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Infection and Immunity, February 1999, p. 826-833, Vol. 67, No. 2
Department of Microbiology and Immunology,
Tulane University Medical Center, New Orleans, Louisiana 70112
Received 13 August 1998/Returned for modification 11 September
1998/Accepted 13 November 1998
The incidence of fungal infections caused by the opportunistic
yeast Candida albicans has increased significantly in
recent years. The ability to vaccinate selected patients against the organism would be advantageous. In this paper we describe a potential anti-C. albicans vaccine consisting of heat-killed C. albicans (HK-CA) in combination with the novel mucosal adjuvant
LT(R192G), a genetically detoxified form of the heat-labile toxin of
enterotoxigenic Escherichia coli. Groups of male CBA/J mice
were immunized intranasally on three occasions at weekly intervals with
2 × 107 HK-CA per dose, alone or in conjunction with
10 µg of LT(R192G) per dose. Two weeks following the last application
of antigen, some animals were challenged intravenously (i.v.) with
104, 105, or 106 viable C. albicans to assess protection as measured by survival and/or
culture. Some groups of animals were footpad tested with C. albicans mannan to assess delayed-type hypersensitivity
(DTH), and all the animals were bled for antibody assays. In two
independent studies, all the animals immunized with HK-CA plus
LT(R192G) were able to eradicate 104 C. albicans completely, as determined by kidney culture 4 weeks after challenge. Animals immunized with HK-CA only had reduced levels
of C. albicans compared to the adjuvant or
saline-only control. Greatly enhanced survival was observed when mice
immunized with HK-CA plus LT(R192G) were challenged with
105 live C. albicans as well. Animals immunized
with HK-CA plus LT(R192G) developed a significant DH response,
while those given HK-CA alone developed only marginal DH responses.
High immunoglobulin G (IgG) levels to cytoplasmic antigens developed in
mice immunized with HK-CA plus LT(R192G), but they were found only
after i.v. challenge. Addition of adjuvant shifted the antibody
isotype production in i.v.-challenged animals to a response
dominated by IgG2a. Clearly, intranasal immunization with killed
C. albicans in conjunction with LT(R192G) afforded
significant levels of protection. This novel approach offers new
possibilities for the development of an effective vaccine against
candidiasis for use in humans.
Candida albicans is a
ubiquitous fungus, which, along with other species of
Candida, may be found as part of the normal flora of humans
(11). Although healthy individuals are regularly colonized with Candida, serious disease seldom occurs unless some
precipitating factor alters the balance in favor of the fungus.
Unfortunately, precipitating factors such as immunosuppressive
therapies and diseases involving down-regulation of the immune system,
including AIDS, are becoming more prevalent. Thus, serious forms of
candidiasis are on the increase, and a vaccine capable of stimulating
immunity in patients prior to the institution of immunosuppressive
therapies could be of considerable value.
A rational approach to the development of a vaccine is problematic
because the specific immune system mechanisms responsible for
protective immunity have not been defined clearly. Moreover, candidiasis is a multifaceted disease which may manifest itself at
multiple levels, including mucocutaneous tissue and internal organs. To
complicate the issue further, the protective mechanisms associated with
different tissues appear to be different. For example, cellular
immunity appears to be critical for the defense of mucocutaneous tissue
(1) but does not appear to be the sole critical factor in
systemic defense (5). T lymphocytes appear to be important
for the development of acquired immunity (7, 25, 44), but a
specific role for T cells has not been established. It is not known,
for example, whether T cells are critical for antibody production, for
the development of cellular immune system phenomena, or for the
production of cytokines that alter the activity of
non-(antigen)-specific phagocytic cells. In fact, antibody, cellular
immunity, and innate immunity as a composite may be responsible for the
protection observed following immunization with the organism. Since
life-threatening forms of candidiasis occur at the systemic level, and
since the precise protective mechanisms at that level are ill defined,
the development of a vaccine for the disease must be empirical.
In addition to our lack of knowledge about the component(s) of the
immune system most responsible for acquired immunity at the systemic
level, the nature of the immunogen(s) that stimulates a protective
responses is also largely unknown. The best protective effects observed
to date have been stimulated by immunization with viable cells from
virulent (26, 40) or avirulent (3) strains of
C. albicans. Killed cells or subcellular components have
been moderately successful (2, 20, 35, 39, 49). Although some evidence has accumulated that antibody to
specific immunogens, e.g., hsp90 (38), hsp75, or non-hsp96
(13), is protective systemically, much of the evidence is circumstantial.
With the advent of newer adjuvants and protocols for immunization, it
seems timely to revisit the issue of immunization against C. albicans. An approach that has been explored recently involves mucosal immunization with inactivated organisms or purified immunogenic components derived from virulent organisms, delivered in combination with a mucosal adjuvant (17, 21).
