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Oxidised mannan as a novel adjuvant inducing mucosal IgA production
Vaccine 20 (2002) 1068–1078
Oxidised mannan as a novel adjuvant inducing mucosal IgA production John Stambas a,∗ , Geoffrey Pietersz b , Ian McKenzie b , Christina Cheers a a
Department of Microbiology and Immunology, University of Melbourne, Parkville 3052, Vic., Australia b Austin Research Institute, Heidelberg 3084, Vic., Australia Received 9 June 2001; received in revised form 17 August 2001; accepted 11 October 2001
Abstract Mannan, oxidatively coupled to recombinant protein antigens, has here been tested as a possible adjuvant for the production of antibody on the mucosa. Given intranasally, but not intraperitoneally, mannan markedly enhanced the production of IgA, IgG1 and IgG2a in the serum, and IgA locally in the lung and at remote mucosal sites, including tears, vaginal and salivary secretions. Oxidative coupling was critical to its action, since neither mannan simply mixed with protein nor mannan–protein conjugates which had been reduced by treatment with sodium borohydride, acted as adjuvants. Oxidatively coupled mannan was compared with the widely studied mucosal adjuvant, cholera toxin (CT). The use of oxidised mannan as an adjuvant induced better responses than CT judged by the induction of IgA in serum, vaginal washings and saliva. Thus, oxidised mannan, which is non-toxic and can be administered without injection, is a suitable adjuvant coupled with protective antigens for vaccinating against a number of infections that occur via the mucous membranes. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Mucosa; IgA; Oxidised mannan
1. Introduction Most infectious agents enter the body through mucosal membranes, and recent vaccine strategies have concentrated on the production of antibodies at these sites to block their entry. The stimulation of secretory immune responses, which includes mucosal IgA, the predominant antibody isotype in mucosal secretions with the capability to neutralise bacteria, bacterial products and viruses, is considered to be crucial to vaccine development [1–4]. Currently most vaccines delivered by injection are not efficient at inducing a mucosal response, and the efficient induction of mucosal immunity requires delivery onto the mucosa. At present, the oral polio vaccine remains the only successful large-scale vaccine that gives protection at the mucosal level. It is a live vaccine whose attenuation was achieved empirically with the attendant risks of back-mutation. Other strategies, including live recombinant bacteria and DNA vaccines, have been used with encouraging results [5,6]. Immunisation with defined protein antigens would have many advantages, including safety in immunosuppressed individuals. However, before such vaccines can be developed there is a need for adjuvants and delivery systems to overcome ∗ Corresponding author. Tel.: +61-8-3445704; fax: +61-9-3471540. E-mail address: j [email protected] (J. Stambas).
the problems associated with mucosal vaccination using defined protein antigens, including poor immunogenicity or the induction of tolerance. Bacterial enterotoxins such as cholera toxin (CT) produced by Vibrio cholerae, and labile toxin from Escherichia coli have been used as adjuvants for mucosal IgA responses when co-administered with antigen [7,8], but they are not appropriate to use in humans due to their toxic properties. Studies using the enterotoxin B subunits (detoxified) of V. cholerae and E. coli have resulted in conflicting views of their adjuvanticity [8]. We now report that oxidised (but not reduced) mannan–antigen complexes, delivered at a mucosal site, give superior IgA responses. Mannan, a polymannose derived from the cell wall of yeast, was shown to enhance immune responses in mice when oxidised and conjugated to human mucin 1, an antigen over-expressed in some cancers [9,10]. Oxidised mannan had different effects depending on the species immunised. In mice, oxidised mannan–mucin 1 injected intraperitoneally (i.p.) induced cellular Th1 responses with CTL, tumour protection and little antibody, whereas in humans and monkeys immunisation with autologous mucin 1 linked to mannan produced large amounts of IgG1 antibody and little cellular immunity. In humans, monkeys and mice oxidised mannan–mucin 1 was found to be non-toxic, making it an attractive candidate adjuvant for vaccine application [11]. By changing the route of administration to mice, we
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have now found that oxidised mannan can be used as adjuvant for mucosal IgA. Two contrasting bacterial proteins were used as antigens: a secreted protein, listeriolysin O (LLO), the immunodominant antigen of Listeria monocytogenes and the 19 kDa protein secreted by Mycobacterium tuberculosis. They were conjugated to oxidised mannan and the antibody responses produced compared with that of CT. Oxidised mannan-conjugated antigen gave superior IgA, IgG1 and IgG2a responses and appears to be a promising choice for the mucosal delivery of antigens.
2. Materials and methods 2.1. Mice Female 6–8-week-old (CBA×BALB/c)F1 and C57BL/10 mice were pedigree inbred and maintained under conventional but infection-free conditions in the animal house of the Department of Microbiology and Immunology, University of Melbourne. 2.2. Production of antigens A pbluescript plasmid containing the gene for LLO from L. monocytogenes (without its leader sequence) was obtained from Richard Strugnell (University of Melbourne, Australia) and subcloned into the E. coli expression vector pGEX-2T [12] in the correct reading frame and orientation. The expression of an 84 kDa LLO glutothione-S-transferase (GST) fusion protein (LLO.FP) was induced with 0.1 mM IPTG (Sigma Chemical Co., MO, USA) at 37 ◦ C for 5 h. Bacteria were collected by centrifugation (1500 × g) for 5 min, washed and lysed by sonication. The pellet was collected after centrifugation and solubilised in 8 M urea (Eastman Kodak Co., Rochester, NY, USA) overnight at 4 ◦ C. After further centrifugation (26,000 × g) for 15 min the supernatant was dialysed in 0.01 M Tris (Sigma) pH 8.0/1 M urea and the BIOCAD perfusion chromatography system (Perceptive Biosystems, Framingham, MA, USA) used to purify the protein by anion exchange. To produce the 19 kDa protein secreted by M. tuberculosis, a recombinant 19 kDa plasmid construct was obtained from Professor Douglas Young (Imperial College School of Medicine, London, UK) and the expression of a 19 kDa HIS tag fusion protein (19 kDa. FP) induced with 0.1 mM IPTG at 37 ◦ C for 5 h. Bacteria were collected by centrifugation (1500×g), washed and lysed by sonication. The supernatant was collected after further centrifugation (26,000 × g) for 15 min and dialysed in 0.01 M Tris pH 8.0/300 mM NaCl/20 mM imidazole and purified under native conditions on a nickel chelate column (Qiagen, Hilden, Germany) using the BIOCAD perfusion chromatography system. Purity of the protein preparations was checked by confirming a single band on SDS-PAGE.
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2.3. Preparation of oxidised or reduced mannan–antigen conjugates Mannan (Sigma) was coupled to the antigens under oxidising conditions [9]. Mannan at 14 mg/ml in 0.1 M phosphate buffer pH 6.0 was oxidised with 0.01 M sodium periodate for 60 min at 4 ◦ C. Ten microlitres of ethandiol (Sigma) was added to quench the reaction and the mixture incubated for 30 min at 4 ◦ C. This mixture was then passed through a PD-10 column (Pharmacia Biotech, Uppsala, Sweden) equilibrated with bicarbonate buffer pH 9.0. The oxidised mannan, eluted in the 2 ml void volume, was mixed with 700 g of LLO.FP or 19 kDa. FP, incubated overnight at room temperature and used without any further purification. Conjugation was confirmed when the conjugates were separated using 12.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and a heterogeneous smear (compared with the single band of uncoupled protein) was observed using Coomassie Blue stain. The heterogeneity was a reflection of the heterogeneous molecular weight of mannan and was confirmation of successful coupling. For comparison in some experiments, the oxidised conjugates were reduced with sodium borohydride (NaBH4 ) (Aldrich, Castle Hill, NSW, Australia) 1 mg/ml overnight at room temperature and used without further purification [9]. 2.4. Immunisation with mannan–antigen conjugates Mice were lightly anaesthetised with penthrane and 50 l of mannan–antigen conjugate (12 g of antigen per mouse in bicarbonate buffer pH 9.0) placed onto the nares to be inhaled by the mouse. The same amount of non-conjugated antigen was similarly applied. Unless stated otherwise, this procedure was performed on days 0, 10 and 17 of the experiments. For intraperitoneal immunisation, mice were given 12 g of mannan–LLO.FP (M–LLO.FP) conjugate in 0.2 ml bicarbonate buffer pH 9.0 on days 0, 10 and 17. Cholera toxin (Sigma) was also used for comparative purposes as an adjuvant to act as a positive control in some experiments. One microgram of CT [13,14] was mixed with 12 g of LLO.FP and administered intranasally (i.n.) in a 50 l volume on days 0, 10 and 17. 2.5. Collection of samples Serum samples were collected after mice were bled by cardiac puncture following euthanasia at the end of each experiment. For time course experiments mice were placed on a heat box, a small incision made in a lateral vein and 200 l of blood collected with a micropippetor. The serum was subsequently separated by centrifugation. Mouth and vaginal washings were collected from anaesthetised mice, by washing with 50 or 100 l of phosphate buffered saline (PBS), respectively, using a micropippetor. Lung washings
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were obtained on day 35 after mice were euthanised with CO2 . Lungs were washed in situ with 0.5 ml PBS through an opening in the trachea. Tear samples were collected from anaesthetised mice on day 24 using a wedge of Whatman no. 1 filter paper [15]. The filter paper was placed on the tear meniscus and in the conjuctival sac for only 5 s, thus, avoiding stimulation of tear production. The volume of tears collected was determined by comparison with the uptake of known volumes of saline on filter paper. The tips of the wedges were cut off and soaked in 25 l Tris-buffered saline 0.05% Tween 20 overnight prior to assay. All samples were stored at −20 ◦ C prior to assay.
dose–response curves with titrations of IgA standard (ICN Biomedicals Inc., Costa Mesa, CA, USA). This allowed for the calculation of antigen-specific IgA as a percentage of total IgA.
