- Email: [email protected]
Monoclonal antibodies for bacterial infection
15. Neeman, N. et al. (1979). Effect of leukocyte hydrolases on bacteria. XIV. Bacteriolytic effects of human sera, synovial fluids, and purulent exudates on Staphylococcus attreus and Streptococcusfaecalis: Modulation by Cohn's fraction II and by polyelectrolytes. Inflammation 3:379-394. 16. Ohno, N., T. Yadomae, and T. Miyazaki. (1982). Enhancement of autolysis of Streptococcus pneumoniae by lysozyme. Microbiol. Immunol. 26:347-352. 17. Rogers, II. L., II. R. Perkins, and J. B. Ward. (1980). The bacterial autolysins, pp. 437-460. In: Microbial Cell Walls and Membranes. London, Chapman and Hall.
18. Schwab, J. H. and S. II. Ohanian. (1967). Degradation of streptococcal cell wall antigens in vivo. J. Bacteriol. 94:1346 19. Sela, M. N. et al. (1975). The effect of leukocyte hydrolases on bacteria. V. Modification of bacteriolysis by antiinflammatory agents and by cationic and anionic polyelectrolytes. Inflammation 1:57-69. 20. Tomasz, A. (1979). The mechanism of the irreversible antimicrobial effects of penicillins: How beta-lactam antibiotics kill and lyse bacteria. Ann. Rev. Microbiol. 33:113-137. 21. Warren, G. It. and J. Gray. (1965). Effect of sublethal concentrations of penicillin on the lysis of bacteria by
lysozyme and trypsin. Proc. Soc. Exp. Biol. Med. 120:504-511. 22. Wecke, J. et al. (1982). Cell wall degradation of Staphylococcus attreus by lysozyme. Arch. Microbiol. 131"116-123. 23. Westmacott, D. and H. P. Perkins. (1979). The effects of lysozyme on Bacillus cereus 569. Rupture of chains of bacteria and enhancement of sensitivity to lysozyme. J. Gen. Microbiol. 115:1-11. 24. Zeya, H. I. and J. K. Spitznagel. (1966). Cationic proteins of polymorphonuclear leukocyte lysosomes. II. Composition, properties and mechanisms of antibacterial action. J. Bacteriol. 91:755-762.
Monoclonal Antibodies for Bacterial Infection
varies from 60 to 600 ng/ml (8); (b) Each of these assays relies on antisera prepared in animals. Variations in the quality of the antisera will affect the sensitivity and specificity of the assay (19); (c) Each of the assays mentioned uses antisera that is directed against group-specific determinants and, therefore, are not suitable for serotyping streptococcal isolates. The importance of specific antibody in the prevention of streptococcal infection was demonstrated initially by Lancefield et al. (13). Rabbit antisera directed against the major carbohydrate or protein determinants of GBS was protective in mice challenged with whole live organisms. The protective role for antibody in human neonates thus far has been demonstrated only for type III GBS (2, 3). The use of high titer antisera for the treatment of infants with life-threatening infections has been limited by the availability of such material. In 1976, Kohler and Milstein described a new technology for the isolation of hybrid myeloma cell lines (hybridomas), which secrete monoclonal antibody of predetermined specificity (12). These cell lines are immortalized and antibody is easily recovered from supernatant or tumor-stimulated ascitic fluid. Polin and Kennett have prepared monoclonal antibodies that react with the major carbohydrate determinants of types Ia/lc, Ib, II, and III group B streptococcus (15).
The purpose of this report is to describe the diagnostic and therapeutic applications of anti-group B streptococcal monoclonal antibodies, including (a) detection of GBS colonization; (b) identification of GBS antigen in body fluid specimens; and (c) protection against fatal infection in mice.
Richard A. Polin, M.D. Division of Neonatology of The Children's Hospital of Philadelphia Philadelphia, Pennsylvania Mary Catherine Harris, M.D. Department of Pediatrics, University of Pennsylvania School of Medicine Philadelphia, Pennsylvania The emergence of group B streptococcus (GBS) as the major pathogen responsible for neonatal sepsis and meningitis has focused attention both on methods for rapid detection of infection and colonization, as well as alternative methods of treatment for infants with invasive disease (2, 3). Employing standard laboratory techniques, the recovery of GBS from culture specimens requires 2 4 - 4 8 hr. The recent introduction of immunodiagnostic assays (immunofluorescence, counterimmunoelectrophoresis, staphylococcal coagglutination, latex particle agglutination) has permitted rapid detection of whole organisms and streptococcal antigens in body fluid specimens and culture media containing mixed flora (7, 9, 17). There are several major limitations, however, with these assays, including: (a) The minimal concentration of streptococcal antigen that can be reliably detected
© 1983by ElsevierSciencePublishingCo.. Inc.
Production of Monoclonal Antibodies Vaccines of formalinized GBS were prepared from the following Lancefield reference strains: Type Ia-090/14; Type Ib-H36B/60/2; Type Ic-1909/14; Type II-18RS21/67/2; Type IIID136C. BALB/c mice were given three immunizations of formalinized bacteria at weekly intervals. The first immunization consisted of antigen (0.5/ml) in incomplete Freund's adjuvant administered intraperitoneally. The second dose (0.5/ml), diluted in saline, was administered intraperitoneally and subcutaneously. The final injection (0.2/ml) was administered intravenously in a tail vein. Cell fusion and cloning were performed according to previously published methods (11). Hybridomas were tested for binding to types Ia, Ib, Ie, II, and III GBS, and type XIV pneumococcus using a binding immunoassay in which monoclonal antibody bound to bacteria was detected with a sheep anti-mouse immunoglobulin conjugated to peroxidase. For our studies four monoclonal antibodies were
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Plate coated with bacterial antigen
Plate washed
Test sample containing bacterial antigen added with rnonoclonal antibody
Plate washed
Addition of peroxidase antiglobulin conjugate
Plate washed
000 0o
°&
Substrata added; no color produced
Fig,re 1. Enzyme finked monoclonal antibody inhibition assay (ELMIA). Specimen contains GBS antigen.
Figure 2. Monoclonal antibody sandwich enzyme assay. Specimen contains GBS antigen.
