Vol. 57, No. 4
INFECTION AND IMMUNITY, Apr. 1989, p. 1113-1118
0019-9567/89/041113-06$02.00/0
Copyright © 1989, American Society for Microbiology
Protective Efficacy of Protein A-Specific Antibody against
Bacteremic Infection Due to Staphylococcus aureus
in an Infant Rat Model
DAVID P. GREENBERG,'* ARNOLD S. BAYER,2 AMBROSE L. CHEUNG,1t AND JOEL I. WARD1
Departments of Pediatrics1 and Medicine,2 Harbor-UCLA Medical Center, University of California at Los Angeles,
Torrance, California 90509
Received 19 September 1988/Accepted 11 January 1989
Staphylococcal protein A (SpA) is a potent antiphagocytic component of the cell wall of most pathogenic
Staphylococcus aureus strains. We studied the in vitro opsonophagocytic and in vivo protective activities of
rabbit immunoglobulin G (IgG) antibody to purified SpA obtained from two unencapsulated S. aureus strains
(Cowan I and 17A). Postimmune serum contained high titers of specific IgG to SpA, as measured by a modified
enzyme-linked immunosorbent assay that blocked nonspecific binding of IgG to SpA. In vitro, both S. ag*reus
strains were efficiently phagocytosed and killed by polymorphonuclear leukocytes in the presence of
nonimmune sera and complement. With one strain (Cowan I), opsonophagocytosis was significantly enhanced
in the presence of SpA antibody, but with the other strain (17A), killing was significantly decreased with
immune serum. We then evaluated the potential protective benefit of SpA antibody in preventing S. aureus
bacteremia in infant rats. Two-day-old rats received saline or various doses of SpA antiserum and were
challenged subcutaneously 1 day later, but even the highest levels of antibody did not significantly reduce
mortality, bacteremia or metastatic infection to lungs or liver (frequency or magnitude). This lack of protective
efficacy was not related to a failure of SpA F(ab')2 to bind to cell surface-exposed epitopes, since F(ab')2
fragments prepared from hyperimmune serum bound avidly to the whole organism in an enzyme-linked
immunosorbent assay.
To evaluate the effect of immunization with SpA antibody
against invasive S. aureus disease, we developed an enzyme-linked immunosorbent assay (ELISA) to quantitate
specific antibody to SpA and modified an infant rat model for
S. aureus bacteremia. The aims of the current study were (i)
to study the effect of specific SpA antibody on in vitro
opsonophagocytosis of S. aureus and (ii) to evaluate the
protective efficacy of SpA antibody in an animal model of S.
aureus bacteremia.
(This study was presented in part at the 27th Interscience
Conference on Antimicrobial Agents and Chemotherapy,
New York, N.Y., 4 to 7 October 1987.)
Staphylococcus aureus is an important cause of serious
nosocomial and community-acquired infections, including
bacteremia, endocarditis, osteomyelitis, septic arthritis, and
pneumonia (3, 27). Populations at risk for staphylococcal
disease include intravenous drug users, surgical patients,
immunocompromised patients, and recipients of prosthetic
heart valves and other implant devices (3, 5, 24, 30). Despite
the current use of bactericidal agents to treat such infections,
the morbidity and mortality remain high (10). In addition,
reports of increased resistance to antistaphylococcal antibiotics have renewed interest in potential immunization strategies for susceptible patients (20).
Previous attempts to prevent S. aureus infection by immunization with toxoid vaccines or whole cells have been
largely unsuccessful (1). However, the feasibility of using
staphylococcal protein A (SpA), the dominant cell wall
protein of the organism, as an immunogen has not previously
been explored. SpA makes up about 7% of the S. aureus cell
wall (12) and is present in over 95% of all strains (13).
Although the precise immunologic and microbiologic functions of SpA are unknown, this protein appears to contribute
to the resistance of the organism to phagocyte-mediated
killing. For example, S. aureus strains with high SpA contents are more resistant to phagocytosis than are strains with
less SpA (23). The antiphagocytic effect is likely due to the
ability of SpA to bind the immunoglobulin G (IgG) of most
mammalian species (including humans) via its Fc-reactive
sites (26). SpA presumably competes with phagocytic cells
for available IgG-Fc sites, thereby diminishing IgG-mediated
opsonization (23).
MATERIALS AND METHODS
Organisms. Two strains of S. aureus were used in this
study: Cowan I (obtained from the American Type Culture
Collection [ATCC 12598]) and 17A (obtained from Per
Oeding [University of Bergen, Bergen, Norway]). Both are
clinical or laboratory strains and were determined to be
nonencapsulated (courtesy of Walter Karakawa, Pennsylvania State University, University Park, Pa.). We chose to
study nonencapsulated strains so as to be able to distinguish
the effects of SpA antibody independently of those of
anticapsular antibody.
