Immunology 1991 74 20-24
Autoantibody to heat-shock protein 90 can mediate protection against systemic candidosis R. C. MATTHEWS, J. P. BURNIE, D. HOWAT,* T. ROWLAND* & F. WALTON* Department of Medical Microbiology, Manchester University Medical School, Manchester and *Celltech Ltd, Slough Acceptedfor publication 3 June 1991
SUMMARY Epitope mapping shows that patients recovering from systemic infection with Candida albicans produce antibodies against both fungal-specific and conserved epitopes of the heat-shock protein (hsp) 90. In a mouse model of systemic candidosis, mortality was halved by prior administration of sera from two infected patients containing antibodies to hsp 90. One of these patients had no other candidal antibodies detectable on immunoblotting. The protective effect was mediated by the immunoglobulin fraction of the immune serum. It was not observed with a normal human serum. A mouse monoclonal antibody raised against one of the conserved peptide epitopes suggested that an autoantibody to hsp 90 could mediate protection against systemic candidosis in the animal model.
INTRODUCTION Heat-shock proteins (hsp) are major targets for the immune system in many infections. As they are highly conserved it has been suggested that cross-reactive immunity to shared epitopes on hsp assures a constantly high level of immunity to a variety of different microbes, bridging the gap between innate immunity and the specific immune response.' However, attempts to induce protective immunity in mice against leprosy and tuberculosis by immunization with mycobacterial hsp 65 failed.2 Immunoblotting shows that patients who recover from systemic candidiosis produce a major antibody response to the 47,000 molecular weight (MW) breakdown product of hsp 90, whereas fatal cases have little antibody or falling titres.3'4 Epitope mapping Candida albicans hsp 90 with patients' sera revealed an epitope, LKVIRK, recognized by all patients with this antibody.5 This epitope was conserved between human and C. albicans hsp 90, indicating that patients recovering from systemic candidiosis commonly produce autoantibodies against self-epitopes. In this paper we show that patients' sera containing these antibodies can protect against systemic candidosis in an animal model. A murine monoclonal antibody raised against the conserved peptide epitope also reduced mortality, indicating that an autoantibody to an hsp can protect against infection.
C. albicans infections who had antibody to the 47,000 MW breakdown product of hsp 90 on immunoblots. Patient number I had multiple blood cultures positive prior to responding to amphotericin B. Patient number 2 had an infected nephrostomy line and positive urine cultures, treated with fluconazole. Also examined was the serum from a normal control with no clinical or cultural evidence of candidal infection and no antibody to the 47,000 MW antigen on immunoblots. Immunoblotting was performed as described previously, with a pressate of C. albicans prepared in an Xpress (LKB, Bromma, Sweden) from the yeast grown overnight on Sabouraud's plates at 37°.3 Briefly, after electrophoresis on a 10% polyacrylamide gel and transblotting onto nitrocellulose, the latter was blocked overnight at 40 in 3% bovine serum albumin (BSA) in buffered saline. It was incubated for 2 hr at room temperature with patient (1:10) or rabbit (1:25) sera diluted in 3% BSA, 0 05% Tween 20. After washing it was incubated for 1 hr in alkaline-phosphatase conjugated anti-human or antirabbit immunoglobulin (Sigma), washed again and stained with NBT-BCIP (Sigma).6 Epitope mapping was performed as previously described5 by the method of Geysen et al.,7 with reagents from CRB, Nantwich Cheshire, U.K. The 395 amino acid residues derived from the sequenced carboxy fragment of C. albicans hsp 90 were synthesized on polyethylene pins as a complete set of overlapping nonapeptides. Peptide 1 consisted of residues 1-9, peptide 2 of residues 2-10, etc. Then the peptides, still coupled to the surface of the pins, were tested against sera by enzyme immunoassay (EIA). Pins were precoated for 1 hr, in microtitre plates containing 1% ovalbumin, 1% BSA in PBS-T (phosphate-buffered saline, 0-1% Tween 20). They were then incubated overnight at 40 in patient (1:80) or rabbit (1:40) antiserum, washed four times with PBS-T and incubated for 1 hr
MATERIALS AND METHODS Patient sera, immunoblotting and epitope mapping Sera were obtained from two patients recovering from severe Correspondence: Dr R. C. Matthews, Dept. of Medical Microbiology, Manchester University Medical School, Oxford Road, Manchester M13 9PT, U.K.
20
Autoantibody to heat-shock protein MW
with horseradish peroxidase-conjugated anti-immunoglobulin (1:1000). After washing again, the pins were immersed for 30 min in ABTS (0 5 mg/ml azino-di-3-ethylbenzthiazodinsulphonate in pH 4-0 citrate buffer with 0-03% hydrogen peroxide) and the A405 measurements taken in an EIA plate reader. Pins were cleaned by sonication. Production of monoclonal antibody and polyvalent antisera A murine monoclonal antibody was raised against the synthesized peptide epitope LKVIRKNIVKKMIE conjugated through cysteine to keyhole limpet haemocyanin (KLH). The peptide was synthesized on an ABI peptide synthesizer model 431A (Applied Biosystems, Warrington, Cheshire, U.K.). BALB/c mice were injected subcutaneously with 50 ,ug of immunogen in sterile complete Freund's adjuvant and thereafter at intervals of 14 days, intraperitoneally with 50 jig immunogen in incomplete Freund's adjuvant until seroconversion. Fusion was performed 4 days after a final intravenous immunization of 50 jig immunogen in sterile physiological saline. Fusion, hybridoma screening, clonal selection and antibody analysis were performed according to standard protocols, essentially as described by St Groth & Scheidegger.8 Selected hybridomas were screened for antibody against the 47,000 MW antigen by immunoblotting against C. albicans. Positive hybridomas were recloned and re-assayed. Rabbit antiserum (R1) was raised against C. albicans by subcutaneous injection with 25 mg pressate mixed with I ml complete Freund's adjuvant. Immunization was repeated at 14 days and serum collected at 28 days. Animal studies C. albicans was grown overnight at 37 in Sabouraud's broth with constant agitation. It was then concentrated 10 times in 15% glycerol in PBS, pH 7 4, divided into I 0-ml aliquots and stored frozen at 70°. As required, aliquots were placed on ice to reduce the stress induced by thawing. A murine model of systemic candidosis was established by injecting I 00 pA of various dilutions of this standardized batch of C. albicans. The following concentrations were examined 5 x 109/ml, I x 109/ml, 5 x 108/ml, 1 x 108/ml and 5 x 107/ml in groups of 48, 36, 24, 30 and 12 mice, respectively. The inoculum selected (100 pil at I x 109/ml) was the minimum dose giving 100% mortality. This challenge dose was then given to mice pretreated with the following: (i) sera from two patients with antibody to the 47,000 MW antigen (patients number 1 and 2, Table 1), collected prior to starting anti-fungal therapy; (ii) the immunoglobulin (Ig) fraction from patient number 1, obtained by 50% ammonium sulphate precipitation; (iii) normal human serum; (iv) the Ig fraction from normal human serum; (v) the monoclonal antibody against epitope LKVIRKNIVKKMIE (CA-Str7-1); (vi) an irrelevant monoclonal antibody L2; and (vii) rabbit antiserum (RI). Patient, rabbit sera and Ig fractions were given as a 0 5 ml bolus 30 min prior to challenge. Monoclonal antibody (3-72 mg/ml) was given as 0-2 ml, 1 hr prior to challenge. Mice were all adult male BALB/c mice. The protection studies were repeated using a less lethal dose of C. albicans. An inoculum of 1 x 107 C. albicans cells was given to two groups with 10 mice in each. One group was pretreated with 0 2 ml monoclonal antibody Ca-Str7-1 I hr before challenge. -
21 12,000
0o, 000 48,000
47,000 40,000
4 3 2 1 Figure 1. Immunoblots of C. albicans grown at 23' (lanes 1 and 3) and 37 (lanes 2 and 4) with the serum from patient number I probed with alkaline phosphatase-conjugated anti-human IgM (lanes 1 and 2) and anti-human IgG (lanes 3 and 4).