2.6. Detection of antibody in the serum and mucosal sites by ELISA
3. Results
Microtitre plates (Nunc Roskilde, Denmark) were coated overnight at 4 ◦ C with 5 g/ml LLO.FP or 19 kDa. FP in carbonate buffer pH 9.1 to detect antigen-specific IgA or with 5 g/ml goat anti-mouse IgA (Sigma) to detect total IgA in the capture ELISA. The wells were then blocked with 2% foetal calf serum (FCS) (Trace Biosciences, Castle Hill, NSW, Australia) in PBS for 1 h at 37 ◦ C. The plates were washed three times with 0.08% Tween 20 (BDH Laboratory Supplies, Poole, England) PBS and appropriately diluted samples in 50 l added and incubated for 2 h at room temperature. After two more washes, antigen-specific IgA was detected by the addition of an anti-mouse IgA affinity purified horseradish peroxidase (HRP) conjugate (Southern Biotechnology Associates Inc., Birmingham, USA) diluted 1/1000 in 0.1% bovine serum albumin (BSA) (CSL, Melbourne, Australia) for 1 h at room temperature. Antigen-specific IgG1 or IgG2a was detected with the addition of a biotinylated anti-mouse IgG1 or IgG2a conjugate (Caltag Laboratories, Burlinggame, CA, USA) diluted 1/1000 in 0.1% BSA. The plates were washed twice more and a streptavidin peroxidase conjugate (Boehringer Mannheim, Mannheim, Germany) added at a 1/1000 dilution in 0.1% BSA. Following a further two washes, the antibody titres of all the subclasses tested were determined when the substrates containing either 0.4 g/l 3,3 ,5,5 -tetramethylbenzidine (TMB) (Kirkgaard and Perry Laboratories, Gaithersburg, MD, USA) and 0.02% H2 O2 or 2,2 -azino-bis(3-ethylbenthiazoline-6-sulphonic acid) (ABTS) (Sigma) and 0.03% H2 O2 were added (50 l per well). Plates were left 10 min for colour to develop and the reaction stopped with 2 M H2 SO4 for the TMB substrate or 0.2 M citric acid for ABTS. OD at 450 nm (TMB) or 405 nm (ABTS) was read in an ELISA reader (Labsystems, Helsinki, Finland). Antibody titres were presented as the highest dilution which yielded an optical density at 450 or 405 nm >0.1 OD units higher than normal serum at 1/100 dilution. For calculation of means, the titre was converted to log10 and a geometric mean with standard deviations was derived. In some experiments antibody concentration (ng/ml) in lung washings was determined by comparing
2.7. Statistics The statistical significance of data was determined by the two-sample ranks test (Wilcoxon–White) on the log10 titre. Differences with P < 0.05 were considered significant.
3.1. LLO-specific antibodies in serum following intranasal immunisation with M–LLO.FP Administration of antigen onto mucosal surfaces generally favours the induction of mucosal antibody, including IgA. To determine whether the intranasal route of immunisation was superior to intraperitoneal in inducing substantial IgA antibody responses, (CBA × BALB/c)F1 mice were immunised i.n. or i.p. on days 0, 10 and 17 with 12 g M–LLO.FP. Serum was obtained from three mice 7 days after the final immunisation and antibody levels measured by ELISA (Fig. 1). Mice immunised i.n. with M–LLO.FP, produced antigen-specific IgA to a geometric mean titre of log10 2.96 ± 0.21 (Fig. 1a), whereas IgA antibody was not detectable after the standard i.p. immunisation. It should be noted that i.p. immunisation could lead to antibody production, as IgG1 titres of log10 2.81 were detected (Fig. 1b). However, even for this isotype i.n. immunisation was superior, inducing titres in excess of log10 3.11. To test whether mannan had a significant adjuvant effect on immunisation, (CBA × BALB/c)F1 mice were immunised i.n. with 12 g of M–LLO.FP conjugate on days 0, 10 and 17 and bled on day 24. The serum antibody levels from individual mice were measured by ELISA (Fig. 2). IgA serum antibody responses in M–LLO.FP immunised mice (log10 3.65 ± 0.28) were significantly higher (P < 0.01) than those obtained from mice immunised i.n. with 12 g of non-conjugated LLO.FP (log10 1.40 ± 0.00) (Fig. 2a). A significant difference (P < 0.01) in the IgG1 response (Fig. 2b) was demonstrated when M–LLO.FP immunised mice (log10 4.87 ± 0.22) were compared to non-conjugated LLO.FP controls (log10 2.14 ± 1.05). The antibody titres for the IgG2a subclass (Fig. 2c) also proved to be significantly different (P < 0.01) when conjugated and non-conjugated LLO.FP were compared, with titres of log10 4.48 ± 0.19 and log10 1.96 ± 0.81, respectively. Infection with L. monocytogenes, an organism known for its induction of cell mediated immunity, resulted in minimal antibody responses. The experiment was repeated with conjugates prepared on different occasions with three different batches of mannan, and proved to be a very reproducible observation.
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Fig. 1. Intranasal vs. intraperitoneal immunisation. (CBA × BALB/c)F1 mice were immunised on days 0, 10, 17 with 12 g of M–LLO.FP intranasally or intraperitoneally and serum obtained from individual mice on day 24. Antigen-specific IgA was detected by ELISA and optical density measured at 450 nm. Closed squares (䊏) indicate intranasally immunised mice, open circles (䊊) intraperitoneally immunised mice and open triangles () the unimmunised controls. Results for individual mice are shown.
3.2. Comparison between oxidised mannan and CT as mucosal adjuvants In previous studies where CT has been used as a mucosal adjuvant in mice, the immunisation regimes adopted have varied in both dose and frequency of immunisation [13,14,16–19]. Therefore, to compare the adjuvanticity of mannan and CT the initial experiment was performed using the schedule previously found optimal for mannan [20] of administering antigen and adjuvant on days 0, 10 and 17. (CBA × BALB/c)F1 mice were given either 12 g of antigen as M–LLO.FP or CT + LLO.FP i.n. Serum, vaginal washings and mouth washings were collected at the times shown on Fig. 3 for titration of IgA. IgA titres in negative control groups, including mannan alone, CT alone and normal serum, were less than the limit of detection
Fig. 2. Subclasses of antibody in the serum of M–LLO.FP immunised mice. (CBA × BALB/c)F1 mice were immunised intranasally with 12 g of M–LLO.FP on days 0, 10 and 17 and serum from five individual mice obtained on day 24. Antigen-specific IgA (a), IgG1 (b) and IgG2a (c) was detected by ELISA. Individual titres are shown as dot plots, with corresponding symbols for different antibody classes from the same mouse. Differences between groups receiving M–LLO.FP or unconjugated LLO.FP were significant for all classes of antibody.
and are not shown. Oxidised mannan was a consistently better mucosal adjuvant than CT when administered with LLO.FP. Immunisation with M–LLO.FP induced a peak geometric mean IgA titre of log10 2.83 ± 0.31 in serum with some titres in excess of log10 3.11. This compared with log10 2.23 ± 0.60 for CT + LLO.FP (Fig. 3a). LLO.FP
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Fig. 3. Antigen-specific IgA in the serum and distant mucosal sites over a 41-day period. (CBA × BALB/c)F1 mice were immunised on days 0, 10 and 17 with 12 g of M–LLO.FP, with 1 g of CT mixed with 12 g of LLO.FP or with 12 g of LLO.FP alone. Serum (a), vaginal washings (b), and mouth washings (c) were collected from five individual mice on days 7, 20, 27 and 41. Antigen-specific IgA titres were determined by ELISA and the progress in individual mice shown.
alone induced a mean titre of only log10 1.84 ± 0.52. The superior efficacy of M–LLO.FP was also observed at distant mucosal sites. IgA titres in vaginal washings (Fig. 3b) were more variable than in serum, with peak mean IgA titres of log10 2.29 ± 0.51 after M–LLO.FP immunisation compared with a mean of log10 1.77 ± 0.53 for CT + LLO.FP. LLO.FP alone induced a mean titre of log10 1.57 ± 0.50. IgA titres in the saliva (Fig. 3c) of M–LLO.FP immunised mice were only detectable on day 41 of the experiment with a mean of log10 0.97 ± 0.34. Titres, although low, were higher than those observed for LLO.FP or CT + LLO.FP. In both instances, CT was quite a poor adjuvant. In a second series of experiments (Fig. 4), the immunisation schedule was extended to 0, 28 and 56 days, with doses as before, to test whether a longer time between prime and boost would favour either adjuvant. Again the response to M–LLO.FP produced higher titres than CT + LLO.FP. The peak geometric mean titres following M–LLO.FP were log10 3.02 ± 0.18 in serum and log10 2.41 ± 0.33 in vaginal washings. This compared with log10 1.50 ± 0.87 and
log10 1.35 ± 0.23, respectively, following CT + LLO.FP. Although differences were often not statistically significant because of the variability of individual mice, oxidised mannan was consistently the better adjuvant under two different immunisation regimes. IgA titres were examined over a considerable time course in these experiments. In general, they did not reach substantial levels until after three injections, whether of M–LLO.FP or CT + LLO.FP. IgA persisted at peak titre in the serum for more than 3 weeks after the last immunisation, particularly of M–LLO.FP (Figs. 3a and 4a). IgA production in the vagina (Figs. 3b and 4b) also required three immunisations and these titres were more variable than in serum, and generally less sustained. 3.3. Detection of IgA in tear and lung washing samples To extend the investigation of mucosal IgA to other sites, (CBA × BALB/c)F1 mice were immunised i.n. with M–LLO.FP as before and tears and lung washings collected
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Fig. 4. Antigen-specific IgA at distant mucosal sites over a 112-day period. (CBA × BALB/c)F1 mice were immunised on days 0, 28 and 56 with 12 g of M–LLO.FP, with 1 g of CT mixed with 12 g of LLO.FP or with 12 g of LLO.FP alone. Serum (a) and vaginal washings (b) were collected from four individual mice on days 31, 74 and 112. Antigen-specific IgA titres were determined by ELISA and the progress in individual mice shown.
on days 24 and 35, respectively. Mice immunised with the M–LLO.FP conjugate produced significantly higher titres of IgA (P < 0.01) in tears when compared to the unconjugated controls (geometric mean titres: M–LLO.FP = log10 2.01 ± 0.29 compared to log10 1.40 ± 0.00 for LLO.FP alone) (Fig. 5a). The same trend was observed in the lungs on day 35 (Fig. 5b). Significantly higher titres of IgA were detected in mice immunised with M–LLO.FP (geometric mean titre = log10 2.70 ± 0.59) compared to unconjugated LLO.FP (geometric mean titre = log10 1.78 ± 0.45) control (P < 0.05).
