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A therapeutic vaccine for mucosal candidiasis
Vaccine 19 (2001) 2516– 2521 www.elsevier.com/locate/vaccine
A therapeutic vaccine for mucosal candidiasis Shokrollah Elahi a, Robert Clancy a,b,*, Gerald Pang b a
Discipline of Immunology and Microbiology, Faculty of Medicine and Health Sciences, Uni6ersity of Newcastle, Newcastle, NSW 2308, Australia b The Vasse Research Institute, Le6el 4, Da6id Maddison Clinical Sciences Building, Royal Newcastle Hospital, Cnr King and Watt Streets, Newcastle, NSW 2300, Australia
Abstract Persistent and recurrent infection of mucosal surfaces with Candida albicans is common, ranging from a nuisance to a life threatening clinical problem. No effective prophylactic or therapeutic vaccine has been developed. We have studied a mouse model of oral candida infection to identify regulatory and effector molecules of T cell activation as parameters of induced immunity, and here describe the use of this model to determine an optimal immunisation strategy. Oral immunisation with the blastospore yeast form (but not subcutaneous immunisation) induced clinical immunity, with a shift in parameters of cytokine response characterised by an early and sustained production of both IFN-g and IL-4 from antigen-stimulated cervical node T lymphocytes. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Candida; Oral; Vaccine
1. Introduction Infection of mucosal surfaces by Candida albicans in man is a common and distressing condition. Most commonly infection involves the oral and vaginal mucosa, though involvement of other mucosal surfaces are well recognised. Oral infection occurs in about two thirds of subjects with permanent dental prosthesis [1]. Recurrent vulvovaginal candidiasis (RVVC) affects 3– 5% of women of reproductive age [2]. Oropharyngeal candidiasis is common in immune suppressed individuals, and is of particular concern as it predisposes to systemic infection. Subjects with impaired T lymphocyte function, including those with HIV infection, are particularly prone to oral infection with C. albicans. Women with RVVC have a reduced pool of circulating Th1 CD4 + T cells, with subsequent impairment of the hormonally-dependent movement of antigen-specific T cells into the reproductive tract mucosa [2]. Thus, the common denominator to establishing a proneness to
* Corresponding author. Tel.: +61-249-236135; fax: + 61-249236998. E-mail address: [email protected] (R. Clancy).
mucosal infection with C. albicans is a reduction in specific T lymphocyte activity. Logically, any attempt to induce immunity through immunisation must sit within a framework defined by a knowledge of protective mechanisms. Thus, a murine model, consisting of ‘infection-resistant’ (BALB/c) and ‘infection-prone’ (DBA/2) mice, has been studied to define protective mechanisms at the level of T cell produced cytokines. Using this model, it has been shown that the cervical node T cell response, which correlates best with rapid oral clearance of C. albicans, is a balanced Tho cytokine response involving early secretion of both IFN-g and IL-4 [3]. Study of reinfection in this model, showed diminished intensity and duration of infection within the oral cavity, which correlated with an earlier appearance of both IFN-g and IL-4 when compared to a primary infection (unpublished observations). Thus, preliminary studies defined parameters of infection and the cytokine response, which could be used to monitor outcome of immunisation strategies. The current study builds on the database obtained from the infection model which utilised ‘infection-resistant’ (BALB/c) and ‘infectionprone’ (DBA/2) mice, to determine whether an effective vaccine could be developed to reduce intensity and duration of C. albicans infection within the oral cavity.
0264-410X/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 0 ) 0 0 4 8 2 - 5
S. Elahi et al. / Vaccine 19 (2001) 2516–2521
2. Materials and methods
2.1. Mice Male BALB/c (H-2d) and DBA/2 mice (H-2d), 6–8 weeks old were purchased from the Animal Resource Centre, Perth, WA. They were housed in groups of three to five and provided with food and water ad libitum. All mice were used after 1 week of acclimatisation.
2.2. Fungal culture C. albicans isolate 3630 was obtained from the National Reference Laboratory, Royal North Shore Hospital, Sydney, Australia. The yeast cells were cultured in sabouraud dextrose broth (Oxoid, Hampshire, UK) for 48 h at 25°C in a shaking water bath. The blastospores were transferred into fresh medium and cultured at 25°C for a further 18 h. The blastospores were collected by centrifugation, washed twice with phosphatebuffered saline (PBS) and then adjusted to 108 blastospores per ml in PBS until use.
