblast cell line transfected with the murine GM-CSF gene. After 10 days, nonadherent DCs were harvested by gentle washing and were typically >85% CD11c+, class II MHCint, CD40low, B7.1low, and B7.2low.

Assessment of Ag-presenting ability

Aliquots of 3 x 106 cells to be used as APCs were plated in 12-well plates or 24-well ultra low adherence plates in 1 ml (Costar) and pulsed with Ag for 2 h at 37°C. For the last 45 min of culture, 50 µg/ml mitomycin C (Sigma-Aldrich) was added before the APCs were recovered and washed four times in RPMI 1640. After washing, APCs were plated in triplicate at 1 x 105 cells/well in 96-well, flat-bottom microtiter plates (Costar) together with 2 x 105 lymph node cells from DO11.10 or OT-II mice in a total volume of 200 µl. To assess T cell proliferation, 1 µCi/well [3H]TdR (West of Scotland Radionucleotide Dispensary) was added for the last 16 h of culture, and cell-bound DNA was harvested onto glass-fiber filter mats (Wallac). [3H]TdR uptake was counted on a Betaplate counter (Wallac).

Statistical analysis

Data were compared using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Optimal conditions for incorporation of Ag and adjuvant into ISCOMs

In a previous study we showed that the CTA1-DD adjuvant could be effectively incorporated into ISCOMs and that the combined vector acquired adjuvant potency greatly surpassing either system used alone (31). In this study we explored the possibility of incorporating unrelated proteins in the same ISCOM particle using the proteins from influenza virus PR8 as a representative infectious agent together with CTA1-DD containing the MHC class II-restricted p323 peptide from OVA (CTA1-OVA-DD). In preliminary studies we established that both CTA1-OVA-DD and PR8 Ags were incorporated optimally at pH 6.0. Electron microscopic inspection and sucrose gradient analysis confirmed the successful construction of ISCOMs that carried both proteins in a balanced ratio of 1:1, with the peak distribution of each component being in fractions 5–7 of the sucrose gradient (Fig. 1). These fractions were subsequently used for the immunization studies.


Figure 1
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  		FIGURE 1. Analysis of CTA1-OVA-DD and PR8 incorporation into ISCOMs. ISCOMs containing CTA1-OVA-DD and PR8 Ags were subjected to sucrose density gradient analysis, and the fractions were analyzed for CTA1-DD/PR8 and saponin content as described in the text. The peaks of incorporation of CTA1-DD, PR8 proteins, and saponin were superimposed, illustrating the successful formation of ISCOMs. The y-axis values represent arbitrary OD units at A450 nm for immunodetection of HRP-labeled CTA1-DD or PR8 (left) and saponin at A214 nm (right) using a spectrophotometer.

 
Immunogenicity of combined CTA1-DD/ISCOM vector containing PR8 Ags

The immunogenicity of the combined vaccine vector was determined after i.n. immunization. Mice were immunized three times with PR8/CTA1-OVA-DD/ISCOMs, and the specific immune responses to recall Ag were assessed in spleen or CLN T cells 8 days after the final immunization. As we mentioned above, mice immunized with CTA1-OVA-DD/ISCOMs showed excellent priming to the OVA peptide, and these responses were much lower in mice receiving the enzymatically inactive form of CTA1R7K-OVA-DD/ISCOMs (Fig. 2). A similar pattern of responses was seen when CLNs or spleen cells were restimulated with PR8 Ags in vitro, confirming that the additional Ags that had been incorporated into the CTA1-DD/ISCOMs were immunogenic. In addition, this process had not altered the enzyme activity of CTA1, because the R7K form was still much less active than the ISCOMs containing the intact CTA1 and induced responses similar to those using PR8/ISCOMs alone. The enhancing effect on T cell priming with the combined vector was regularly 3- to 8-fold higher than that observed with the ISCOMs or the mutant inactive PR8/CTA1R7K-OVA-DD/ISCOMs (Fig. 2). For comparison, mice immunized with PR8 and the CTA1-DD protein alone responded to the same magnitude as PR8/ISCOMs or the mutant combined PR8/CTA1R7K-OVA-DD/ISCOM vector (Fig. 2, C and D).


Figure 2
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  		FIGURE 2. Intranasal immunization with PR8/CTA1-OVA-DD/ISCOMs primes T cells at both systemic and mucosal sites. Priming of OVA- and PR8-specific proliferative responses in spleen (A and C) or cervical lymph nodes (B and D) by i.n. immunization was performed. Mice were immunized three times i.n. with PR8/ISCOMs, PR8/CTA1-OVA-DD/ISCOMs, the enzymatically inactive PR8/CTA1R7K-OVA-DD/ISCOMs, or PR8 alone (/) or were given PR8 together with the CTA1-DD adjuvant. Isolated lymphocytes were stimulated 7 days after the final immunization with recall Ag, OVA323–339 or PR8 Ag in vitro. The results shown are from five individual mice per group and are representative of three identical experiments with similar results. Data are expressed as the mean cpm ± SEM (**, p < 0.01; *, p < 0.05).