RESULTS
Characterization of patients' and rabbit sera Immunoblotting showed that serum from patient number 1 contained antibodies to bands at 40,000, 47,000, 48,000, 60,000 and 92,000 MW (Fig. 1). Serum from patient number 2 only had detectable antibody to the 40,000 and 47,000 MW bands. Epitope mapping of serum from patient number I showed reactivity with multiple epitopes, at 8-13 (DEPAGE), 105-109 (TMSSY), 115-119 (IMKAQ), 287-292 (TAFSKN), 317-322 (LKVIRK), 329-333 (LSREM) and 358-362 (FITDD) amino acid residues from the carboxy terminus.5 In contrast, serum from patient number 2 only had antibody to two epitopes, 8-13 (DEPAGE) and 317-322 (LKVIRK) amino acid residues from the carboxy terminus. The rabbit antiserum (R1) raised against C. albicans recognized numerous bands on the immunoblot, including those at 40,000, 47,000, 60,000 and 92,000 MW. Epitope mapping demonstrated reactivity with epitopes at 8-13 (DEPAGE) and 329-333 (LSREM) amino acid residues from the carboxy terminus.
Characterization of the monoclonal antibody A novel IgG hybridoma cell line (CA-Str7- 1) was produced which, on immunoblotting, detected bands at 34,000, 47,000 and 92,000 MW. Epitope mapping confirmed the monoclonal's reactivity with the epitope it was raised against, LKVIRK, 317322 amino acid residues from the carboxy terminus. Mouse model of systemic candidosis The standardized inoculum had a yeast cell count of 5 x 109 cells/ml and a cell viability of 1 5 x 109 cells/ml. It was found that 100 ,ul of 5 x 109 cells/ml gave 100% mortality at 23 hr postinoculation, 1 x 109 cells/ml gave 100% mortality at 28 hr, 5 x 108 cells/ml gave a 45%/0 mortality (36 hr), 1 x 108 cells/ml gave a 40%/0 mortality (115 hr) and 5 x 107 cells/ml gave an 8%/ mortality (20 hr). Therefore 100 p1 of 1 x 109 cells/ml (1 x 108 cells) were selected as the minimum dose giving 100% mortality.
Mouse protection studies In the first experiment (Fig. 2) three groups of seven mice each were challenged with a lethal dose of 1 x 1 08 cells. The first group
22
R. C. Matthews et al. Table 1. Effects of patients' sera, normal serum, rabbit antiserum and monoclonal antibodies following challenge with a lethal dose of C. albicans
7
R6 z
O/o mortality
llllll
(I I:3
-'
0.-
.1
1
I
0 2 4 6 8 10 12 14 Time (days) post-inoculation
36
Figure 2. Survival ofthree groups of seven mice each after challenge with lethal dose of I x 108 yeast cells. Group 1 (*) was unprotected. Group 2 (0) received serum from patient number I prior to challenge. Group 3 (0) received serum from patient number 2 prior to challenge. a
Agent given before challenge
n
at 24 hr
0 Normal human serum Ig fraction from normal human serum
36 6 6
100 100 100
| 7
43
Patient I serum
P 5
40 50 50 66 100 83
7 6 6 6 6
Patient 2 serum Ig fraction from patient 1 mAb CA-Str7-I mAb L2 Rabbit I antiserum, Rl n = number of mice.
6
,, 5
C,,4 E2
Table 2. Effect of monoclonal antibody CA-Str7-1 following challenge with a less lethal dose of C. albicans
0
> 3
(02 0
% mortality at
6 1 z
b 0
2
4
I
. I ,I I I 6 8 10 12 14 16 18 20 22 Time (days) post-inoculation
I
25
Figure 3. Survival of two groups of six mice each after challenge with a lethal dose of 1 x 108 yeast cells. Group 1 (m) was unprotected. Group 2 (0) received serum from patient number 1 prior to challenge.
Agent given before challenge
n
16 hr
24 hr
0 mAb CA-Str7-1
10 10
40 20
60 20
n = number of mice.