2.3. Oral infection Mice were anaesthetised by intraperitoneal injection with 75 ml of ketamine:xylazil (100:20 mg/ml). They were orally inoculated with the blastospores according to the method described by Chakir et al. [4]. Briefly, 108/ml of blastospores in PBS were centrifuged at 14 000×g for 5 min. The pellet was recovered on a fine-tip sterile swab (Corsham, Wiltshire, UK) which was then used for oral inoculation by topical application.
2.4. Quantitation of oral infection Groups of mice (3 – 5 per group) were sacrificed at various time points to determine the number of C. albicans in the oral mucosa. The oral cavity (i.e. cheek, tongue and soft palate) was completely swabbed using a fine-tipped cotton swab. After swabbing, the cotton end was cut off and then placed in an eppendorf tube containing 1 ml PBS. The yeast cells were resuspended by mixing on a vortex mixer before culture in serial tenfold dilutions on sabouraud dextrose agar (Oxoid, UK) supplemented with chloramphenicol (0.05 g/l) for 48 h at 37°C.
2.5. Cytokine assay CLN cells in RPMI 1640 medium supplemented with 10% FCS were cultured at 4×106 cells per well in the presence of 2.5 mg/ml of C. albicans antigen in a 24-well plate for 3 days (as described above). The culture
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supernatants were collected and then assayed for IL-4, IL-12 and IFN-g by ELISA using matched-antibody pairs and recombinant cytokines as standards (Pharmingen, San Diego, CA). Briefly, immunopolysorb microtitre plates (Nunc, Denmark) were coated with capture rat monoclonal anti-IL-4 (IgG1), IL-12 (IgG2a) or IFN-g (IgG1) antibody at 1 mg/ml in sodium bicarbonate buffer (pH 9.6) overnight at 4°C. The wells were washed and then blocked with 1% BSA before the culture supernatants and the appropriate standard were added to each well. Biotinylated rat monoclonal anti-IL-4, IL-12 or IFN-g antibody at 2 mg/ml was added as the second antibody. Detection was done with streptavidin peroxidase (AMRAD, Melbourne, Australia) and TMB (Sigma-Aldrich). The sensitivity of ELISA were 31 pg/ml for IFN-g in the culture supernatant, 5 pg/ml in saliva and 10 pg/ml for IL-4.
2.6. Immunisation schedule Formalin-fixed C. albicans vaccine was prepared by fixing C. albicans in 4% paraformaldehyde solution for 2 h at room temperature after which time the cells were washed several times with PBS by centrifugation at 4°C. After the final wash, the yeasts were resuspended in PBS at 1× 109 cells per ml and stored at 40°C until use. Heat-killed vaccine was prepared by resuspending C. albicans in sterile PBS and then autoclave at 121°C for 30 min after which time the suspension was cooled to room temperature. After washing in PBS, the yeasts were resuspended at 5× 108 per ml and then stored at 4°C until use. The hyphae form was prepared by incubating C. albicans in foetal calf serum at 37°C for 7 days. The vaccine was prepared by washing the hyphae form with PBS and then inactivated by autoclaving as described above. DBA/2 mice were immunised by subcutaneous injection of 1× 106 C. albicans in PBS and then boosted with a similar dose by the same route 14 days later before challenge. Oral immunisation was carried by intragastric feeding with 2× 108 C. albicans blastospores or hyphae in 0.2 ml PBS every 2 days for 20 days before challenge.
3. Results
3.1. Pattern of C. albicans colonisation with oral infection in DBA/2 mice immunised s/cut with C. albicans No significant reduction of colonisation could be detected between control and immunised mice until day 10 (Fig. 1).
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3.2. Pattern of C. albicans colonisation with oral infection in DBA/2 mice immunised orally with C. albicans blastospores Significant differences in colonisation were detected from day 2 following oral infection (Fig. 2).
3.3. Pattern of C. albicans colonisation with oral infection in DBA/2 mice immunised orally with C. albicans hyphae Significant differences in colonisation were detected from day 6 following oral infection (Fig. 3).
3.4. Cytokine patterns from stimulated lymph node cells With respect to IFN-g levels, higher, more stable, and more protracted levels were detected in DBA/2 mice when stimulated with blastospores compared with hyphae (standardised at the same dose using total protein content) (Figs. 4 – 7). With respect to IL-4, in hyphae-immunised mice, no difference from unimmunised controls could be detected until day 8. At days 8 and 10, significantly higher levels of IL-4 were in fact detected in control mice. Following immunisation with blastospores, an increasing, higher and more sustained level of IL-4 was noted, reaching statistical significance from day 1 to day 10.