SpA. Purified SpA of S. aureus Cowan I was obtained
commercially (Calbiochem-Behring, La Jolla, Calif.), and
SpA from strain 17A was purified in our laboratory as
previously described (6). Briefly, S. aureus was grown in
peptone yeast extract medium (Difco Laboratories, Detroit,
Mich.) at 37°C to the late-log phase (11 h). Whole cells were
harvested by centrifugation, washed with Tris buffer (0.05
M, pH 7.8), and then disrupted with 0.1-mm diameter glass
beads in a Braun homogenizer (B. Braun Co., Melsungen,
Federal Republic of Germany). After the bacterial suspen-
* Corresponding author.
t Present address: The Rockefeller University, New York, NY
10021.
1113
1114
GREENBERG ET AL.
sion was heated to 75°C for 10 min to inactivate autolytic
activity, the cell walls were recovered by differential centrifugation (7) and washed with Tris buffer. This procedure has
been shown to yield cell wall-rich preparations of S. aureus
(7). The cell wall preparation was then extracted twice with
2% Triton X-100 at room temperature for 30 min to remove
residual membrane fractions, pelleted, and then solubilized
with lysostaphin (50 ,ug/ml) at 37°C for 2 h (7). After removal
of the insoluble material by centrifugation, the remaining
supernatant was dialyzed and SpA was further purified on an
IgG-Sepharose affinity column (6).
Immunization with SpA. New Zealand White rabbits were
immunized with purified SpA prepared from Cowan I and
17A. Rabbits received four weekly intramuscular injections
(100 ,ug each) of SpA in Freund complete adjuvant (Sigma
Chemical Co., St. Louis, Mo.), followed 1 week later with a
subcutaneous injection of SpA (100 ,g) without adjuvant.
Animals were bled before immunization and weekly during
the immunization sequence. One month later, rabbits were
bled and the adequacy of the immunization procedure was
proven by demonstration of antibody titers of .1:16 to the
homologous SpA by double immunodiffusion (21).
ELISA. To quantitate specific antibody to SpA, a modified
ELISA was developed. To overcome the nonimmune Fc
binding of IgG to SpA, purified human IgG-Fc fragments
(Protos Laboratories, San Francisco, Calif.) were first used
to block SpA-Fc-binding sites. Human Fc (10 ,ug/ml) was
added to purified SpA of strain Cowan I (130 ng/ml in Tris
buffer, ph 7.4; Calbiochem-Behring) and incubated at room
temperature for 5 min. The SpA-human Fc complex (100 Il)
was then adsorbed onto microtiter wells (Immulon 1;
Dynatech Laboratories, Inc., Alexandria, Va.) after incubation for 2 h at 37°C. The wells were washed three times with
Tris buffer with 0.05% Tween (pH 7.4; Tris-Tween buffer),
and 100 [lI of diluted test serum (10' to 10-5 in Tris-Tween
buffer) was added and incubated in the wells overnight at
4°C. After three additional washes with Tris-Tween buffer,
100 RIu of diluted alkaline phosphatase-conjugated, affinitypurified F(ab')2 goat anti-rabbit IgG-F(ab')2 (Pel-Freeze Biologicals, Rogers, Ark.) was incubated in the wells for 2 h at
37°C. After three additional washes, 100 ,ul of p-nitrophenylphosphate substrate (1 mg/ml in 10% diethanolamine
buffer; Sigma) was added. After 30 to 60 min of incubation at
room temperature, the A405 was measured (Titertek Multiskan; Flow Laboratories, Inc., McLean, Va.). The control
wells included SpA alone, human Fc alone, or rabbit IgG
alone, and these were always negative. For each assay,
adequate blocking of SpA-Fc-binding sites was assessed by
the binding of nonimmune rabbit IgG to SpA alone but not to
the SpA-human Fc complex. SpA antibody was quantitated
by the method of Zollinger and Boslego (31) by using an IgG
standard curve to calculate antibody concentrations in test
serum with the lower limit of detection at 0.01 ,ug/ml.