 
Intranasal immunization with the combined vector also stimulated strong Ag-specific Ab responses in serum, with the enzymatically active PR8/CTA1-OVA-DD/ISCOMs stimulating ~10-fold higher serum anti-PR8 IgG Ab titers compared with PR8/ISCOMs alone (Fig. 3). For comparison, mice immunized with PR8 together with CTA1-DD alone gave significantly better specific IgG responses than mice immunized with only PR8 Ag, but had titers comparable to those of mice immunized with PR8/ISCOMs (Fig. 3A). Moreover, marked bronchioalveolar and genital tract IgA responses were observed in mice immunized with the combined vector (Fig. 3), which, again, were enhanced 10-fold compared with ISCOMs alone. Interestingly, the mutant CTA1R7K-OVA-DD/ISCOM vector enhanced anti-PR8 titers 3-fold (p < 0.05) above those of mice given PR8/ISCOMs alone in both serum and secretions, suggesting that the adjuvant function of the combined vector was not exclusively dependent on the enzymatic activity (Fig. 3).


Figure 3
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  		FIGURE 3. Induction of influenza-specific systemic and mucosal Ab production by immunization with PR8/CTA1-OVA-DD/ISCOMs. Serum (A), bronchial lavage (B), or vaginal secretions (C) were taken 7 days after the last of three i.n. immunizations with PR8/CTA1-OVA-DD/ISCOMs, the enzymatically inactive PR8/CTA1R7K-OVA-DD/ISCOMs, or PR8/ISCOMs alone. PR8-specific Abs were measured by ELISA and are expressed as log10 titers for individual mice; the mean is shown by the bar (**, p < 0.01; *, p < 0.05). The content of total IgA in the bronchial lavage and genital tract secretions was similar in the respective groups, indicating that the total IgA production was not differently affected by the immunizations. Thus, there was 86 ± 24, 43 ± 20, and 40 ± 16 µg/ml total IgA (in the order given above) in lavage and 1.9 ± 0.7, 1.8 ± 1.0, and 1.5 ± 0.8 µg/ml total IgA in genital tract secretions in the respective groups. This is one representative experiment of three with similar results.

 
CTA1-DD-ISCOMs combined vector induced balanced Th1- and Th2-type response

The CT adjuvant is known to skew CD4 T cell priming toward a Th2-type response, whereas ISCOMs stimulate strong Th1 immunity, with IFN-{gamma} and CTL activity (39, 40, 41). Therefore, we predicted that the combined vector might promote a balanced Th1 and Th2 type of response to influenza Ags. In support of this, ISCOMs promoted higher relative PR8-specific IgG2a Ab responses than those stimulated by the combined vector, whereas specific IgG2a titers were lower compared with IgG1 titers in CT-immunized mice (Fig. 4A). Also, total serum IgE levels were higher in mice immunized i.n. with CT than in those given the combined vector or ISCOMs alone (Fig. 4). In addition, spleen and CLN (data not shown) cells restimulated in vitro with PR8 Ag produced high levels of both Th1- and Th2-dependent cytokines, which were much enhanced compared with those of cells obtained from mice immunized with enzymatically inactive PR8/CTA1R7K-OVA-DD or PR8/ISCOMs (Fig. 4).


Figure 4
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  		FIGURE 4. The combined PR8/CTA1-OVA-DD/ISCOM vector induces a balanced Th1 and Th2 response. Mice were immunized three times i.n. with PR8/CTA1-OVA-DD/ISCOMs, the enzymatically inactive PR8/CTA1R7K-OVA-DD/ISCOMs, PR8/ISCOMs alone, or PR8 Ag plus CT, and anti-PR8 IgG1 ({blacksquare}) and IgG2a ({square}) serum titers as well as total serum IgE (nanograms per milliliter) responses were measured by ELISA 7–9 days after the final immunization (A and B). Spleen cells were restimulated for 96 h with PR8 Ag in vitro and IL-10 (C), IL-5 (D), and IFN-{gamma} (E) concentrations in culture supernatants were determined by ELISA. The results (picograms per milliliter) are expressed as the mean ± SEM of individual samples from five mice per group (*, p < 0.05). This is one of three experiments with similar results.

 
CTA1-DD/ISCOM vector stimulates germinal center reactions

In search of an early in vivo marker that reflected the adjuvant effect of the combined vector, we investigated the ability of CTA1-OVA-DD/ISCOMs to stimulate GC formations in the regional lymph nodes. We found strong GC reactions 14 days after a single i.n. immunization with PR8/CTA1-OVA-DD/ISCOMs, whereas the enzymatically inactive PR8/CTA1R7K-OVA-DD/ISCOMs induced smaller and fewer GC reactions in the CLNs, similar to those seen after treatment with ISCOMs alone (Fig. 5). The size and frequency of GCs stimulated by the CTA1-OVA-DD/ISCOM vector were comparable with those seen after i.n. immunization with intact CT holotoxin, which is known to induce prominent GC reactions (Fig. 5) (42, 43, 44). A flow cytometric analysis of GL7+ B cells from CLNs also revealed the difference between immunizations with the combined vector and CT, on the one hand, and ISCOMs alone or the mutant vector, on the other (Fig. 5C). Thus, the combined vector was as effective as CT in stimulating GC formations in CLNs in i.n. immunized mice.