Plate coated with antibacterial monoclonal
antibody
Plate washed
Addition of test sample containing baclerial antigen Plate washed
Addition of peroxidase labelled antibacterial monoclonal antibody Plate washed
Addition of substrata, color produced
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chosen that exhibited a high affinity in the enzyme immunoassay for one of the GBS serotypes; anti-GBS Ia/Ic, Ib, II, III. The anti-GBS Ia/Ic, II, and III monoclonal antibodies were ILK, whereas the anti-lb monoclonal antibody was y2aK. To detect GBS antigen in body fluid specimens [blood, urine, or cerebraspinal fluid (CSF)], two different enzyme assays were developed; a competitive enzyme immunoassay (Fig. I) and a sandwich enzyme assay (Fig. 2). In contrast with commercially available assays, both of these tests employed monoclonal antibodies instead of antisera. The protocol for the enzyme linked monoclonal antibody inhibition assay (ELMIA) follows. Briefly, 25 I.tl of a phosphate buffer or 25 Ixl of a body fluid specimen was placed in wells of a polyvinylchloride plate coated with type-specific GBS. Serial dilutions of hybridoma supernatants containing type-specific GBS antibody beginning at 1:4 were added to the wells. The plates were incubated for 2 hr at room temperature and washed three times in phosphate buffered saline (PBS) by immersion flicking. One hundred microliters of peroxidase-labeled anti-mouse IgG was added to each test well and the plates were incubated 2 hr at room temperature. Following the incubations, the wells were washed and two drops of substrate added [10 ml citrate buffer, pH 4.5; 10 mg orthophenylene diamine (2 p.l 13% hydrogen peroxide)]. The color of each test well was evaluated 30 min after substrate was added. A specimen was interpreted as positi,;,e (i.e., containing type-specific GBS antigen) if it inhibited the binding of the type-specific GBS antibody to the bacteria attached to the wells. When antigen was present in the fluid tested and inhibition occurred, the optical density of the solution was significantly less than that of comparable wells containing only antibody. To obtain quantitative readings, all plates were read on an 8-channel photometer. The ELMIA has been used successfully to identify type III GBS native antigen in 20 infants with late onset group B streptococcal meningitis in whom the concentration of antigen
Clinical Immunology Newsletter
Table 1 Analysis o f a C e r e b r o s p i n a l F l u i d C o n t a i n i n g T y p e I I l S t r e p t o c o c c a l A n t i g e n s : P h o t o m e t e r P r i n t o u t Antibody Dihaions
A. B. C. D. E.
Antibody Antibody Antibody Antibody Antibody
+ + + + +
Undihaed
1.'4
1:8
1:16
1:32
1:64
1:128
1:256
6 4 5 5 6
6 3 6 5 6
5 2 5 5 5
4 1 3 3 4
2 1 2 2 3
1 1 I 1 2
1 1 0 0 1
1 0 0 0 1
1
2
3
4
5
6
7
8
PBS diluent CSF ( G B S + ) control CSF control CSF control CSF
Row Number
Four spinal fluids were analyzed in this experiment. The photometer was programmed to compare the optical density of each test well to a reference optical density of substrate reaction mixture containing no enzyme activity. The numbers shown represent the amount of antibody remaining available for reaction with the glutaraldehyde-fixed type III GBS. Row A contained antibody dilutions ranging from 1:4 to 1:256 incubated without any spinal fluid; row B contained spinal fluid from a proven case of GBS meningitis in addition to antibody dilutions; and rows C, D, and E contained antibody plus three spinal fluids from infants without any meningeal infection. In row B there was a significant decrease in the optical density of wells 2 to 4. This spinal fluid was known to contain type 11I GBS antigen.
ranged from 0.8 to 12.8 i.tg/ml. The assay however, was able to detect native antigen at a concentration o f 10 ng/ml; a value 6 - 6 0 times lower than is detectable by conventional assays. A sample printout from an assay o f a single positive spinal fluid is shown in Table 1. Although the E L M I A was sensitive and specific, there were two drawbacks to this method. First of all, the assay did not prove as reliable for urine specimens as it was for spinal fluid. Urine from some individuals who were clinically well contained substances that interfered with the binding o f the monoclonal antibody to the bottom o f the test well. This produced false-positive reactions that could not be prevented by boiling the specimen or adjusting the urine pH. Secondly, the E L M I A is a competitive inhibition assay and, therefore, is not as easily interpreted visually as other assays that have increased color production as their endpoint. The sandwich enzyme assay was developed in response to these criticisms. As shown in Figure 2, Immulon I test plates were coated with 1:250 dilutions of ascites containing type-specific monoclonal antibody. Twenty-five microliters o f a body fluid specimen presumed to contain bacterial antigen or 25 I.tl of T o d d - H e w i t t broth from an actively growing culture o f GBS was placed in the test well. Following a 60-rain incubation, the plates were washed and a 1:100 dilution o f the identical monoclonal antibody conju-
© 1983by ElsevierSciencePublishingCo., Inc.
gated to peroxidase was added to each well. The plates were incubated an additional hour, washed, and substrate was added. A positive body fluid specimen or culture in this enzyme assay turned the substrate a bright yellow color. The sandwich enzyme assay proved as reliable as the E L M I A for detection o f type III GBS native antigen in C S F specimens. In contrast to the E L M I A , however, the sandwich assay detected type III GBS native antigen at a concentration o f 1 ng/ml. Therefore, the sandwich assay was at least tenfold more sensitive than the competitive inhibition assay. The monoclonal antibody sandwich enzyme
assay also proved useful for detection o f streptococcal antigen in T o d d Hewitt broth. The number o f microorganisms detectable with this assay along with the optical densities (450 nm) that correlated with these bacterial counts is shown in Table 2. Therefore, the sandwich assay offers promise as a rapid detection system for women colonized with group B streptococcus during labor. Functional Properties of GBS Monoclonal Antibodies The role of specific antibody in the protection and treatment o f life-threatening GBS infections was demon-
Table 2 Sandwich Enzyme Assay Microorganisms Detected (cfithnl)a
Optical Density b
la
107 5 x 106 l × 106
1;50 1,50 1.180
Ib
107 5 × 106 1 × lO6
1.180 0.733 0.410
II
lO7 5 × lO6 l × 106
1.370 1.170 0.573
10 7
0.990 0.230
GBS Serot)pe
III
5 x 106 I × 106
'~Numbers of GBS detected with the monoclonal antibody sandwich enzyme assay. b Background "control" optical density = 0.056.
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! 55
Table 3 Agglutination GBS Serotype
Monclonal Antibody
Anti-GBS Anti-GBS Anti-GBS Anti-GBS SP2/0 Ag
Ia/Ic Ib II III 14
la
Ib
lc
+ -
+
+
11 111
+
+
+ , Agglutination; - , no agglutination.
strated more than two decades ago by Lancefield (13). During the 1970s, Baker and Kasper demonstrated a similar protective role for type III GBS antibody in human infections (3, 4). To determine whether the anti-GBS antibodies might be useful as therapeutic agents, we have investigated the functional properties (agglutination, complement fixation and opsonization) of each monoclonal antibody and have evaluated the antibodies for their
Table 4
Complement Fixation Sera Monclonal Antibody
Anti-GBS Anti-GBS Anti-GBS Anti-GBS SP2/0 Ag
Untreated
Mg EGTA Treated
Heat-Inactivated
+ + + + -
-
* * * * *
Ia/lc lb II III 14
+ , Antibodies fixed complement, hemolysis absent; - , no complement fixation, hemolysis present; *, both complement pathways inactivated; hemolysis absent.