In order to assess the effectiveness of the mucosal adjuvant in terms of its ability to enhance the amount of antigen-specific IgA, as well as to compensate for variations in collection volumes of samples and dilutions, experiments were carried out to determine antigen-specific IgA as a percentage of total IgA (Table 1). Immunisation with M–LLO.FP resulted in an increase in total IgA secretion when compared to the unconjugated control (P < 0.01), as well as an increase in antigen-specific IgA, rising from undetectable levels to 32.2 ± 19.6 ng per well when compared to LLO.FP alone and unimmunised controls.
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Fig. 5. Antigen-specific IgA in tears and lung washings. (CBA × BALB/c)F1 mice were immunised on days 0, 10 and 17 with 12 g of M–LLO.FP or with 12 g of LLO.FP alone. Mice were anaesthetised on day 24 of the experiment and tears (1 l) collected from five mice per group (a). Mice were euthanised on day 35 of the experiment and lung washings (0.5 ml) collected (b). Antigen-specific IgA titres were determined by ELISA and individual titres are shown as dot plots. Differences between groups receiving M–LLO.FP or unconjugated LLO.FP were significant (P < 0.01) in tear samples and (P < 0.05) for lung washings.
Table 1 Antigen-specific IgA as a percentage of total IgA Groupa M–LLO.FP LLO.FP Normal
Captured IgA (ng per well)b 371 ± 204d 51.0 ± 17.9 48.4 ± 5.03
Antigen-specific IgA (ng per well)b
Percentage-specific IgAl total IgAc
32.2 ± 19.6 NDe ND
11.7 ± 7.70 ND ND
(CBA × BALB/c)F1 mice were immunised intranasally on days 0, 10 and 17 with 12 g of M–LLO.FP or with 12 g of LLO.FP alone. Mice were euthanised on day 35 of the experiment and lung washings (0.5 ml) collected. b Total IgA was detected using a capture ELISA. Antigen-specific IgA was determined by a standard ELISA. The concentration of IgA was derived from a standard curve and the values from individual mice used to calculate means and standard deviations. c Antigen-specific IgA as a percentage of total IgA was calculated for individual mice and their values were then used to calculate a mean and standard deviation. d Significantly different from LLO.FP and unimmunised mice, P < 0.01. e ND: not detected. a
3.4. Influence of conjugation of mannan to antigen on IgA induction To investigate whether there was a need for the mannan to be conjugated to LLO.FP or whether the adjuvant effect could be achieved when mannan was simply mixed with antigen, mice were immunised with the M–LLO.FP conjugate or with a mannan + LLO.FP mixture (mannan and 12 g of LLO.FP mixed together). (CBA × BALB/c)F1 mice were immunised i.n. on days 0, 10 and 17. Serum and vaginal washings were taken from individual mice on day 24 of the experiment. IgA titres were subsequently determined by ELISA (Table 2). The results clearly showed that a conjugate was required for adjuvant effect. The geometric mean antibody titre in the serum for conjugated LLO.FP was log10 3.00 ± 0.19. This was significantly higher than the mannan + LLO.FP mixture which produced an average titre of log10 1.53 ± 0.49. A similar pattern was seen
Table 2 Requirement for conjugation of mannan and LLO.FP for IgA production Antigen/adjuvanta
M–LLO.FP (linked) M + LLO.FP (mixed) LLO.FP Mannan Normal
Log10 IgA antibody titre Serumb
Vaginal washingsb
3.00 ± 0.19c 1.53 ± 0.49 1.15 ± 0.30 <1 <1
2.29 1.65 1.15 1.12 1.08
± ± ± ± ±
0.70c 0.48 0.11 0.15 0.10
a (CBA × BALB/c)F1 mice were immunised intranasally with M–LLO.FP, LLO.FP, mannan mixed with LLO.FP (unconjugated) or mannan alone on days 0, 10 and 17. Serum samples and vaginal washings obtained on day 24 from four individual mice. b Individual IgA titres were determined by ELISA. Antibody titres were converted to log10 and geometric means and standard deviations were derived. c Significantly different from mannan + LLO.FP (mixed), P < 0.05.
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in the antibody response at a distant mucosal site, namely the vagina, with titres of log10 2.29 ± 0.70 for the conjugate and log 1.65 ± 0.48 for the mixture. All negative controls produced undetectable levels of IgA with small titres of antibody observed in vaginal washings for the LLO.FP alone group (log10 1.15 ± 0.11).
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Earlier experiments with mannan-conjugated proteins injected i.p. showed that conjugates produced under reduced conditions were better at inducing antibody than were oxidised conjugates [9]. Therefore, (CBA × BALB/c)F1 mice were immunised i.n. with M–LLO.FP conjugate (oxidised form) or M–LLO.FP conjugate (reduced form). The results indicated that the oxidised form of the M–LLO.FP conjugate given i.n. induced higher titres of IgA, IgG1 and IgG2a in the serum (Fig. 6a, b and c) compared with the reduced form. All four mice immunised with M–LLO.FP (oxidised form) had higher titres than the corresponding four mice given the reduced form of M–LLO.FP, with the exception of one high responder mouse in the group given reduced mannan. Significant differences (P < 0.05) applied to all the subclasses assayed (geometric mean titres: IgA = log10 3.15 ± 0.30 for oxidised, log10 2.46±0.39 for reduced; IgG1 = log10 3.52± 0.15 for oxidised, log10 2.90 ± 0.43 for reduced; IgG2a = log10 3.22±0.25 for oxidised, log10 2.25±0.51 for reduced). 3.5. Use of oxidised mannan as adjuvant for other antigens To demonstrate that the use of mannan as a mucosal adjuvant was applicable to other antigens, oxidised mannan was conjugated to the 19 kDa protein secreted by M. tuberculosis. C57BL/10 mice were immunised i.n. with 12 g of M–19.FP conjugate on days 0, 10 and 17. On day 24 of the experiment the mice were euthanised and bled by cardiac puncture. Serum was separated and IgA titres determined by ELISA (Fig. 7). The four mice immunised with M–19.FP produced significantly higher titres of IgA (geometric mean log10 3.19 ± 0.36) when compared with mice
Fig. 6. Requirement for the M–LLO.FP conjugate to be in the oxidised form. (CBA×BALB/c)F1 mice were immunised intranasally with 12 g of M–LLO.FP in the oxidised or reduced form on days 0, 10 and 17. Serum from five individual mice was collected on day 24 and antigen-specific IgA (a), IgG1 (b) and IgG2a (c) was detected by ELISA. Individual titres are shown as dot plots, with corresponding symbols for different antibody classes from the same mouse. Differences between groups receiving oxidised or reduced M–LLO.FP were significant (P < 0.05) for all classes of antibody.
Fig. 7. Use of oxidised mannan as adjuvant for mycobacterial 19 kDa. FP. Groups of four C57BL/10 mice were immunised intranasally with 12 g of M-19.FP or 12 g of 19 kDa.FP on days 0, 10 and 17. Serum was collected on day 24 and antigen-specific IgA determined by ELISA. Individual titres are shown as dot plots. Differences between groups receiving M-19.FP and 19 kDa.FP alone were significant (P < 0.05).
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given 12 g of 19 kDa.FP (mean titres of log10 2.83 ± 0.31) (P < 0.05). 4. Discussion These experiments present oxidised mannan as a novel mucosal adjuvant which, when administered intranasally, induced high mucosal and serum titres of IgA, IgG1 and IgG2a specific for recombinant protein antigens with which it was administered. Oxidised mannan was previously used conjugated to the breast cancer antigen mucin 1 and injected i.p. into mice where it induced CTL and Th1 cytokines, protecting against tumours expressing mucin 1 [9,10]. In that context it induced a poor antibody response, dominated by IgG2a. It was the change to intranasal administration in the current experiments which resulted in production of IgA and other classes of antibody in serum and mucosal secretions. Mucosal antibodies and particularly IgA, have been shown to protect against influenza in the lung [21], against Helicobacter pylori in the stomach [22], against Haemophilis influenzae in the ear [14] and against Chlamydia trachomatis in the genital tract [23] amongst other examples. Induction of mucosal antibodies following intramuscular or subcutaneous immunisation with conventional vaccines is poor [16,24]. Therefore, oxidised mannan may be a valuable adjuvant if coupled to protective antigens from a wide variety of infectious agents. Induction of mucosal immunity begins with the uptake of antigen by membranous (M) cells (specialised epithelial cells) on the mucosal surface. These cells either process and present antigen to underlying T cells or B cells themselves or transport antigen to parenchymal macrophages, dendritic cells and B cells. Once interaction of the antigen presenting cell (APC) with T and B lymphocytes has occurred, an immune response or mucosal tolerance may result. Immune responses generally involve antibody production, with IgA the predominant antibody isotype. Antigen sensitised immune cells are then circulated to other systemic and mucosal sites for expansion of effector mechanisms [25]. The fact that there is a preferential circulation of T cells activated at mucosal sites to return to the same or other mucosal sites accounts for the induction of IgA at remote mucosal surfaces. Our results support these general observations. Cholera toxin has become the most extensively studied mucosal adjuvant in experimental models, although its relative toxicity has so far precluded it from approval for use as a human adjuvant [26]. In the present experiments, immunisation with mannan-conjugated LLO intranasally-induced superior IgA and IgG2a responses in the mucosa and serum compared with CT plus LLO or LLO alone. The difference in the IgG1 response was less marked, but still higher for the mannan adjuvant. Titres of antibody following immunisation with mannan conjugates were not only higher than
with CT, they were more sustained. Mannan has the further advantage over CT in being non-toxic and has been extensively tested in human trials of cancer immunotherapy [11]. As with the present observations on mannan, a mixture of the B subunit of CT (CTB) with antigen resulted in higher titres of IgA in the serum and mucosa when given to mice intranasally rather than intraperitoneally [16]. It is widely believed that this influence of the route of exposure is a function of the mucosal antigen presenting cells. Two interesting observations were made when analysing the form of the antigen/adjuvant in the present experiments. The first showed that oxidised mannan must be conjugated to the antigen (LLO.FP or 19 kDa FP) in order to facilitate the adjuvant effect. This confirms the observations with the mucin 1 studies where conjugation of oxidised mannan to the mucin 1 antigen was necessary in order to induce better immune responses [9]. The second observation contrasts with the mucin 1 studies because the oxidised form of the M–LLO.FP was required for maximum antibody production. Intraperitoneal immunisation with the oxidised form of mannan–mucin 1 resulted in low antibody levels [9]. Immunisation with oxidised mannan not only increased the amount of total IgA secreted as observed in the lung washings but also increased the amount of specific IgA secreted at this mucosal site. This type of response to immunisation with a mucosal adjuvant has also been observed in other systems. Intranasal delivery of AgI/II of Streptococcus mutans with the B subunit of cholera toxin resulted in an increase in both the total and antigen-specific salivary IgA response [27]. The ability of oxidised mannan to create such increases justifies further work in this area concentrating specifically on mucosal infection models. The fact that this effect persists for as long as 18 days after the last immunisation with M–LLO.FP suggests that this is not an acute inflammatory effect. How, then, does mannan act as an adjuvant? It has been shown that antigens bearing mannose residues are bound to the mannose receptors on APC, facilitating uptake into the MHC class II pathway for efficient antigen presentation [28–30]. Oxidised mannan–mucin 1 induced both class I restricted CTL and a Th1 response [10]. It has been suggested that oxidised mannan–mucin 1, by virtue of the aldehyde residues created by the oxidation process, escapes the phagocytic pathway and is transported into the MHC class I pathway inducing CTL [31]. This pathway would not be open to reduced complexes, where aldehyde residues are reduced to alcohols by the action of borohydride. The exact mechanism by which aldehyde acts is unknown. Curiously, we did not detect the induction of class I-restricted CTL in experiments with M–LLO.FP injected i.p. (Stambas et al., unpublished data). Furthermore, the cytokine response to our antigens given intranasally was not classically either Th1 or Th2, since IgG1 and IgA (generally considered Th2 responses) and IgG2a (Th1) were all elevated. This was confirmed by cytokine assays where both IFN-␥ and IL-4 were elevated (Stambas et al., unpublished data).