Fig. 1. Patterns of colonisation with C. albicans in DBA/2 mice following subcutaneous immunisation with formalin-fixed C. albicans. DBA/2 mice were immunised by subcutaneous injection of formalinfixed C. albicans (1 ×106 in 0.2 ml PBS). After 2 weeks mice were given a booster injection of fixed C. albicans and then followed 2 days later with a challenge by topical application of live C. albicans (1 ×108) to the oral mucosa. At various times after challenge, mice were killed and the level of colonisation assessed by swabbing the oral cavity. Results shown are the means 9 S.E.M. for five mice. **PB 0.01 denotes significant difference compared with values from control mice.
Fig. 2. Patterns of colonisation with C. albicans in DBA/2 mice following oral immunisation with C. albicans blastospores. DBA/2 mice were immunised orally with heat-killed C. albicans blastospores (2 ×108 in 0.2 ml PBS) every other day for 20 days. Following the last dose, mice were challenged with live C. albicans. At various times after challenge, mice were killed and the level of colonisation was assessed by swabbing the oral cavity. Data shown are the means 9 S.E.M. for five mice. *P B 0.05; **PB0.000l compared with values from control mice.
3.5. IFN-k in sali6a Following blastospore immunisation (oral) significant levels of IFN-g could be detected in saliva prior to oral infection. A sharp increase in levels immediately followed challenge with C. albicans, with significantly
Fig. 3. Patterns of colonisation with C. albicans in DBA/2 mice following oral immunisation with hyphae form. DBA/2 mice were immunised orally with C. albicans hyphae form every 2 days for 20 days. Following the last dose, mice were challenged with C. albicans. At various times indicated, the level of colonisation was assessed by swabbing the oral cavity. Data shown are the means 9 S.E.M. for five mice. *P B 0.05, **PB 0.01 compared with values from control mice.
S. Elahi et al. / Vaccine 19 (2001) 2516–2521
Fig. 4. IFN-g production in culture of cervical lymph node cells stimulated with C. albicans from mice orally immunised with heatkilled C. albicans. Cervical lymph node cells (5 ×106 cells per ml) obtained from DBA/2 mice at various times after oral immunisation were stimulated with C. albicans antigens for 3 days after which the culture supernatants were assessed for IFN-g production by ELISA. Data shown are the means 9 S.E.M. for five mice. *PB 0.05, **PB 0.0001 compared with values from control mice.
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Fig. 6. IL-4 production by CLN cells stimulated with C. albicans from mice after oral immunisation with C. albicans blastospores. CLN cells obtained at various times from control (open square) and immunised (filled square) DBA/2 mice were stimulated with C. albicans antigens for 3 days after which time the culture supernatants were assessed for IL-4 production by ELISA. Data shown are the means9S.E.M. for five mice. *PB 0.05; **PB 0.0001 compared with values from control mice.
4. Discussion
lower levels in the non-immunised control group (Fig. 8). In the early period following infection (the first 4 days) when the patterns of colonisation differed between those unimmunised with blastospores and hyphae, particular differences are noted in cytokine levels between the immunisation groups.
This study demonstrates the value of infection and cytokine markers in assessing outcome, the selective advantage of blastospores over hyphal forms, and the advantage of oral immunisation over systemic immunisation as the route of administration. Study of a model of ‘infection-prone’ (DBA/2) and ‘infection-resistant’ (BALB/c) strains of mice defined an early and sustained secretion of both IFN-g and IL-4 as the cytokine pattern that best defined resistance to oral infection with C. albicans [3]. These observations were reinforced by studies of reinfection (unpublished obser-
Fig. 5. IFN-g production by cervical lymph node cells stimulated with C. albicans from mice after oral immunisation with C. albicans hyphae form. CLN cells obtained at various times from control and immunised DBA/2 mice were stimulated with C. albicans antigens after which the culture supernatants were assessed for IFN-g production by ELISA. Data shown are the means 9 S.E.M. for five mice. *PB 0.05 compared with values from control mice.
Fig. 7. IL-4 production by CLN cells stimulated with C. albicans from mice orally immunised with C. albicans hyphae form. CLN cells obtained at various times from control and immunised DBA/2 mice were stimulated with C. albicans antigens for 3 days after which the culture supernatants were assessed for IL-4 production by ELISA. Results shown are the means 9S.E.M. for five mice. *PB 0.05 compared with values from control mice.