~ < p <
4 O3 ,J .M
_J
L~ 3 tj
13
%
.02
~±
i
p<.00~
~o
o=
•
p<.05 p<.05
z
~
I
0
A 5-
(~ ..J .J ILl (J
%
2¸ 0
1.56
40 60 TIME (MINUTES)
20
40 60 TIME (MINUTES)
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0
80 B
~
0
C
20
05
80
20
40 60 TIME (MINUTES)
80
Figure 3. (A) Radiolabelled bacterial uptake: Type lc GBS. Resuhs of preincubation with anti-GBS lallc monoclonal antibody ((3) versus SP2 control (0). (B) Radiolabelled bacterial uptake: Type H GBS. Results of preincubation with anti-GBS type H monoclonal antibody (0) versus SP2 control (0). (C) Radiolabelled bacterial uptake: Type 111 GBS. Results of prehwubation with anti-GBS t)pe 111 monoclonal antibody (C)) versus SP2 control (0).
ability to protect mice against lethal group B streptococcal infection (10). Bacterial agglutination was performed by reacting type-specific group B streptococcus with each hybrid myeloma supernatant in a microtiter plate. As illustrated in Table 3, each of the monoclonal antibodies agglutinated GBS of identical serotype specificity, but not bacteria of a different serotype. Complement fixation was evaluated by incubating type-specific monoclonal antibody and serum in each well of a polyvinylchloride plate, and precoated with type-specific GBS of identical specificity to the antibody. After an overnight incubation, sheep red blood cells sensitized with hemolysin were added to the test wells; hemolysis was assessed macroscopically after 30 min. As shown in Table 4, all the monoclonal antibodies were complement fixing. Inhibition of the classical complement pathway (pretreatment of serum with MgEGTA) prevented complement fixation, indicating that the monoclonal antibodies interacted with the classical, but not the alternate, complement pathway. Phagocytic uptake was determined using a radiolabeled bacterial uptake technique (1, 14). Radiolabeled (3Hleucine) group B streptococci were opsonized with monoclonal antibody and incubated with neutrophil monolayers adherent to glass coverslips. AntiGBS Ia/Ic, II, and III monoclonal antibodies were opsonic for GBS of identical serotype specificity (Fig. 3 a - c ) . anti-GBS Ia/Ic antibody, however, was not opsonic for type Ia GBS and the monoclonal antibody against Ib GBS was not an effective opsonin for that serotype. This indicates that certain monoclonal antibodies may not possess the functional characteristics to make them useful as diagnostic or therapeutic reagents. Each monoclonal antibody was also evaluated for its ability to confer protection against fatal GBS infection. BALB/c mice were administered an intracapsular injection of hybridoma cells secreting monoclonal antibody with one of the following specificities: anti-GBS Ia/Ic, Ib, or II. (Type III GBS was not studied because it does not easily infect adult BALB/c mice.)
Clinical Immunology Newsletter
Table 5 Mouse Protection Experiments Antibody Produced by Tumor
GBS SeroO'pe hljected hztraperitoneally la
Ic
lb
H
Anti-GBS Ia/Ic Anti-GBS Ib Anti-GBS II
5/5a -0/5
515 ---
-5/5 --
0/5 -5/5
Control Mice (no tumors)
0/5
0/5
0/5
0/5
specific functional properties (agglutination, complement fixation, and opsonization) that confer protection against experimental infection in mice. Although murine monoclonal antibodies could be purified for administration to septic neonates, recent advances in hybridoma technology offer the possibility of developing a human vaccine to this pathogen.
" Number of survivors/numbertested.
Tumors were visible within 2 wk. After 14 days, mice with tumors and an equal number of controls were challenged with an intraperitoneal injection of live GBS of identical specificity to the tumor. One hundred percent of mice with tumors that were challenged with GBS of identical specificity to the tumor survived (Table 5); controls died within 24 hr. All surviving tumor mice had anti-GBS titers of > I0,000. Discussion Antisera directed against bacterial antigen have several important clinical applications. In the diagnostic microbiology laboratory, antisera can be used to identify bacteria isolated from clinical specimens and have been incorporated into immunoassays to detect bacterial antigens in body fluid specimens. In addition, antisera have been administered to infants with overwhelming infection to decrease neonatal morbidity and mortality. Although conventional immunoassays can detect the presence of GBS antigen before the organism is isolated by the microbiology laboratory, the ELMIA and the sandwich assay offer added sensitivity. These assays can identify GBS antigen reliably at a concentration of 10 ng/ml and 1 ng/ml, respectively. More important, however, monoclonal antibodies can be used to identify women who are colonized with GBS during labor and delivery. Most neonates acquire GBS during passage through the birth canal (5, 6). Infection begins with colonization of the maternal genital tract. Pathogenic bacteria spread upward through the cervix into the amniotic cavity, resulting in amnionitis. Susceptible infants either inhale or
© 1983by ElsevierSciencePuNishingCo.. Inc.