had five dead at 16 hr and the remaining two dead at 18 hr postinoculation. The next group received serum from patient number 1 prior to challenge and had one death at 16 hr, a further two at 23 hr, one at 70 hr, one at 328 hr, one at 336 hr and one alive at 36 days. The next group, receiving serum from patient number 2, had one death at 16 hr, a further two deaths at 23 hr and the remaining four did not die until 70 hr post-inoculation. A repeat experiment (Fig. 3) with two groups of six mice had all six dead at 24 hr in the unprotected group. In the other group, which received serum from patient number 1 before challenge, one mouse died at 18 hr, a further two at 21 hr, one at 45 hr, one at 17 days and one was still alive at 25 days. In the next experiment involving three groups, with six mice in each, the first group received normal human serum 30 min prior to challenge and all six mice were dead at 24 hr. The second group received the immunoglobulin fraction isolated from normal human serum and again all six mice died, four at 18 hr and two at 21 hr. The third group received the immunoglobulin fraction isolated from patient number I and one mouse died at 18 hr, two at 21 hr, two at 45 hr and one was still alive at 25 days. The protective effect of the monoclonal antibody CA-Str7-1 was compared with that of an irrelevant monoclonal antibody, L2. In a control unprotected group all six mice were dead at 19 hr. In the group receiving L2 all six were dead at 19 hr. In the group given Ca-Str7-1, four were dead at 19 hr, one further mouse died at 43 hr and the remaining mouse died at 139 hr. In a subsequent experiment using a less lethal dose of C. albicans
(1 x 107 cells), given to two groups with 10 mice in each, four mice in the unprotected group were dead at 16 hr and a further two died at 23-5 hr. In the group receiving the monoclonal antibody CA-Str7-1 only two died, both at 16 hr postinoculation. These results are summarized, as percentage mortality at 24 hr, in Tables 1 and 2. The rabbit antiserum (RI) had very little affect on mortality, only one out of the six mice surviving to 48 hr. DISCUSSION In this study serum taken from two patients, prior to receiving anti-fungal chemotherapy, who recovered from severe candidal infections, was given prophylactically in an animal model of systemic candidosis. It halved the mortality. This protective effect was mediated by the immunoglobulin fraction of the serum and not observed with normal serum. Previously it has been demonstrated that resistance to murine candidosis is greater in mice given murine immune serum than those given normal serum, but the serum component responsible was not identified.9 Systemic candidosis has a high mortality, up to 75 %, even in patients given anti-fungal therapy.'0 We suggested, on the basis of immunoblotting patients' sera and clinical observation, that antibody to the immunodominant 47,000 MW antigen of C.
23
Autoantibody to heat-shock protein albicans plays a role in protection against systemic candidosis." '2 In systemically infected patients who seroconvert to C. albicans, 92% have antibody to the 47,000 MW band, 40% to a 60,000 MW band, 27% to a 92,000 MW band and 20% to a 40,000 MW band on immunoblots.'3 All survivors produced a major antibody response to the 47,000 MW band whereas fatal cases had little or no antibody or a falling titre. The 47,000 MW antigen circulates in the sera of these patients as a heat-stable breakdown product of larger more heat-labile antigens. It has now been reported by a further three independent groups'4-'6 and appears to be distinct from the 48,000-52,000 MW enolase antigen described by Buckley's group, which has a different pattern of antibody reactivity.7- 19 Antibodies raised against the 47,000 MW antigen cross-reacted with a 92,000 MW antigen. Cloning and sequencing the 47,000 MW antigen revealed that it is a breakdown product of hsp 90.4 C. albicans hsp 90 has an approximate MW of 92,000 on immunoblots. The 40,000 MW band, like the 47,000 MW, appears to be a breakdown product since a rabbit antiserum raised against the carboxy terminal epitope (DEPAGE) crossreacted with the 92,000 and 40,000 MW bands, and all patients with antibody to DEPAGE had antibody to the 40,000 MW antigen.5 Therefore, the only detectable candidal antibody in patient number 2 was to hsp 90. Patient number 1 also had antibodies to the 48,000 and 60,000 MW bands. The latter may correspond to Saccharomyces cerevisiae hsp 60, which shows extensive sequence homology to the hsp 65 family.20 Members of the hsp 65 family are major targets for antibodies in many infections, and elevated antibodies to this protein have recently been demonstrated in patients with superficial candidal infections.2122 A monoclonal antibody to the 60,000 MW antigen of C. albicans was not protective in mice.6 Patient number 1 had antibodies reacting to seven epitopes of hsp 90, four of which (LKVIRK, TAFSKN, KEFED and IMKAQ) showed 100% direct homology with comparable sites on human hsp 90. One epitope (LSREM) showed a high degree of homology with the human counterpart (ISREM), the change from L to I representing a conserved amino acid change. The remaining two epitopes were nearer the less-conserved carboxy terminus, with the more terminal epitope (DEPAGE) showing the least homology with the same site on human hsp 90 (DDDTSR). The other epitope was TMSSY, which compares with TMGYM in human hsp 90. Patient number 2 had antibodies reacting with only two epitopes, the conserved LKVIRK epitope and the candida-specific DEPAGE epitope. Previously we found that all patients with antibody to C. albicans hsp 90 recognized the conserved epitope LKVIRK.s This epitope was not recognized by uninfected control patients or hyperimmune rabbit antisera against C. albicans. The second most commonly recognized epitope, LSERM, also had a high degree of homology with human hsp 90. The C. albicansspecific, carboxy terminal epitope DEPAGE was the third most commonly recognized, by about 50% of patients with antibody to hsp 90. Both epitopes LKVIRK and DEPAGE were immunogenic, whereas the synthesized peptide LSREM was not. The latter may be a carbohydrate binding site, as suggested for this site in human hsp 90.23 Autoantibodies to conserved epitopes of hsp 90 are commonly produced in patients recovering from systemic candidosis, without any clinical evidence of autoimmune sequelae. This is surprising since self-tolerance should lead to conserved
epitopes being weakly immunogenic, if at all. However, T cells to self-epitopes of hsp 65 have been shown to exist in normal individuals.24'25 Hsp 65 is a major target for both antibodies and T cells in mycobacterial infections.26 Autoimmune T cells to mycobacterial hsp 65 have been linked to autoimmune diseases (reviewed in refs 1 and 27) and increased antibodies to hsp have been observed in rheumatoid arthritis (hsp 65), ankylosing spondylitis (hsp 90) and systemic lupus erythematosus (hsp 70, hsp 90 and ubiquitin).28-33 Despite the dominance of hsp in the immune response to infection and its overlap as an important antigen in autoimmunity, symptomatic autoimmune disease is a relatively rare event after infection. In order to determine whether these autoantibodies to C. albicans hsp 90 might be contributing to the protective effect of patients' sera, a monoclonal antibody (CA-Str7- 1) was raised against the synthesized peptide epitope LKVIRK. When given to a group of 10 mice prior to challenge with 1 x 107 yeast cells, mortality at 24 hr fell from 60% to 20%. When mice were given the full lethal dose (1 x 108 cells) this protective effect was less marked but mortality at 24 hr was reduced from 100%, with the L2 monoclonal, to 66% with CA-Str7-1. The rabbit antiserum, which contained antibodies to many different candidal antigens as well as hsp 90, had very little effect on mortality (83%). It reacted with the species-specific carboxy terminal epitope, DEPAGE, and the partially conserved epitope LSREM but not the fully conserved epitope LKVIRK against which CA-Str7-1 had been raised. Controversy as to the protective effect of candidal antibody has sometimes arisen following studies with rabbit antiserum.34'35 This may be due to some species-dependent variability in the immunogenicity of different epitopes of hsp 90. None of four rabbit hyperimmune antisera to C. albicans produced antibody to the epitope LKVIRK.s This could be why RI antiserum had relatively little protective effect, despite the presence of high titre antibody to the 47,000 MW antigen and many other antigens on immunoblotting. The protective effect observed with serum from patient number 2 is therefore more likely to be due to its reactivity with LKVIRK than DEPAGE. Many different arms of the immune system are involved in host defence against C. albicans and their relative importance may depend on the species of the infected host as well as whether the infection is superficial or systemic.'"2 Antibody-mediated protection is likely to involve antibodies against discontinuous epitopes as well as the continuous epitopes detected by epitope mapping. This makes it particularly interesting that a single monoclonal antibody raised against a conserved epitope of hsp 90 should have a protective effect against systemic candidosis.