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The mechanism by which M–LLO.FP and M–19.FP induce such excellent antibody responses after i.n. immunisation is not presently known. Others have shown preferential induction of Th2 responses following intranasal instillation of leishmania antigens, even in strains of mouse which are genetically constrained to produce a typical Th1 response when these organisms are injected parenterally [32]. Perhaps alveolar macrophages or dendritic cells, which have been shown to present antigens efficiently in other systems [33], are playing a significant role in the induction of these IgA responses. Further studies following the gross and intracellular location of antigen will be required in order to determine the pathway of antigen presentation. Intranasal administration of oxidised mannan conjugates may advance development of human vaccines for sexually transmitted diseases such as Chlamydia or human immunodeficiency virus, other mucosally acquired infections like influenza, rota virus and trachoma, as well as for veterinary applications. The fact that oxidised mannan is a novel, non-toxic adjuvant that induces systemic and mucosal antibody responses superior to the standard mucosal adjuvant CT, warrants further investigation. References [1] Williams RC, Gibbons RJ. Inhibition of bacterial adherence by secretory immunoglobulin A: a mechanism of antigen disposal. Science 1972;177:697–9. [2] Hajishengallis G, Nikolova E, Russell MW. Inhibition of Streptococcus mutans adherence to saliva-coated hydroxyapatite by human secretory immunoglobulin A (S-IgA) antibodies to cell surface protein antigen I/II: reversal by IgA1 protease cleavage. Infect Immunol 1992;60:5057–64. [3] Gilbert JV, Plaut AG, Longmaid B, Lamm ME. Inhibition of microbial IgA proteases by human secretory IgA and serum. Mol Immunol 1983;20:1039–49. [4] Outlaw MC, Dimmock NJ. Mechanisms of neutralization of influenza virus on mouse tracheal epithelial cells by mouse monoclonal polymeric IgA and polyclonal IgM directed against the viral haemagglutinin. J Gen Virol 1990;71:69–76. [5] Chatfield S, Roberts M, Londono P, Cropley I, Douce G, Dougan G. The development of oral vaccines based on live attenuated Salmonella strains. FEMS Immunol Med Microbiol 1993;7:1–7. [6] Ulmer JB, Donnelly JJ, Parker SE, Rhodes GH, Felgner PL, Dwarki VJ, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 1993;259:1745–9. [7] Elson CO, Ealding W. Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin. J Immunol 1984;132:2736–41. [8] de Haan L, Verweij W, Agsteribbe E, Wilschut J. The role of ADP-ribosylation and G(M1)-binding activity in the mucosal immunogenicity and adjuvanticity of the Escherichia coli heat-labile enterotoxin and Vibrio cholerae cholera toxin. Immunol Cell Biol 1998;76:270–9. [9] Apostolopoulos V, Pietersz GA, Loveland BE, Sandrin MS, McKenzie IF. Oxidative/reductive conjugation of mannan to antigen selects for T1 or T2 immune responses. Proc Natl Acad Sci USA 1995;92:10128–32. [10] Apostolopoulos V, Loveland BE, Pietersz GA, McKenzie IF. CTL in mice immunized with human mucin 1 are MHC-restricted. J Immunol 1995;155:5089–94.
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[11] Karanikas V, Hwang LA, Pearson J, Ong CS, Apostolopoulos V, Vaughan H, et al. Antibody and T cell responses of patients with adenocarcinoma immunized with mannan–mucin 1 fusion protein. J Clin Invest 1997;100:2783–92. [12] Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione-Stransferase. Gene 1988;67:31–40. [13] Martin M, Metzger DJ, Michalek SM, Connell TD, Russell MW. Comparative analysis of the mucosal adjuvanticity of the type II heat-labile enterotoxins LT-IIa and LT-IIb. Infect Immunol 2000;68:281–7. [14] Kodama S, Suenaga S, Hirano T, Suzuki M, Mogi G. Induction of specific immunoglobulin A and Th2 immune responses to P6 outer membrane protein of nontypeable Haemophilus influenzae in middle ear mucosa by intranasal immunization. Infect Immunol 2000;68:2294–300. [15] Preston MJ, Kernacki KA, Berk JM, Hazlett LD, Berk RS. Kinetics of serum, tear, and corneal antibody responses in resistant and susceptible mice intracorneally infected with Pseudomonas aeruginosa. Infect Immunol 1992;60:885–91. [16] Hirabayashi Y, Kurata H, Funato H, Nagamine T, Aizawa C, Tamura S, et al. Comparison of intranasal inoculation of influenza HA vaccine combined with cholera toxin B subunit with oral or parenteral vaccination. Vaccine 1990;8:243–8. [17] Falero-Diaz G, Challacombe S, Banerjee D, Douce G, Boyd A, Ivanyi J. Intranasal vaccination of mice against infection with Mycobacterium tuberculosis. Vaccine 2000;18:3223–9. [18] Sabirov A, Kodama S, Hirano T, Suzuki M, Mogi G. Intranasal immunization enhances clearance of nontypeable Haemophilus influenzae and reduces stimulation of tumor necrosis factor alpha production in the murine model of otitis media. Infect Immunol 2001;69:2964–71. [19] Vadolas J, Davies JK, Wright PJ, Strugnell RA. Intranasal immunization with liposomes induces strong mucosal immune responses in mice. Eur J Immunol 1995;25:969–75. [20] Pietersz GA, Li W, Popovski V, Caruana JA, Apostolopoulos V, McKenzie IF. Parameters for using mannan–mucin 1 fusion protein to induce cellular immunity. Cancer Immunol Immunotherap 1998;45:321–6. [21] Tamura S, Funato H, Hirabayashi Y, Suzuki Y, Nagamine T, Aizawa C, et al. Cross-protection against influenza A virus infection by passively transferred respiratory tract IgA antibodies to different hemagglutinin molecules. Eur J Immunol 1991;21:1337–44. [22] Goto T, Nishizono A, Fujioka T, Ikewaki J, Mifune K, Nasu M. Local secretory immunoglobulin A and postimmunization gastritis correlate with protection against Helicobacter pylori infection after oral vaccination of mice. Infect Immunol 1999;67:2531–9. [23] Su H, Feilzer K, Caldwell HD, Morrison RP. Chlamydia trachomatis genital tract infection of antibody-deficient gene knockout mice. Infect Immunol 1997;65:1993–9. [24] Muszkat M, Yehuda AB, Schein MH, Friedlander Y, Naveh P, Greenbaum E, et al. Local and systemic immune response in community-dwelling elderly after intranasal or intramuscular immunization with inactivated influenza vaccine. J Med Virol 2000;61:100–6. [25] Mestecky J. The common mucosal immune system and current strategies for induction of immune responses in external secretions. J Clin Immunol 1987;7:265–76. [26] Levine MM. Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. J Infect Dis 1987;155:377–89. [27] Wu HY, Russell MW. Induction of mucosal immunity by intranasal application of a streptococcal surface protein antigen with the cholera toxin B subunit. Infect Immunol 1993;61:314–22. [28] Sallusto F, Cella M, Danieli C, Lanzavecchia A. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II
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compartment: downregulation by cytokines and bacterial products. J Exp Med 1995;182:389–400. [29] Tan MC, Mommaas AM, Drijfhout JW, Jordens R, Onderwater JJ, Verwoerd D, et al. Mannose receptor-mediated uptake of antigens strongly enhances HLA class II-restricted antigen presentation by cultured dendritic cells. Eur J Immunol 1997;27: 2426–35. [30] Engering AJ, Cella M, Fluitsma D, Brockhaus M, Hoefsmit EC, Lanzavecchia A, et al. The mannose receptor functions as a high capacity and broad specificity antigen receptor in human dendritic cells. Eur J Immunol 1997;27:2417–25.
[31] Apostolopoulos V, Pietersz GA, Gordon S, Martinez-Pomares L, McKenzie IF. Aldehyde–mannan antigen complexes target the MHC class I antigen-presentation pathway. Eur J Immunol 2000;30:1714– 23. [32] Constant SL, Lee KS, Bottomly K. Site of antigen delivery can influence T cell priming: pulmonary environment promotes preferential Th2-type differentiation. Eur J Immunol 2000;30:840–7. [33] Hamilton-Easton A, Eichelberger M. Virus-specific antigen presentation by different subsets of cells from lung and mediastinal lymph node tissues of influenza virus infected mice. J Virol 1995;69:6359–66.