swallow infected amniotic fluid and develop generalized infection. Recent attempts to decrease the incidence of GBS disease have focused on the treatment of maternal carders with penicillin (18). This approach has not been satisfactory because of an inability to predict which women will have genital colonization with GBS during labor and delivery. The sandwich assay, however, should permit detection of GBS colonization within a short period of time and, therefore, identify women at risk for delivering infants with invasive GBS disease. The newborn infant is dependent on active placental transport of maternal IgG for protection against fatal GSB infection. Baker and Kasper demonstrated that the majority of women who deliver infants that are asymptomatically colonized with GBS have demonstrable serum antibodies, whereas infected neonates and their mothers have no detectable titer (2, 3). Newborn infants can be supplied with antibody either by actively immunizing mothers with purified polysaccharide vaccines or by administering sera containing specific antibodies to GBS to mothers or infants (16). Many investigators have suggested the use of plasma or exchange transfusions to treat neonatal sepsis; however, controlled data are lacking. Somatic cell hybridization and hybridoma technology offer the possibility of making unlimited quantities of highly specific antibody against GBS. This antibody has potential use prenatally for mothers who are antibody deficient, or postnatally for the treatment of infected infants. We have demonstrated that the anti-group B streptococcal monoclonal antibodies possess
References 1. AIIred, C. D., A. O. Shigeoka, and H. R. Hill. (1979). Evaluation of group B streptococcal opsonins by radiolabelled bacterial uptake. J. Immunol. Methods 26:355-363. 2. Baker, C. J. and D. L. Kasper (1977). Immunological investigations of infants with septicemia or meningitis due to group B streptococcus. J. Infect. Dis. 136:$98-S105. 3. Baker, C. J. and D. L. Kasper. (1976). Correlation of maternal antibody deficiency with susceptibility to neonatal group B streptococcal infection. N. Engl. J. Med. 294:753-756. 4. Baker, C. J., M. S. Edwards, and D. L. Kasper. (1978). Immunogenicity of polysaccharides from type III group B streptococcus. J. Clin. Invest. 61:1107-1110. 5. Baker, C. J. (1979). Group B streptococcal infection in neonates. Pcdiat. Rev. 1:5-15. 6. Blanc, W. A. (1961). Pathways of fetal and early neonatal infection. J. Pediat. 59:473-496. 7. Edwards, M. S. and C. J. Baker. (1979). Prospective diagnosis of early onset group B streptococcal infection by countercurrent immunoelectrophoresis. J. Pediat. 94:286-298. 8. Edwards, M. S., D. L. Kaspar, and C. J. Baker. (1979). Rapid diagnosis of type III group B streptococcal meningitis by latex particle agglutination. J. Pediat. 95:202-205. 9. Feigin, R. D. et al. (1976). Countercurrentimmunoelectrophoresis of urine as well as of CSF and blood for diagnosis of bacterial meningitis. J. Pediat. 89:773-775. 10. Harris, M. C. et al. (1982). Functional properties of anti-group B streptococcal monoclonal antibodies. Clin. Immunol. Immunopathol. 24:342350. 11. Kennett, R. H. (1980). Enzyme linked antibody assay with cells attached to PVC plates. In: R. H. Kennett, T. J. McKearn, and K. B. Bechtol (eds.), Monoclonal Antibodies. New York, Plenum.
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n ii
12. Kohter, G. and C. Milstein. (1975). Continuous cultures of fused cells making antibody of predefined specificity. Nature 256:495-497. 13. Lancefield, R. C., M. McCarty, and W. N. Everly. (1975). Multiple mouse protective antibodies directed against group B streptococci. J. Exp. Med. 142:165-179. 14. Mandell, G. L. (1975). Effect of temperature on phagocytosis by human polymorphonuclear neutrophils. Infect. Immunol. 12:221-223.
Complement Activation During Fungal Infections Thomas R. Kozel, Ph D. Professor and Chairnzan Department of Microbiology University of Nevada, Reno The reaction products of the complement cascade form essential components of host-parasite interaction in a broad spectrum of infectious diseases. The reaction of microbial antigens with appropriate classes of antibody leads to binding of complement component CI to the antigen-antibody complex and activation of the classical complement pathway. In an alternative mechanism for complement activation, the surfaces of some bacteria, fungi, and erythrocytes of certain species are able to activate the complement cascade without the need for antibody or CI. Recent studies have suggested that the chemical characteristics of these particulate activators determine whether components of the alternative pathway will be focused and stabilized at the cell surface. Regardless of whether activation occurs via the classical or alternative pathways, the reaction products of the ensuing complement cascade have biologic activities that may influence host-parasite interaction. Completion of the cascade may lead to lysis of target cells. Deposition of C3b at the surface of target cells may promote phagocytosis of the target by phagocytes with C3b receptors. The small peptide C3a causes mast cells to de-
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15. Polin, R. A. and R. Kennett. (1980). Use of monoclonal antibodies in an enzyme immunoassay for rapid identification of types II and III group B streptococcus. J. Clin. Microbiol. 11:332-336. 16. Shigeoka, A. O., R. T. Hall, and H. R. ltili. (1978). Blood-transfusion in group B streptococcal sepsis. Lancet 1:636-638. 17. Siegel, J. D. and G. H. McCracken. (1978). Detection of group B streptococcal antigens in body fluids of neonates. J. Pediat. 93:491-492.
18. Steigman, A. J., E. J. Bottone, and B. A. Hanna. (1975). Does intramuscular penicillin at delivery prevent group B beta hemolytic streptococcal disease of newborn infants? J. Pediat. 87:496-497. 19. Thirumoorthi, M. C. and A. S. Dajani. (1979). Comparision of staphylococcal coagglutination, latex agglutination, and countercurrentimmunoelectrophoresis for bacterial antigen detection. J. Clin. Microbiol. 9:28-32.
granulate with subsequent release of pharmacologic mediators, such as histamine. Other soluble cleavage products, such as C5a, are chemotactic for neutrophils and may contribute to inflammatory responses to microbial infections. The potential for activation of the complement cascade by fungi was noted in 1954 when Pillemer et al. (11) reported that zymosan, an insoluble residue of yeast cell walls, could initiate inactivation Of the third component of complement without the need for specific antibody. Since that time, there has been an increased awareness of the role of complement in fungal disease.
Animal studies using complement deficient or depleted mice and guinea pigs have amply demonstrated the importance of an intact complement system for host defense. C5-deficient mice have an increased susceptibility to experimental candidiasis and cryptococcosis (14). In the case of experimental candidiasis, the increased susceptibility to infection was linked to deficient opsonic activity of C5-deficient serum (8). These results are collaborated by studies of guinea pigs decomplemented with purified cobra venom factor. Animals rendered deficient in alternative pathway and terminal complement components had an increased susceptibility to candidiasis (4) and cryptococcosis (2). It should be noted that an intact complement cascade may not be important for all fungal infections, since susceptibility to experimental blastomycosis was not linked to the absence of complement component C5 (10).
O p s o n i z a t i o n of Yeasts Phagocytosis of both Candida albicans and Cryptococcus neoformans is dependent on the presence of heat-labile complement components. Whole yeasts, such as Histoplasma capsulatttm (12), C. albicans (13), and C. neoformans (5), are able to activate the alternative complement pathway. This alternative pathway activation leads to deposition of C3b at the yeast surface, which may act as an opsonic ligand for interaction with phagocyte C3b receptors. Alternative pathway activation without the need for specific antibody plays an important role in natural immunity. There is also evidence that the classical pathway participates in opsonization of C. albicans (16) and C. neoformans (3). Thus, opsonization of these yeasts probably involves C3b activated by both the classical and alternative pathways.