Figure 5
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  		FIGURE 5. The combined CTA1-DD/ISCOM vector augments GC formations. Mice were given a single i.n. immunization with PR8/CTA1-OVA-DD/ISCOMs, enzymatically inactive PR8/CTA1R7K-OVA-DD/ISCOMs, PR8/ISCOMs alone, or CT plus PR8 micelles (2 µg of each). A, Fourteen days later, frozen sections of the CLNs were prepared and double labeled with Texas Red-conjugated anti-IgM (red) and FITC-labeled GL-7 (green) to detect GC reactions (green/yellow). B, the mean area of GL-7+ cells in the B cell follicles of four CLNs per mouse from five mice in each group was calculated. C, These calculations were complemented with FACS analysis of the frequency of GL-7+ cells per total B220+ CLN cells from five mice for the respective groups, using the background labeling of GC-negative naive CLN cells as a control (/). *, p < 0.05. This experiment represents one of three identical experiments with similar results.

 
Contrary to ISCOMs, the combined vector acts on B cells

We next examined how the enhanced immune responses found after immunization with the CTA1-OVA-DD/ISCOM vector correlated with the APC population involved in their uptake. Purified BM, DCs, or derived spleen B cells were pulsed for different time periods with CFSE-labeled CTA1-OVA-DD/ISCOMs or CFSE-labeled ISCOMs containing equimolar amounts of the OVA323–339 peptide, and the uptake was analyzed by FACS. These studies showed that although DCs took up the OVA/ISCOMs and CTA1-OVA-DD/ISCOMs with equal efficiency, B cells only took up the combined vector and not the modified ISCOMs (Fig. 6A). In parallel, BM-derived DCs presented the OVA peptide to DO11.10 OVA-specific CD4+ T cells in vitro with identical efficiency when pulsed with all the different ISCOM constructs, whereas purified B cells presented OVA peptide only when pulsed with the combined vectors containing CTA1-OVA-DD and could not present OVA/ISCOMs themselves (Fig. 6, B and D). The ability of B cells to present Ag in the combined vector in vitro appeared not to depend on the CTA1 enzyme, because the mutant CTA1R7K-OVA-DD/ISCOMs also triggered peptide-specific T cell proliferation. Thus, the combined CTA1-OVA-DD/ISCOM vector gains access to and can be presented by a broader repertoire of APCs than conventional ISCOMs.


Figure 6
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  		FIGURE 6. Uptake and presentation of ISCOM-associated Ag by DCs and B cells in vitro. Purified spleen B cells (A) or BM-derived DCs (C) from BALB/c mice were pulsed with either CFSE-labeled CTA1-OVA-DD/ISCOMs or CFSE-labeled OVA/ISCOMs for 1 h, and uptake was analyzed by FACS analysis. Although DCs took up both forms of ISCOMs equally above background levels (open curve), B cells only acquired the combined CTA1-OVA-DD/ISCOM vector, not the OVA/ISCOMs. To assess the Ag-presenting ability, spleen B cells (B) or BM-derived DCs (D) were pulsed for 2 h with 10 µg/ml OVA/ISCOMs or a molar equivalent, based on the OVA323–339 peptide content, of CTA1-OVA-DD/ISCOMs or CTAR7K-OVA-DD/ISCOMs or of OVA323–339 peptide (0.32 µg/ml) alone. The APC function of DCs and B cells were determined after 72 h in culture with DO11.10 T cells. Results are from one representative experiment of three and are expressed as the mean [3H]TdR incorporation ± SD from triplicate cultures (**, p < 0.01 vs B cells pulsed with OVA/ISCOMs or OVA peptide alone).

 
Differential distribution of CTA1-DD/ISCOMs and ISCOMs in draining lymph nodes

To confirm these properties of the combined vector in vivo, mice were injected s.c. into the footpad with CFSE-labeled ISCOMs or CFSE-labeled CTA1-OVA-DD/ISCOMs, and their distribution in the draining popliteal lymph nodes was examined at various time points. Two hours after injection, both OVA/ISCOMs and CTA1-OVA-DD/ISCOMs could be found in the deep cortical regions of the lymph node, consistent with access via the afferent lymphatics and subcapsular sinuses (Fig. 7). Thereafter, the CTA1-OVA-DD/ISCOMs began to concentrate in the B cell follicles, whereas the ISCOMs never appeared in follicles and were progressively lost from the lymph node, with only small amounts remaining in the deep cortex after 24 h. These results were also confirmed by FACS analysis of isolated cells from the draining lymph node, which showed that CFSE-labeled CTA1-OVA-DD/ISCOMs could be detected in CD19+ B cells, whereas both the combined vector and the normal ISCOMs were found in DCs (data not shown).


Figure 7
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  		FIGURE 7. CTA1-DD/ISCOMs and ISCOMs localize in different anatomical compartments of the lymph node. Mice were injected with 5 µg of CFSE-labeled CTA1-OVA-DD/ISCOMs or OVA/ISCOMs into the footpad. Two and 24 h after injection, the popliteal lymph nodes were excised, and frozen sections were stained with Texas Red-labeled anti-B220 (red; A–D) to highlight B cell follicles or with Texas Red-labeled anti-CD4 (red; E and F)/7-amino-4-methylcoumarin-3-acetic acid-labeled anti-CD4 (blue; H) to identify the T cell-dependent areas (TDA). CFSE-labeled ISCOMs (green) of both types localized initially in the deep cortical areas between follicles (A and B). Thereafter, CTA1-OVA-DD/ISCOMs accumulated in the B cell follicles (C and E), whereas only small amounts of OVA/ISCOMs remained in the TDA (blue) by 24 h (G and H). This is one representative experiment of three with similar results.