ACKNOWLEDGMENTS R. C. Matthews is funded by the Wellcome Trust. We thank ABI for synthesis of peptides. REFERENCES 1. KAUFMANN S.H.E. (1990) Heat shock proteins and the immune response. Immunol. Today, 11, 129. 2. WATSON J.D. (1989) Leprosy: understanding protective immunity. Immunol. Today, 10, 218. 3. MATTHEWS R.C., BURNIE J.P. & TABAQCHALI S. (1984) Immunoblot analysis of the serological response in systemic candidosis. Lancet, ;;, 1415.
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4. MATTHEWS R.C. & BURNIE J.P. (1989) Cloning of a DNA sequence encoding a major fragment of the 47 kilodalton stress protein homologue of Candida albicans. FEMS Microbiol. Letts. 60, 25. 5. MATTHEWS R.C., BURNIE J.P. & LEE W. (1991) The application of epitope mapping to the development of a new serological test for systemic candidosis. J. immunol. Meth. (in press). 6. MATTHEWS R.C., BURNIE J.P., Fox A., BASKERVILLE A., WELLS C., STRACHAN S. & CLARK I. (1989) The diagnostic and therapeutic potential of a monoclonal antibody to the 60 kilodalton nuclear antigen of Candida albicans. Serodiag. Immunother. 3, 75. 7. GEYSEN H.M., RODDA S.J., MASON T.J., TRIBBICK G. & SCHOOFS P.G. (1987) Strategies for epitope analysis using peptide synthesis. J. immunol. Meth. 102, 259. 8. DE ST GROTH F. & SCHEIDEGGER D. (1980) The production of monoclonal antibodies: strategy and tactics. J. immunol. Meth. 35, 1.
9. PEARSALL N.N., ADAMS B.L. & BUNNI R. (1978) Immunological responses to Candida albicans III. Effect of passive transfer of lymphoid cells or serum on murine candidosis. J. Immunol. 120, 1176. 10. HORN R.B., WONG B., KIEHN T.E. & ARMSTRONG D. (1985) Fugemia in a cancer hospital: changing frequency, earlier onset and results of therapy. Rev. Infect. Dis. 7, 646. 11. MATTHEWS R.C., BURNIE J.P., SMITH D., CLARK I., MIDGLEY J., CONOLLY M. & GAZZARD B. (1988) Candida and AIDS: evidence for protective antibody. Lancet, i, 253. 12. MATTHEWS R.C. (1988) Immunotherapy of systemic candidiasis. Serodiag. Immunother. 2, 1. 13. MATTHEWS R.C., BURNIE J.P. & TABAQCHALI S. (1987) Isolation of immunodominant antigens from sera of patients with systemic candidiasis and characterization of serological response to Candida albicans. J. clin. Microbiol. 25, 230. 14. AU-YOUNG J.K., TROY F.A. & GOLDSTEIN E. (1985) Serologic analysis of antigen-specific reactivity in patients with systemic candidiasis. Diag. Microbiol. Infect. Dis. 3, 419. 15. NEALE T.J., MUIR J.C. & DRAKE B. (1987) The immunochemical characterisation of circulating immune complex constituents in Candida albicans osteomyelitis by isoelectric focusing, immunoblot and immunoprint. Aust. NZ J. Med. 17, 201. 16. FERREIRA R.P., YU B., NIKI J. & ARMSTRONG D. (1990) Detection of Candida antigenuria in disseminated candidiasis by immunoblotting. J. clin. Microbiol. 28, 1075. 17. STOCKBINE N.A., JARGEN M.T. & BUCKLEY H.R. (1984) Production and characterisation of three monoclonal antibodies to Candida albicans proteins. Infect. Immun. 43, 1012. 18. STROCKBINE N.A., JARGEN M.T., ZWEIBEL S.M. & BUCKLEY H.R. (1984) Identification and molecular weight characterisation of antigens from Candida albicans that are recognised by human sera. Infect. Immun. 43, 715. 19. SAFRANEK W.W. & BUCKLEY H.R. (1990) Characterisation of Candida albicans enolase. Poster Session, 2nd Conterence on Candida and Candidiasis, Philadelphia, USA. 20. READING D.S., HALLBERG R.L. & MYERS A.M. (1989) Characterization of the yeast hsp 60 gene coding for a mitochondrial assembly factor. Nature, 337, 655.