I n f l a m m a t o r y R e s p o n s e to Fungi The ability of fungi to activate complement cleavage fragments that are chemotactic for neutrophils undoubtedly contributes to the inflammatory response to fungi and fungal products. For example, experimental and clinical cutaneous candidiasis is characterized by an accumulation of neutrophilic granulocytes within the epidermis and beneath the stratum corneum (13). Deposits of C3 are observed in association with these lesions (15). It is most likely that these complement deposits are formed as a consequence of alternative pathway activation, because im-
Clinical Immunology Newsletter
18. Schwab, J. H. and S. II. Ohanian. (1967). Degradation of streptococcal cell wall antigens in vivo. J. Bacteriol. 94:1346 19. Sela, M. N. et al. (1975). The effect of leukocyte hydrolases on bacteria. V. Modification of bacteriolysis by antiinflammatory agents and by cationic and anionic polyelectrolytes. Inflammation 1:57-69. 20. Tomasz, A. (1979). The mechanism of the irreversible antimicrobial effects of penicillins: How beta-lactam antibiotics kill and lyse bacteria. Ann. Rev. Microbiol. 33:113-137. 21. Warren, G. It. and J. Gray. (1965). Effect of sublethal concentrations of penicillin on the lysis of bacteria by
lysozyme and trypsin. Proc. Soc. Exp. Biol. Med. 120:504-511. 22. Wecke, J. et al. (1982). Cell wall degradation of Staphylococcus attreus by lysozyme. Arch. Microbiol. 131"116-123. 23. Westmacott, D. and H. P. Perkins. (1979). The effects of lysozyme on Bacillus cereus 569. Rupture of chains of bacteria and enhancement of sensitivity to lysozyme. J. Gen. Microbiol. 115:1-11. 24. Zeya, H. I. and J. K. Spitznagel. (1966). Cationic proteins of polymorphonuclear leukocyte lysosomes. II. Composition, properties and mechanisms of antibacterial action. J. Bacteriol. 91:755-762.
Monoclonal Antibodies for Bacterial Infection
varies from 60 to 600 ng/ml (8); (b) Each of these assays relies on antisera prepared in animals. Variations in the quality of the antisera will affect the sensitivity and specificity of the assay (19); (c) Each of the assays mentioned uses antisera that is directed against group-specific determinants and, therefore, are not suitable for serotyping streptococcal isolates. The importance of specific antibody in the prevention of streptococcal infection was demonstrated initially by Lancefield et al. (13). Rabbit antisera directed against the major carbohydrate or protein determinants of GBS was protective in mice challenged with whole live organisms. The protective role for antibody in human neonates thus far has been demonstrated only for type III GBS (2, 3). The use of high titer antisera for the treatment of infants with life-threatening infections has been limited by the availability of such material. In 1976, Kohler and Milstein described a new technology for the isolation of hybrid myeloma cell lines (hybridomas), which secrete monoclonal antibody of predetermined specificity (12). These cell lines are immortalized and antibody is easily recovered from supernatant or tumor-stimulated ascitic fluid. Polin and Kennett have prepared monoclonal antibodies that react with the major carbohydrate determinants of types Ia/lc, Ib, II, and III group B streptococcus (15).
The purpose of this report is to describe the diagnostic and therapeutic applications of anti-group B streptococcal monoclonal antibodies, including (a) detection of GBS colonization; (b) identification of GBS antigen in body fluid specimens; and (c) protection against fatal infection in mice.
Richard A. Polin, M.D. Division of Neonatology of The Children's Hospital of Philadelphia Philadelphia, Pennsylvania Mary Catherine Harris, M.D. Department of Pediatrics, University of Pennsylvania School of Medicine Philadelphia, Pennsylvania The emergence of group B streptococcus (GBS) as the major pathogen responsible for neonatal sepsis and meningitis has focused attention both on methods for rapid detection of infection and colonization, as well as alternative methods of treatment for infants with invasive disease (2, 3). Employing standard laboratory techniques, the recovery of GBS from culture specimens requires 2 4 - 4 8 hr. The recent introduction of immunodiagnostic assays (immunofluorescence, counterimmunoelectrophoresis, staphylococcal coagglutination, latex particle agglutination) has permitted rapid detection of whole organisms and streptococcal antigens in body fluid specimens and culture media containing mixed flora (7, 9, 17). There are several major limitations, however, with these assays, including: (a) The minimal concentration of streptococcal antigen that can be reliably detected
© 1983by ElsevierSciencePublishingCo.. Inc.
Production of Monoclonal Antibodies Vaccines of formalinized GBS were prepared from the following Lancefield reference strains: Type Ia-090/14; Type Ib-H36B/60/2; Type Ic-1909/14; Type II-18RS21/67/2; Type IIID136C. BALB/c mice were given three immunizations of formalinized bacteria at weekly intervals. The first immunization consisted of antigen (0.5/ml) in incomplete Freund's adjuvant administered intraperitoneally. The second dose (0.5/ml), diluted in saline, was administered intraperitoneally and subcutaneously. The final injection (0.2/ml) was administered intravenously in a tail vein. Cell fusion and cloning were performed according to previously published methods (11). Hybridomas were tested for binding to types Ia, Ib, Ie, II, and III GBS, and type XIV pneumococcus using a binding immunoassay in which monoclonal antibody bound to bacteria was detected with a sheep anti-mouse immunoglobulin conjugated to peroxidase. For our studies four monoclonal antibodies were
0197-1859,'83/S0+02.00
153
Plate coated with bacterial antigen
Plate washed
Test sample containing bacterial antigen added with rnonoclonal antibody
Plate washed
Addition of peroxidase antiglobulin conjugate
Plate washed
000 0o
°&
Substrata added; no color produced
Fig,re 1. Enzyme finked monoclonal antibody inhibition assay (ELMIA). Specimen contains GBS antigen.
Figure 2. Monoclonal antibody sandwich enzyme assay. Specimen contains GBS antigen.