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Fig. 8. IFN-g production in saliva from mice after oral immunisation with C. albicans blastospores. Saliva collected at various times from control and immunised DBA/2 mice were assessed for IFN-g by ELISA. Data shown are the means 9 S.E.M. for five mice. *PB 0.05, **PB 0.01 compared with values from control mice.
vation). This pattern was reproduced following successful oral immunisation with blastospores, but not with the hyphal form. Thus, the cytokine pattern that reflected optimal protection [3] is consistent with these studies. Additional markers identified in the dual mouse model as being valuable in monitoring protection are IFN-g and nitric oxide levels in saliva (unpublished observations). Again the appearance and profile of IFN-g in saliva following oral immunisation. These indices would be of particular value in the assessment of vaccine trials in man. The improved patterns of clearance and levels of IFN-g and IL-4 in those immunised with the blastospore form of C. albicans may reflect antigen handling differences by the gut-associated lymphoid tissue, or a different array of surface antigens. Blastospores approximate the optimal size for entry into the Peyer’s patch [5]. The difference between the two yeast forms was most marked in analysing cytokine production from antigen-stimulated cervical lymph node T cells, providing a mechanism for determining optimal formulation and dose of C. albicans antigens in this model. Systemic immunisation by subcutaneous injection of C. albicans failed to alter the clearance pattern of the microbe from the oral cavity, suggesting the major source of protective T cells is the gut associated lymphoid tissue. These observations are the first that suggest the oral cavity participates within the ‘common mucosal system’. An alternate explanation of these events, is that clearance of infection is largely mediated by effector molecules produced within salivary glands, organs known to participate within the common mucosal system. It is relevant, therefore, that studies in the dual mouse model (above) have shown levels of IFN-g
and nitric oxide in mixed saliva that correlate inversely with the level of oral mucosal colonisation, with an enhancement of colonisation occurring when nitric oxide production is blocked (unpublished observations). It is not clear at this stage, however, whether effector molecules came from cells within the oral mucosa or the salivary glands, though the demonstration of secretion of IFN-g from specific T cells within cervical lymph nodes favours a contribution from the oral mucosa. What potential exists for developing an oral vaccine for man? The successful vaccine experiments in the DBA/2 mouse model provide proof-of-concept to encourage studies in man, as well as providing information as to how to conduct the studies. The blastospore yeast form induces the best level of immunity. Of particular value is the recognition of parameters of immunity, likely to be present in human saliva (IFN-g and nitric oxide), as these molecules are easy to measure and enable a quick assessment in normal subjects of dose and formulation effectivity. The vaccine operates by activating specific T lymphocytes, with both local and systemic antibody appearing after the infection is resolved in the murine model [3]. This maybe a limiting factor in man, as many of the clinical circumstances where a vaccine would be desirable (e.g. in those with HIV disease and in immunosuppressed individuals) may respond poorly to a T cell-dependent vaccine. Response would likely be better in subjects with dental prostheses whose immune system is more intact. A group of particular interest would be women with RVVC who have a depleted T cell pool [2]. The oral vaccine could expand this pool and reverse the cytokine defect that underpins this disease. A further clinical question is whether an oral vaccine could act as a therapeutic vaccine for an established mucosal infection, as well as a prophylactic vaccine. This concept requires testing, but studies are encouraged by the success of oral vaccines containing killed non-typable H. influenzae in man, where a significant reduction of episodes of acute bronchitis follows oral immunisation of subjects with chronic lung disease who are colonised by this bacterium [6]. Studies in the recurrent bronchitis model suggested caution in the use of added adjuvants, given the downregulated nature of mucosal immune mechanisms [6]. These questions can be addressed by extending vaccine studies into man.
References [1] Webb BC, Thomas CJ, Willcox MDP, Harty DWS, Knox KW. Candida associated denture stomatitis. Aetiology and management: a review. Aust Dent J 1998;43(1):45– 50. [2] Corrigan EM, Clancy RL, Dunkley ML, Eyers FM, Beagley KW. Cellular immunity in recurrent vulvovaginal candidiasis. Clin Exp Immunol 1998;111:574– 8.
S. Elahi et al. / Vaccine 19 (2001) 2516–2521 [3] Elahi S, Pang G, Clancy R, Ashman RB. Cellular and cytokine correlates of mucosal protection in a murine model of oral candidiasis. Infect Immun 2000;68:5771–7. [4] Chakir J, Cote L, Coulombe C, Delasriers N. Differential pattern of infection and immune response during experimental oral candidiasis in BALB/c and DBA/2 (H-24) mice. Oral Microbiol Immunol 1994;9:88– 94.
.
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[5] McGhee JR, Czerkinsky C, Mestecky J. Mucosal vaccines: an overview. In: Ogra PL, Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR, editors. Mucosal Immunology, 2nd edn. San Diego: Academic Press, 1999:741– 57. [6] Clancy RL, Pang G, Dunkley M, Taylor D, Cripps A. Acute on chronic bronchitis: a model of mucosal immunology. Immunol Cell Biol 1995;73:414– 7.