Oxidised mannan as a novel adjuvant inducing mucosal IgA production John Stambas a,∗ , Geoffrey Pietersz b , Ian McKenzie b , Christina Cheers a a
Department of Microbiology and Immunology, University of Melbourne, Parkville 3052, Vic., Australia b Austin Research Institute, Heidelberg 3084, Vic., Australia Received 9 June 2001; received in revised form 17 August 2001; accepted 11 October 2001
Abstract Mannan, oxidatively coupled to recombinant protein antigens, has here been tested as a possible adjuvant for the production of antibody on the mucosa. Given intranasally, but not intraperitoneally, mannan markedly enhanced the production of IgA, IgG1 and IgG2a in the serum, and IgA locally in the lung and at remote mucosal sites, including tears, vaginal and salivary secretions. Oxidative coupling was critical to its action, since neither mannan simply mixed with protein nor mannan–protein conjugates which had been reduced by treatment with sodium borohydride, acted as adjuvants. Oxidatively coupled mannan was compared with the widely studied mucosal adjuvant, cholera toxin (CT). The use of oxidised mannan as an adjuvant induced better responses than CT judged by the induction of IgA in serum, vaginal washings and saliva. Thus, oxidised mannan, which is non-toxic and can be administered without injection, is a suitable adjuvant coupled with protective antigens for vaccinating against a number of infections that occur via the mucous membranes. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Mucosa; IgA; Oxidised mannan
1. Introduction Most infectious agents enter the body through mucosal membranes, and recent vaccine strategies have concentrated on the production of antibodies at these sites to block their entry. The stimulation of secretory immune responses, which includes mucosal IgA, the predominant antibody isotype in mucosal secretions with the capability to neutralise bacteria, bacterial products and viruses, is considered to be crucial to vaccine development [1–4]. Currently most vaccines delivered by injection are not efficient at inducing a mucosal response, and the efficient induction of mucosal immunity requires delivery onto the mucosa. At present, the oral polio vaccine remains the only successful large-scale vaccine that gives protection at the mucosal level. It is a live vaccine whose attenuation was achieved empirically with the attendant risks of back-mutation. Other strategies, including live recombinant bacteria and DNA vaccines, have been used with encouraging results [5,6]. Immunisation with defined protein antigens would have many advantages, including safety in immunosuppressed individuals. However, before such vaccines can be developed there is a need for adjuvants and delivery systems to overcome ∗ Corresponding author. Tel.: +61-8-3445704; fax: +61-9-3471540. E-mail address: j [email protected] (J. Stambas).
the problems associated with mucosal vaccination using defined protein antigens, including poor immunogenicity or the induction of tolerance. Bacterial enterotoxins such as cholera toxin (CT) produced by Vibrio cholerae, and labile toxin from Escherichia coli have been used as adjuvants for mucosal IgA responses when co-administered with antigen [7,8], but they are not appropriate to use in humans due to their toxic properties. Studies using the enterotoxin B subunits (detoxified) of V. cholerae and E. coli have resulted in conflicting views of their adjuvanticity [8]. We now report that oxidised (but not reduced) mannan–antigen complexes, delivered at a mucosal site, give superior IgA responses. Mannan, a polymannose derived from the cell wall of yeast, was shown to enhance immune responses in mice when oxidised and conjugated to human mucin 1, an antigen over-expressed in some cancers [9,10]. Oxidised mannan had different effects depending on the species immunised. In mice, oxidised mannan–mucin 1 injected intraperitoneally (i.p.) induced cellular Th1 responses with CTL, tumour protection and little antibody, whereas in humans and monkeys immunisation with autologous mucin 1 linked to mannan produced large amounts of IgG1 antibody and little cellular immunity. In humans, monkeys and mice oxidised mannan–mucin 1 was found to be non-toxic, making it an attractive candidate adjuvant for vaccine application [11]. By changing the route of administration to mice, we
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have now found that oxidised mannan can be used as adjuvant for mucosal IgA. Two contrasting bacterial proteins were used as antigens: a secreted protein, listeriolysin O (LLO), the immunodominant antigen of Listeria monocytogenes and the 19 kDa protein secreted by Mycobacterium tuberculosis. They were conjugated to oxidised mannan and the antibody responses produced compared with that of CT. Oxidised mannan-conjugated antigen gave superior IgA, IgG1 and IgG2a responses and appears to be a promising choice for the mucosal delivery of antigens.
2. Materials and methods 2.1. Mice Female 6–8-week-old (CBA×BALB/c)F1 and C57BL/10 mice were pedigree inbred and maintained under conventional but infection-free conditions in the animal house of the Department of Microbiology and Immunology, University of Melbourne. 2.2. Production of antigens A pbluescript plasmid containing the gene for LLO from L. monocytogenes (without its leader sequence) was obtained from Richard Strugnell (University of Melbourne, Australia) and subcloned into the E. coli expression vector pGEX-2T [12] in the correct reading frame and orientation. The expression of an 84 kDa LLO glutothione-S-transferase (GST) fusion protein (LLO.FP) was induced with 0.1 mM IPTG (Sigma Chemical Co., MO, USA) at 37 ◦ C for 5 h. Bacteria were collected by centrifugation (1500 × g) for 5 min, washed and lysed by sonication. The pellet was collected after centrifugation and solubilised in 8 M urea (Eastman Kodak Co., Rochester, NY, USA) overnight at 4 ◦ C. After further centrifugation (26,000 × g) for 15 min the supernatant was dialysed in 0.01 M Tris (Sigma) pH 8.0/1 M urea and the BIOCAD perfusion chromatography system (Perceptive Biosystems, Framingham, MA, USA) used to purify the protein by anion exchange. To produce the 19 kDa protein secreted by M. tuberculosis, a recombinant 19 kDa plasmid construct was obtained from Professor Douglas Young (Imperial College School of Medicine, London, UK) and the expression of a 19 kDa HIS tag fusion protein (19 kDa. FP) induced with 0.1 mM IPTG at 37 ◦ C for 5 h. Bacteria were collected by centrifugation (1500×g), washed and lysed by sonication. The supernatant was collected after further centrifugation (26,000 × g) for 15 min and dialysed in 0.01 M Tris pH 8.0/300 mM NaCl/20 mM imidazole and purified under native conditions on a nickel chelate column (Qiagen, Hilden, Germany) using the BIOCAD perfusion chromatography system. Purity of the protein preparations was checked by confirming a single band on SDS-PAGE.
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2.3. Preparation of oxidised or reduced mannan–antigen conjugates Mannan (Sigma) was coupled to the antigens under oxidising conditions [9]. Mannan at 14 mg/ml in 0.1 M phosphate buffer pH 6.0 was oxidised with 0.01 M sodium periodate for 60 min at 4 ◦ C. Ten microlitres of ethandiol (Sigma) was added to quench the reaction and the mixture incubated for 30 min at 4 ◦ C. This mixture was then passed through a PD-10 column (Pharmacia Biotech, Uppsala, Sweden) equilibrated with bicarbonate buffer pH 9.0. The oxidised mannan, eluted in the 2 ml void volume, was mixed with 700 g of LLO.FP or 19 kDa. FP, incubated overnight at room temperature and used without any further purification. Conjugation was confirmed when the conjugates were separated using 12.5% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and a heterogeneous smear (compared with the single band of uncoupled protein) was observed using Coomassie Blue stain. The heterogeneity was a reflection of the heterogeneous molecular weight of mannan and was confirmation of successful coupling. For comparison in some experiments, the oxidised conjugates were reduced with sodium borohydride (NaBH4 ) (Aldrich, Castle Hill, NSW, Australia) 1 mg/ml overnight at room temperature and used without further purification [9]. 2.4. Immunisation with mannan–antigen conjugates Mice were lightly anaesthetised with penthrane and 50 l of mannan–antigen conjugate (12 g of antigen per mouse in bicarbonate buffer pH 9.0) placed onto the nares to be inhaled by the mouse. The same amount of non-conjugated antigen was similarly applied. Unless stated otherwise, this procedure was performed on days 0, 10 and 17 of the experiments. For intraperitoneal immunisation, mice were given 12 g of mannan–LLO.FP (M–LLO.FP) conjugate in 0.2 ml bicarbonate buffer pH 9.0 on days 0, 10 and 17. Cholera toxin (Sigma) was also used for comparative purposes as an adjuvant to act as a positive control in some experiments. One microgram of CT [13,14] was mixed with 12 g of LLO.FP and administered intranasally (i.n.) in a 50 l volume on days 0, 10 and 17. 2.5. Collection of samples Serum samples were collected after mice were bled by cardiac puncture following euthanasia at the end of each experiment. For time course experiments mice were placed on a heat box, a small incision made in a lateral vein and 200 l of blood collected with a micropippetor. The serum was subsequently separated by centrifugation. Mouth and vaginal washings were collected from anaesthetised mice, by washing with 50 or 100 l of phosphate buffered saline (PBS), respectively, using a micropippetor. Lung washings
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were obtained on day 35 after mice were euthanised with CO2 . Lungs were washed in situ with 0.5 ml PBS through an opening in the trachea. Tear samples were collected from anaesthetised mice on day 24 using a wedge of Whatman no. 1 filter paper [15]. The filter paper was placed on the tear meniscus and in the conjuctival sac for only 5 s, thus, avoiding stimulation of tear production. The volume of tears collected was determined by comparison with the uptake of known volumes of saline on filter paper. The tips of the wedges were cut off and soaked in 25 l Tris-buffered saline 0.05% Tween 20 overnight prior to assay. All samples were stored at −20 ◦ C prior to assay.
dose–response curves with titrations of IgA standard (ICN Biomedicals Inc., Costa Mesa, CA, USA). This allowed for the calculation of antigen-specific IgA as a percentage of total IgA.