 
CTA1-DD/ISCOMs target B cells as APCs in vivo

To address the role of B cells in the immunogenicity of the combined vector, we purified B cells from the popliteal lymph nodes of mice immunized with OVA/ISCOMs or CTA1-OVA-DD/ISCOMs and examined their ability to present OVA to TCR-transgenic OT-II CD4+ T cells in vitro. This showed that B cells from mice injected with the combined CTA1-OVA-DD/ISCOM vector, but not those injected with OVA/ISCOMs alone, could present Ag to the specific T cells (Fig. 8A).


Figure 8
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  		FIGURE 8. The combined CTA1-DD/ISCOM vector is presented by B cells ex vivo and requires B cells for its enhanced adjuvant activity in vivo. A, Ex vivo presentation of ISCOM-associated Ag by B cells. C57BL/6 mice were immunized with ISCOMs containing 2.5 µg of either OVA or a molar equivalent of CTA1-OVA-DD in each rear footpad. After 4 h, B cells were purified by MACS and cultured together with OT-II T lymphocytes for 72 h. The results from one representative experiment of three are shown as the mean [3H]TdR incorporation ± 1 SD from triplicate cultures (**, p < 0.01 vs OVA ISCOMs). BALB/c (B) or µMT (B cell-deficient; C) mice were immunized twice i.n. with PR8/CTA1-OVA-DD/ISCOMs or PR8/ISCOMs; 7 days after the last immunization, spleen cells were restimulated for 72 h with PR8 Ag in vitro. The results from one representative experiment of three are shown as the mean incorporation of [3H]TdR ± 1 SD for triplicate cultures with five individual mice/group (**, p < 0.01 vs PR8/ISCOMs).

 
Finally, we determined whether B cells were needed for the immunogenicity of the combined CTA1-OVA-DD/ISCOMs in vivo by immunizing µMT, B cell-deficient mice i.n. with PR8/CTA1-OVA-DD/ISCOMs or PR8/ISCOMs. When restimulated and cultured with PR8 Ag in vitro, spleen cells from wild-type and µMT mice showed equivalent responses after immunization with PR8/ISCOMs. However, in striking contrast to wild-type mice, µMT mice demonstrated no enhancement of T cell priming induced by the combined CTA1-OVA-DD/ISCOM vector (Fig. 8B). These findings also support the idea that B cells play a central role in vivo in the immunoenhancing effect of the combined CTA1-OVA-DD/ISCOMS vector.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study we have confirmed and extended our previous findings that a potent mucosal vaccine vector can be constructed by combining the distinctive adjuvants CTA1-DD and ISCOMs. We show that the combined vector can be exploited to host additional Ags, such as influenza surface proteins, and the resulting formulation not only induces strong mucosal and systemic immune responses to all incorporated Ags, but also retains the enzymatic function of CTA1, which is required for its biological function. Our results also indicate that the explanation for the enhanced adjuvant function of ISCOMs containing CTA1-DD compared with conventional ISCOMs is that the CTA1-DD component allows additional targeting to B cells as APCs.

Previous studies have shown that many protein Ags, including those prepared from influenza PR8, are highly immunogenic when incorporated into ISCOMs (39, 40, 41, 42, 43, 44) as is the CTA1-DD construct containing the OVA323–339 peptide (31). We demonstrate that CTA1-DD/ISCOMs can be modified to incorporate PR8 Ags, and the resulting vector induces mucosal and systemic immune responses when given i.n. Not only was the combined vector highly immunogenic, but it was also, because, as batch-to-batch quality control responses to the integrated OVA peptide reflected, a stable, reproducible, and effective vaccine vector for strongly enhanced immune responses (31). This occurs using very low doses of Ag, with as little as 2 µg of PR8 protein and the equivalent of 150 ng of OVA peptide per dose being immunogenic by the nasal or parenteral routes (31). In addition, the combined CTA1-DD/ISCOM vector induces a balanced Th1 and Th2 response, which comprises IFN-{gamma} and IL-5 production as well as T cell-mediated immune responses, such as delayed-type hypersensitivity and CTL activity and serum IgG and local IgA Abs (31), with no evidence of priming of IgE production. These properties distinguish the combined vector from its individual components, first by enabling much smaller doses of Ag to be used. In addition, the use of CT is frequently associated with Th2 polarization and potentially harmful IgE production, whereas ISCOMs stimulate marked Th1 and CD8+ T cell responses when used alone (39, 40, 41, 45, 46). These features were also confirmed in the present study and demonstrated in the combined vector’s balanced IgG2a- and IgG1-specific serum responses in contrast to the relative skewing of these responses toward Th1 and Th2 by ISCOMs and CT, respectively. In fact, in this regard the combined vector mimicked the balanced Th1 and Th2 responses seen with CTA1-DD adjuvant alone (22). Together, these findings highlight the potential usefulness of CTA1-DD/ISCOMs as practical mucosal vaccine vectors that will provide a flexible and stable means of inducing protective immunity against a variety of pathogens.