Cholera toxin (CT) produced by various strains of Vibrio
cholerae and heat-labile enterotoxin (LT) produced by some
enterotoxigenic strains of Escherichia coli (10, 22,
36, 53, 54) are the two bacterial products with the greatest
potential to function as mucosal adjuvants. Recent studies have
examined the potential of CT and LT as mucosal adjuvants against a
variety of bacterial and viral pathogens in vaccines containing whole
killed organisms or purified subunits of relevant virulence
determinants from these organisms. Representative examples include
tetanus toxoid (52-54), inactivated influenza virus
(28, 31), recombinant urease from Helicobacter
spp. (34, 50), pneumococcal surface protein A from
Streptococcus pneumoniae (51), Norwalk virus
capsid protein (37), synthetic peptides from measles virus
(29), and the human immunodeficiency virus type 1 C4/V3
peptide T1SP10 MN(A) (48). There are many other examples,
and it is clear that both LT and CT have significant potential
for use as adjuvants for mucosally administered antigens (see
references 17 and 21 for
recent reviews). However, the fact that these two proteins are
toxic for humans and animals, even at low doses, precludes their
practical use in vaccines.
A series of mutants of LT and CT have been developed in an effort to
dissociate the adjuvant properties of these molecules from their toxic
effects. One of these LT mutants, designated LT(R192G), was
constructed by using site-directed mutagenesis to create a single amino
acid substitution in the biologically active domain (A subunit). This
mutation rendered the toxin insensitive to trypsin activation and
consequently greatly diminished its toxicity without altering the
intrinsic adjuvanticity characteristic of the native molecule
(16). A number of reports published recently have evaluated
the efficacy of LT(R192G) as an effective mucosal adjuvant (8,
32, 41).
In this paper, we report the effectiveness of a vaccine composed of
heat-inactivated C. albicans and LT(R192G) to
potentiate a protective immune response against intravenous
(i.v.) challenge with live, virulent C. albicans.
Mice.
Male CBA/J mice, 6 to 8 weeks of age, obtained from
Jackson Laboratory (Bar Harbor, Maine) were housed in bioclean hoods
and provided with food and water ad lib. All mice were allowed a 1-week acclimatization period before the experiments were begun.
Vaccine preparation.
C. albicans 20A, a serotype A
isolate originally obtained from Errol Reiss at the Centers for Disease
Control and Prevention, was used for all experiments. This strain was
maintained at 4°C with monthly transfer on Sabouraud dextrose agar.
Live C. albicans used for challenge and for intradermal
(i.d.) inoculations was prepared as follows. Cultures of C. albicans were incubated for 24 h on slants of Sabouraud
dextrose agar at 37°C, inoculated into Trypticase soy dialysate broth
(43), and incubated at 37°C on a gyratory shaker operating
at 165 rpm. The cells were harvested after 18 h and washed
three times in nonpyrogenic saline (NPS). The final pellet was
resuspended in NPS, and the cells were counted in a hemocytometer and
diluted to the appropriate concentration in NPS. The viability of the
culture was determined by plate count. C. albicans used for
immunization was prepared as described above, except that the cell
suspension was heated at 60°C for 2 h. The lack of viability of
this preparation was confirmed by plating 109 cells on
Sabouraud dextrose agar and incubating them at 37°C overnight.
Heat-killed C. albicans is designated HK-CA below.
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Effectiveness of a Vaccine Composed of Heat-Killed
Candida albicans and a Novel Mucosal Adjuvant, LT(R192G),
against Systemic Candidiasis
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials and methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
Immunization procedures and method. Two types of experiments were performed. For intranasal (i.n.) immunizations, the mice were lightly anesthetized with Metofane (Pitman-Moore, Mundelein, Ill.) and the inoculum was delivered as two applications of 5 µl to each nostril for a total volume of 20 µl per dose.
The first set of experiments was carried out to determine whether vaccination as above protected immunized animals against C. albicans challenge. Three groups of 7 to 12 animals each were immunized i.n. on three occasions at weekly intervals with 2 × 107 HK-CA in conjunction with 10 µg of LT(R192G) per dose or with either 10 µg of LT(R192G) or NPS alone. Some experiments also included animals that were immunized twice i.d. with 2 × 106 viable C. albicans or once intragastrically (i.g.) with 2 × 107 viable C. albicans followed by a single i.d. inoculation with 2 × 106 viable organisms as described previously (18, 25). In one experiment, a group of animals was immunized twice with 2 × 107 HK-CA administered intragastrically. Two weeks after the last immunization, all the animals were challenged i.v. via the lateral tail vein with 104, 105, or 106 viable C. albicans. Studies to determine protection following i.v. challenge were conducted three times. In the first two experiments, protection was assessed by culturing the kidneys and brains of surviving animals for the presence of viable C. albicans 4 weeks after challenge with 104 live C. albicans. In the third experiment, protection was assessed by monitoring survival for 100 days after challenge with 105 or 106 live C. albicans. At the end of this period, all the surviving animals were sacrificed and blood was collected for serological analysis. A second set of experiments was performed to analyze the levels of delayed-type hypersensitivity (DTH) and antibody responses 2 weeks following the completion of the immunization protocol, i.e., at the time when i.v. challenge was performed in the experiments described above. In these experiments, groups of five or six mice were immunized with HK-CA plus adjuvant or with HK-CA, adjuvant, or NPS alone, as described above. A control group was inoculated intradermally twice with 106 live C. albicans. These animals were then footpad tested to detect DTH and subsequently bled to determine antibody responses.DTH. The development of DTH in response to each immunization regimen was determined by the footpad assay as described previously (12, 14). Footpad thickness was measured 24 and 48 h after the antigen (mannan) was injected, and the mean thickness was calculated by subtracting preinjection measurements from postinjection measurements. Each mouse was inoculated in the footpad with 5 µg of C. albicans mannan as described previously (24).