21. WELLER B.I., SIMMONS P.D. & IVANYI L. (1990) Identification of immunodominant antigens of Candida albicans in patients with superficial candidosis. Clin. Immunol. Immunopathol. 54, 347. 22. IVANYI L. & IVANYI J. (1990) Elevated antibody levels to mycobacterial 65 kDa stress protein in patients with superficial candidiasis. J. infect. Dis. 1262, 519. 23. YAMAZAKI M., AKAOGI K., MIWA T., IMAI T., SOEDA E. & YOKOYAMA K. (1989) Nucleotide sequence of a full-length cDNA for 90 kDa heat-shock protein from human peripheral blood lymphocytes. Nucl. Acid Res. 17, 7108. 24. LAMB J.R., BAL V., MENDEZ-SAMPERIO P., MEHLERT A., So A., ROTHBARD J., JINDAL S., YOUNG R.A. & YOUNG D.B. (1989) Stress proteins may provide the link between the immune response to infection and autoimmunity. Int. Immunol. 1, 191. 25. MUNK M.E., SCHOEL B., MODROW S., KARR R.W., YOUNG R.A. & KAUFMANN S.H.E. (1989) T lymphocytes from healthy individuals with specificity to self-epitopes shared by the mycobacterial and human 65-kilodalton heat shock protein. J. Immunol. 143, 2844. 26. KAUFMANN S.H.E., VATH U., THOLE J.E.R., VAN EMBDEN J.D.A. & EMMRICH F. (1987) Enumeration of T-cells reactive with Mycobacterium tuberculosis organisms and specific for the recombinant mycobacterial 64 kilodalton protein. Eur. J. Immunol. 17, 351. 27. COHEN I.R. (1990) Microbial heat-shock protein and autoimmunity. In: New, Antibacterial Strategies (ed. H. C. Neu), p. 263. Churchill Livingstone, New York. 28. BAHR G.M., ROOK G.A.W., AL-SAFFAR M., VAN EMBDEN J., STANFORD J. L. & BEHBEHANI K. (1988) Antibody levels to mycobacteria in relation to HLA type: evidence for non-HLA-linked high levels of antibody to the 65 kD heat shock protein of M. boris in rheumatoid arthritis. Clin. exp. Immunol. 74, 211. 29. MINOTA S., CAMERON B., WELCH W.J. & WINFIELD J.B. (1988) Autoantibodies to the constitutive 73-kD member of the hsp 70 family of heat shock proteins in systemic lupus erythematosus. J. exp. Med. 168, 1475. 30. MINOTA S., KOYASU S., JAHARA I. & WINFIELD J. (1988) Autoantibodies to the heat-shock protein 90 in systemic lupus erythematosus. J. clin. Invest. 81, 106. 31. MULLER S., BRIAN J.P. & VAN REGENMORTEL M.H.V. (1988) Presence of antibodies to ubiquitin during the autoimmune response associated with systemic lupus erythematosus. Proc. natl. Acad. Sci. U.S.A., 85, 8176. 32. LAKOMEK H.J., WILL H., ZECH M. & KRUSKEMPER H.L. (1984) A new serological marker in ankylosing spondylitis. Arthritis Rheum. 27, 961. 33. TSOULFA G., ROOK G.A., VAN EMBDEN J.D., YOUNG D.B., MEHLERT A., ISENBERG D.A., HAY F.C. & LYDYARD P.M. (1988) Raised serum IgG and IgA antibodies to mycobacterial antigens in rheumatoid arthritis. Ann. Rheum. Dis. 48, 118. 34. WINNER H.I. (1956) Immunity in experimental moniliasis. J. Pathol. Bacteriol. 71, 234. 35. AL-DOORY Y. (1970) An immune factor in baboon anti-candida serum. Sabouraudia, 8, 41.
Autoantibody to heat-shock protein 90 can mediate protection against systemic candidosis R. C. MATTHEWS, J. P. BURNIE, D. HOWAT,* T. ROWLAND* & F. WALTON* Department of Medical Microbiology, Manchester University Medical School, Manchester and *Celltech Ltd, Slough Acceptedfor publication 3 June 1991
SUMMARY Epitope mapping shows that patients recovering from systemic infection with Candida albicans produce antibodies against both fungal-specific and conserved epitopes of the heat-shock protein (hsp) 90. In a mouse model of systemic candidosis, mortality was halved by prior administration of sera from two infected patients containing antibodies to hsp 90. One of these patients had no other candidal antibodies detectable on immunoblotting. The protective effect was mediated by the immunoglobulin fraction of the immune serum. It was not observed with a normal human serum. A mouse monoclonal antibody raised against one of the conserved peptide epitopes suggested that an autoantibody to hsp 90 could mediate protection against systemic candidosis in the animal model.
INTRODUCTION Heat-shock proteins (hsp) are major targets for the immune system in many infections. As they are highly conserved it has been suggested that cross-reactive immunity to shared epitopes on hsp assures a constantly high level of immunity to a variety of different microbes, bridging the gap between innate immunity and the specific immune response.' However, attempts to induce protective immunity in mice against leprosy and tuberculosis by immunization with mycobacterial hsp 65 failed.2 Immunoblotting shows that patients who recover from systemic candidiosis produce a major antibody response to the 47,000 molecular weight (MW) breakdown product of hsp 90, whereas fatal cases have little antibody or falling titres.3'4 Epitope mapping Candida albicans hsp 90 with patients' sera revealed an epitope, LKVIRK, recognized by all patients with this antibody.5 This epitope was conserved between human and C. albicans hsp 90, indicating that patients recovering from systemic candidiosis commonly produce autoantibodies against self-epitopes. In this paper we show that patients' sera containing these antibodies can protect against systemic candidosis in an animal model. A murine monoclonal antibody raised against the conserved peptide epitope also reduced mortality, indicating that an autoantibody to an hsp can protect against infection.
C. albicans infections who had antibody to the 47,000 MW breakdown product of hsp 90 on immunoblots. Patient number I had multiple blood cultures positive prior to responding to amphotericin B. Patient number 2 had an infected nephrostomy line and positive urine cultures, treated with fluconazole. Also examined was the serum from a normal control with no clinical or cultural evidence of candidal infection and no antibody to the 47,000 MW antigen on immunoblots. Immunoblotting was performed as described previously, with a pressate of C. albicans prepared in an Xpress (LKB, Bromma, Sweden) from the yeast grown overnight on Sabouraud's plates at 37°.3 Briefly, after electrophoresis on a 10% polyacrylamide gel and transblotting onto nitrocellulose, the latter was blocked overnight at 40 in 3% bovine serum albumin (BSA) in buffered saline. It was incubated for 2 hr at room temperature with patient (1:10) or rabbit (1:25) sera diluted in 3% BSA, 0 05% Tween 20. After washing it was incubated for 1 hr in alkaline-phosphatase conjugated anti-human or antirabbit immunoglobulin (Sigma), washed again and stained with NBT-BCIP (Sigma).6 Epitope mapping was performed as previously described5 by the method of Geysen et al.,7 with reagents from CRB, Nantwich Cheshire, U.K. The 395 amino acid residues derived from the sequenced carboxy fragment of C. albicans hsp 90 were synthesized on polyethylene pins as a complete set of overlapping nonapeptides. Peptide 1 consisted of residues 1-9, peptide 2 of residues 2-10, etc. Then the peptides, still coupled to the surface of the pins, were tested against sera by enzyme immunoassay (EIA). Pins were precoated for 1 hr, in microtitre plates containing 1% ovalbumin, 1% BSA in PBS-T (phosphate-buffered saline, 0-1% Tween 20). They were then incubated overnight at 40 in patient (1:80) or rabbit (1:40) antiserum, washed four times with PBS-T and incubated for 1 hr
MATERIALS AND METHODS Patient sera, immunoblotting and epitope mapping Sera were obtained from two patients recovering from severe Correspondence: Dr R. C. Matthews, Dept. of Medical Microbiology, Manchester University Medical School, Oxford Road, Manchester M13 9PT, U.K.