As we have found previously (31), the improved adjuvant properties of CTA1-DD incorporated into ISCOMs were largely dependent on the presence of enzymatically active toxin, because ISCOMs containing the R7K mutant of CTA1-DD were barely more immunogenic than ISCOMs with PR8 alone. This underlines the critical role of enzyme function in the activity of CT-related adjuvants (47). Nevertheless, ISCOMs containing enzymatically inactive CTA1R7K-DD retained some enhanced adjuvant function, such as specific Ab production in vivo and presentation of Ag by B cells in vitro. Together, these results indicate the potentially beneficial effects of targeting B cells as additional APCs. In vitro studies have shown that ISCOM particles themselves are taken up and presented preferentially by DCs (27, 28, 48), a finding we have extended in this study by showing that ISCOMs accumulate in DCs in vitro and in the DC-rich, T cell-dependent areas of lymph nodes in vivo. Interestingly, the pattern of uptake in vivo was consistent with localization in the fibroreticular conduits of T cell areas, which others have shown to be important sites of accumulation of Ag-loaded DCs and initial interactions between DCs and T cells (49, 50, 51, 52). In contrast, ISCOMs containing CTA1-DD were taken up very efficiently by B cells as well as by DCs in vitro and were presented by both APCs to CD4+ T cells, presumably reflecting the ability of the DD portion to bind to B cells selectively via their surface Ig. Furthermore, s.c. injected CTA1-DD/ISCOMs had a unique ability to accumulate in B cell follicles of lymph nodes, where they were retained for at least 24 h, by which time conventional ISCOMs were virtually undetectable in the lymph node. Most importantly, the augmented adjuvant properties of CTA1-DD/ISCOMs were largely absent in B cell-deficient, µMT mice. For these reasons, we propose that this novel combined vector is so effective because it targets both DCs and B cells in vivo.

Unfortunately, attempts to document the same effects of the combined vector on B cells in the nasal-associated lymphoid tissues or CLNs after i.n. immunizations failed because no CFSE-labeled DCs and B cells were detected. We have previously experienced this problem when using labeled CTA1-DD or CT given i.n. (22, 53). Despite this, we have no reason to believe that DCs or B cells were not targeted in the nasal-associated lymphoid tissues and CLNs by the combined vector, especially because distinct GCs developed in the CLNs, as shown in the present study. Also, µMT mice failed to exhibit augmented responses after i.n. immunization with the combined vector, suggesting a relative dependence on B cells for the effect.

The ISCOM particle provides a stable formulation that interacts efficiently with and activates DCs (27, 28, 48), whereas CTA1-DD allows preferential delivery into B cells. These B cells are likely to be activated as a result of both the binding of sIg and the presence of pharmacologically active CTA1 enzyme. Activated B cells have been shown to be highly efficient APCs in other systems (52, 54) and together with the efficient localization of CTA1-DD/ISCOMs in B cell follicles, these properties presumably encourage cognate interactions between Ag-specific T and B cells in evolving germinal centers. This idea is supported by the fact that GC formations were greatly enhanced after administration of CTA1-DD/ISCOMs compared with ISCOMs alone. As well as enhancing primary immune responses, as we show in this study, recent work demonstrates that such interactions are of crucial importance in sustaining memory T and B cell responses (55, 56, 57). This would be a major factor in the success of any vaccine vector, and we are currently studying the effects of B cell-targeted ISCOMs on the induction of immunological memory.

A particular asset to the combined vector was its strong augmenting effect on mucosal IgA responses. Contrary to ISCOMs themselves, which are fairly poor inducers of mucosal IgA (58), combination with the CTA1-DD adjuvant rendered ISCOMs much better IgA-stimulating properties. We consistently observed >10-fold stronger mucosal IgA responses in CTA1-OVA-DD/ISCOMs compared with i.n. ISCOM-immunized mice. Importantly, regardless of the total IgA production at the mucosal sites, the specific responses were truly augmented and were not a reflection of polyclonal stimulation of IgA production. In vaccine formulations, such strong mucosal IgA immunity may be critical for protection against many infectious diseases.

In conclusion, we have shown that a combined vector comprising CTA1-DD incorporated into ISCOMs has considerable potential as a vaccine for mucosal immunization with a variety of protein Ags. By targeting the CTA1 adjuvant to both DCs and B cells as APCs, it allows powerful immune responses to be induced using low doses of Ags and points the way to a new generation of rationally designed vaccines.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Swedish Research Council; the Swedish Cancer Foundation, the Sahlgrenska University Hospital Foundation, European Union Grants QLK2-CT-2001-01702, QLK2-CT-1999-00228, and LSHP-CT-2003-503240, and the Welcome Trust. Back

2 Address correspondence and reprint requests to Dr. Nils Lycke, Department of Clinical Immunology, University of Goteborg, 413 46 Goteborg, Sweden. E-mail address: nils.lycke{at}microbio.gu.se Back

3 Abbreviations used in this paper: i.n, intranasal; BM, bone marrow; CLN, cervical lymph node; CT, cholera toxin; DC, dendritic cell; GC, germinal center; LT, E. coli heat-labile toxin; ISCOM, QuilA-containing immune-stimulating complex. Back