A therapeutic vaccine for mucosal candidiasis Shokrollah Elahi a, Robert Clancy a,b,*, Gerald Pang b a
Discipline of Immunology and Microbiology, Faculty of Medicine and Health Sciences, Uni6ersity of Newcastle, Newcastle, NSW 2308, Australia b The Vasse Research Institute, Le6el 4, Da6id Maddison Clinical Sciences Building, Royal Newcastle Hospital, Cnr King and Watt Streets, Newcastle, NSW 2300, Australia
Abstract Persistent and recurrent infection of mucosal surfaces with Candida albicans is common, ranging from a nuisance to a life threatening clinical problem. No effective prophylactic or therapeutic vaccine has been developed. We have studied a mouse model of oral candida infection to identify regulatory and effector molecules of T cell activation as parameters of induced immunity, and here describe the use of this model to determine an optimal immunisation strategy. Oral immunisation with the blastospore yeast form (but not subcutaneous immunisation) induced clinical immunity, with a shift in parameters of cytokine response characterised by an early and sustained production of both IFN-g and IL-4 from antigen-stimulated cervical node T lymphocytes. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Candida; Oral; Vaccine
1. Introduction Infection of mucosal surfaces by Candida albicans in man is a common and distressing condition. Most commonly infection involves the oral and vaginal mucosa, though involvement of other mucosal surfaces are well recognised. Oral infection occurs in about two thirds of subjects with permanent dental prosthesis [1]. Recurrent vulvovaginal candidiasis (RVVC) affects 3– 5% of women of reproductive age [2]. Oropharyngeal candidiasis is common in immune suppressed individuals, and is of particular concern as it predisposes to systemic infection. Subjects with impaired T lymphocyte function, including those with HIV infection, are particularly prone to oral infection with C. albicans. Women with RVVC have a reduced pool of circulating Th1 CD4 + T cells, with subsequent impairment of the hormonally-dependent movement of antigen-specific T cells into the reproductive tract mucosa [2]. Thus, the common denominator to establishing a proneness to
* Corresponding author. Tel.: +61-249-236135; fax: + 61-249236998. E-mail address: [email protected] (R. Clancy).
mucosal infection with C. albicans is a reduction in specific T lymphocyte activity. Logically, any attempt to induce immunity through immunisation must sit within a framework defined by a knowledge of protective mechanisms. Thus, a murine model, consisting of ‘infection-resistant’ (BALB/c) and ‘infection-prone’ (DBA/2) mice, has been studied to define protective mechanisms at the level of T cell produced cytokines. Using this model, it has been shown that the cervical node T cell response, which correlates best with rapid oral clearance of C. albicans, is a balanced Tho cytokine response involving early secretion of both IFN-g and IL-4 [3]. Study of reinfection in this model, showed diminished intensity and duration of infection within the oral cavity, which correlated with an earlier appearance of both IFN-g and IL-4 when compared to a primary infection (unpublished observations). Thus, preliminary studies defined parameters of infection and the cytokine response, which could be used to monitor outcome of immunisation strategies. The current study builds on the database obtained from the infection model which utilised ‘infection-resistant’ (BALB/c) and ‘infectionprone’ (DBA/2) mice, to determine whether an effective vaccine could be developed to reduce intensity and duration of C. albicans infection within the oral cavity.
0264-410X/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 0 ) 0 0 4 8 2 - 5
S. Elahi et al. / Vaccine 19 (2001) 2516–2521
2. Materials and methods
2.1. Mice Male BALB/c (H-2d) and DBA/2 mice (H-2d), 6–8 weeks old were purchased from the Animal Resource Centre, Perth, WA. They were housed in groups of three to five and provided with food and water ad libitum. All mice were used after 1 week of acclimatisation.
2.2. Fungal culture C. albicans isolate 3630 was obtained from the National Reference Laboratory, Royal North Shore Hospital, Sydney, Australia. The yeast cells were cultured in sabouraud dextrose broth (Oxoid, Hampshire, UK) for 48 h at 25°C in a shaking water bath. The blastospores were transferred into fresh medium and cultured at 25°C for a further 18 h. The blastospores were collected by centrifugation, washed twice with phosphatebuffered saline (PBS) and then adjusted to 108 blastospores per ml in PBS until use.
2.3. Oral infection Mice were anaesthetised by intraperitoneal injection with 75 ml of ketamine:xylazil (100:20 mg/ml). They were orally inoculated with the blastospores according to the method described by Chakir et al. [4]. Briefly, 108/ml of blastospores in PBS were centrifuged at 14 000×g for 5 min. The pellet was recovered on a fine-tip sterile swab (Corsham, Wiltshire, UK) which was then used for oral inoculation by topical application.