Binding of F(ab')2 to SpA and whole cells. To further
evaluate specific SpA antibody responses, F(ab')2 fragments
were prepared and isolated from pre- and postimmune sera
as previously described (11). Briefly, immunoglobulin from
the pre- and postimmune sera (immunized with Cowan I
SpA) was precipitated three times with 33% ammonium
sulfate and then dialyzed extensively against sodium acetate
buffer (0.07 M, pH 4.0, in 0.05 M NaCI). Pepsin was then
added (3 mg per 100 mg of pooled immunoglobulin) and
incubated overnight at 37°C. Following pepsin digestion, the
pH was adjusted to 8.0 and the specimen was dialyzed
against phosphate-buffered saline (0.01 M, pH 7.4). A portion of this sample was passed over a G-100 Sephadex
INFECT. IMMUN.
column, and fractions were collected. Fractions containing
F(ab')2 fragments (visualized on sodium dodecyl sulfatepolyacrylamide gel electrophoresis were pooled, and all preand postimmune samples were adjusted for equal concentrations of protein (measured spectrophotometrically by the
optical density at 280 nm).
The relative binding of F(ab')2 fragments prepared from
pre- and postimmune sera to homologous SpA or whole S.
aureus cells was measured by ELISA. For this ELISA, the
conditions were as previously described, except that there
was no need to block SpA-Fc-binding sites with human
IgG-Fc fragments. To assess binding to the whole organism,
the homologous S. aureus strain was grown to the log phase,
washed three times with sterile normal saline, and then heat
killed at 60°C for 2 h. The heat-killed inoculum was adjusted
to -107 (optical density at 540 nm, 1.0), and 100 ,u was
adsorbed to microtiter wells by incubation at 4°C overnight.
Conditions for the rest of the whole-cell ELISA were as
previously described.
Opsonophagocytic assay. A modification of the opsonophagocytoic assay of Hirsch and Strauss was used (17). Fresh
human polymorphonuclear leukocytes (PMNs) were isolated
by Ficoll-Hypaque density centrifugation (Flow Laboratories) and suspended in a balanced salt solution (minimal
essential medium [MEM]; GIBCO Laboratories, Grand Island, N.Y.). This suspension consistently yielded >90%
PMNs with >95% viability shown by trypan blue exclusion.
The PMN suspension was diluted to 108 cells per ml by
hemacytometer count. An overnight culture of S. aureus
was diluted 1:10 in fresh Todd-Hewitt broth (Difco) and
grown to the log phase. Bacterial cells were pelleted, washed
with normal saline, and suspended in MEM to 108 cells per
ml. The following were added to polypropylene tubes
(Fisher Scientific Co., Pittsburgh, Pa.): 106 PMNs (10 RI),
10 RI of test serum, 106 homologous S. aureus cells (10 ,u),
10 RI (diluted 1:5) of infant human cord serum (a single
complement source was used for all assays), and 60 RI of
MEM (total volume, 100 [lI). A potentially important variable in these assays is the concentration of IgG in the
mixture, since high concentrations of IgG create SpA-Fc
interactions and may decrease S. aureus opsonization (23).
Since the IgG concentration can thereby influence the efficiency of phagocytosis, the total IgG concentrations in preand postimmune sera were measured by a standard ELISA
(2) and diluted to maintain equal concentrations before
addition to the opsonic assay. Assays with pre- and postimmune sera were performed in eight opsonic tubes. Control
tubes with S. aureus in the presence of PMNs, test serum, or
complement alone were included in the assays. Assay tubes
were rotated at 37°C, and 10-pA portions were removed at 0,
60, and 120 min for quantitative bacterial cultures; dilutions
were performed in sterile distilled water to lyse PMNs. The
percentage of bacterial survival was defined at each time
point as (number of viable bacterial/original inoculum) x
100.
Animal model. Outbred, pregnant, pathogen-free SpragueDawley rats (Charles River Breeding Laboratories, Inc.,
Wilmington, Mass.) were used to evaluate the pharmacokinetics and protective efficacy of SpA antibody. Two-day-old
infant rats were given intraperitoneal passive immunization
with various concentrations of rabbit anti-SpA hyperimmune
serum or saline. The mean SpA antibody concentrations in
infant rat sera measured by ELISA over time were as
follows: preimmunization <0.5 ,ug/ml; 6 h postimmunization, 20.5 jig/ml; 24 h postimmunization, 39.9 p.g/ml; 48 h
postimmunization, 27.7 ,ug/ml; 72 h postimmunization, 21.9
VOL. 57, 1989
-
PROTEIN A ANTIBODY IN AN S. AUREUS ANIMAL MODEL
,ug/ml. Since SpA antibody levels were maximal at 24 h
postimmunization, we challenged the animals with the homologous live S. aureus strain 24 h after administration of
antibody in all subsequent studies. Preliminary studies demonstrated that the 90% lethal dose for strain 17A in this
animal model was 108 CFU. In addition, pilot experiments
revealed that subcutaneous administration of 108 CFU of S.
aureus 17A resulted in consistent bacteremia within 24 h and
death of -90% of the animals within 4 days postchallenge.