Plate coated with antibacterial monoclonal
antibody
Plate washed
Addition of test sample containing baclerial antigen Plate washed
Addition of peroxidase labelled antibacterial monoclonal antibody Plate washed
Addition of substrata, color produced
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chosen that exhibited a high affinity in the enzyme immunoassay for one of the GBS serotypes; anti-GBS Ia/Ic, Ib, II, III. The anti-GBS Ia/Ic, II, and III monoclonal antibodies were ILK, whereas the anti-lb monoclonal antibody was y2aK. To detect GBS antigen in body fluid specimens [blood, urine, or cerebraspinal fluid (CSF)], two different enzyme assays were developed; a competitive enzyme immunoassay (Fig. I) and a sandwich enzyme assay (Fig. 2). In contrast with commercially available assays, both of these tests employed monoclonal antibodies instead of antisera. The protocol for the enzyme linked monoclonal antibody inhibition assay (ELMIA) follows. Briefly, 25 I.tl of a phosphate buffer or 25 Ixl of a body fluid specimen was placed in wells of a polyvinylchloride plate coated with type-specific GBS. Serial dilutions of hybridoma supernatants containing type-specific GBS antibody beginning at 1:4 were added to the wells. The plates were incubated for 2 hr at room temperature and washed three times in phosphate buffered saline (PBS) by immersion flicking. One hundred microliters of peroxidase-labeled anti-mouse IgG was added to each test well and the plates were incubated 2 hr at room temperature. Following the incubations, the wells were washed and two drops of substrate added [10 ml citrate buffer, pH 4.5; 10 mg orthophenylene diamine (2 p.l 13% hydrogen peroxide)]. The color of each test well was evaluated 30 min after substrate was added. A specimen was interpreted as positi,;,e (i.e., containing type-specific GBS antigen) if it inhibited the binding of the type-specific GBS antibody to the bacteria attached to the wells. When antigen was present in the fluid tested and inhibition occurred, the optical density of the solution was significantly less than that of comparable wells containing only antibody. To obtain quantitative readings, all plates were read on an 8-channel photometer. The ELMIA has been used successfully to identify type III GBS native antigen in 20 infants with late onset group B streptococcal meningitis in whom the concentration of antigen
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Table 1 Analysis o f a C e r e b r o s p i n a l F l u i d C o n t a i n i n g T y p e I I l S t r e p t o c o c c a l A n t i g e n s : P h o t o m e t e r P r i n t o u t Antibody Dihaions
A. B. C. D. E.
Antibody Antibody Antibody Antibody Antibody
+ + + + +
Undihaed
1.'4
1:8
1:16
1:32
1:64
1:128
1:256
6 4 5 5 6
6 3 6 5 6
5 2 5 5 5
4 1 3 3 4
2 1 2 2 3
1 1 I 1 2
1 1 0 0 1
1 0 0 0 1
1
2
3
4
5
6
7
8
PBS diluent CSF ( G B S + ) control CSF control CSF control CSF
Row Number
Four spinal fluids were analyzed in this experiment. The photometer was programmed to compare the optical density of each test well to a reference optical density of substrate reaction mixture containing no enzyme activity. The numbers shown represent the amount of antibody remaining available for reaction with the glutaraldehyde-fixed type III GBS. Row A contained antibody dilutions ranging from 1:4 to 1:256 incubated without any spinal fluid; row B contained spinal fluid from a proven case of GBS meningitis in addition to antibody dilutions; and rows C, D, and E contained antibody plus three spinal fluids from infants without any meningeal infection. In row B there was a significant decrease in the optical density of wells 2 to 4. This spinal fluid was known to contain type 11I GBS antigen.
ranged from 0.8 to 12.8 i.tg/ml. The assay however, was able to detect native antigen at a concentration o f 10 ng/ml; a value 6 - 6 0 times lower than is detectable by conventional assays. A sample printout from an assay o f a single positive spinal fluid is shown in Table 1. Although the E L M I A was sensitive and specific, there were two drawbacks to this method. First of all, the assay did not prove as reliable for urine specimens as it was for spinal fluid. Urine from some individuals who were clinically well contained substances that interfered with the binding o f the monoclonal antibody to the bottom o f the test well. This produced false-positive reactions that could not be prevented by boiling the specimen or adjusting the urine pH. Secondly, the E L M I A is a competitive inhibition assay and, therefore, is not as easily interpreted visually as other assays that have increased color production as their endpoint. The sandwich enzyme assay was developed in response to these criticisms. As shown in Figure 2, Immulon I test plates were coated with 1:250 dilutions of ascites containing type-specific monoclonal antibody. Twenty-five microliters o f a body fluid specimen presumed to contain bacterial antigen or 25 I.tl of T o d d - H e w i t t broth from an actively growing culture o f GBS was placed in the test well. Following a 60-rain incubation, the plates were washed and a 1:100 dilution o f the identical monoclonal antibody conju-
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gated to peroxidase was added to each well. The plates were incubated an additional hour, washed, and substrate was added. A positive body fluid specimen or culture in this enzyme assay turned the substrate a bright yellow color. The sandwich enzyme assay proved as reliable as the E L M I A for detection o f type III GBS native antigen in C S F specimens. In contrast to the E L M I A , however, the sandwich assay detected type III GBS native antigen at a concentration o f 1 ng/ml. Therefore, the sandwich assay was at least tenfold more sensitive than the competitive inhibition assay. The monoclonal antibody sandwich enzyme
assay also proved useful for detection o f streptococcal antigen in T o d d Hewitt broth. The number o f microorganisms detectable with this assay along with the optical densities (450 nm) that correlated with these bacterial counts is shown in Table 2. Therefore, the sandwich assay offers promise as a rapid detection system for women colonized with group B streptococcus during labor. Functional Properties of GBS Monoclonal Antibodies The role of specific antibody in the protection and treatment o f life-threatening GBS infections was demon-
Table 2 Sandwich Enzyme Assay Microorganisms Detected (cfithnl)a
Optical Density b
la
107 5 x 106 l × 106
1;50 1,50 1.180
Ib
107 5 × 106 1 × lO6
1.180 0.733 0.410
II
lO7 5 × lO6 l × 106
1.370 1.170 0.573
10 7
0.990 0.230
GBS Serot)pe
III
5 x 106 I × 106
'~Numbers of GBS detected with the monoclonal antibody sandwich enzyme assay. b Background "control" optical density = 0.056.
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Table 3 Agglutination GBS Serotype
Monclonal Antibody
Anti-GBS Anti-GBS Anti-GBS Anti-GBS SP2/0 Ag
Ia/Ic Ib II III 14
la
Ib
lc
+ -
+
+
11 111
+
+
+ , Agglutination; - , no agglutination.
strated more than two decades ago by Lancefield (13). During the 1970s, Baker and Kasper demonstrated a similar protective role for type III GBS antibody in human infections (3, 4). To determine whether the anti-GBS antibodies might be useful as therapeutic agents, we have investigated the functional properties (agglutination, complement fixation and opsonization) of each monoclonal antibody and have evaluated the antibodies for their
Table 4
Complement Fixation Sera Monclonal Antibody
Anti-GBS Anti-GBS Anti-GBS Anti-GBS SP2/0 Ag
Untreated
Mg EGTA Treated
Heat-Inactivated
+ + + + -
-
* * * * *
Ia/lc lb II III 14
+ , Antibodies fixed complement, hemolysis absent; - , no complement fixation, hemolysis present; *, both complement pathways inactivated; hemolysis absent.