2.4. Quantitation of oral infection Groups of mice (3 – 5 per group) were sacrificed at various time points to determine the number of C. albicans in the oral mucosa. The oral cavity (i.e. cheek, tongue and soft palate) was completely swabbed using a fine-tipped cotton swab. After swabbing, the cotton end was cut off and then placed in an eppendorf tube containing 1 ml PBS. The yeast cells were resuspended by mixing on a vortex mixer before culture in serial tenfold dilutions on sabouraud dextrose agar (Oxoid, UK) supplemented with chloramphenicol (0.05 g/l) for 48 h at 37°C.
2.5. Cytokine assay CLN cells in RPMI 1640 medium supplemented with 10% FCS were cultured at 4×106 cells per well in the presence of 2.5 mg/ml of C. albicans antigen in a 24-well plate for 3 days (as described above). The culture
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supernatants were collected and then assayed for IL-4, IL-12 and IFN-g by ELISA using matched-antibody pairs and recombinant cytokines as standards (Pharmingen, San Diego, CA). Briefly, immunopolysorb microtitre plates (Nunc, Denmark) were coated with capture rat monoclonal anti-IL-4 (IgG1), IL-12 (IgG2a) or IFN-g (IgG1) antibody at 1 mg/ml in sodium bicarbonate buffer (pH 9.6) overnight at 4°C. The wells were washed and then blocked with 1% BSA before the culture supernatants and the appropriate standard were added to each well. Biotinylated rat monoclonal anti-IL-4, IL-12 or IFN-g antibody at 2 mg/ml was added as the second antibody. Detection was done with streptavidin peroxidase (AMRAD, Melbourne, Australia) and TMB (Sigma-Aldrich). The sensitivity of ELISA were 31 pg/ml for IFN-g in the culture supernatant, 5 pg/ml in saliva and 10 pg/ml for IL-4.
2.6. Immunisation schedule Formalin-fixed C. albicans vaccine was prepared by fixing C. albicans in 4% paraformaldehyde solution for 2 h at room temperature after which time the cells were washed several times with PBS by centrifugation at 4°C. After the final wash, the yeasts were resuspended in PBS at 1× 109 cells per ml and stored at 40°C until use. Heat-killed vaccine was prepared by resuspending C. albicans in sterile PBS and then autoclave at 121°C for 30 min after which time the suspension was cooled to room temperature. After washing in PBS, the yeasts were resuspended at 5× 108 per ml and then stored at 4°C until use. The hyphae form was prepared by incubating C. albicans in foetal calf serum at 37°C for 7 days. The vaccine was prepared by washing the hyphae form with PBS and then inactivated by autoclaving as described above. DBA/2 mice were immunised by subcutaneous injection of 1× 106 C. albicans in PBS and then boosted with a similar dose by the same route 14 days later before challenge. Oral immunisation was carried by intragastric feeding with 2× 108 C. albicans blastospores or hyphae in 0.2 ml PBS every 2 days for 20 days before challenge.
3. Results
3.1. Pattern of C. albicans colonisation with oral infection in DBA/2 mice immunised s/cut with C. albicans No significant reduction of colonisation could be detected between control and immunised mice until day 10 (Fig. 1).
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3.2. Pattern of C. albicans colonisation with oral infection in DBA/2 mice immunised orally with C. albicans blastospores Significant differences in colonisation were detected from day 2 following oral infection (Fig. 2).
3.3. Pattern of C. albicans colonisation with oral infection in DBA/2 mice immunised orally with C. albicans hyphae Significant differences in colonisation were detected from day 6 following oral infection (Fig. 3).
3.4. Cytokine patterns from stimulated lymph node cells With respect to IFN-g levels, higher, more stable, and more protracted levels were detected in DBA/2 mice when stimulated with blastospores compared with hyphae (standardised at the same dose using total protein content) (Figs. 4 – 7). With respect to IL-4, in hyphae-immunised mice, no difference from unimmunised controls could be detected until day 8. At days 8 and 10, significantly higher levels of IL-4 were in fact detected in control mice. Following immunisation with blastospores, an increasing, higher and more sustained level of IL-4 was noted, reaching statistical significance from day 1 to day 10.