Received for publication August 2, 2005. Accepted for publication December 29, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Medina, E., C. A. Guzman. 2000. Modulation of immune responses following antigen administration by mucosal route. FEMS Immunol. Med. Microbiol. 27: 305-311. [Medline]
  2. Del Giudice, G., M. Pizza, R. Rappuoli. 1999. Mucosal delivery of vaccines. Methods 19: 148-155. [Medline]
  3. Garside, P., A. M. Mowat. 2001. Oral tolerance. Semin. Immunol. 13: 177-185. [Medline]
  4. Salmond, R. J., J. A. Luross, N. A. Williams. 2002. Immune modulation by the cholera-like enterotoxins. Expert Rev. Mol. Med. 2002: 1-16. [Medline]
  5. Lavelle, E. C., A. Jarnicki, E. McNeela, M. E. Armstrong, S. C. Higgins, O. Leavy, K. H. Mills. 2004. Effects of cholera toxin on innate and adaptive immunity and its application as an immunomodulatory agent. J. Leukocyte Biol. 75: 756-763. [Abstract/Free Full Text]
  6. Harandi, A. M.. 2004. The potential of immunostimulatory CpG DNA for inducing immunity against genital herpes: opportunities and challenges. J. Clin. Virol. 30: 207-210. [Medline]
  7. Sajic, D., A. J. Patrick, K. L. Rosenthal. 2005. Mucosal delivery of CpG oligodeoxynucleotides expands functional dendritic cells and macrophages in the vagina. Immunology 114: 213-224. [Medline]
  8. McCluskie, M. J., H. L. Davis. 1999. CpG DNA as mucosal adjuvant. Vaccine 18: 231-237. [Medline]
  9. Bacon, A., J. Makin, P. J. Sizer, I. Jabbal-Gill, M. Hinchcliffe, L. Illum, S. Chatfield, M. Roberts. 2000. Carbohydrate biopolymers enhance antibody responses to mucosally delivered vaccine antigens. Infect. Immun. 68: 5764-5770. [Abstract/Free Full Text]
  10. Baldridge, J. R., Y. Yorgensen, J. R. Ward, J. T. Ulrich. 2000. Monophosphoryl lipid A enhances mucosal and systemic immunity to vaccine antigens following intranasal administration. Vaccine 18: 2416-2425. [Medline]
  11. Marciani, D. J.. 2003. Vaccine adjuvants: role and mechanisms of action in vaccine immunogenicity. Drug Discov. Today 8: 934-943. [Medline]
  12. Spangler, B. D.. 1992. Structure and function of cholera toxin and the related Escherichia coli heat-labile enterotoxin. Microbiol. Rev. 56: 622[Abstract/Free Full Text]
  13. Kawamura, Y. I., R. Kawashima, Y. Shirai, R. Kato, T. Hamabata, M. Yamamoto, K. Furukawa, K. Fujihashi, J. R. McGhee, H. Hayashi, et al 2003. Cholera toxin activates dendritic cells through dependence on GM1-ganglioside which is mediated by NF-{kappa}B translocation. Eur. J. Immunol. 33: 3205-3212. [Medline]
  14. Rappuoli, R., M. Pizza, G. Douce, G. Dougan. 1999. Structure and mucosal adjuvanticity of cholera and Escherichia coli heat-labile enterotoxins. Immunol. Today 20: 493-500. [Medline]
  15. Soriani, M., L. Bailey, T. R. Hirst. 2002. Contribution of the ADP-ribosylating and receptor-binding properties of cholera-like enterotoxins in modulating cytokine secretion by human intestinal epithelial cells. Microbiology 148: 667-676. [Abstract/Free Full Text]
  16. Wu, A. L., W. A. Walker. 1976. Immunological control mechanism against cholera toxin: interference with toxin binding to intestinal receptors. Infect. Immun. 14: 1034-1042. [Abstract/Free Full Text]
  17. van Ginkel, F. W., J. R. McGhee, J. M. Watt, A. Campos-Torres, L. A. Parish, D. E. Briles. 2003. Pneumococcal carriage results in ganglioside-mediated olfactory tissue infection. Proc. Natl. Acad. Sci. USA 100: 14363-14367. [Abstract/Free Full Text]
  18. van Ginkel, F. W., R. J. Jackson, Y. Yuki, J. R. McGhee. 2000. Cutting edge: the mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues. J. Immunol. 165: 4778-4782. [Abstract/Free Full Text]
  19. Fujihashi, K., T. Koga, F. W. van Ginkel, Y. Hagiwara, J. R. McGhee. 2002. A dilemma for mucosal vaccination: efficacy versus toxicity using enterotoxin-based adjuvants. Vaccine 20: 2431-2438. [Medline]
  20. Mutsch, M., W. Zhou, P. Rhodes, M. Bopp, R. T. Chen, T. Linder, C. Spyr, R. Steffen. 2004. Use of the inactivated intranasal influenza vaccine and the risk of Bell’s palsy in Switzerland. N. Engl. J. Med. 350: 896-903. [Abstract/Free Full Text]
  21. Gluck, R., R. Mischler, P. Durrer, E. Furer, A. B. Lang, C. Herzog, S. J. Cryz, Jr. 2000. Safety and immunogenicity of intranasally administered inactivated trivalent virosome-formulated influenza vaccine containing Escherichia coli heat-labile toxin as a mucosal adjuvant. J. Infect. Dis. 181: 1129-1132. [Medline]