~ < p <
4 O3 ,J .M
_J
L~ 3 tj
13
%
.02
~±
i
p<.00~
~o
o=
•
p<.05 p<.05
z
~
I
0
A 5-
(~ ..J .J ILl (J
%
2¸ 0
1.56
40 60 TIME (MINUTES)
20
40 60 TIME (MINUTES)
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0
80 B
~
0
C
20
05
80
20
40 60 TIME (MINUTES)
80
Figure 3. (A) Radiolabelled bacterial uptake: Type lc GBS. Resuhs of preincubation with anti-GBS lallc monoclonal antibody ((3) versus SP2 control (0). (B) Radiolabelled bacterial uptake: Type H GBS. Results of preincubation with anti-GBS type H monoclonal antibody (0) versus SP2 control (0). (C) Radiolabelled bacterial uptake: Type 111 GBS. Results of prehwubation with anti-GBS t)pe 111 monoclonal antibody (C)) versus SP2 control (0).
ability to protect mice against lethal group B streptococcal infection (10). Bacterial agglutination was performed by reacting type-specific group B streptococcus with each hybrid myeloma supernatant in a microtiter plate. As illustrated in Table 3, each of the monoclonal antibodies agglutinated GBS of identical serotype specificity, but not bacteria of a different serotype. Complement fixation was evaluated by incubating type-specific monoclonal antibody and serum in each well of a polyvinylchloride plate, and precoated with type-specific GBS of identical specificity to the antibody. After an overnight incubation, sheep red blood cells sensitized with hemolysin were added to the test wells; hemolysis was assessed macroscopically after 30 min. As shown in Table 4, all the monoclonal antibodies were complement fixing. Inhibition of the classical complement pathway (pretreatment of serum with MgEGTA) prevented complement fixation, indicating that the monoclonal antibodies interacted with the classical, but not the alternate, complement pathway. Phagocytic uptake was determined using a radiolabeled bacterial uptake technique (1, 14). Radiolabeled (3Hleucine) group B streptococci were opsonized with monoclonal antibody and incubated with neutrophil monolayers adherent to glass coverslips. AntiGBS Ia/Ic, II, and III monoclonal antibodies were opsonic for GBS of identical serotype specificity (Fig. 3 a - c ) . anti-GBS Ia/Ic antibody, however, was not opsonic for type Ia GBS and the monoclonal antibody against Ib GBS was not an effective opsonin for that serotype. This indicates that certain monoclonal antibodies may not possess the functional characteristics to make them useful as diagnostic or therapeutic reagents. Each monoclonal antibody was also evaluated for its ability to confer protection against fatal GBS infection. BALB/c mice were administered an intracapsular injection of hybridoma cells secreting monoclonal antibody with one of the following specificities: anti-GBS Ia/Ic, Ib, or II. (Type III GBS was not studied because it does not easily infect adult BALB/c mice.)
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Table 5 Mouse Protection Experiments Antibody Produced by Tumor
GBS SeroO'pe hljected hztraperitoneally la
Ic
lb
H
Anti-GBS Ia/Ic Anti-GBS Ib Anti-GBS II
5/5a -0/5
515 ---
-5/5 --
0/5 -5/5
Control Mice (no tumors)
0/5
0/5
0/5
0/5
specific functional properties (agglutination, complement fixation, and opsonization) that confer protection against experimental infection in mice. Although murine monoclonal antibodies could be purified for administration to septic neonates, recent advances in hybridoma technology offer the possibility of developing a human vaccine to this pathogen.
" Number of survivors/numbertested.
Tumors were visible within 2 wk. After 14 days, mice with tumors and an equal number of controls were challenged with an intraperitoneal injection of live GBS of identical specificity to the tumor. One hundred percent of mice with tumors that were challenged with GBS of identical specificity to the tumor survived (Table 5); controls died within 24 hr. All surviving tumor mice had anti-GBS titers of > I0,000. Discussion Antisera directed against bacterial antigen have several important clinical applications. In the diagnostic microbiology laboratory, antisera can be used to identify bacteria isolated from clinical specimens and have been incorporated into immunoassays to detect bacterial antigens in body fluid specimens. In addition, antisera have been administered to infants with overwhelming infection to decrease neonatal morbidity and mortality. Although conventional immunoassays can detect the presence of GBS antigen before the organism is isolated by the microbiology laboratory, the ELMIA and the sandwich assay offer added sensitivity. These assays can identify GBS antigen reliably at a concentration of 10 ng/ml and 1 ng/ml, respectively. More important, however, monoclonal antibodies can be used to identify women who are colonized with GBS during labor and delivery. Most neonates acquire GBS during passage through the birth canal (5, 6). Infection begins with colonization of the maternal genital tract. Pathogenic bacteria spread upward through the cervix into the amniotic cavity, resulting in amnionitis. Susceptible infants either inhale or
© 1983by ElsevierSciencePuNishingCo.. Inc.