20
Autoantibody to heat-shock protein MW
with horseradish peroxidase-conjugated anti-immunoglobulin (1:1000). After washing again, the pins were immersed for 30 min in ABTS (0 5 mg/ml azino-di-3-ethylbenzthiazodinsulphonate in pH 4-0 citrate buffer with 0-03% hydrogen peroxide) and the A405 measurements taken in an EIA plate reader. Pins were cleaned by sonication. Production of monoclonal antibody and polyvalent antisera A murine monoclonal antibody was raised against the synthesized peptide epitope LKVIRKNIVKKMIE conjugated through cysteine to keyhole limpet haemocyanin (KLH). The peptide was synthesized on an ABI peptide synthesizer model 431A (Applied Biosystems, Warrington, Cheshire, U.K.). BALB/c mice were injected subcutaneously with 50 ,ug of immunogen in sterile complete Freund's adjuvant and thereafter at intervals of 14 days, intraperitoneally with 50 jig immunogen in incomplete Freund's adjuvant until seroconversion. Fusion was performed 4 days after a final intravenous immunization of 50 jig immunogen in sterile physiological saline. Fusion, hybridoma screening, clonal selection and antibody analysis were performed according to standard protocols, essentially as described by St Groth & Scheidegger.8 Selected hybridomas were screened for antibody against the 47,000 MW antigen by immunoblotting against C. albicans. Positive hybridomas were recloned and re-assayed. Rabbit antiserum (R1) was raised against C. albicans by subcutaneous injection with 25 mg pressate mixed with I ml complete Freund's adjuvant. Immunization was repeated at 14 days and serum collected at 28 days. Animal studies C. albicans was grown overnight at 37 in Sabouraud's broth with constant agitation. It was then concentrated 10 times in 15% glycerol in PBS, pH 7 4, divided into I 0-ml aliquots and stored frozen at 70°. As required, aliquots were placed on ice to reduce the stress induced by thawing. A murine model of systemic candidosis was established by injecting I 00 pA of various dilutions of this standardized batch of C. albicans. The following concentrations were examined 5 x 109/ml, I x 109/ml, 5 x 108/ml, 1 x 108/ml and 5 x 107/ml in groups of 48, 36, 24, 30 and 12 mice, respectively. The inoculum selected (100 pil at I x 109/ml) was the minimum dose giving 100% mortality. This challenge dose was then given to mice pretreated with the following: (i) sera from two patients with antibody to the 47,000 MW antigen (patients number 1 and 2, Table 1), collected prior to starting anti-fungal therapy; (ii) the immunoglobulin (Ig) fraction from patient number 1, obtained by 50% ammonium sulphate precipitation; (iii) normal human serum; (iv) the Ig fraction from normal human serum; (v) the monoclonal antibody against epitope LKVIRKNIVKKMIE (CA-Str7-1); (vi) an irrelevant monoclonal antibody L2; and (vii) rabbit antiserum (RI). Patient, rabbit sera and Ig fractions were given as a 0 5 ml bolus 30 min prior to challenge. Monoclonal antibody (3-72 mg/ml) was given as 0-2 ml, 1 hr prior to challenge. Mice were all adult male BALB/c mice. The protection studies were repeated using a less lethal dose of C. albicans. An inoculum of 1 x 107 C. albicans cells was given to two groups with 10 mice in each. One group was pretreated with 0 2 ml monoclonal antibody Ca-Str7-1 I hr before challenge. -
21 12,000
0o, 000 48,000
47,000 40,000
4 3 2 1 Figure 1. Immunoblots of C. albicans grown at 23' (lanes 1 and 3) and 37 (lanes 2 and 4) with the serum from patient number I probed with alkaline phosphatase-conjugated anti-human IgM (lanes 1 and 2) and anti-human IgG (lanes 3 and 4).
RESULTS
Characterization of patients' and rabbit sera Immunoblotting showed that serum from patient number 1 contained antibodies to bands at 40,000, 47,000, 48,000, 60,000 and 92,000 MW (Fig. 1). Serum from patient number 2 only had detectable antibody to the 40,000 and 47,000 MW bands. Epitope mapping of serum from patient number I showed reactivity with multiple epitopes, at 8-13 (DEPAGE), 105-109 (TMSSY), 115-119 (IMKAQ), 287-292 (TAFSKN), 317-322 (LKVIRK), 329-333 (LSREM) and 358-362 (FITDD) amino acid residues from the carboxy terminus.5 In contrast, serum from patient number 2 only had antibody to two epitopes, 8-13 (DEPAGE) and 317-322 (LKVIRK) amino acid residues from the carboxy terminus. The rabbit antiserum (R1) raised against C. albicans recognized numerous bands on the immunoblot, including those at 40,000, 47,000, 60,000 and 92,000 MW. Epitope mapping demonstrated reactivity with epitopes at 8-13 (DEPAGE) and 329-333 (LSREM) amino acid residues from the carboxy terminus.
Characterization of the monoclonal antibody A novel IgG hybridoma cell line (CA-Str7- 1) was produced which, on immunoblotting, detected bands at 34,000, 47,000 and 92,000 MW. Epitope mapping confirmed the monoclonal's reactivity with the epitope it was raised against, LKVIRK, 317322 amino acid residues from the carboxy terminus. Mouse model of systemic candidosis The standardized inoculum had a yeast cell count of 5 x 109 cells/ml and a cell viability of 1 5 x 109 cells/ml. It was found that 100 ,ul of 5 x 109 cells/ml gave 100% mortality at 23 hr postinoculation, 1 x 109 cells/ml gave 100% mortality at 28 hr, 5 x 108 cells/ml gave a 45%/0 mortality (36 hr), 1 x 108 cells/ml gave a 40%/0 mortality (115 hr) and 5 x 107 cells/ml gave an 8%/ mortality (20 hr). Therefore 100 p1 of 1 x 109 cells/ml (1 x 108 cells) were selected as the minimum dose giving 100% mortality.
Mouse protection studies In the first experiment (Fig. 2) three groups of seven mice each were challenged with a lethal dose of 1 x 1 08 cells. The first group
22
R. C. Matthews et al. Table 1. Effects of patients' sera, normal serum, rabbit antiserum and monoclonal antibodies following challenge with a lethal dose of C. albicans
7
R6 z
O/o mortality
llllll
(I I:3
-'
0.-
.1
1
I
0 2 4 6 8 10 12 14 Time (days) post-inoculation
36
Figure 2. Survival ofthree groups of seven mice each after challenge with lethal dose of I x 108 yeast cells. Group 1 (*) was unprotected. Group 2 (0) received serum from patient number I prior to challenge. Group 3 (0) received serum from patient number 2 prior to challenge. a
Agent given before challenge
n
at 24 hr
0 Normal human serum Ig fraction from normal human serum
36 6 6
100 100 100
| 7
43
Patient I serum
P 5
40 50 50 66 100 83
7 6 6 6 6
Patient 2 serum Ig fraction from patient 1 mAb CA-Str7-I mAb L2 Rabbit I antiserum, Rl n = number of mice.
