1 Laboratories of Microbiology, Institute for Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal
2 Immunology, Institute for Biomedical Sciences Abel Salazar (ICBAS), University of Porto, Porto, Portugal
3 Institute of Molecular and Cellular Biology (IBMC), Porto, Portugal
4 Gulbenkian Institute of Sciences, Oeiras, Portugal
Correspondence
Delfina Tavares
dtavares{at}icbas.up.pt
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ABSTRACT |
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These authors contributed equally to this work.
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INTRODUCTION |
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Lifelong immunoprotection against the aetiological micro-organisms of infectious disorders may occur after natural clearance of infectious agents for which there are no available treatments, as in the case of measles, German measles, mumps or hepatitis A and B. Subclinical infections caused by very small numbers of pathogenic microbes may also explain the higher levels of resistance to infections that are found among some populations of health professionals. Similarly, naturally acquired high protection can be achieved in cattle or in guinea pigs after clearance of sublethal inocula of bacteria (Blander & Horwitz, 1989; Pal & Horwitz, 1992
; Stevens et al., 1995
), fungi (Timoney et al., 1988
) and protozoa (Urquhart et al., 1998
). It is also pertinent to recall that the exceptionally efficient smallpox vaccine was made of virus particles that were not fully attenuated, since skin infectious vesicles and subsequent scars usually occurred after the vaccination.
In experimental candidiasis, the best protective effects have been achieved by immunization with viable cells from virulent (Cassone et al., 1995; Giger et al., 1978
; Mourad & Friedman, 1961
) strains of Candida albicans, while immunization with killed cells or subcellular components of the organism has been moderately successful (Banerjee et al., 1985
; Eckstein et al., 1997
; Levy et al., 1985
; Mencacci et al., 1994
).
We have previously observed that bacteria (Ferreira et al., 1997; Lima et al., 1992
; Santarem et al., 1987
), viruses (Arala-Chaves et al., 1988
) and fungi (Tavares et al., 1993
) secrete virulence-associated immunomodulatory proteins (VIPs). These VIPs are only produced by micro-organisms in their pathogenic form. They are immunomodulatory because they are simultaneously mitogens for T- and B-lymphocytes and immunosuppressive for foreign antigens (Arala-Chaves et al., 1988
; Ferreira et al., 1988
; Lima et al., 1992
; Pal & Horwitz, 1992
; Ribeiro et al., 1991
; Soares et al., 1990
; Tavares et al., 1993
). We have shown the importance of VIPs for the survival of pathogenic micro-organisms to be due to the fact that they potentiate microbial growth (Lima et al., 1992
; Santarem et al., 1987
; Tavares et al., 1993
) and because mice immunized with VIPs become resistant to infection (Soares et al., 1990
; Tavares et al., 1995
).
In the last decade, other authors have also isolated microbial VIPs from different bacteria and viruses. Thus, virulent (Balaban et al., 1998; Pal & Horwitz, 1992
), immunosuppressive (Kierszenbaum et al., 1990
; Klapproth et al., 1995
; Sun et al., 1998
), T-lymphocyte (Bruno et al., 1998
; Itoh et al., 1992
; Miyoshi-Akiyama et al., 1993
; Mody et al., 1995
) and B-lymphocyte (Dugas et al., 1991
; Feng & Lo, 1994
; Ying & Weis, 1993
) mitogenic proteins have been described. Moreover, immunoprotection achieved with proteins secreted by Mycobacterium tuberculosis (Horwitz et al., 1995
) and Staphylococcus aureus (Balaban et al., 1998
) has also been reported.
All of the aforementioned observations have led us to investigate whether naturally acquired immunoprotection against systemic candidiasis, developed in mice that recovered from a fungal inoculation with 106 Candida blastoconidia, was due to immunoneutralization of p43, a VIP produced by C. albicans.
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METHODS |
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C. albicans infection and/or priming protocols.
Pathogenic C. albicans was isolated and maintained as described previously (Tavares et al., 1993). Some of the pathogenic C. albicans blastoconidia were heat-killed at 100 °C for 30 min. The attenuated form of the same fungal strain was obtained after 60 passages in Sabouraud glucose agar (Difco) over 8 months, as previously described in detail (Tavares et al., 1993
). Naïve BALB/c mice were primed with 106 Candida blastoconidia by the intraperitoneal (i.p.) route with pathogenic C. albicans, the attenuated fungus or the heat-killed fungus. Control mice received the same volume of vehicle (PBS minus C. albicans) as the test mice via the i.p. route. One month later, all groups of mice were infected i.p. with an inoculum of 107 pathogenic C. albicans blastoconidia. Kidneys were removed aseptically at selected times after fungal priming or after infection with 107 pathogenic C. albicans blastoconidia. The organs were homogenized in 3 ml of PBS and the homogenates were serially diluted 10-fold. Plating of the homogenates was started at an initial dilution of 1 : 100. Colony-forming units (c.f.u.) of C. albicans were counted after 48 h incubation at 37 °C on mycobiotic agar (Difco) in duplicate cultures for each serial dilution. Only the number of c.f.u. found in the kidneys is shown because kidney is the organ of maximum fungal growth after systemic C. albicans infection (Louria, 1985
). Furthermore, as shown in Table 1
, there was a good correlation between fungal load in the kidneys and in the liver, the spleen and several other organs scored (data not shown).