Animals challenged with smaller inocula of this strain frequently had only localized infections and did not develop
bacteremia or die. In contrast, studies with Cowan I showed
this strain to be somewhat less virulent than strain 17A, with
lower mortality rates, despite persistent bacteremia and
infection of visceral organs following a similar subcutaneous
challenge. Pilot studies also confirmed that 3-day-old (versus
4- to 7-day-old) infant rats were maximally susceptible to S.
aureus bacteremia following subcutaneous challenge.
At 2 days after birth, infant rats were randomly assigned to
receive an intraperitoneal dose of rabbit SpA antiserum or
normal saline (0.05 ml). At 24 h after passive immunization
with antiserum or saline, each rat pup was bled (100 [.l) and
SpA antibody was quantitated by ELISA. All of the pups
were then challenged subcutaneously with -10' salinewashed log-phase cells of the homologous S. aurelis strain.
At 24 h after bacterial challenge, each live pup was bled (100
RlI) for quantitative bacterial culture. Mortality rates were
recorded and tabulated for 4 days postchallenge. After death
or sacrifice on day 4, quantitative cultures of rat lungs and
livers were obtained, since these organs represented common sites of metastatic infection observed in our pilot
studies with this model (D. P. Greenberg, A. L. Cheung, J.
Peters, A. S. Bayer, and J. I. Ward, Program Abstr. 27th
Intersci. Conf. Antimicrob. Agents Chemother. abstr. no.
510, 1987). Lung and liver tissues were removed aseptically,
weighed, homogenized, serially diluted in normal saline, and
cultured quantitatively in Mueller-Hinton agar.
Statistics. For opsonophagocytic assays, the percentages
of bacterial survival at 60 and 120 min were compared
between the pre- and postimmune sera with a two-tailed
Student t test and with the Mann-Whitney-Wilcoxon rank
test. For the protection studies, the frequencies of bacteremia, metastatic organ infection, and death among the treatment groups were compared by a two-tailed Fisher exact
test. The bacterial counts of blood and organ cultures in the
different treatment groups were compared by a two-tailed
Student t test and by nonparametric tests. P values of s0.05
were considered significant.
RESULTS
SpA antibody ELISA. Figure 1 shows a representative
dilution curve for rabbit antibody to Cowan I SpA in pre- and
postimmune whole sera by ELISA. At serum dilutions
greater than 1:10, SpA antibody was not detected in preimmune serum (or in pooled normal rabbit IgG preparations;
data not shown). In contrast, SpA antibody was measured in
immune rabbit serum at titers as great as 1:10,000 (Fig. 1).
Antibody to Cowan I SpA bound similarly to ELISA wells
with purified 17A SpA and to wells with Cowan I SpA (data
not shown). Inhibition studies with purified homologous SpA
removed all detectable antibody from immune serum (Fcand Fab-binding antibody; data not shown). SpA antibody
levels in postimmune rabbit sera were typically in the range
of 500 to 2,000 ,ug of IgG equivalent per ml. The SpA
antibody level of a single test serum was reproducible with
'15% day-to-day variability.
E
0
2.0
-
0 -0 Preimmune Serum
A - -A Postimmune Serum
1.8
1.6
X
1.4
0Ln
1.2
z
1.0
0.8
Li
1115
A
0.6
n-
o
0.42\
0.2-A
--
0.0
10-1
i0-2
10-3
10-
io-5
SERUM DILUTION
FIG. 1. Representative dilution curves by ELISA of pre- and
postimmune sera from a rabbit immunized with purified SpA. The
optical density (OD) is proportional to concentrations of antibody to
SpA. With an IgG standard curve, 500 to 2,000 ,ug of SpA IgG
antibody per ml was detected in postimmune rabbit sera (data from
four rabbits).
Binding of F(ab')2 to SpA and whole cells. Figure 2 shows
the relative binding of F(ab')2 fragments prepared from preand postimmune sera to Cowan I SpA and whole cells.
Preimmune F(ab')2 bound minimally or not at all to these
antigens, whereas postimmune F(ab')2 bound to SpA and
whole cells avidly and could be measured easily to a 1:1,000
dilution.
Opsonophagocytic assays. S. aureus Cowan I and 17A were
readily phagocytized and killed in the presence of pre- and
postimmune sera and complement (Table 1), although neither strain was killed in the presence of PMNs alone (without
serum and complement) or heat-inactivated serum alone
(without PMNs and complement). Strain Cowan I was
phagocytized and killed more efficiently with postimmune
serum than with preimmune serum (P < 0.002 at 60 and 120
min). However, strain 17A was phagocytized and killed
more efficiently with preimmune serum than with postimmune serum (P < 0.005 at 60 and 120 min).