2.6. Detection of antibody in the serum and mucosal sites by ELISA
3. Results
Microtitre plates (Nunc Roskilde, Denmark) were coated overnight at 4 ◦ C with 5 g/ml LLO.FP or 19 kDa. FP in carbonate buffer pH 9.1 to detect antigen-specific IgA or with 5 g/ml goat anti-mouse IgA (Sigma) to detect total IgA in the capture ELISA. The wells were then blocked with 2% foetal calf serum (FCS) (Trace Biosciences, Castle Hill, NSW, Australia) in PBS for 1 h at 37 ◦ C. The plates were washed three times with 0.08% Tween 20 (BDH Laboratory Supplies, Poole, England) PBS and appropriately diluted samples in 50 l added and incubated for 2 h at room temperature. After two more washes, antigen-specific IgA was detected by the addition of an anti-mouse IgA affinity purified horseradish peroxidase (HRP) conjugate (Southern Biotechnology Associates Inc., Birmingham, USA) diluted 1/1000 in 0.1% bovine serum albumin (BSA) (CSL, Melbourne, Australia) for 1 h at room temperature. Antigen-specific IgG1 or IgG2a was detected with the addition of a biotinylated anti-mouse IgG1 or IgG2a conjugate (Caltag Laboratories, Burlinggame, CA, USA) diluted 1/1000 in 0.1% BSA. The plates were washed twice more and a streptavidin peroxidase conjugate (Boehringer Mannheim, Mannheim, Germany) added at a 1/1000 dilution in 0.1% BSA. Following a further two washes, the antibody titres of all the subclasses tested were determined when the substrates containing either 0.4 g/l 3,3 ,5,5 -tetramethylbenzidine (TMB) (Kirkgaard and Perry Laboratories, Gaithersburg, MD, USA) and 0.02% H2 O2 or 2,2 -azino-bis(3-ethylbenthiazoline-6-sulphonic acid) (ABTS) (Sigma) and 0.03% H2 O2 were added (50 l per well). Plates were left 10 min for colour to develop and the reaction stopped with 2 M H2 SO4 for the TMB substrate or 0.2 M citric acid for ABTS. OD at 450 nm (TMB) or 405 nm (ABTS) was read in an ELISA reader (Labsystems, Helsinki, Finland). Antibody titres were presented as the highest dilution which yielded an optical density at 450 or 405 nm >0.1 OD units higher than normal serum at 1/100 dilution. For calculation of means, the titre was converted to log10 and a geometric mean with standard deviations was derived. In some experiments antibody concentration (ng/ml) in lung washings was determined by comparing
2.7. Statistics The statistical significance of data was determined by the two-sample ranks test (Wilcoxon–White) on the log10 titre. Differences with P < 0.05 were considered significant.
3.1. LLO-specific antibodies in serum following intranasal immunisation with M–LLO.FP Administration of antigen onto mucosal surfaces generally favours the induction of mucosal antibody, including IgA. To determine whether the intranasal route of immunisation was superior to intraperitoneal in inducing substantial IgA antibody responses, (CBA × BALB/c)F1 mice were immunised i.n. or i.p. on days 0, 10 and 17 with 12 g M–LLO.FP. Serum was obtained from three mice 7 days after the final immunisation and antibody levels measured by ELISA (Fig. 1). Mice immunised i.n. with M–LLO.FP, produced antigen-specific IgA to a geometric mean titre of log10 2.96 ± 0.21 (Fig. 1a), whereas IgA antibody was not detectable after the standard i.p. immunisation. It should be noted that i.p. immunisation could lead to antibody production, as IgG1 titres of log10 2.81 were detected (Fig. 1b). However, even for this isotype i.n. immunisation was superior, inducing titres in excess of log10 3.11. To test whether mannan had a significant adjuvant effect on immunisation, (CBA × BALB/c)F1 mice were immunised i.n. with 12 g of M–LLO.FP conjugate on days 0, 10 and 17 and bled on day 24. The serum antibody levels from individual mice were measured by ELISA (Fig. 2). IgA serum antibody responses in M–LLO.FP immunised mice (log10 3.65 ± 0.28) were significantly higher (P < 0.01) than those obtained from mice immunised i.n. with 12 g of non-conjugated LLO.FP (log10 1.40 ± 0.00) (Fig. 2a). A significant difference (P < 0.01) in the IgG1 response (Fig. 2b) was demonstrated when M–LLO.FP immunised mice (log10 4.87 ± 0.22) were compared to non-conjugated LLO.FP controls (log10 2.14 ± 1.05). The antibody titres for the IgG2a subclass (Fig. 2c) also proved to be significantly different (P < 0.01) when conjugated and non-conjugated LLO.FP were compared, with titres of log10 4.48 ± 0.19 and log10 1.96 ± 0.81, respectively. Infection with L. monocytogenes, an organism known for its induction of cell mediated immunity, resulted in minimal antibody responses. The experiment was repeated with conjugates prepared on different occasions with three different batches of mannan, and proved to be a very reproducible observation.
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Fig. 1. Intranasal vs. intraperitoneal immunisation. (CBA × BALB/c)F1 mice were immunised on days 0, 10, 17 with 12 g of M–LLO.FP intranasally or intraperitoneally and serum obtained from individual mice on day 24. Antigen-specific IgA was detected by ELISA and optical density measured at 450 nm. Closed squares (䊏) indicate intranasally immunised mice, open circles (䊊) intraperitoneally immunised mice and open triangles () the unimmunised controls. Results for individual mice are shown.
3.2. Comparison between oxidised mannan and CT as mucosal adjuvants In previous studies where CT has been used as a mucosal adjuvant in mice, the immunisation regimes adopted have varied in both dose and frequency of immunisation [13,14,16–19]. Therefore, to compare the adjuvanticity of mannan and CT the initial experiment was performed using the schedule previously found optimal for mannan [20] of administering antigen and adjuvant on days 0, 10 and 17. (CBA × BALB/c)F1 mice were given either 12 g of antigen as M–LLO.FP or CT + LLO.FP i.n. Serum, vaginal washings and mouth washings were collected at the times shown on Fig. 3 for titration of IgA. IgA titres in negative control groups, including mannan alone, CT alone and normal serum, were less than the limit of detection
Fig. 2. Subclasses of antibody in the serum of M–LLO.FP immunised mice. (CBA × BALB/c)F1 mice were immunised intranasally with 12 g of M–LLO.FP on days 0, 10 and 17 and serum from five individual mice obtained on day 24. Antigen-specific IgA (a), IgG1 (b) and IgG2a (c) was detected by ELISA. Individual titres are shown as dot plots, with corresponding symbols for different antibody classes from the same mouse. Differences between groups receiving M–LLO.FP or unconjugated LLO.FP were significant for all classes of antibody.
and are not shown. Oxidised mannan was a consistently better mucosal adjuvant than CT when administered with LLO.FP. Immunisation with M–LLO.FP induced a peak geometric mean IgA titre of log10 2.83 ± 0.31 in serum with some titres in excess of log10 3.11. This compared with log10 2.23 ± 0.60 for CT + LLO.FP (Fig. 3a). LLO.FP
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Fig. 3. Antigen-specific IgA in the serum and distant mucosal sites over a 41-day period. (CBA × BALB/c)F1 mice were immunised on days 0, 10 and 17 with 12 g of M–LLO.FP, with 1 g of CT mixed with 12 g of LLO.FP or with 12 g of LLO.FP alone. Serum (a), vaginal washings (b), and mouth washings (c) were collected from five individual mice on days 7, 20, 27 and 41. Antigen-specific IgA titres were determined by ELISA and the progress in individual mice shown.
alone induced a mean titre of only log10 1.84 ± 0.52. The superior efficacy of M–LLO.FP was also observed at distant mucosal sites. IgA titres in vaginal washings (Fig. 3b) were more variable than in serum, with peak mean IgA titres of log10 2.29 ± 0.51 after M–LLO.FP immunisation compared with a mean of log10 1.77 ± 0.53 for CT + LLO.FP. LLO.FP alone induced a mean titre of log10 1.57 ± 0.50. IgA titres in the saliva (Fig. 3c) of M–LLO.FP immunised mice were only detectable on day 41 of the experiment with a mean of log10 0.97 ± 0.34. Titres, although low, were higher than those observed for LLO.FP or CT + LLO.FP. In both instances, CT was quite a poor adjuvant. In a second series of experiments (Fig. 4), the immunisation schedule was extended to 0, 28 and 56 days, with doses as before, to test whether a longer time between prime and boost would favour either adjuvant. Again the response to M–LLO.FP produced higher titres than CT + LLO.FP. The peak geometric mean titres following M–LLO.FP were log10 3.02 ± 0.18 in serum and log10 2.41 ± 0.33 in vaginal washings. This compared with log10 1.50 ± 0.87 and
log10 1.35 ± 0.23, respectively, following CT + LLO.FP. Although differences were often not statistically significant because of the variability of individual mice, oxidised mannan was consistently the better adjuvant under two different immunisation regimes. IgA titres were examined over a considerable time course in these experiments. In general, they did not reach substantial levels until after three injections, whether of M–LLO.FP or CT + LLO.FP. IgA persisted at peak titre in the serum for more than 3 weeks after the last immunisation, particularly of M–LLO.FP (Figs. 3a and 4a). IgA production in the vagina (Figs. 3b and 4b) also required three immunisations and these titres were more variable than in serum, and generally less sustained. 3.3. Detection of IgA in tear and lung washing samples To extend the investigation of mucosal IgA to other sites, (CBA × BALB/c)F1 mice were immunised i.n. with M–LLO.FP as before and tears and lung washings collected
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Fig. 4. Antigen-specific IgA at distant mucosal sites over a 112-day period. (CBA × BALB/c)F1 mice were immunised on days 0, 28 and 56 with 12 g of M–LLO.FP, with 1 g of CT mixed with 12 g of LLO.FP or with 12 g of LLO.FP alone. Serum (a) and vaginal washings (b) were collected from four individual mice on days 31, 74 and 112. Antigen-specific IgA titres were determined by ELISA and the progress in individual mice shown.
on days 24 and 35, respectively. Mice immunised with the M–LLO.FP conjugate produced significantly higher titres of IgA (P < 0.01) in tears when compared to the unconjugated controls (geometric mean titres: M–LLO.FP = log10 2.01 ± 0.29 compared to log10 1.40 ± 0.00 for LLO.FP alone) (Fig. 5a). The same trend was observed in the lungs on day 35 (Fig. 5b). Significantly higher titres of IgA were detected in mice immunised with M–LLO.FP (geometric mean titre = log10 2.70 ± 0.59) compared to unconjugated LLO.FP (geometric mean titre = log10 1.78 ± 0.45) control (P < 0.05).