  22. Eriksson, A. M., K. M. Schön, N. Y. Lycke. 2004. The cholera toxin-derived CTA1-DD vaccine adjuvant administered intranasally does not cause inflammation or accumulate in the nervous tissues. J. Immunol. 173: 3310-3319. [Abstract/Free Full Text]
  23. Ågren, L., B. Löwenadler, N. Lycke. 1998. A novel concept in mucosal adjuvanticity: the CTA1-DD adjuvant is a B cell-targeted fusion protein that incorporates the enzymatically active cholera toxin A1 subunit. Immunol. Cell Biol. 76: 280-287. [Medline]
  24. Uhlen, M., B. Guss, B. Nilsson, S. Gatenbeck, L. Philipson, M. Lindberg. 1984. Complete sequence of the staphylococcal gene encoding protein A. A gene evolved through multiple duplications. J. Biol. Chem. 259: 1695-1702. [Abstract/Free Full Text]
  25. Ljungberg, U. K., B. Jansson, U. Niss, R. Nilsson, B. E. Sandberg, B. Nilsson. 1993. The interaction between different domains of staphylococcal protein A and human polyclonal IgG, IgA, IgM and F(ab')2: separation of affinity from specificity. Mol. Immunol. 30: 1279-1285. [Medline]
  26. Ågren, L. C., L. Ekman, B. Löwenadler, N. Y. Lycke. 1997. Genetically engineered nontoxic vaccine adjuvant that combines B cell targeting with immunomodulation by cholera toxin A1 subunit. J. Immunol. 158: 3936-3946. [Abstract]
  27. Robson, N. C., H. Beacock-Sharp, A. M. Donachie, A. M. Mowat. 2003. The role of antigen-presenting cells and interleukin-12 in the priming of antigen-specific CD4+ T cells by immune stimulating complexes. Immunology 110: 95-104. [Medline]
  28. Robson, N. C., H. Beacock-Sharp, A. M. Donachie, A. M. Mowat. 2003. Dendritic cell maturation enhances CD8+ T-cell responses to exogenous antigen via a proteasome-independent mechanism of major histocompatibility complex class I loading. Immunology 109: 374-383. [Medline]
  29. Ågren, L., E. Sverremark, L. Ekman, K. Schön, B. Löwenadler, C. Fernandez, N. Lycke. 2000. The ADP-ribosylating CTA1-DD adjuvant enhances T cell-dependent and independent responses by direct action on B cells involving anti-apoptotic Bcl-2- and germinal center-promoting effects. J. Immunol. 164: 6276-6286. [Abstract/Free Full Text]
  30. Eriksson, A., N. Lycke. 2003. The CTA1-DD vaccine adjuvant binds to human B cells and potentiates their T cell stimulating ability. Vaccine 22: 185-193. [Medline]
  31. Mowat, A. M., A. M. Donachie, S. Jägewall, K. Schön, B. Löwenadler, K. Dalsgaard, P. Kaastrup, N. Lycke. 2001. CTA1-DD-immune stimulating complexes: a novel, rationally designed combined mucosal vaccine adjuvant effective with nanogram doses of antigen. J. Immunol. 167: 3398-3405. [Abstract/Free Full Text]
  32. Moschos, S. A., V. W. Bramwell, S. Somavarapu, H. O. Alpar. 2005. Comparative immunomodulatory properties of a chitosan-MDP adjuvant combination following intranasal or intramuscular immunisation. Vaccine 23: 1923-1930. [Medline]
  33. Baudner, B. C., M. M. Giuliani, J. C. Verhoef, R. Rappuoli, H. E. Junginger, G. D. Giudice. 2003. The concomitant use of the LTK63 mucosal adjuvant and of chitosan-based delivery system enhances the immunogenicity and efficacy of intranasally administered vaccines. Vaccine 21: 3837-3844. [Medline]
  34. Haneberg, B., D. Kendall, H. M. Amerongen, F. M. Apter, J. P. Kraehenbuhl, M. R. Neutra. 1994. Induction of specific immunoglobulin A in the small intestine, colon-rectum, and vagina measured by a new method for collection of secretions from local mucosal surfaces. Infect. Immun. 62: 15-23. [Abstract/Free Full Text]
  35. Bromander, A. K., L. Ekman, M. Kopf, J. G. Nedrud, N. Y. Lycke. 1996. IL-6-deficient mice exhibit normal mucosal IgA responses to local immunizations and Helicobacter felis infection. J. Immunol. 156: 4290-4297. [Abstract]
  36. Ågren, L. C., L. Ekman, B. Löwenadler, J. G. Nedrud, N. Y. Lycke. 1999. Adjuvanticity of the cholera toxin A1-based gene fusion protein, CTA1-DD, is critically dependent on the ADP-ribosyltransferase and Ig-binding activity. J. Immunol. 162: 2432-2440. [Abstract/Free Full Text]
  37. Tsuji, T., T. Inoue, A. Miyama, M. Noda. 1991. Glutamatic acid-112 of the A-subunit of heat-labile enterotoxin from enterotoxigenic Escherichia coli is important for ADP-ribosyltransferase activity. FEBS Lett. 291: 319-321. [Medline]
  38. Gärdby, E., P. Lane, N. Y. Lycke. 1998. Requirements for B7-CD28 costimulation in mucosal IgA responses: paradoxes observed in CTLA4-H{gamma}1 transgenic mice. J. Immunol. 161: 49-59. [Abstract/Free Full Text]
  39. Smith, R. E., A. M. Donachie, A. M. Mowat. 1998. Immune stimulating complexes as mucosal adjuvants. Immunol. Cell Biol. 76: 263-269. [Medline]