Animal studies. Table 2 shows the results of passive
protection studies with rabbit immune serum to SpA (17A
and Cowan I) in infant rats, assessing the incidences of S.
aureus bacteremia, metastatic infection, and death. As expected, high levels of SpA antibody were measured in the
sera of infant rats passively immunized with immune rabbit
serum; lower concentrations of SpA antibody were observed
in rats given diluted immune serum. S. aureus bacteremia
and death were observed in most animals challenged with
strain 17A, irrespective of passive immunization with SpA
antiserum. Mean bacterial counts in blood and quantitative
lung-liver cultures were also similar among all of the groups
challenged with strain 17A. Thus, despite high levels of SpA
antibody, bacteremia, visceral-organ dissemination, and
death were not prevented in this model.
As noted in our pilot studies, strain Cowan I resulted in a
less lethal infection than did strain 17A, with few animals
dying postchallenge, irrespective of immunization status or
development of bacteremia (Table 2). It is interesting that
despite high SpA antibody levels, significantly more passively immunized than saline-treated rats developed bacteremia (P <0.05). Immunized and unimmunized animals also
had similar rates of metastatic lung-liver S. aureus infections.
1116
GREENBERG ET AL.
2.0
INFECT. IMMUN.
-
*-A SpA-immune F(ob')
0-*Preimmune F(ob')
1.8 1
1.6-
B
*-*SpA-immune F(ab')2
I
E lb
-
c
Ln 1.4-_ _
r) 1.2
U
_4_
-
f
0)
c) 0.8
-
4-
-
_
4-
o 0.6-_4-
o 0.6 -
.
0U.,+
O
-
0.20.0
-
_
1.01.0 -
_4-
-
1.4-
0*
CO 1.2
4-
0
:t 1.0
*- *Preimmune F(ab')
1.8 +-
_
n A -_
U.4
0.2 -1
10b
10
-2
F(aob' )2
1
3
10t
Dilution
1'-4
10
0.0
-
I
10
-
1072
F(ab')2
10CF3
1
(-4
Dilution
FIG. 2. Relative binding by ELISA of pre- and postimmune F(ab')2 fragments to purified SpA (A) or S. aureus whole cells (B). OD, Optical
density.
DISCUSSION
Humans and most vertebrate animals possess a high
degree of natural immunity to S. aureus infection (1); antibodies to teichoic acid (4), peptidoglycan (29), and alphatoxin (15) have been detected in infected and noninfected
individuals. Despite this natural immunity, S. aureus infections continue to be a difficult and common clinical problem
for certain patients (3, 5, 24, 30). Strategies for preventing S.
aureus infection have included immunization with staphylococcal toxins and cell wall antigens, such as coagulase,
alpha-toxin, beta-toxin, and whole cells, yielding disappointing or conflicting results (1).
SpA, known to play a role in antistaphylococcal host
defense mechanisms, has not previously been evaluated as a
TABLE 1. Opsonophagocytocytosis of S. aureus
with SpA antiseraa
Mean
t
SEM % survival
Strain and serum
60 min
Cowan I
PMN alone
Preimmune alone
Preimmune (PMN + C,)b
Postimmune alone
Postimmune (PMN + C')
131
64
33
87
7.1
± 13.0
± 4.5
± 3.6
+ 24
± 0.8c
120 min
62 ± 1.3
50 ± 3.3
9.9 ± 1.6c
316 ± 53
1.9 ± 0.3c
17A
PMN alone
Preimmune alone
Preimmune (PMN + C')
Postimmune alone
Postimmune (PMN + C')
111 ± 47
117 + 27
30 ± 2.7d
139 ± 25
56 5.2d
63
241
8.8
343
28
± 5.1
± 61
+ 0.7d
± 17
± 4.8d
a Two or three assays were done with PMN or serum alone, and eight
assays were done with pre- or postimmune serum plus PMN and complement.
Assays designated PMN alone were done without serum and complement;
assays designated pre- or postimmune alone were done without PMN and
complement.
b
PMN + C', PMN and complement source included.