In order to assess the effectiveness of the mucosal adjuvant in terms of its ability to enhance the amount of antigen-specific IgA, as well as to compensate for variations in collection volumes of samples and dilutions, experiments were carried out to determine antigen-specific IgA as a percentage of total IgA (Table 1). Immunisation with M–LLO.FP resulted in an increase in total IgA secretion when compared to the unconjugated control (P < 0.01), as well as an increase in antigen-specific IgA, rising from undetectable levels to 32.2 ± 19.6 ng per well when compared to LLO.FP alone and unimmunised controls.
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Fig. 5. Antigen-specific IgA in tears and lung washings. (CBA × BALB/c)F1 mice were immunised on days 0, 10 and 17 with 12 g of M–LLO.FP or with 12 g of LLO.FP alone. Mice were anaesthetised on day 24 of the experiment and tears (1 l) collected from five mice per group (a). Mice were euthanised on day 35 of the experiment and lung washings (0.5 ml) collected (b). Antigen-specific IgA titres were determined by ELISA and individual titres are shown as dot plots. Differences between groups receiving M–LLO.FP or unconjugated LLO.FP were significant (P < 0.01) in tear samples and (P < 0.05) for lung washings.
Table 1 Antigen-specific IgA as a percentage of total IgA Groupa M–LLO.FP LLO.FP Normal
Captured IgA (ng per well)b 371 ± 204d 51.0 ± 17.9 48.4 ± 5.03
Antigen-specific IgA (ng per well)b
Percentage-specific IgAl total IgAc
32.2 ± 19.6 NDe ND
11.7 ± 7.70 ND ND
(CBA × BALB/c)F1 mice were immunised intranasally on days 0, 10 and 17 with 12 g of M–LLO.FP or with 12 g of LLO.FP alone. Mice were euthanised on day 35 of the experiment and lung washings (0.5 ml) collected. b Total IgA was detected using a capture ELISA. Antigen-specific IgA was determined by a standard ELISA. The concentration of IgA was derived from a standard curve and the values from individual mice used to calculate means and standard deviations. c Antigen-specific IgA as a percentage of total IgA was calculated for individual mice and their values were then used to calculate a mean and standard deviation. d Significantly different from LLO.FP and unimmunised mice, P < 0.01. e ND: not detected. a
3.4. Influence of conjugation of mannan to antigen on IgA induction To investigate whether there was a need for the mannan to be conjugated to LLO.FP or whether the adjuvant effect could be achieved when mannan was simply mixed with antigen, mice were immunised with the M–LLO.FP conjugate or with a mannan + LLO.FP mixture (mannan and 12 g of LLO.FP mixed together). (CBA × BALB/c)F1 mice were immunised i.n. on days 0, 10 and 17. Serum and vaginal washings were taken from individual mice on day 24 of the experiment. IgA titres were subsequently determined by ELISA (Table 2). The results clearly showed that a conjugate was required for adjuvant effect. The geometric mean antibody titre in the serum for conjugated LLO.FP was log10 3.00 ± 0.19. This was significantly higher than the mannan + LLO.FP mixture which produced an average titre of log10 1.53 ± 0.49. A similar pattern was seen
Table 2 Requirement for conjugation of mannan and LLO.FP for IgA production Antigen/adjuvanta
M–LLO.FP (linked) M + LLO.FP (mixed) LLO.FP Mannan Normal
Log10 IgA antibody titre Serumb
Vaginal washingsb
3.00 ± 0.19c 1.53 ± 0.49 1.15 ± 0.30 <1 <1
2.29 1.65 1.15 1.12 1.08
± ± ± ± ±
0.70c 0.48 0.11 0.15 0.10
a (CBA × BALB/c)F1 mice were immunised intranasally with M–LLO.FP, LLO.FP, mannan mixed with LLO.FP (unconjugated) or mannan alone on days 0, 10 and 17. Serum samples and vaginal washings obtained on day 24 from four individual mice. b Individual IgA titres were determined by ELISA. Antibody titres were converted to log10 and geometric means and standard deviations were derived. c Significantly different from mannan + LLO.FP (mixed), P < 0.05.
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in the antibody response at a distant mucosal site, namely the vagina, with titres of log10 2.29 ± 0.70 for the conjugate and log 1.65 ± 0.48 for the mixture. All negative controls produced undetectable levels of IgA with small titres of antibody observed in vaginal washings for the LLO.FP alone group (log10 1.15 ± 0.11).
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Earlier experiments with mannan-conjugated proteins injected i.p. showed that conjugates produced under reduced conditions were better at inducing antibody than were oxidised conjugates [9]. Therefore, (CBA × BALB/c)F1 mice were immunised i.n. with M–LLO.FP conjugate (oxidised form) or M–LLO.FP conjugate (reduced form). The results indicated that the oxidised form of the M–LLO.FP conjugate given i.n. induced higher titres of IgA, IgG1 and IgG2a in the serum (Fig. 6a, b and c) compared with the reduced form. All four mice immunised with M–LLO.FP (oxidised form) had higher titres than the corresponding four mice given the reduced form of M–LLO.FP, with the exception of one high responder mouse in the group given reduced mannan. Significant differences (P < 0.05) applied to all the subclasses assayed (geometric mean titres: IgA = log10 3.15 ± 0.30 for oxidised, log10 2.46±0.39 for reduced; IgG1 = log10 3.52± 0.15 for oxidised, log10 2.90 ± 0.43 for reduced; IgG2a = log10 3.22±0.25 for oxidised, log10 2.25±0.51 for reduced). 3.5. Use of oxidised mannan as adjuvant for other antigens To demonstrate that the use of mannan as a mucosal adjuvant was applicable to other antigens, oxidised mannan was conjugated to the 19 kDa protein secreted by M. tuberculosis. C57BL/10 mice were immunised i.n. with 12 g of M–19.FP conjugate on days 0, 10 and 17. On day 24 of the experiment the mice were euthanised and bled by cardiac puncture. Serum was separated and IgA titres determined by ELISA (Fig. 7). The four mice immunised with M–19.FP produced significantly higher titres of IgA (geometric mean log10 3.19 ± 0.36) when compared with mice
Fig. 6. Requirement for the M–LLO.FP conjugate to be in the oxidised form. (CBA×BALB/c)F1 mice were immunised intranasally with 12 g of M–LLO.FP in the oxidised or reduced form on days 0, 10 and 17. Serum from five individual mice was collected on day 24 and antigen-specific IgA (a), IgG1 (b) and IgG2a (c) was detected by ELISA. Individual titres are shown as dot plots, with corresponding symbols for different antibody classes from the same mouse. Differences between groups receiving oxidised or reduced M–LLO.FP were significant (P < 0.05) for all classes of antibody.
Fig. 7. Use of oxidised mannan as adjuvant for mycobacterial 19 kDa. FP. Groups of four C57BL/10 mice were immunised intranasally with 12 g of M-19.FP or 12 g of 19 kDa.FP on days 0, 10 and 17. Serum was collected on day 24 and antigen-specific IgA determined by ELISA. Individual titres are shown as dot plots. Differences between groups receiving M-19.FP and 19 kDa.FP alone were significant (P < 0.05).
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given 12 g of 19 kDa.FP (mean titres of log10 2.83 ± 0.31) (P < 0.05). 4. Discussion These experiments present oxidised mannan as a novel mucosal adjuvant which, when administered intranasally, induced high mucosal and serum titres of IgA, IgG1 and IgG2a specific for recombinant protein antigens with which it was administered. Oxidised mannan was previously used conjugated to the breast cancer antigen mucin 1 and injected i.p. into mice where it induced CTL and Th1 cytokines, protecting against tumours expressing mucin 1 [9,10]. In that context it induced a poor antibody response, dominated by IgG2a. It was the change to intranasal administration in the current experiments which resulted in production of IgA and other classes of antibody in serum and mucosal secretions. Mucosal antibodies and particularly IgA, have been shown to protect against influenza in the lung [21], against Helicobacter pylori in the stomach [22], against Haemophilis influenzae in the ear [14] and against Chlamydia trachomatis in the genital tract [23] amongst other examples. Induction of mucosal antibodies following intramuscular or subcutaneous immunisation with conventional vaccines is poor [16,24]. Therefore, oxidised mannan may be a valuable adjuvant if coupled to protective antigens from a wide variety of infectious agents. Induction of mucosal immunity begins with the uptake of antigen by membranous (M) cells (specialised epithelial cells) on the mucosal surface. These cells either process and present antigen to underlying T cells or B cells themselves or transport antigen to parenchymal macrophages, dendritic cells and B cells. Once interaction of the antigen presenting cell (APC) with T and B lymphocytes has occurred, an immune response or mucosal tolerance may result. Immune responses generally involve antibody production, with IgA the predominant antibody isotype. Antigen sensitised immune cells are then circulated to other systemic and mucosal sites for expansion of effector mechanisms [25]. The fact that there is a preferential circulation of T cells activated at mucosal sites to return to the same or other mucosal sites accounts for the induction of IgA at remote mucosal surfaces. Our results support these general observations. Cholera toxin has become the most extensively studied mucosal adjuvant in experimental models, although its relative toxicity has so far precluded it from approval for use as a human adjuvant [26]. In the present experiments, immunisation with mannan-conjugated LLO intranasally-induced superior IgA and IgG2a responses in the mucosa and serum compared with CT plus LLO or LLO alone. The difference in the IgG1 response was less marked, but still higher for the mannan adjuvant. Titres of antibody following immunisation with mannan conjugates were not only higher than
with CT, they were more sustained. Mannan has the further advantage over CT in being non-toxic and has been extensively tested in human trials of cancer immunotherapy [11]. As with the present observations on mannan, a mixture of the B subunit of CT (CTB) with antigen resulted in higher titres of IgA in the serum and mucosa when given to mice intranasally rather than intraperitoneally [16]. It is widely believed that this influence of the route of exposure is a function of the mucosal antigen presenting cells. Two interesting observations were made when analysing the form of the antigen/adjuvant in the present experiments. The first showed that oxidised mannan must be conjugated to the antigen (LLO.FP or 19 kDa FP) in order to facilitate the adjuvant effect. This confirms the observations with the mucin 1 studies where conjugation of oxidised mannan to the mucin 1 antigen was necessary in order to induce better immune responses [9]. The second observation contrasts with the mucin 1 studies because the oxidised form of the M–LLO.FP was required for maximum antibody production. Intraperitoneal immunisation with the oxidised form of mannan–mucin 1 resulted in low antibody levels [9]. Immunisation with oxidised mannan not only increased the amount of total IgA secreted as observed in the lung washings but also increased the amount of specific IgA secreted at this mucosal site. This type of response to immunisation with a mucosal adjuvant has also been observed in other systems. Intranasal delivery of AgI/II of Streptococcus mutans with the B subunit of cholera toxin resulted in an increase in both the total and antigen-specific salivary IgA response [27]. The ability of oxidised mannan to create such increases justifies further work in this area concentrating specifically on mucosal infection models. The fact that this effect persists for as long as 18 days after the last immunisation with M–LLO.FP suggests that this is not an acute inflammatory effect. How, then, does mannan act as an adjuvant? It has been shown that antigens bearing mannose residues are bound to the mannose receptors on APC, facilitating uptake into the MHC class II pathway for efficient antigen presentation [28–30]. Oxidised mannan–mucin 1 induced both class I restricted CTL and a Th1 response [10]. It has been suggested that oxidised mannan–mucin 1, by virtue of the aldehyde residues created by the oxidation process, escapes the phagocytic pathway and is transported into the MHC class I pathway inducing CTL [31]. This pathway would not be open to reduced complexes, where aldehyde residues are reduced to alcohols by the action of borohydride. The exact mechanism by which aldehyde acts is unknown. Curiously, we did not detect the induction of class I-restricted CTL in experiments with M–LLO.FP injected i.p. (Stambas et al., unpublished data). Furthermore, the cytokine response to our antigens given intranasally was not classically either Th1 or Th2, since IgG1 and IgA (generally considered Th2 responses) and IgG2a (Th1) were all elevated. This was confirmed by cytokine assays where both IFN-␥ and IL-4 were elevated (Stambas et al., unpublished data).