6
,, 5
C,,4 E2
Table 2. Effect of monoclonal antibody CA-Str7-1 following challenge with a less lethal dose of C. albicans
0
> 3
(02 0
% mortality at
6 1 z
b 0
2
4
I
. I ,I I I 6 8 10 12 14 16 18 20 22 Time (days) post-inoculation
I
25
Figure 3. Survival of two groups of six mice each after challenge with a lethal dose of 1 x 108 yeast cells. Group 1 (m) was unprotected. Group 2 (0) received serum from patient number 1 prior to challenge.
Agent given before challenge
n
16 hr
24 hr
0 mAb CA-Str7-1
10 10
40 20
60 20
n = number of mice.
had five dead at 16 hr and the remaining two dead at 18 hr postinoculation. The next group received serum from patient number 1 prior to challenge and had one death at 16 hr, a further two at 23 hr, one at 70 hr, one at 328 hr, one at 336 hr and one alive at 36 days. The next group, receiving serum from patient number 2, had one death at 16 hr, a further two deaths at 23 hr and the remaining four did not die until 70 hr post-inoculation. A repeat experiment (Fig. 3) with two groups of six mice had all six dead at 24 hr in the unprotected group. In the other group, which received serum from patient number 1 before challenge, one mouse died at 18 hr, a further two at 21 hr, one at 45 hr, one at 17 days and one was still alive at 25 days. In the next experiment involving three groups, with six mice in each, the first group received normal human serum 30 min prior to challenge and all six mice were dead at 24 hr. The second group received the immunoglobulin fraction isolated from normal human serum and again all six mice died, four at 18 hr and two at 21 hr. The third group received the immunoglobulin fraction isolated from patient number I and one mouse died at 18 hr, two at 21 hr, two at 45 hr and one was still alive at 25 days. The protective effect of the monoclonal antibody CA-Str7-1 was compared with that of an irrelevant monoclonal antibody, L2. In a control unprotected group all six mice were dead at 19 hr. In the group receiving L2 all six were dead at 19 hr. In the group given Ca-Str7-1, four were dead at 19 hr, one further mouse died at 43 hr and the remaining mouse died at 139 hr. In a subsequent experiment using a less lethal dose of C. albicans
(1 x 107 cells), given to two groups with 10 mice in each, four mice in the unprotected group were dead at 16 hr and a further two died at 23-5 hr. In the group receiving the monoclonal antibody CA-Str7-1 only two died, both at 16 hr postinoculation. These results are summarized, as percentage mortality at 24 hr, in Tables 1 and 2. The rabbit antiserum (RI) had very little affect on mortality, only one out of the six mice surviving to 48 hr. DISCUSSION In this study serum taken from two patients, prior to receiving anti-fungal chemotherapy, who recovered from severe candidal infections, was given prophylactically in an animal model of systemic candidosis. It halved the mortality. This protective effect was mediated by the immunoglobulin fraction of the serum and not observed with normal serum. Previously it has been demonstrated that resistance to murine candidosis is greater in mice given murine immune serum than those given normal serum, but the serum component responsible was not identified.9 Systemic candidosis has a high mortality, up to 75 %, even in patients given anti-fungal therapy.'0 We suggested, on the basis of immunoblotting patients' sera and clinical observation, that antibody to the immunodominant 47,000 MW antigen of C.
23
Autoantibody to heat-shock protein albicans plays a role in protection against systemic candidosis." '2 In systemically infected patients who seroconvert to C. albicans, 92% have antibody to the 47,000 MW band, 40% to a 60,000 MW band, 27% to a 92,000 MW band and 20% to a 40,000 MW band on immunoblots.'3 All survivors produced a major antibody response to the 47,000 MW band whereas fatal cases had little or no antibody or a falling titre. The 47,000 MW antigen circulates in the sera of these patients as a heat-stable breakdown product of larger more heat-labile antigens. It has now been reported by a further three independent groups'4-'6 and appears to be distinct from the 48,000-52,000 MW enolase antigen described by Buckley's group, which has a different pattern of antibody reactivity.7- 19 Antibodies raised against the 47,000 MW antigen cross-reacted with a 92,000 MW antigen. Cloning and sequencing the 47,000 MW antigen revealed that it is a breakdown product of hsp 90.4 C. albicans hsp 90 has an approximate MW of 92,000 on immunoblots. The 40,000 MW band, like the 47,000 MW, appears to be a breakdown product since a rabbit antiserum raised against the carboxy terminal epitope (DEPAGE) crossreacted with the 92,000 and 40,000 MW bands, and all patients with antibody to DEPAGE had antibody to the 40,000 MW antigen.5 Therefore, the only detectable candidal antibody in patient number 2 was to hsp 90. Patient number 1 also had antibodies to the 48,000 and 60,000 MW bands. The latter may correspond to Saccharomyces cerevisiae hsp 60, which shows extensive sequence homology to the hsp 65 family.20 Members of the hsp 65 family are major targets for antibodies in many infections, and elevated antibodies to this protein have recently been demonstrated in patients with superficial candidal infections.2122 A monoclonal antibody to the 60,000 MW antigen of C. albicans was not protective in mice.6 Patient number 1 had antibodies reacting to seven epitopes of hsp 90, four of which (LKVIRK, TAFSKN, KEFED and IMKAQ) showed 100% direct homology with comparable sites on human hsp 90. One epitope (LSREM) showed a high degree of homology with the human counterpart (ISREM), the change from L to I representing a conserved amino acid change. The remaining two epitopes were nearer the less-conserved carboxy terminus, with the more terminal epitope (DEPAGE) showing the least homology with the same site on human hsp 90 (DDDTSR). The other epitope was TMSSY, which compares with TMGYM in human hsp 90. Patient number 2 had antibodies reacting with only two epitopes, the conserved LKVIRK epitope and the candida-specific DEPAGE epitope. Previously we found that all patients with antibody to C. albicans hsp 90 recognized the conserved epitope LKVIRK.s This epitope was not recognized by uninfected control patients or hyperimmune rabbit antisera against C. albicans. The second most commonly recognized epitope, LSERM, also had a high degree of homology with human hsp 90. The C. albicansspecific, carboxy terminal epitope DEPAGE was the third most commonly recognized, by about 50% of patients with antibody to hsp 90. Both epitopes LKVIRK and DEPAGE were immunogenic, whereas the synthesized peptide LSREM was not. The latter may be a carbohydrate binding site, as suggested for this site in human hsp 90.23 Autoantibodies to conserved epitopes of hsp 90 are commonly produced in patients recovering from systemic candidosis, without any clinical evidence of autoimmune sequelae. This is surprising since self-tolerance should lead to conserved