Preimmune versus immune; P < 0.002 at 60 and 120 min.
d Preimmune versus immune; P < 0.005 at 60 and 120 min.
c
potential protective immunogen against bacteremic S. aureus infection. SpA, found in the cell walls of nearly all
pathogenic S. aureus strains (13), avidly binds IgG and other
immunoglobulins of mammalian species by its Fc-reactive
sites (four sites per molecule) (14, 26). When added to fresh
serum, SpA activates and depletes complement primarily via
the classical pathway (28). In addition, cell-bound SpA
inhibits staphylococcal phagocytosis by human neutrophils;
in the presence of fresh human serum, S. aureus strains with
greater amounts of SpA are more resistant to phagocytosis
than are strains with less SpA (23). This phenomenon is
likely due to cell-bound SpA, which binds IgG (Fc fragment)
and thereby reduces available IgG-Fc sites for antibodymediated opsonization. Furthermore, when low levels of
soluble SpA are present in serum containing complement,
phagocytosis is inhibited (9); this phenomenon is probably
related to the formation of soluble SpA-IgG complexes or
random SpA-mediated complement activaiton. This latter
event renders complement unavailable for activation at the
bacterial surface, an important step in the opsonophagocytosis of S. aureus.
We recently evaluated the potential protective efficacy of
active immunization with whole cells of S. aureus for the
prevention of bacteremia and endocarditis in rabbits (16).
Whole-cell-induced S. aureus antibody did not prevent or
modify any stage of the development of endocarditis in
rabbits, including clearance of bacteremia, attachment of
bacteria to aortic valves, or metastatic renal infection. We
observed, however, that the SpA antibody response induced
by whole-cell immunization was less than 10% of that which
occurred with active immunization with purified SpA (16).
Our hypothesis for these studies was that specific SpA
antibody might block nonimmune Fc binding of IgG to S.
aureus and thereby block the antiphagocytic characteristics
of cell-bound SpA. We postulated that high concentrations
of SpA-specific Fab-binding antibody might prevent the
ability of SpA to inhibit phagocytosis and thus enhance
phagocytosis by classic antigen (SpA)-antibody (anti-SpA)
immune complex mechanisms. We were encouraged in this
regard by the study of Pankey et al. which demonstrated the
VOL. 57, 1989
PROTEIN A ANTIBODY IN AN S. AUREUS ANIMAL MODEL
1117
TABLE 2. Infant rat protection studies with antibody to SpA
Challenge
strain and
infant rat group
(no. of rats)
17A
1 (11)
2 (10)
3 (12)
4 (12)
5 (12)
Cowan I
1 (12)
2 (10)
(kg) of
i..SpA
Amt
antibod
28
2.8
0.28
0.028
OC
114
OC
Arithmetic
mean
SpA antibody
level at 24 h
postdose (p.g/ml)
No. of positive
bodcultures!
blood cultures/
13.6
0.98
0.09
<0.01
<0.01
42.8
<0.01
Geometric mean ± SEM CFU/g in
positive cultures
Lung
10/11
9/9
6/11
8/12b
12/12
7/12b
1/10
5.09
5.43
4.95
4.89
5.73
±
±
±
±
O0.26a
0.50
0.16
0.25
0.29
4.01 ± 0.55a
3.25 ± 0.32
No. of
deaths/total
Liver
5.25
6.37
4.93
5.10
5.73
± 0.30a
± 0.83
± 0.30
± 0.31
± 0.22
3.86 ± 0.47a
2.73 ± 0.37
10/11la
10/11
12/12
10/12
12/12
1/12"
1/10
a For each strain, no significant differences were found between any of the groups.
b
The number of positive blood cultures in this group differed significantly from that of the saline group (P < 0.05; Fisher exact test).
c Saline control.
limited benefit of active immunization with SpA for the
prevention of bovine mastitis (a localized infection without
bacteremia) (22); immunized cows had a significantly higher
spontaneous cure rate of experimentally induced S. aureus
mastitis than did unimmunized cows.
In this study, we found that complement-mediated opsonophagocytosis of two unencapsulated strains of S. aureus
was effective in the presence of pre- or postimmune rabbit
serum. Other investigators have also demonstrated active
phagocytosis and killing of unencapsulated S. aureus when
complement and normal serum were present (1). Our demonstration of greater opsonophagocytosis of strain 17A with
preimmune serum compared with postimmune serum is not
easily explained but is relatively unimportant, since killing
was effective with either serum.
Despite effective in vitro opsonophagocytosis of S. aureus
with immune SpA antiserum, this antiserum, when passively
administered to rats, failed to modify any stage in the
development of bacteremic infection following subcutaneous
challenge with a homologous strain. One potential explanation for the lack of protective efficacy in this model might be
that the antibody elicited by active immunization with purified SpA was specific for SpA epitopes not surface exposed
on the intact organism. We evaluated this prospect by
demonstrating that immune F(ab')2 fragments bound avidly
to whole S. aureus cells at titers 100- 1,000-fold greater than
those of nonimmune F(ab')2 fragments. Nevertheless, it
remains possible that polyclonal antibody does not recognize
important SpA-associated epitopes which are surface exposed in vivo. It is also possible that the lack of protective
efficacy in this animal model can be explained by relative
differences in the binding affinities of F(ab')2 and Fc to SpA.