Serological analysis.
All the animals were sacrificed by
CO2 inhalation, and blood was immediately collected by
cardiac puncture. Serum samples were separated by centrifugation in
Microtainer tubes (Becton Dickinson & Co., Franklin Lakes, N.J.) and
stored at 
20°C until analyzed. Each serum sample was individually
analyzed by enzyme-linked immunosorbent assays (ELISA). For all ELISAs,
96-well plates were coated with 5 µg of soluble cytoplasmic
substances (SCS) per well prepared from viable C. albicans as described previously (19) or with a
dithiothreitol extract (DTTE) from cell walls (42) and
incubated overnight at 4°C. All subsequent steps were carried out at room temperature. After blocking nonspecific sites with 1%
bovine serum albumin, twofold serial dilutions of serum from the
experimental animals were added. Alkaline phosphatase-conjugated rabbit
anti-mouse immunoglobulin G (IgG) or anti-mouse IgA (Sigma Chemical
Co., St. Louis, Mo.) was used for determination of the total
concentration of IgG or IgA in serum. Biotinylated anti-mouse IgG1,
IgG2a, IgG2b, or IgG3 (PharMingen, San Diego, Calif.) followed by
alkaline phosphatase-conjugated streptavidin (Southern Biotechnology Associates, Birmingham, Ala.) was used to quantify antibody isotypes. The optical density at 405 nm was determined with an ELISA reader (Bio-Tek Instruments, Inc., Winooski, Vt.) after addition of 100 µl
of a 1-mg/ml solution of p-nitrophenyl phosphate (Sigma).
Statistical analysis.
The total colony-forming units per
organ were determined by standard dilution calculations and expressed
as logarithmic numbers. Thereafter, logarithmic numbers for the
respective groups were averaged to obtain geometric means. Since there
is no logarithmic value for zero, it was assumed that a single organism
could have been missed in the manipulations of the homogenates.
Consequently, we used 0.4, the logarithmic value for 1, for the animals
that were negative by culture. The Mann-Whitney test was used to
determine statistical significance for culture and footpad data.
P 
0.05 were considered significant.
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RESULTS |
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Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Protective immunity following immunization. The first set of experiments was designed to determine whether i.n. administration of nonviable C. albicans in conjunction with LT(R192G) would stimulate protective immunity. Animals immunized i.n. with HK-CA in conjunction with LT(R192G), as well as those immunized with HK-CA, LT(R192G), or NPS alone, were challenged i.v. with 104 viable C. albicans 2 weeks following the last application of immunogen. Survival was monitored over a 28-day period, and then the animals were sacrificed and their kidneys and brains were cultured quantitatively. Animals immunized by previously proven methods of immunization, i.e., by i.g. and/or i.d. inoculations of viable C. albicans (18, 26), were included as controls.
As seen in Fig. 1, the results of two independent experiments demonstrate clearly that animals vaccinated i.n. with HK-CA plus LT(R192G) were able to eradicate C. albicans completely from their kidneys. Some of the animals immunized i.n. with HK-CA alone displayed reduced numbers of C. albicans in their kidneys the first time the experiment was performed; however, this observation was not a reproducible phenomenon in subsequent experiments. Animals immunized by i.g. followed by i.d. inoculation of viable C. albicans also had significantly (P 0.05) reduced levels of
the yeast in their kidneys, but the reductions were not nearly as
dramatic as those observed in animals immunized i.n. with HK-CA in
conjunction with LT(R192G). The levels in the i.g.-i.d. group of
animals were consistent with those previously demonstrated for this
type of immunization (18, 26). As expected, control animals
immunized i.n. with LT(R192G) alone or NPS alone had disseminated
candidiasis, as indicated by the presence of large numbers of
C. albicans in their kidneys. Moreover, as demonstrated
in the experiment Fig. 1A, animals immunized i.g. with nonviable
C. albicans did not develop protective responses. The
inability of inactivated Candida to induce protection when
administered i.g. has been previously observed in our laboratory.
Consequently, i.g. immunization was not included the second experiment.
The brains of all immunized and challenged animals were also cultured.
Tissues from animals immunized i.n. with HK-CA plus adjuvant were
negative, whereas the average levels of C. albicans
found in the brains of animals in all the other groups were not
significantly different (P 0.05) from those in the
NPS controls (data not shown).