  40. Sjölander, A., D. Drane, E. Maraskovsky, J. P. Scheerlinck, A. Suhrbier, J. Tennent, M. Pearse. 2001. Immune responses to ISCOM formulations in animal and primate models. Vaccine 19: 2661-2665. [Medline]
  41. Mowat, A. M., K. J. Maloy, A. M. Donachie. 1993. Immune stimulating complexes as adjuvants for inducing local and systemic immunity after oral immunization with protein antigens. Immunology 80: 527-534. [Medline]
  42. Mowat, A. M., G. Reid. 2001. The preparation of immune stimulating complexes (ISCOMS) as adjuvants for local and systemic immunisation with protein antigens. J. E. Coligan, Jr, and A. M. Kruisbeek, Jr, and D. Margulies, Jr, and E. Shevach, Jr, and W. Strober, Jr, eds. Current Protocols in Immunology 2.11.11-2.11.12. Wiley & Sons, New York.
  43. Maloy, K. J., A. M. Donachie, D. T. O’Hagan, A. M. Mowat. 1994. Induction of mucosal and systemic immune-responses by immunization with ovalbumin entrapped in poly(lactide-co-glycolide) microparticles. Immunology 81: 661-667. [Medline]
  44. Maloy, K. J., A. M. Donachie, A. M. Mowat. 1995. Induction of Th1 and Th2 CD4+ T cell responses by oral or parenteral immunization with ISCOMS. Eur. J. Immunol. 25: 2835-2841. [Medline]
  45. Mowat, A. M., A. M. Donachie, G. Reid, O. Jarrett. 1991. Immune stimulating complexes containing Quil A and protein antigen prime class I MHC-restricted T lymphocytes in vivo and are active by the oral route. Immunology 72: 317-322. [Medline]
  46. Takahashi, H., T. Takeshita, B. Morein, S. Putney, R. N. Germain, J. Berzofsky. 1990. Induction of CD8+ cytotoxic T cells by immunisation with purified HIV-1 envelope protein in ISCOMS. Nature 344: 873-875. [Medline]
  47. Lycke, N.. 2005. From toxin to adjuvant: basic mechanisms for the control of mucosal IgA immunity and tolerance. Immunol. Lett. 97: 193-198. [Medline]
  48. Beacock-Sharp, H., A. M. Donachie, N. C. Robson, A. M. Mowat. 2003. A role for dendritic cells in the priming of antigen-specific CD4+ and CD8+ T lymphocytes by immune-stimulating complexes in vivo. Int. Immunol. 15: 711-720. [Abstract/Free Full Text]
  49. Lindquist, R. L., G. Shakhar, D. Dudziak, H. Wardemann, T. Eisenreich, M. L. Dustin, M. C. Nussenzweig. 2004. Visualizing dendritic cell networks in vivo. Nat. Immunol. 5: 1243-1250. [Medline]
  50. Katakai, T., T. Hara, J. H. Lee, H. Gonda, M. Sugai, A. Shimizu. 2004. A novel reticular stromal structure in lymph node cortex: an immuno-platform for interactions among dendritic cells, T cells and B cells. Int. Immunol. 16: 1133-1142. [Abstract/Free Full Text]
  51. Itano, A. A., S. J. McSorley, R. L. Reinhardt, B. D. Ehst, E. Ingulli, A. Y. Rudensky, M. K. Jenkins. 2003. Distinct dendritic cell populations sequentially present antigen to CD4 T cells and stimulate different aspects of cell-mediated immunity. Immunity 19: 47-57. [Medline]
  52. Yan, J., M. J. Wolff, J. Unternaehrer, I. Mellman, M. J. Mamula. 2005. Targeting antigen to CD19 on B cells efficiently activates T cells. Int. Immunol. 17: 869-877. [Abstract/Free Full Text]
  53. Grdic, D., L. Ekman, K. Schön, K. Lindgren, J. Mattsson, K. E. Magnusson, P. Ricciardi-Castagnoli, N. Lycke. 2005. Splenic marginal zone dendritic cells mediate the cholera toxin adjuvant effect: dependence on the ADP-ribosyltransferase activity of the holotoxin. J. Immunol. 175: 5192-5202. [Abstract/Free Full Text]
  54. Lanzavecchia, A.. 1990. Receptor-mediated antigen uptake and its effect on antigen presentation to class II-restricted T lymphocytes. Annu. Rev. Immunol. 8: 773-793. [Medline]
  55. Gourley, T. S., E. J. Wherry, D. Masopust, R. Ahmed. 2004. Generation and maintenance of immunological memory. Semin. Immunol. 16: 323-333. [Medline]
  56. Gaspal, F. M., M. Y. Kim, F. M. McConnell, C. Raykundalia, V. Bekiaris, P. J. Lane. 2005. Mice deficient in OX40 and CD30 signals lack memory antibody responses because of deficient CD4 T cell memory. J. Immunol. 174: 3891-3896. [Abstract/Free Full Text]
  57. Smith, K. M., J. M. Brewer, A. M. Mowat, Y. Ron, P. Garside. 2004. The influence of follicular migration on T-cell differentiation. Immunology 111: 248-251. [Medline]
  58. Grdic, D., R. Smith, A. Donachie, M. Kjerrulf, E. Hörnquist, A. Mowat, N. Lycke. 1999. The mucosal adjuvant effects of cholera toxin and immune-stimulating complexes differ in their requirement for IL-12, indicating different pathways of action. Eur. J. Immunol. 29: 1774-1784. [Medline]



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