epitopes being weakly immunogenic, if at all. However, T cells to self-epitopes of hsp 65 have been shown to exist in normal individuals.24'25 Hsp 65 is a major target for both antibodies and T cells in mycobacterial infections.26 Autoimmune T cells to mycobacterial hsp 65 have been linked to autoimmune diseases (reviewed in refs 1 and 27) and increased antibodies to hsp have been observed in rheumatoid arthritis (hsp 65), ankylosing spondylitis (hsp 90) and systemic lupus erythematosus (hsp 70, hsp 90 and ubiquitin).28-33 Despite the dominance of hsp in the immune response to infection and its overlap as an important antigen in autoimmunity, symptomatic autoimmune disease is a relatively rare event after infection. In order to determine whether these autoantibodies to C. albicans hsp 90 might be contributing to the protective effect of patients' sera, a monoclonal antibody (CA-Str7- 1) was raised against the synthesized peptide epitope LKVIRK. When given to a group of 10 mice prior to challenge with 1 x 107 yeast cells, mortality at 24 hr fell from 60% to 20%. When mice were given the full lethal dose (1 x 108 cells) this protective effect was less marked but mortality at 24 hr was reduced from 100%, with the L2 monoclonal, to 66% with CA-Str7-1. The rabbit antiserum, which contained antibodies to many different candidal antigens as well as hsp 90, had very little effect on mortality (83%). It reacted with the species-specific carboxy terminal epitope, DEPAGE, and the partially conserved epitope LSREM but not the fully conserved epitope LKVIRK against which CA-Str7-1 had been raised. Controversy as to the protective effect of candidal antibody has sometimes arisen following studies with rabbit antiserum.34'35 This may be due to some species-dependent variability in the immunogenicity of different epitopes of hsp 90. None of four rabbit hyperimmune antisera to C. albicans produced antibody to the epitope LKVIRK.s This could be why RI antiserum had relatively little protective effect, despite the presence of high titre antibody to the 47,000 MW antigen and many other antigens on immunoblotting. The protective effect observed with serum from patient number 2 is therefore more likely to be due to its reactivity with LKVIRK than DEPAGE. Many different arms of the immune system are involved in host defence against C. albicans and their relative importance may depend on the species of the infected host as well as whether the infection is superficial or systemic.'"2 Antibody-mediated protection is likely to involve antibodies against discontinuous epitopes as well as the continuous epitopes detected by epitope mapping. This makes it particularly interesting that a single monoclonal antibody raised against a conserved epitope of hsp 90 should have a protective effect against systemic candidosis.
ACKNOWLEDGMENTS R. C. Matthews is funded by the Wellcome Trust. We thank ABI for synthesis of peptides. REFERENCES 1. KAUFMANN S.H.E. (1990) Heat shock proteins and the immune response. Immunol. Today, 11, 129. 2. WATSON J.D. (1989) Leprosy: understanding protective immunity. Immunol. Today, 10, 218. 3. MATTHEWS R.C., BURNIE J.P. & TABAQCHALI S. (1984) Immunoblot analysis of the serological response in systemic candidosis. Lancet, ;;, 1415.
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21. WELLER B.I., SIMMONS P.D. & IVANYI L. (1990) Identification of immunodominant antigens of Candida albicans in patients with superficial candidosis. Clin. Immunol. Immunopathol. 54, 347. 22. IVANYI L. & IVANYI J. (1990) Elevated antibody levels to mycobacterial 65 kDa stress protein in patients with superficial candidiasis. J. infect. Dis. 1262, 519. 23. YAMAZAKI M., AKAOGI K., MIWA T., IMAI T., SOEDA E. & YOKOYAMA K. (1989) Nucleotide sequence of a full-length cDNA for 90 kDa heat-shock protein from human peripheral blood lymphocytes. Nucl. Acid Res. 17, 7108. 24. LAMB J.R., BAL V., MENDEZ-SAMPERIO P., MEHLERT A., So A., ROTHBARD J., JINDAL S., YOUNG R.A. & YOUNG D.B. (1989) Stress proteins may provide the link between the immune response to infection and autoimmunity. Int. Immunol. 1, 191. 25. MUNK M.E., SCHOEL B., MODROW S., KARR R.W., YOUNG R.A. & KAUFMANN S.H.E. (1989) T lymphocytes from healthy individuals with specificity to self-epitopes shared by the mycobacterial and human 65-kilodalton heat shock protein. J. Immunol. 143, 2844. 26. KAUFMANN S.H.E., VATH U., THOLE J.E.R., VAN EMBDEN J.D.A. & EMMRICH F. (1987) Enumeration of T-cells reactive with Mycobacterium tuberculosis organisms and specific for the recombinant mycobacterial 64 kilodalton protein. Eur. J. Immunol. 17, 351. 27. COHEN I.R. (1990) Microbial heat-shock protein and autoimmunity. In: New, Antibacterial Strategies (ed. H. C. Neu), p. 263. Churchill Livingstone, New York. 28. BAHR G.M., ROOK G.A.W., AL-SAFFAR M., VAN EMBDEN J., STANFORD J. L. & BEHBEHANI K. (1988) Antibody levels to mycobacteria in relation to HLA type: evidence for non-HLA-linked high levels of antibody to the 65 kD heat shock protein of M. boris in rheumatoid arthritis. Clin. exp. Immunol. 74, 211. 29. MINOTA S., CAMERON B., WELCH W.J. & WINFIELD J.B. (1988) Autoantibodies to the constitutive 73-kD member of the hsp 70 family of heat shock proteins in systemic lupus erythematosus. J. exp. Med. 168, 1475. 30. MINOTA S., KOYASU S., JAHARA I. & WINFIELD J. (1988) Autoantibodies to the heat-shock protein 90 in systemic lupus erythematosus. J. clin. Invest. 81, 106. 31. MULLER S., BRIAN J.P. & VAN REGENMORTEL M.H.V. (1988) Presence of antibodies to ubiquitin during the autoimmune response associated with systemic lupus erythematosus. Proc. natl. Acad. Sci. U.S.A., 85, 8176. 32. LAKOMEK H.J., WILL H., ZECH M. & KRUSKEMPER H.L. (1984) A new serological marker in ankylosing spondylitis. Arthritis Rheum. 27, 961. 33. TSOULFA G., ROOK G.A., VAN EMBDEN J.D., YOUNG D.B., MEHLERT A., ISENBERG D.A., HAY F.C. & LYDYARD P.M. (1988) Raised serum IgG and IgA antibodies to mycobacterial antigens in rheumatoid arthritis. Ann. Rheum. Dis. 48, 118. 34. WINNER H.I. (1956) Immunity in experimental moniliasis. J. Pathol. Bacteriol. 71, 234. 35. AL-DOORY Y. (1970) An immune factor in baboon anti-candida serum. Sabouraudia, 8, 41.