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Cellular immunity in immunized animals. As an initial step to examining the nature of the protective immune response in vaccinated animals, groups of five or six mice were studied for the antibody and cellular immune responses developed after vaccination but prior to challenge. DTH was assessed by the footpad assay 2 weeks following the last of three immunizations with the fungus-adjuvant mixture. These mice were not challenged with live Candida at any point. The results depicted in Fig. 3 indicate that mice immunized with adjuvant alone developed no DTH response whereas those immunized with HK-CA alone developed minimal responses. Those responses were not statistically significant. In contrast, animals immunized with HK-CA plus LT(R192G) developed substantial and highly significant DTH reactions. The responses of the latter animals were comparable to those detected in animals inoculated i.d. or i.g.-i.d. with live C. albicans (18, 26). The immunological reactivity observed in the group immunized with HK-CA plus LT(R192G) indicates the development of a vigorous antigen-specific T-cell response, which correlated with protection and was elicited marginally by inactivated C. albicans in the absence of the mucosal adjuvant.
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Humoral immunity in immunized animals. The levels of specific anti-Candida antibodies in serum were determined following i.n. immunization with the LT(R192G)-containing vaccine in animals treated in two different ways. First, footpad-tested animals from the previous experiment were sacrificed and bled 3 days after footpad testing. Footpad testing as described above was performed 2 weeks after the last of three applications of the vaccine. Thus, this first group of animals had not been challenged i.v. No antibody was detected in these animals, and when their responses were compared to those of unchallenged animals immunized i.g.-i.d., only two of eight animals in the latter group had demonstrable serum anti-Candida IgG 2 weeks following the second exposure to viable C. albicans (data not shown).
The second group of animals analyzed was that containing the mice that had been challenged i.v. with 104 viable C. albicans 2 weeks after the last application of vaccine and then sacrificed 4 weeks after the i.v. challenge. A control group, consisting of animals immunized i.g.-i.d. with viable Candida and challenged i.v. with viable C. albicans, as indicated above, was included in these assays. C. albicans-specific antibodies were measured by ELISA with anti-IgG whole molecule or sera specific for IgG1, IgG2a, IgG2b, IgG3, or IgA. Antibody detection was assessed with either the cytoplasmic extract, SCS, or the cell wall extract, DTTE. As shown in Fig. 4A, the highest levels of IgG in serum against SCS were found in animals that had been immunized with HK-CA in conjunction with LT(R192G) and then challenged i.v. with viable C. albicans. These animals had the highest level of protection of any of the vaccine groups examined. There were detectable levels of IgG2a and IgG2b (data not shown) but no detectable IgG3 antibodies to SCS in these mice. Animals immunized with HK-CA alone and then challenged i.v. with viable C. albicans also developed antibodies to SCS, but the levels were much lower than those in animals that received HK-CA plus LT(R192G), and two of the animals had low levels of IgG1 antibodies specific for the SCS. No antibodies were detected in the negative control animals, i.e., animals given only adjuvant or NPS as the immunogen and then challenged i.v. with viable C. albicans. No IgG antibodies were detected against the cell wall antigen, DTTE, in any of the groups of mice, and none of the immunized and challenged animals had detectable IgA antibodies in serum specific for SCS (data not shown). Interestingly, the positive control group, i.e., the animals immunized by the i.g. and i.d. routes with viable C. albicans and then challenged i.v. with viable C. albicans, had lower total IgG levels but higher IgG1 levels compared to the group that received HK-CA plus LT(R192G).
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DISCUSSION |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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We have shown clearly that i.n. immunization with whole killed C. albicans mixed with the novel mucosal adjuvant LT(R192G) stimulated high levels of protection against systemic challenge with a highly virulent strain of C. albicans. Protective immunity was associated with substantial levels of DTH and with high levels of anti-SCS IgG in serum. A predominantly IgG2a response to soluble cytoplasmic antigens was observed in animals that survived a challenge with 105 C. albicans, and there were low levels of IgG1 and IgG2b in most of these mice as well. Antibody was not detectable to DTTE of the cell wall of the same strain of C. albicans used to immunize and infect animals.
The level of protection observed in the animals immunized with inactivated cells and adjuvant and then challenged with 105 viable C. albicans was remarkable in that 100% of the adjuvant- or NPS-only animals had died by 18 days whereas only 1 of 10 mice immunized with the organism plus adjuvant died over a course of 100 days. Similarly, although 80% of the immunized animals challenged with 106 organisms died over a period of 12 days, animals treated with adjuvant or NPS only were all dead within 2 to 3 days. When the kidneys and brains of animals immunized with HK-CA plus LT(R192G) were cultured 28 days following i.v. challenge, none of the animals examined in either of two independent experiments had any organisms detectable in the tissue homogenates. In the past, nonviable C. albicans has not been highly successful as an immunogen (25, 26, 40, 47). Considerably more success was attained when viable virulent C. albicans (26, 40) or an avirulent strain (3) was used, and there have been three reports of significant levels of protective immunity stimulated by C. albicans ribosomes (45), a mannoprotein (39), and antibody to an extracellular candidal protein, p43 (49).