If Fc binds to SpA with significantly greater avidity than
does F(ab')2, then irrespective of the presence of a specific
antibody, Fab-binding sites on SpA would be concealed in a
complex of Fc-bound IgG. Indeed, this could be the primary
biologic function of SpA, thereby enabling S. aureus to
escape opsonophagocytic killing in vivo (23). With about
80,000 Fc-binding sites per organism (19), SpA binding of
IgG nonspecifically may conceal other important cell surface
antigens with a blanket of Fc-bound IgG and protect these
antigens from host defense mechanisms.
Hyperimmune SpA antiserum to strain Cowan I was
associated with highly efficient opsonophagocytosis and
killing of the homologous strain in vitro. However, animals
given passively administered Cowan I SpA antiserum paradoxically had a significantly greater frequency of positive
blood cultures than did saline-immunized controls. This
suggests a detrimental influence of specific hyperimmune
serum in vivo. The reason for this deleterious effect of
hyperimmune serum on the clearance of bacteremia is unknown, but the effect may be due to blockade of the
reticuloendothelial system by excess immunoglobulin. In
this regard, Derby and Rogers have demonstrated impaired
bloodstream clearance of staphylococci after pharmacological blockade of the reticuloendothelial system (i.e., by
Thorotrast [8]). Also, others have shown a detrimental effect
following high-dose immunoglobulin administration in animals challenged with group B streptococci (18), Escherichia
coli (18), and Haemophilus influenzae (J. Schreiber, C.
Basker, C. Priehs, and G. Siber, Prog. Soc. Ped. Res.
21:334A, 1987). Alternatively, SpA antibody may directly
block S. aureus killing, as has been observed with Neisseria
gonorrhoeae, in which IgG to protein III blocked killing of
the organism by other specific antibodies (25).
ACKNOWLEDGMENTS
This work was supported in part by American Heart Association
(Greater Los Angeles Affiliate) grant 853-G1-1 and by Public Health
Service training grant 5T32 HD07245 from the National Institutes of
Health.
We thank Troy Anderson and Debra Turner for technical assistance, Chung-Yin Chiu for statistical assistance, and Mary Magee
for secretarial support.
LITERATURE CITED
1. Adlam, C., and C. S. F. Easman. 1983. Immunity and hypersensitivity to staphylococcal infection, p. 275-323. In C. S. F.
Easman and C. Adlam (ed.), Staphylococci and staphylococcal
infections. Academic Press, Inc., New York.
2. Anthony, B. F., N. F. Concepcion, and K. F. Concepcion. 1985.
Human antibody to group-specific polysaccharide of group B
Streptococcus. J. Infect. Dis. 151:221-226.
3. Bayer, A. S. 1982. Staphylococcal bacteremia and endocarditis.
Arch. Intern. Med. 142:1169-1177.
4. Bayer, A. S., D. B. Tillman, N. Concepcion, and L. B. Guze.
1980. Clinical value of teichoic acid antibody titers in the
diagnosis and management of the staphylococcemias. West. J.
Med. 132:294-300.
5. Chambers, H. F., 0. M. Korzeniowski, and M. A. Sande. 1983.
Staphylococcus aureus endocarditis: clinical manifestations in
addicts and non-addicts. Medicine 62:170-177.
6. Cheung, A. L., A. S. Bayer, J. Peters, and J. I. Ward. 1987.
Analysis by gel electrophoresis, Western blot, and peptide
mapping of protein A heterogeneity in Staphylococcus aureus
strains. Infect. Immun. 55:843-847.
1118
GREENBERG ET AL.
7. Cheung, A. L., A. S. Bayer, J. Peters, and J. I. Ward. 1988.
Surface proteins of Staphylococcus aureus. Rev. Infect. Dis.
10(Suppl. 2):S351-S355.
8. Derby, B. G., and D. E. Rogers. 1961. Studies of bacteremia: V.
The effect of simultaneous leukopenia and reticuloendothelial
blockade on the early bloodstream clearance of staphylococci
and Escherichia coli. J. Exp. Med. 113:1053-1066.
9. Dossett, J. H., G. Kronvall, R. C. Williams, Jr., and P. G. Quie.
1969. Antiphagocytic effects of staphylococcal protein A. J.
Immunol. 103:1405-1410.
10. Espersen, F., and N. Frimodt-Moller. 1986. Staphylococcus
aureus endocarditis: a review of 119 cases. Arch. Intern. Med.
146:1118-1121.