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Antibody detection.
A standard ELISA was done to quantify specific antibodies in mice sera. Briefly, polystyrene microtitre plates (Nunc) were coated with p43 (10 µg ml-1) or Cs overnight at 4 °C. The wells were saturated for 30 min with 1 % (w/v) BSA in PBS at room temperature. Appropriate serial dilutions of the serum samples were then incubated in the plates for 2 h at room temperature. After washing, the bound antibodies were revealed by the addition of peroxidase-labelled goat-anti-mouse IgG (Southern Biotechnology Associates) for 1 h at room temperature. O-Phenylenediamine (Sigma) and H2O2 were used to develop the reaction; it was stopped by the addition of SDS. The colorimetric change was measured with a Biotek Chromoscan at 450 nm. The ELISA antibody titre was expressed as the reciprocal of the highest dilution giving an A450 value of 0·1 above that of the control (no serum added).
Immunizations with p43 or Cs and passive serum administration.
BALB/c mice were immunized twice with intradermal injections of 10 µg of p43 or 10 µg of Cs in 0·2 ml PBS with a 3-week interval between injections. p43-Immunized mice were treated i.p. with 0·5 ml of sterile Ig enriched fraction (IgF) (ammonium sulphate precipitation) obtained from the same strain of mice immunized with p43, or unimmunized, at the same time (day 0) and one day after (day+1) p43 immunization. One month later, both groups of mice were infected i.p. with 107 pathogenic Candida blastoconidia. Mice primed with heat-killed Candida blastoconidia were treated i.p. with 0·5 ml of IgF obtained from the same strain of mice immunized with p43 or Cs, or unimmunized one day before (day -1), at the same time (day 0) and one day after (day+1) infection with 107 pathogenic Candida blastoconidia. Each injection of IgF was obtained from 0·25 to 0·45 ml of mice sera. The IgF obtained from immunized mice was adjusted with balanced salt solution to 2500 ELISA titres against the respective antigen. IgF from unimmunized mice was adjusted to the same protein concentration as the anti-p43 IgF (27 mg ml-1), whereas the concentration of IgF with anti-Cs was 18 mg protein ml-1.
Statistical analysis.
In all cases, each experiment was repeated at least three times. Data were statistically analysed by Student's t-test. P values were considered statistically significant if they were less than 0·05.
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RESULTS |
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Resistance to systemic candidiasis is induced in BALB/c mice primed with a pathogenic small fungal inoculum
To evaluate the importance of fungal pathogenicity on priming of mice with Candida blastoconidia, we infected BALB/c mice with 107 pathogenic Candida blastoconidia 1 month after the animals were primed with 106 pathogenic, attenuated or heat-killed blastoconidia.
As shown in Fig. 1(a), mice primed i.p. 1 month before with 106 heat-killed or attenuated C. albicans blastoconidia were as heavily infected as control (non-primed) mice, 5 days after i.p. infection with the large inoculum of pathogenic Candida blastoconidia. In contrast, the numbers of C. albicans c.f.u. recovered from the kidneys were markedly reduced in all mice primed with the small inoculum of pathogenic Candida blastoconidia. As depicted in Fig. 1(b)
, all control mice or mice primed with heat-killed or attenuated Candida blastoconidia were colonized to a similar extent or even more colonized at 15 days after infection than 5 days after the infection. In contrast, all mice primed with the pathogenic inoculum had cleared the fungus 15 days after infection (Fig. 1b
).
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Passive administration of anti-p43 but not of anti-Cs antibodies induces resistance to systemic fungal infection
To further analyse the role of antibodies against p43 in the immunoprotection against systemic candidiasis, the fungal load was evaluated in BALB/c mice that passively received anti-p43, anti-Cs or unrelated antibodies 30 days after priming with heat-killed Candida blastoconidia. The fungal load was markedly decreased in the group of mice that received IgF containing anti-p43 antibodies (1·2x103 c.f.u. in both kidneys) as compared to mice that received IgF containing anti-Cs (1·0x105 c.f.u. in both kidneys) or unrelated antibodies (between 1x104 and 1x105 c.f.u. in both kidneys). Candida cells were no longer detectable in the kidneys of half of the mice treated with IgF anti-p43 antibodies 5 days after infection with the larger fungal inoculum, whereas the fungus was only barely detectable in the remaining half of the mice of this group. In contrast, the majority of mice in each of the two groups that were treated with IgF, containing either anti-Cs or unrelated antibodies, showed a considerably higher fungal load in the kidney on the same day of observation. Even the mice of these two groups that presented a milder fungal infection showed a higher number of Candida cells in the kidneys than that found in mice treated with IgF containing anti-p43 antibodies. The differences between the mice that received the IgF containing unrelated antibodies or anti-p43 were statistically significant (P<0·05; n=6), whereas those between the mice that received IgF containing unrelated antibodies or anti-Cs were not (P>0·1; n=6).