The development of immunization protocols for candidiasis is complicated further by the fact that the mechanisms responsible for protection at the mucosal level appear to be different from those operative at the systemic level. In addition, when exploring protective mechanisms against candidiasis, it is imperative to define clearly whether the assessment of protection to a first exposure to viable C. albicans is being investigated or whether protection following immunization (acquired immunity) is under consideration. Most people have been exposed to C. albicans, usually in the gastrointestinal tract, since a high percentage of the population is colonized with the organism (11). Mucosal exposure stimulates protective systemic immunity, as demonstrated in an animal model (18) and as evidenced by the fact that most humans, despite lifelong colonization with the organism, never develop systemic disease. T lymphocytes are clearly involved in protective mechanisms, at both the mucosal (5) and systemic (7, 26, 46) levels, but the precise role of these T cells has not been defined.
The role of antibody in protection is controversial (6) and may depend upon whether one is evaluating the response to a first systemic exposure to viable C. albicans (27), a mucosal infection such as that in the vagina (15), or development of antibody to antigens that may actually enhance the infection (4). In our experiments with HK-CA and LT(R192G), the animals produced antibody against a number of SCS antigens, including a small antigen, i.e., approximately 29 kDa. A strong response to the 29-kDa antigen, in comparison to the other antigenic components of the SCS, was noted in the animals immunized with HK-CA plus LT(R192G) that were sacrificed 28 days following i.v. challenge with 104 live C. albicans (data not shown). Others (30, 33) have noted that patients with superficial or vaginal candidiasis have antibody responses to antigens that migrate in this region as well. While the evidence for involvement of antibodies to this antigen in any kind of protective mechanism is purely circumstantial, it may be profitable to examine this particular antigen in more detail for a role in the protective response.
We have shown that LT(R192G) can induce protective immunity when coadministered with whole inactivated bacteria (8) or viruses (32) or subunits of relevant virulence determinants from these pathogens (41). This adjuvant promotes the development of both humoral (antibody) and cell-mediated immune responses against the pathogen in both the systemic and mucosal compartments.
In a recent study by Chong et al. (8), the function of
LT(R192G) in protection against typhoid-like disease was to
upregulate or enhance the Th1 arm of the immune response against killed
organisms. Specifically, mice immunized orally with killed
Salmonella dublin in conjunction with LT(R192G) were
protected against lethal challenge and had higher gamma interferon
(IFN-
) interleukin-2, and IgG responses than did mice immunized
orally with killed S. dublin alone, which were not
protected. Conclusive evidence for an association of IFN- with
adjuvant-induced protection was provided in the studies in which
neutralization of endogenous IFN- with anti-IFN- (monoclonal
antibody R4-6A2) resulted in loss of protection against lethal oral challenge.
In the present study, we examined the ability of LT(R192G) to enhance the humoral and cellular immune responses against C. albicans and to induce protection against colonization and lethal i.v. challenge with wild-type C. albicans. Solid protection against colonization and lethal i.v. challenge was achieved following i.n. immunization with heat-killed whole organisms in conjunction with this adjuvant. Both the humoral and cellular immune responses against C. albicans were enhanced. A strong DTH response to mannan was observed in animals vaccinated with the mixture of HK-CA and LT(R192G). Moreover, isotype analysis of anti-Candida antibodies in protected animals revealed a predominance of antibodies of the IgG2a isotype, suggesting a strong Th1-type cytokine response. Studies to characterize the cytokine pattern observed in protected animals, using antigen restimulation studies and reverse transcriptase PCR analyses, are under way.
Currently, there are no vaccines available for human mycoses, and there is an urgent need to develop measures of prophylactic immunointervention against fungal pathogens. This is especially important if one considers a mass prophylactic treatment for immunocompromised human hosts. On the one hand, vaccines containing a live, replicating organism are associated with an inherent risk of infection, even when the vaccine strain is attenuated or exhibits low virulence. On the other hand, although subunit vaccines are safer, the cost and labor involved in the purification process of proteins or immunogenic carbohydrates represent a hindrance to the development of such vaccines. The concept of using inactivated C. albicans as a component of a potential anti-Candida vaccine is attractive because of its safety and the ease and low cost associated with the preparation of large numbers of cells of the inactivated yeast.
In future studies, we intend to determine if the protection conferred by this vaccine can be passively transferred to naive and immunocompromised mice and whether immunological protection supersedes the induction of immunosuppression in experimental animals. Concomitantly, our studies will determine if protection can be achieved in animals that are colonized with Candida prior to vaccination and if cross-protection against other Candida species can be achieved by using this mucosal immunization strategy. With the information obtained in the proposed studies, future vaccine strategies can be designed by using similar vaccination procedures for a variety of fungal pathogens. These are important issues because they take us beyond the phenomenological observations of "enhanced immunity" to a clearer understanding of the mechanisms of protection against Candida and the practical implications of the development of antifungal vaccines.
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ACKNOWLEDGMENTS |
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This investigation was supported by Public Health Service grants AI12806 and AI42777 from the National Institutes of Health and by a grant from SmithKline Beecham Biologics.
We are grateful to Robert Johnson for assistance with statistical analyses.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Immunology, Tulane University Medical Center, 1430 Tulane Ave., New Orleans, LA 70112. Phone: (504) 587-2171. Fax: (504) 588-5144. E-mail: lfreyta{at}mailhost.tcs.tulane.edu.