Fig. 1. Patterns of colonisation with C. albicans in DBA/2 mice following subcutaneous immunisation with formalin-fixed C. albicans. DBA/2 mice were immunised by subcutaneous injection of formalinfixed C. albicans (1 ×106 in 0.2 ml PBS). After 2 weeks mice were given a booster injection of fixed C. albicans and then followed 2 days later with a challenge by topical application of live C. albicans (1 ×108) to the oral mucosa. At various times after challenge, mice were killed and the level of colonisation assessed by swabbing the oral cavity. Results shown are the means 9 S.E.M. for five mice. **PB 0.01 denotes significant difference compared with values from control mice.
Fig. 2. Patterns of colonisation with C. albicans in DBA/2 mice following oral immunisation with C. albicans blastospores. DBA/2 mice were immunised orally with heat-killed C. albicans blastospores (2 ×108 in 0.2 ml PBS) every other day for 20 days. Following the last dose, mice were challenged with live C. albicans. At various times after challenge, mice were killed and the level of colonisation was assessed by swabbing the oral cavity. Data shown are the means 9 S.E.M. for five mice. *P B 0.05; **PB0.000l compared with values from control mice.
3.5. IFN-k in sali6a Following blastospore immunisation (oral) significant levels of IFN-g could be detected in saliva prior to oral infection. A sharp increase in levels immediately followed challenge with C. albicans, with significantly
Fig. 3. Patterns of colonisation with C. albicans in DBA/2 mice following oral immunisation with hyphae form. DBA/2 mice were immunised orally with C. albicans hyphae form every 2 days for 20 days. Following the last dose, mice were challenged with C. albicans. At various times indicated, the level of colonisation was assessed by swabbing the oral cavity. Data shown are the means 9 S.E.M. for five mice. *P B 0.05, **PB 0.01 compared with values from control mice.
S. Elahi et al. / Vaccine 19 (2001) 2516–2521
Fig. 4. IFN-g production in culture of cervical lymph node cells stimulated with C. albicans from mice orally immunised with heatkilled C. albicans. Cervical lymph node cells (5 ×106 cells per ml) obtained from DBA/2 mice at various times after oral immunisation were stimulated with C. albicans antigens for 3 days after which the culture supernatants were assessed for IFN-g production by ELISA. Data shown are the means 9 S.E.M. for five mice. *PB 0.05, **PB 0.0001 compared with values from control mice.
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Fig. 6. IL-4 production by CLN cells stimulated with C. albicans from mice after oral immunisation with C. albicans blastospores. CLN cells obtained at various times from control (open square) and immunised (filled square) DBA/2 mice were stimulated with C. albicans antigens for 3 days after which time the culture supernatants were assessed for IL-4 production by ELISA. Data shown are the means9S.E.M. for five mice. *PB 0.05; **PB 0.0001 compared with values from control mice.
4. Discussion
lower levels in the non-immunised control group (Fig. 8). In the early period following infection (the first 4 days) when the patterns of colonisation differed between those unimmunised with blastospores and hyphae, particular differences are noted in cytokine levels between the immunisation groups.
This study demonstrates the value of infection and cytokine markers in assessing outcome, the selective advantage of blastospores over hyphal forms, and the advantage of oral immunisation over systemic immunisation as the route of administration. Study of a model of ‘infection-prone’ (DBA/2) and ‘infection-resistant’ (BALB/c) strains of mice defined an early and sustained secretion of both IFN-g and IL-4 as the cytokine pattern that best defined resistance to oral infection with C. albicans [3]. These observations were reinforced by studies of reinfection (unpublished obser-
Fig. 5. IFN-g production by cervical lymph node cells stimulated with C. albicans from mice after oral immunisation with C. albicans hyphae form. CLN cells obtained at various times from control and immunised DBA/2 mice were stimulated with C. albicans antigens after which the culture supernatants were assessed for IFN-g production by ELISA. Data shown are the means 9 S.E.M. for five mice. *PB 0.05 compared with values from control mice.
Fig. 7. IL-4 production by CLN cells stimulated with C. albicans from mice orally immunised with C. albicans hyphae form. CLN cells obtained at various times from control and immunised DBA/2 mice were stimulated with C. albicans antigens for 3 days after which the culture supernatants were assessed for IL-4 production by ELISA. Results shown are the means 9S.E.M. for five mice. *PB 0.05 compared with values from control mice.