swallow infected amniotic fluid and develop generalized infection. Recent attempts to decrease the incidence of GBS disease have focused on the treatment of maternal carders with penicillin (18). This approach has not been satisfactory because of an inability to predict which women will have genital colonization with GBS during labor and delivery. The sandwich assay, however, should permit detection of GBS colonization within a short period of time and, therefore, identify women at risk for delivering infants with invasive GBS disease. The newborn infant is dependent on active placental transport of maternal IgG for protection against fatal GSB infection. Baker and Kasper demonstrated that the majority of women who deliver infants that are asymptomatically colonized with GBS have demonstrable serum antibodies, whereas infected neonates and their mothers have no detectable titer (2, 3). Newborn infants can be supplied with antibody either by actively immunizing mothers with purified polysaccharide vaccines or by administering sera containing specific antibodies to GBS to mothers or infants (16). Many investigators have suggested the use of plasma or exchange transfusions to treat neonatal sepsis; however, controlled data are lacking. Somatic cell hybridization and hybridoma technology offer the possibility of making unlimited quantities of highly specific antibody against GBS. This antibody has potential use prenatally for mothers who are antibody deficient, or postnatally for the treatment of infected infants. We have demonstrated that the anti-group B streptococcal monoclonal antibodies possess
References 1. AIIred, C. D., A. O. Shigeoka, and H. R. Hill. (1979). Evaluation of group B streptococcal opsonins by radiolabelled bacterial uptake. J. Immunol. Methods 26:355-363. 2. Baker, C. J. and D. L. Kasper (1977). Immunological investigations of infants with septicemia or meningitis due to group B streptococcus. J. Infect. Dis. 136:$98-S105. 3. Baker, C. J. and D. L. Kasper. (1976). Correlation of maternal antibody deficiency with susceptibility to neonatal group B streptococcal infection. N. Engl. J. Med. 294:753-756. 4. Baker, C. J., M. S. Edwards, and D. L. Kasper. (1978). Immunogenicity of polysaccharides from type III group B streptococcus. J. Clin. Invest. 61:1107-1110. 5. Baker, C. J. (1979). Group B streptococcal infection in neonates. Pcdiat. Rev. 1:5-15. 6. Blanc, W. A. (1961). Pathways of fetal and early neonatal infection. J. Pediat. 59:473-496. 7. Edwards, M. S. and C. J. Baker. (1979). Prospective diagnosis of early onset group B streptococcal infection by countercurrent immunoelectrophoresis. J. Pediat. 94:286-298. 8. Edwards, M. S., D. L. Kaspar, and C. J. Baker. (1979). Rapid diagnosis of type III group B streptococcal meningitis by latex particle agglutination. J. Pediat. 95:202-205. 9. Feigin, R. D. et al. (1976). Countercurrentimmunoelectrophoresis of urine as well as of CSF and blood for diagnosis of bacterial meningitis. J. Pediat. 89:773-775. 10. Harris, M. C. et al. (1982). Functional properties of anti-group B streptococcal monoclonal antibodies. Clin. Immunol. Immunopathol. 24:342350. 11. Kennett, R. H. (1980). Enzyme linked antibody assay with cells attached to PVC plates. In: R. H. Kennett, T. J. McKearn, and K. B. Bechtol (eds.), Monoclonal Antibodies. New York, Plenum.
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12. Kohter, G. and C. Milstein. (1975). Continuous cultures of fused cells making antibody of predefined specificity. Nature 256:495-497. 13. Lancefield, R. C., M. McCarty, and W. N. Everly. (1975). Multiple mouse protective antibodies directed against group B streptococci. J. Exp. Med. 142:165-179. 14. Mandell, G. L. (1975). Effect of temperature on phagocytosis by human polymorphonuclear neutrophils. Infect. Immunol. 12:221-223.
Complement Activation During Fungal Infections Thomas R. Kozel, Ph D. Professor and Chairnzan Department of Microbiology University of Nevada, Reno The reaction products of the complement cascade form essential components of host-parasite interaction in a broad spectrum of infectious diseases. The reaction of microbial antigens with appropriate classes of antibody leads to binding of complement component CI to the antigen-antibody complex and activation of the classical complement pathway. In an alternative mechanism for complement activation, the surfaces of some bacteria, fungi, and erythrocytes of certain species are able to activate the complement cascade without the need for antibody or CI. Recent studies have suggested that the chemical characteristics of these particulate activators determine whether components of the alternative pathway will be focused and stabilized at the cell surface. Regardless of whether activation occurs via the classical or alternative pathways, the reaction products of the ensuing complement cascade have biologic activities that may influence host-parasite interaction. Completion of the cascade may lead to lysis of target cells. Deposition of C3b at the surface of target cells may promote phagocytosis of the target by phagocytes with C3b receptors. The small peptide C3a causes mast cells to de-
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15. Polin, R. A. and R. Kennett. (1980). Use of monoclonal antibodies in an enzyme immunoassay for rapid identification of types II and III group B streptococcus. J. Clin. Microbiol. 11:332-336. 16. Shigeoka, A. O., R. T. Hall, and H. R. ltili. (1978). Blood-transfusion in group B streptococcal sepsis. Lancet 1:636-638. 17. Siegel, J. D. and G. H. McCracken. (1978). Detection of group B streptococcal antigens in body fluids of neonates. J. Pediat. 93:491-492.
18. Steigman, A. J., E. J. Bottone, and B. A. Hanna. (1975). Does intramuscular penicillin at delivery prevent group B beta hemolytic streptococcal disease of newborn infants? J. Pediat. 87:496-497. 19. Thirumoorthi, M. C. and A. S. Dajani. (1979). Comparision of staphylococcal coagglutination, latex agglutination, and countercurrentimmunoelectrophoresis for bacterial antigen detection. J. Clin. Microbiol. 9:28-32.
granulate with subsequent release of pharmacologic mediators, such as histamine. Other soluble cleavage products, such as C5a, are chemotactic for neutrophils and may contribute to inflammatory responses to microbial infections. The potential for activation of the complement cascade by fungi was noted in 1954 when Pillemer et al. (11) reported that zymosan, an insoluble residue of yeast cell walls, could initiate inactivation Of the third component of complement without the need for specific antibody. Since that time, there has been an increased awareness of the role of complement in fungal disease.
Animal studies using complement deficient or depleted mice and guinea pigs have amply demonstrated the importance of an intact complement system for host defense. C5-deficient mice have an increased susceptibility to experimental candidiasis and cryptococcosis (14). In the case of experimental candidiasis, the increased susceptibility to infection was linked to deficient opsonic activity of C5-deficient serum (8). These results are collaborated by studies of guinea pigs decomplemented with purified cobra venom factor. Animals rendered deficient in alternative pathway and terminal complement components had an increased susceptibility to candidiasis (4) and cryptococcosis (2). It should be noted that an intact complement cascade may not be important for all fungal infections, since susceptibility to experimental blastomycosis was not linked to the absence of complement component C5 (10).
O p s o n i z a t i o n of Yeasts Phagocytosis of both Candida albicans and Cryptococcus neoformans is dependent on the presence of heat-labile complement components. Whole yeasts, such as Histoplasma capsulatttm (12), C. albicans (13), and C. neoformans (5), are able to activate the alternative complement pathway. This alternative pathway activation leads to deposition of C3b at the yeast surface, which may act as an opsonic ligand for interaction with phagocyte C3b receptors. Alternative pathway activation without the need for specific antibody plays an important role in natural immunity. There is also evidence that the classical pathway participates in opsonization of C. albicans (16) and C. neoformans (3). Thus, opsonization of these yeasts probably involves C3b activated by both the classical and alternative pathways.
I n f l a m m a t o r y R e s p o n s e to Fungi The ability of fungi to activate complement cleavage fragments that are chemotactic for neutrophils undoubtedly contributes to the inflammatory response to fungi and fungal products. For example, experimental and clinical cutaneous candidiasis is characterized by an accumulation of neutrophilic granulocytes within the epidermis and beneath the stratum corneum (13). Deposits of C3 are observed in association with these lesions (15). It is most likely that these complement deposits are formed as a consequence of alternative pathway activation, because im-
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