Editor: T. R. Kozel
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REFERENCES |
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Abstract
Introduction
Materials and methods
Results
Discussion
References
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| 1. | Balish, E., J. Jensen, T. Warner, J. Brekke, and B. Leonard. 1993. Mucosal and disseminated candidiasis in gnotobiotic SCID mice. J. Med. Vet. Mycol. 31:143-154[Medline]. |
| 2. | Banerjee, U., L. N. Mohapatra, and R. Kumar. 1985. Effect of immunization with formalin killed cells in complete Freund's adjuvant in experimental candidosis. Indian J. Med. Res. 81:454-458[Medline]. |
| 3. |
Bistoni, F.,
A. Vecchiarelli,
E. Cenci,
P. Puccetti,
P. Marconi, and A. Cassone.
1986.
Evidence for macrophage-mediated protection against lethal Candida albicans infection.
Infect. Immun.
51:668-674 |
| 4. |
Bromuro, C.,
R. La Valle,
S. Sandini,
F. Urbani,
C. M. Ausiello,
L. Morelli,
C. Fe d'Ostiani,
L. Romani, and A. Cassone.
1998.
A 70-kilodalton recombinant heat shock protein of Candida albicans is highly immunogenic and enhances systemic murine candidiasis.
Infect. Immun.
66:2154-2162 |
| 5. |
Cantorna, M. T., and E. Balish.
1991.
Role of CD4+ lymphocytes in resistance to mucosal candidiasis.
Infect. Immun.
59:2447-2455 |
| 6. | Casadevall, A. 1995. Antibody immunity and invasive fungal infections. Infect. Immun. 63:4211-4218[Medline]. |
| 7. |
Cenci, E.,
L. Romani,
A. Vecchiarelli,
P. Puccetti, and F. Bistoni.
1989.
Role of L3T4+ lymphocytes in protective immunity to systemic Candida albicans infection in mice.
Infect. Immun.
57:3581-3587 |
| 8. | Chong, C., M. Frieberg, and J. D. Clements. 1998. LT(R192G), a non-toxic mutant of the heat-labile enterotoxin of Escherichia coli elicits enhanced humoral and cellular immune responses associated with protection against lethal oral challenge with Salmonella spp. Vaccine 16:732-740[Medline]. |
| 9. |
Clements, J. D., and R. A. Finkelstein.
1979.
Isolation and characterization of homogeneous heat-labile enterotoxins with high specific activity from Escherichia coli cultures.
Infect. Immun.
24:760-769 |
| 10. | Clements, J. D., N. M. Hartzog, and F. L. Lyon. 1988. Adjuvant activity of Escherichia coli heat-labile enterotoxin and effect on the induction of oral tolerance in mice to unrelated protein antigens. Vaccine 6:269-277[Medline]. |
| 11. | Cohen, R., F. J. Roth, E. Delgado, D. G. Ahearn, and M. H. Kalser. 1969. Fungal flora of the normal human small and large intestine. N. Engl. J. Med. 280:638-641. |
| 12. | Cooper, M. G. 1972. Delayed-type hypersensitivity in the mouse. I. Induction and elicitation by Salmonella adelaide flagellin and its derivatives. Scand. J. Immunol. 1:167-178[Medline]. |
| 13. |
Costantino, P. J.,
K. M. Franklyn,
N. F. Gare, and J. R. Warmington.
1994.
Production of antibodies to antigens of Candida albicans in CBA/H mice.
Infect. Immun.
62:1400-1405 |
| 14. | Crowle, A. J. 1975. Delayed hypersensitivity in the mouse. Adv. Immunol. 20:197-264[Medline]. |
| 15. | De Bernardis, F., M. Boccanera, D. Adriani, E. Spreghini, G. Santoni, and A. Cassone. 1997. Protective role of antimannan and anti-aspartyl proteinase antibodies in an experimental model of Candida albicans vaginitis in rats. Infect. Immun. 65:3399-3405[Abstract]. |
| 16. | Dickinson, B. L., and J. D. Clements. 1995. Dissociation of Escherichia coli heat-labile enterotoxin adjuvanticity from ADP-ribosyltransferase activity. Infect. Immun. 63:1617-1623[Abstract]. |
| 17. | Dickinson, B. L., and J. D. Clements. 1996. Use of Escherichia coli heat-labile enterotoxin as an oral adjuvant, p. 73-87. In H. Kiyono, P. L. Ogra, and J. R. McGhee (ed.), Mucosal vaccines. Academic Press, Inc., San Diego, Calif. |
| 18. |
Domer, J. E., and R. F. Hector.
1987.
Enhanced immune responses in mice treated with penicillin-tetracycline or trimethoprim-sulfamethoxazole when colonized intragastrically with Candida albicans.
Antimicrob. Agents Chemother.
31:691-697 |
| 19. |
Domer, J. E., and S. A. Moser.
1978.
Experimental murine candidiasis: cell-mediated immunity after cutaneous challenge.
Infect. Immun.