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The mechanism by which M–LLO.FP and M–19.FP induce such excellent antibody responses after i.n. immunisation is not presently known. Others have shown preferential induction of Th2 responses following intranasal instillation of leishmania antigens, even in strains of mouse which are genetically constrained to produce a typical Th1 response when these organisms are injected parenterally [32]. Perhaps alveolar macrophages or dendritic cells, which have been shown to present antigens efficiently in other systems [33], are playing a significant role in the induction of these IgA responses. Further studies following the gross and intracellular location of antigen will be required in order to determine the pathway of antigen presentation. Intranasal administration of oxidised mannan conjugates may advance development of human vaccines for sexually transmitted diseases such as Chlamydia or human immunodeficiency virus, other mucosally acquired infections like influenza, rota virus and trachoma, as well as for veterinary applications. The fact that oxidised mannan is a novel, non-toxic adjuvant that induces systemic and mucosal antibody responses superior to the standard mucosal adjuvant CT, warrants further investigation. References [1] Williams RC, Gibbons RJ. Inhibition of bacterial adherence by secretory immunoglobulin A: a mechanism of antigen disposal. Science 1972;177:697–9. [2] Hajishengallis G, Nikolova E, Russell MW. Inhibition of Streptococcus mutans adherence to saliva-coated hydroxyapatite by human secretory immunoglobulin A (S-IgA) antibodies to cell surface protein antigen I/II: reversal by IgA1 protease cleavage. Infect Immunol 1992;60:5057–64. [3] Gilbert JV, Plaut AG, Longmaid B, Lamm ME. Inhibition of microbial IgA proteases by human secretory IgA and serum. Mol Immunol 1983;20:1039–49. [4] Outlaw MC, Dimmock NJ. Mechanisms of neutralization of influenza virus on mouse tracheal epithelial cells by mouse monoclonal polymeric IgA and polyclonal IgM directed against the viral haemagglutinin. J Gen Virol 1990;71:69–76. [5] Chatfield S, Roberts M, Londono P, Cropley I, Douce G, Dougan G. The development of oral vaccines based on live attenuated Salmonella strains. FEMS Immunol Med Microbiol 1993;7:1–7. [6] Ulmer JB, Donnelly JJ, Parker SE, Rhodes GH, Felgner PL, Dwarki VJ, et al. Heterologous protection against influenza by injection of DNA encoding a viral protein. Science 1993;259:1745–9. [7] Elson CO, Ealding W. Generalized systemic and mucosal immunity in mice after mucosal stimulation with cholera toxin. J Immunol 1984;132:2736–41. [8] de Haan L, Verweij W, Agsteribbe E, Wilschut J. The role of ADP-ribosylation and G(M1)-binding activity in the mucosal immunogenicity and adjuvanticity of the Escherichia coli heat-labile enterotoxin and Vibrio cholerae cholera toxin. Immunol Cell Biol 1998;76:270–9. [9] Apostolopoulos V, Pietersz GA, Loveland BE, Sandrin MS, McKenzie IF. Oxidative/reductive conjugation of mannan to antigen selects for T1 or T2 immune responses. Proc Natl Acad Sci USA 1995;92:10128–32. [10] Apostolopoulos V, Loveland BE, Pietersz GA, McKenzie IF. CTL in mice immunized with human mucin 1 are MHC-restricted. J Immunol 1995;155:5089–94.
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[11] Karanikas V, Hwang LA, Pearson J, Ong CS, Apostolopoulos V, Vaughan H, et al. Antibody and T cell responses of patients with adenocarcinoma immunized with mannan–mucin 1 fusion protein. J Clin Invest 1997;100:2783–92. [12] Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione-Stransferase. Gene 1988;67:31–40. [13] Martin M, Metzger DJ, Michalek SM, Connell TD, Russell MW. Comparative analysis of the mucosal adjuvanticity of the type II heat-labile enterotoxins LT-IIa and LT-IIb. Infect Immunol 2000;68:281–7. [14] Kodama S, Suenaga S, Hirano T, Suzuki M, Mogi G. Induction of specific immunoglobulin A and Th2 immune responses to P6 outer membrane protein of nontypeable Haemophilus influenzae in middle ear mucosa by intranasal immunization. Infect Immunol 2000;68:2294–300. [15] Preston MJ, Kernacki KA, Berk JM, Hazlett LD, Berk RS. Kinetics of serum, tear, and corneal antibody responses in resistant and susceptible mice intracorneally infected with Pseudomonas aeruginosa. Infect Immunol 1992;60:885–91. [16] Hirabayashi Y, Kurata H, Funato H, Nagamine T, Aizawa C, Tamura S, et al. Comparison of intranasal inoculation of influenza HA vaccine combined with cholera toxin B subunit with oral or parenteral vaccination. Vaccine 1990;8:243–8. [17] Falero-Diaz G, Challacombe S, Banerjee D, Douce G, Boyd A, Ivanyi J. Intranasal vaccination of mice against infection with Mycobacterium tuberculosis. Vaccine 2000;18:3223–9. [18] Sabirov A, Kodama S, Hirano T, Suzuki M, Mogi G. Intranasal immunization enhances clearance of nontypeable Haemophilus influenzae and reduces stimulation of tumor necrosis factor alpha production in the murine model of otitis media. Infect Immunol 2001;69:2964–71. [19] Vadolas J, Davies JK, Wright PJ, Strugnell RA. Intranasal immunization with liposomes induces strong mucosal immune responses in mice. Eur J Immunol 1995;25:969–75. [20] Pietersz GA, Li W, Popovski V, Caruana JA, Apostolopoulos V, McKenzie IF. Parameters for using mannan–mucin 1 fusion protein to induce cellular immunity. Cancer Immunol Immunotherap 1998;45:321–6. [21] Tamura S, Funato H, Hirabayashi Y, Suzuki Y, Nagamine T, Aizawa C, et al. Cross-protection against influenza A virus infection by passively transferred respiratory tract IgA antibodies to different hemagglutinin molecules. Eur J Immunol 1991;21:1337–44. [22] Goto T, Nishizono A, Fujioka T, Ikewaki J, Mifune K, Nasu M. Local secretory immunoglobulin A and postimmunization gastritis correlate with protection against Helicobacter pylori infection after oral vaccination of mice. Infect Immunol 1999;67:2531–9. [23] Su H, Feilzer K, Caldwell HD, Morrison RP. Chlamydia trachomatis genital tract infection of antibody-deficient gene knockout mice. Infect Immunol 1997;65:1993–9. [24] Muszkat M, Yehuda AB, Schein MH, Friedlander Y, Naveh P, Greenbaum E, et al. Local and systemic immune response in community-dwelling elderly after intranasal or intramuscular immunization with inactivated influenza vaccine. J Med Virol 2000;61:100–6. [25] Mestecky J. The common mucosal immune system and current strategies for induction of immune responses in external secretions. J Clin Immunol 1987;7:265–76. [26] Levine MM. Escherichia coli that cause diarrhea: enterotoxigenic, enteropathogenic, enteroinvasive, enterohemorrhagic, and enteroadherent. J Infect Dis 1987;155:377–89. [27] Wu HY, Russell MW. Induction of mucosal immunity by intranasal application of a streptococcal surface protein antigen with the cholera toxin B subunit. Infect Immunol 1993;61:314–22. [28] Sallusto F, Cella M, Danieli C, Lanzavecchia A. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II
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compartment: downregulation by cytokines and bacterial products. J Exp Med 1995;182:389–400. [29] Tan MC, Mommaas AM, Drijfhout JW, Jordens R, Onderwater JJ, Verwoerd D, et al. Mannose receptor-mediated uptake of antigens strongly enhances HLA class II-restricted antigen presentation by cultured dendritic cells. Eur J Immunol 1997;27: 2426–35. [30] Engering AJ, Cella M, Fluitsma D, Brockhaus M, Hoefsmit EC, Lanzavecchia A, et al. The mannose receptor functions as a high capacity and broad specificity antigen receptor in human dendritic cells. Eur J Immunol 1997;27:2417–25.
[31] Apostolopoulos V, Pietersz GA, Gordon S, Martinez-Pomares L, McKenzie IF. Aldehyde–mannan antigen complexes target the MHC class I antigen-presentation pathway. Eur J Immunol 2000;30:1714– 23. [32] Constant SL, Lee KS, Bottomly K. Site of antigen delivery can influence T cell priming: pulmonary environment promotes preferential Th2-type differentiation. Eur J Immunol 2000;30:840–7. [33] Hamilton-Easton A, Eichelberger M. Virus-specific antigen presentation by different subsets of cells from lung and mediastinal lymph node tissues of influenza virus infected mice. J Virol 1995;69:6359–66.