Immunoprotection against systemic candidiasis induced by p43 immunization is neutralized by the passive administration of anti-p43 antibodies at the same time of immunization
To show the crucial role of anti-p43 antibodies against systemic candidiasis in mice, BALB/c mice were immunized with p43 or immunized with p43 and treated with anti-p43 antibodies in order to neutralize the production of anti-p43 antibodies. All mice immunized with p43 and treated with IgF from serum containing specific anti-p43 antibodies became susceptible to the fungal infection, whereas all mice immunized with p43 and treated with IgF from sera of unimmunized mice were immunoprotected against systemic candidiasis. Thus, the c.f.u. count from the kidneys of mice immunized with p43 and treated with anti-p43 antibodies was comparable to that of unimmunized mice [1x105 c.f.u. in both kidneys 5 days after infection with 107 pathogenic Candida blastoconidia; results between these two groups were not significant (P>0·5; n=6)]. Differences between unimmunized mice and those immunized with p43 and passively transferred with IgF with normal serum were statistically significant (P<0·05; n=6).
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DISCUSSION |
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Most likely, the higher increase in the serum antibodies against Cs observed in mice primed with a small inoculum of pathogenic C. albicans was due to the polyclonal B-cell non-specific effect induced by p43 (Tavares et al., 1993). However, the values of the ratio between the anti-p43 and the anti-Cs antibodies seem to be more important than the absolute titres of anti-p43 antibodies to identify immunoprotection against the fungal infection. This is evident when comparing mice primed with heat-killed or attenuated C. albicans with control mice, since the first groups developed higher titres against p43 than the second and were equally susceptible to C. albicans infection. In agreement with these results, we have previously reported that specific immunization against VIPs (but not against other antigenic components of the fungus) results in impaired protection and adoptive transfer of anti-p43 antibodies whereas antibodies against Cs facilitated systemic C. albicans infection (Tavares et al., 1995
). Our results provide further evidence in support of the crucial role of anti-p43 antibodies in the protection against C. albicans infection. Therefore, immunoprotection against candidiasis can be induced in mice primed with an inoculum of heat-killed fungus by treating the mice with anti-p43 antibodies, whereas treatments with anti-Cs or irrelevant antibodies are not protective against the infection. Also, the resistance to systemic candidiasis induced by the active immunization with p43 was abrogated by the neutralization of anti-p43 antibody production.
The role of the humoral immune response in resistance to systemic candidiasis is controversial. In clinical systemic candidiasis, the titres of serum antibodies detected are mainly against Cs, and specific antibodies against C. albicans antigens have been described as non-protective (Elin et al., 1974; Hector et al., 1982
; Mahanty et al., 1988
; Reitnauer et al., 1986
). Moreover, we have previously observed (Tavares et al., 1995
) in experimental systemic candidiasis that passive administration of high titres of anti-Cs antibodies markedly increased the susceptibility to C. albicans infection. Also, as it is well known, high levels of antibody anti-micro-organism epitopes facilitating microbial growth may reflect, in some cases, easier opsonization of the micro-organism in the target phagocytic cells. However, several studies on experimental candidiasis have shown that certain antibodies might be protective (Casadevall, 1995
; Casadevall et al., 1998
; Mathews & Burnie, 1996
). It has also been described that immunization of mice with C. albicans surface mannan complexes or with mannanprotein conjugates induced a protective antibody response that could be passively transferred to naïve animals (Han & Cutler, 1995
, 1997
; Han et al., 1998
, 2000
). It is well known that mannan adhesins responsible for the attachment of C. albicans to the host cells are an important factor in virulence; thus, antibodies specific for the adhesin fraction could be responsible for the protection. We demonstrated, some years ago, that p43 also was an important virulence factor for C. albicans by immunomodulation of the host immune system (Tavares et al., 1993
). Furthermore, a close relationship between the biological effects of p43 and the pathogenicity of C. albicans was reported (Tavares et al., 2000
). In this study, we have demonstrated that the clearance of systemic candidiasis seems to be associated with the development of an immune response against p43, which elicits an effector instead of a suppressive response to the fungus. Immunization against microbial VIPs may also explain the immunoprotection that occurs after the spontaneous healing of an infection with the pathogenic fungus.
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ACKNOWLEDGEMENTS |
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Received 30 July 2002;
revised 18 October 2002;
accepted 31 October 2002.
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