20:88-98 |
| 20. | Eckstein, M., Y. Barenholz, L. K. Bar, and E. Segal. 1997. Liposomes containing Candida albicans ribosomes as a prophylactic vaccine against disseminated candidiasis in mice. Vaccine 15:220-224[Medline]. |
| 21. | Elson, C. 1996. Cholera toxin as a mucosal adjuvant, p. 59-72. In H. Kiyono, P. L. Ogra, and J. R. McGhee (ed.), Mucosal vaccines. Academic Press, Inc., San Diego, Calif. |
| 22. | Elson, C. O. 1989. Cholera toxin and its subunits as potential oral adjuvants. Immunol. Today 146:29-33. |
| 23. | Gallagher, S., S. E. Winston, S. A. Fullerr, and J. G. R. Hurrell. 1997. Immunoblotting and immunodetection, p. 10.8. In F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl (ed.), Current protocols in molecular biology. John Wiley & Sons, Inc., New York, N.Y. |
| 24. |
Garner, R. E.,
U. Kuruganti,
C. W. Czarniecki,
H. H. Chiu, and J. E. Domer.
1989.
In vivo immune responses to Candida albicans modified by treatment with recombinant murine gamma interferon.
Infect. Immun.
57:1800-1808 |
| 25. | Giger, D. K., J. E. Domer, and J. T. McQuitty, Jr. 1978. Experimental murine candidiasis: pathological and immune responses to cutaneous inoculation with Candida albicans. Infect. Immun. 19:496-509. |
| 26. |
Giger, D. K.,
J. E. Domer,
S. A. Moser, and J. T. McQuitty, Jr.
1978.
Experimental murine candidiasis: pathological and immune responses in T-lymphocyte-depleted mice.
Infect. Immun.
21:729-737 |
| 27. | Han, Y., and J. E. Cutler. 1995. Antibody response that protects against disseminated candidiasis. Infect. Immun. 63:2714-2719[Abstract]. |
| 28. | Hashigucci, K., H. Ogawa, T. Ishidate, R. Yamashita, H. Kamiya, K. Watanabe, N. Hattori, T. Sato, Y. Suzuki, T. Nagamine, C. Aizawa, S. Tamura, T. Kurata, and A. Oya. 1996. Antibody responses in volunteers induced by nasal influenza vaccine combined with Escherichia coli heat-labile enterotoxin B subunit containing a trace amount of the holotoxin. Vaccine 14:113-119[Medline]. |
| 29. | Hathaway, L. J., C. D. Partidos, P. Vohra, and M. W. Steward. 1995. Induction of systemic immune responses to measles virus synthetic peptides administered intranasally. Vaccine 13:1495-1500[Medline]. |
| 30. | Ishiguro, A., M. Homma, T. Sukai, K. Higashide, S. Torii, and K. Tanaka. 1992. Immunoblotting analysis of sera from patients with candidal vaginitis and healthy females. J. Med. Vet. Mycol. 30:281-292[Medline]. |
| 31. | Katz, J. M., X. Lu, S. A. Young, and J. C. Galphin. 1997. Adjuvant activity of the heat-labile enterotoxin from enterotoxigenic Escherichia coli for oral administration of inactivated influenza virus vaccine. J. Infect. Dis. 175:352-363[Medline]. |
| 32. | Katz, J. M., X. Lu, J. C. Galphin, and J. D. Clements. 1996. Heat-labile enterotoxin from E. coli as an adjuvant for oral influenza vaccination, p. 292-297. In L. E. Brown, A. W. Hampson, and R. G. Webster (ed.), Options for the control of influenza, vol. III. Elsevier Science Publishing, Inc., New York, N.Y. |
| 33. | Keller, B. I., P. D. Simmons, and L. Ivanyl. 1990. Identification of immunodominant antigens of Candida albicans in patients with superficial candidosis. Clin. Immunol. Immunopathol. 54:347-353[Medline]. |
| 34. | Lee, C. K., R. Weltzin, W. D. Thomas, Jr., H. Kleanthous, T. H. Ermak, G. Soman, J. E. Hill, S. K. Ackerman, and T. P. Monath. 1995. Oral immunization with recombinant Helicobacter pylori urease induces secretory IgA antibodies and protects mice from challenge with Helicobacter felis. J. Infect. Dis. 172:161-172[Medline]. |
| 35. | Levy, R., E. Segal, and L. Barr-Nea. 1985. Systemic candidiasis in mice immunized with Candida albicans ribosomes. Mycopathologia 91:17-22[Medline]. |
| 36. | Lycke, N., T. Tsuji, and J. Holmgren. 1992. The adjuvant effect of Vibrio cholerae and Escherichia coli heat-labile enterotoxins is linked to their ADP-ribosyltransferase activity. Eur. J. Immunol. 22:2277-2281[Medline]. |
| 37. |
Mason, H. S.,
J. M. Ball,
J. J. Shi,
X. Jiang,
M. K. Estes, and C. J. Arntzen.
1996.
Expression of Norwalk virus capsid protein in transgenic tobacco and potato and its oral immunogenicity in mice.