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Fig. 8. IFN-g production in saliva from mice after oral immunisation with C. albicans blastospores. Saliva collected at various times from control and immunised DBA/2 mice were assessed for IFN-g by ELISA. Data shown are the means 9 S.E.M. for five mice. *PB 0.05, **PB 0.01 compared with values from control mice.
vation). This pattern was reproduced following successful oral immunisation with blastospores, but not with the hyphal form. Thus, the cytokine pattern that reflected optimal protection [3] is consistent with these studies. Additional markers identified in the dual mouse model as being valuable in monitoring protection are IFN-g and nitric oxide levels in saliva (unpublished observations). Again the appearance and profile of IFN-g in saliva following oral immunisation. These indices would be of particular value in the assessment of vaccine trials in man. The improved patterns of clearance and levels of IFN-g and IL-4 in those immunised with the blastospore form of C. albicans may reflect antigen handling differences by the gut-associated lymphoid tissue, or a different array of surface antigens. Blastospores approximate the optimal size for entry into the Peyer’s patch [5]. The difference between the two yeast forms was most marked in analysing cytokine production from antigen-stimulated cervical lymph node T cells, providing a mechanism for determining optimal formulation and dose of C. albicans antigens in this model. Systemic immunisation by subcutaneous injection of C. albicans failed to alter the clearance pattern of the microbe from the oral cavity, suggesting the major source of protective T cells is the gut associated lymphoid tissue. These observations are the first that suggest the oral cavity participates within the ‘common mucosal system’. An alternate explanation of these events, is that clearance of infection is largely mediated by effector molecules produced within salivary glands, organs known to participate within the common mucosal system. It is relevant, therefore, that studies in the dual mouse model (above) have shown levels of IFN-g
and nitric oxide in mixed saliva that correlate inversely with the level of oral mucosal colonisation, with an enhancement of colonisation occurring when nitric oxide production is blocked (unpublished observations). It is not clear at this stage, however, whether effector molecules came from cells within the oral mucosa or the salivary glands, though the demonstration of secretion of IFN-g from specific T cells within cervical lymph nodes favours a contribution from the oral mucosa. What potential exists for developing an oral vaccine for man? The successful vaccine experiments in the DBA/2 mouse model provide proof-of-concept to encourage studies in man, as well as providing information as to how to conduct the studies. The blastospore yeast form induces the best level of immunity. Of particular value is the recognition of parameters of immunity, likely to be present in human saliva (IFN-g and nitric oxide), as these molecules are easy to measure and enable a quick assessment in normal subjects of dose and formulation effectivity. The vaccine operates by activating specific T lymphocytes, with both local and systemic antibody appearing after the infection is resolved in the murine model [3]. This maybe a limiting factor in man, as many of the clinical circumstances where a vaccine would be desirable (e.g. in those with HIV disease and in immunosuppressed individuals) may respond poorly to a T cell-dependent vaccine. Response would likely be better in subjects with dental prostheses whose immune system is more intact. A group of particular interest would be women with RVVC who have a depleted T cell pool [2]. The oral vaccine could expand this pool and reverse the cytokine defect that underpins this disease. A further clinical question is whether an oral vaccine could act as a therapeutic vaccine for an established mucosal infection, as well as a prophylactic vaccine. This concept requires testing, but studies are encouraged by the success of oral vaccines containing killed non-typable H. influenzae in man, where a significant reduction of episodes of acute bronchitis follows oral immunisation of subjects with chronic lung disease who are colonised by this bacterium [6]. Studies in the recurrent bronchitis model suggested caution in the use of added adjuvants, given the downregulated nature of mucosal immune mechanisms [6]. These questions can be addressed by extending vaccine studies into man.
References [1] Webb BC, Thomas CJ, Willcox MDP, Harty DWS, Knox KW. Candida associated denture stomatitis. Aetiology and management: a review. Aust Dent J 1998;43(1):45– 50. [2] Corrigan EM, Clancy RL, Dunkley ML, Eyers FM, Beagley KW. Cellular immunity in recurrent vulvovaginal candidiasis. Clin Exp Immunol 1998;111:574– 8.
S. Elahi et al. / Vaccine 19 (2001) 2516–2521 [3] Elahi S, Pang G, Clancy R, Ashman RB. Cellular and cytokine correlates of mucosal protection in a murine model of oral candidiasis. Infect Immun 2000;68:5771–7. [4] Chakir J, Cote L, Coulombe C, Delasriers N. Differential pattern of infection and immune response during experimental oral candidiasis in BALB/c and DBA/2 (H-24) mice. Oral Microbiol Immunol 1994;9:88– 94.
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[5] McGhee JR, Czerkinsky C, Mestecky J. Mucosal vaccines: an overview. In: Ogra PL, Mestecky J, Lamm ME, Strober W, Bienenstock J, McGhee JR, editors. Mucosal Immunology, 2nd edn. San Diego: Academic Press, 1999:741– 57. [6] Clancy RL, Pang G, Dunkley M, Taylor D, Cripps A. Acute on chronic bronchitis: a model of mucosal immunology. Immunol Cell Biol 1995;73:414– 7.