11. Forni, L. 1979. Reagents for immunofluorescence and their use
for studying lymphoid cell product, p. 151-167. In I. Lefkovits
and B. Pernis (ed.), Immunological methods. Academic Press,
Inc., New York.
12. Forsgren, A. 1969. Protein A from Staphylococcus aureus.
Production of protein A by bacterial and L forms of S. aureus.
Acta Pathol. Microbiol. Scand. 75:481-490.
13. Forsgren, A. 1970. Significance of protein A production by
staphylococci. Infect. Immun. 2:672-673.
14. Forsgren, A., V. Ghetie, R. Lindmark, and J. Sjoquist. 1983.
Protein A and its exploitation, p. 429-480. In C. S. F. Easman
and C. Adlam (ed.), Staphylococci and staphylococcal infections. Academic Press, Inc., New York.
15. Granstrom, M., I. Julander, and R. Molby. 1983. Serological
diagnosis of deep Staphylococcus aureus infections by enzymelinked immunosorbent assay (ELISA) for staphylococcal hemolysins and teichoic acid. Scand. J. Infect. Dis. Suppl. 41:
132-139.
16. Greenberg, D. P., J. I. Ward, and A. S. Bayer. 1987. Influence
of Staphylococcus aureus antibody on experimental endocarditis in rabbits. Infect. Immun. 55:3030-3034.
17. Hirsch, J. G., and B. Strauss. 1964. Studies on heat-labile
opsonin in rabbit serum. J. Immunol. 92:145-154.
18. Kim, K. S. 1987. Use of intravenous immunoglobulins in bacterial diseases. Ann. Intern. Med. 107:369-371.
19. Kronvall, G., P. G. Quie, and R. C. Williams, Jr. 1970. Quantitation of staphylococcal protein A: determination of equilibrium constant and number of protein A residues on bacteria. J.
INFECT. IMMUN.
Immunol. 104:273-278.
20. Myers, J. P., and C. C. Linneman, Jr. 1982. Bacteremia due to
methicillin-resistant Staphylococcous aureus. J. Infect. Dis.
145:532-536.
21. Ouchterlony, 0. 1949. Antigen-antibody reactions in gels. Acta
Pathol. Microbiol. Scand. 26:507-515.
22. Pankey, J. W., N. T. Brodie, J. L. Watts, and S. C. Nickerson.
1985. Evaluation of protein A and a commercial bacterin as
vaccines against Staphylococcus aureus mastitis by experimental challenge. J. Dairy Sci. 68:726-731.
23. Peterson, P. K., J. Verhoef, L. D. Sabath, and P. G. Quie. 1977.
Effect of protein A on staphylococcal opsonization. Infect.
Immun. 15:760-764.
24. Quie, P. G., E. L. Mills, K. L. Cates, J. S. Abramson, W. E.
Regelman, and P. K. Peterson. 1983. Functional defects of
granulocytes and susceptibility to staphylococcal disease, p.
243-273. In C. S. F. Easman and C. Adlam (ed.), Staphylococci
and staphylococcal infections. Academic Press, Inc., New
York.
25. Rice, P. A., H. E. Vayo, M. R. Tam, and M. S. Blake. 1986.
Immunoglobulin G antibodies directed against protein III block
killing of serum-resistant Neisseria gonorrhoeae by immune
serum. J. Exp. Med. 164:1735-1748.
26. Richman, D. D., P. K. Cleveland, M. N. Oxman, and K. M.
Johnson. 1982. The binding of staphylococcal protein A by the
sera of different animal species. J. Immunol. 128:2300-2305.
27. Sheagren, J. N. 1984. Staphylococcus aureus-the persistent
pathogen. N. Engl. J. Med. 310:1368-1373.
28. Sjoquist, J., and G. Stalenheim. 1969. Protein A from Staphylococcus aureus. IX. Complement-fixing activity of protein A-IgG
complexes. J. Immunol. 103:467-473.
29. Verbrugh, H. A., R. Peters, M. Rozenberg-Arska, P. K. Peterson, and J. Verhoef. 1981. Antibodies to cell wall peptidoglycan
of Staphylococcus aureus in patients with serious staphylococcal infections. J. Infect. Dis. 144:1-9.
30. Wilson, W. R., P. M. Jaumin, G. K. Danielson, E. R. Gilviani,
J. A. Washington II, and J. E. Geraci. 1975. Prosthetic valve
endocarditis. Ann. Intern. Med. 82:751-761.
31. Zollinger, W. D., and J. W. Boslego. 1981. A general approach to
standardization of the solid-phase radioimmunoassay for quantitation of class-specific antibodies. J. Immunol. 46:129-140.