Proc. Natl. Acad. Sci. USA
93:5335-5340 |
| 38. | Matthews, R. C., J. P. Burnie, D. Howat, T. Rowland, and F. Walton. 1991. Autoantibody to heat-shock protein 90 can mediate protection against systemic candidosis. Immunology 74:20-24[Medline]. |
| 39. |
Mencacci, A.,
A. Torosantucci,
R. Spaccapelo,
L. Romani,
F. Bistoni, and A. Cassone.
1994.
A mannoprotein constituent of Candida albicans that elicits different levels of delayed-type hypersensitivity, cytokine production, and anticandidal protection in mice.
Infect. Immun.
62:5353-5360 |
| 40. | Mourad, S., and L. Friedman. 1961. Active immunization of mice against Candida albicans. Proc. Soc. Exp. Biol. Med. 106:570-572. |
| 41. |
O'Neal, C. M.,
J. D. Clements,
M. K. Estes, and M. E. Conner.
1998.
Rotavirus 2/6 viruslike particles administered intranasally with cholera toxin, Escherichia coli heat-labile toxin (LT), and LT-R192G induce protection from rotavirus challenge.
J. Virol.
72:3390-3393 |
| 42. |
Reiss, E.,
S. H. Stone, and H. F. Hasenclever.
1974.
Serological and cellular immune activity of peptidoglucomannan fractions of Candida albicans cell walls.
Infect. Immun.
9:881-890 |
| 43. |
Restrepo-Moreno, A., and J. D. Schneidau, Jr.
1967.
Nature of the skin-reactive principle in culture filtrates prepared from Paracoccidioides brasiliensis.
J. Bacteriol.
93:1741-1748 |
| 44. | Romani, L., A. Mencacci, E. Cenci, P. Mosci, G. Vitellozzi, U. Grohmann, P. Puccetti, and F. Bistoni. 1992. Course of primary candidiasis in T cell-depleted mice infected with attenuated variant cells. J. Infect. Dis. 166:1384-1392[Medline]. |
| 45. | Segal, E., S. Nussbaum, and L. Barr-Nea. 1985. Protection against systemic infections with various Candida species elicited by vaccination with Candida albicans ribosomes. Sabouraudia 23:275-285[Medline]. |
| 46. |
Sieck, T. G.,
M. A. Moors,
H. R. Buckley, and K. J. Blank.
1993.
Protection against murine disseminated candidiasis mediated by a Candida albicans-specific T-cell line.
Infect. Immun.
61:3540-3543 |
| 47. | Soles, P., L. Y. Lim, and D. B. Louria. 1967. Active immunity in experimental candidiasis in mice. Sabouraudia 5:315-322[Medline]. |
| 48. | Staats, H. F., W. G. Nichols, and T. J. Palker. 1996. Mucosal immunity to HIV-1: systemic and vaginal antibody responses after intranasal immunization with the HIV-1 C4/V3 peptide T1SP10 MN(A). J. Immunol. 157:462-472[Abstract]. |
| 49. |
Tavares, D.,
P. Ferreira,
M. Vilanova,
A. Videira, and M. Arala-Chaves.
1995.
Immunoprotection against systemic candidiasis in mice.
Int. Immunol.
7:785-796 |
| 50. | Weltzin, R., H. Kleanthous, F. Guirakhoo, T. P. Monath, and C. K. Lee. 1997. Novel intranasal immunization techniques for antibody induction and protection of mice against gastric Helicobacter felis infection. Vaccine 15:370-376[Medline]. |
| 51. | Wu, H. Y., M. H. Nahm, Y. Guo, M. W. Russell, and D. E. Briles. 1997. Intranasal immunization of mice with PspA (pneumococcal surface protein A) can prevent intranasal carriage, pulmonary infection, and sepsis with Streptococcus pneumoniae. J. Infect. Dis. 175:839-846[Medline]. |
| 52. | Xu-Amano, J., R. J. Jackson, K. Fujihashi, H. Kiyono, H. F. Staats, and J. R. McGhee. 1994. Helper Th1 and Th2 cell responses following mucosal or systemic immunization with cholera toxin. Vaccine 12:903-911[Medline]. |
| 53. |
Xu-Amano, J.,
H. Kiyono,
R. J. Jackson,
H. F. Staats,
K. Fujihashi,
P. D. Burrows,
C. O. Elson,
S. Pillai, and J. R. McGhee.
1993.
Helper T cell subsets for immunoglobulin A responses: oral immunization with tetanus toxoid and cholera toxin as adjuvant selectively induces Th2 cells in mucosal associated tissues.
J. Exp. Med.
178:1309-1320 |
| 54. | Yamamoto, M., J. L. Vancott, N. Okahashi, M. Marinaro, H. Kiyono, K. Fujihashi, R. J. Jackson, S. N. Chatfield, H. Bluethmann, and J. R. McGhee. 1996. The role of Th1 and Th2 cells for mucosal IgA responses. Ann. N. Y. Acad. Sci. 778:64-71[Medline]. |
Infection and Immunity, February 1999, p. 826-833, Vol. 67, No. 2
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