INFECTION AND IMMUNITY, May 2006, p. 2742–2750
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 5
Characterization of the Opsonic and Protective Activity against
Staphylococcus aureus of Fully Human Monoclonal Antibodies Specific
for the Bacterial Surface Polysaccharide Poly-N-Acetylglucosamine
Casie Kelly-Quintos,1,2Lisa A. Cavacini,1,3Marshall R. Posner,1,3Donald Goldmann,1,4
and Gerald B. Pier1,2*
Harvard Medical School, Boston, Massachusetts 021151; Channing Laboratory, Department of Medicine, Brigham and
Women’s Hospital, Boston, Massachusetts 021152; Division of Hematology-Oncology, Department of Medicine,
Beth Israel Deaconess Medical Center, Boston, Massachusetts 021153; and Division of Infectious Diseases,
Department of Medicine, Children’s Hospital, Boston, Massachusetts 021154
Received 9 November 2005/Returned for modification 17 January 2006/Accepted 17 February 2006
Carbohydrate antigens are important targets of the immune system in clearing bacterial pathogens. Al-
though the immune system almost exclusively uses antibodies in response to foreign carbohydrates, there is
still much to learn about the role of different epitopes on the carbohydrate as targets of protective immunity.
We examined the role of acetyl group-dependent and -independent epitopes on the staphylococcal surface of
polysaccharide poly-N-acetylated glucosamine (PNAG) by use of human monoclonal antibodies (MAbs) spe-
cific for such epitopes. We utilized hybridoma technology to produce fully human immunoglobulin G2 (IgG2)
MAbs from B cells of an individual post-Staphylococcus aureus infection and cloned the antibody variable
regions to produce an IgG1 form of each original MAb. Specificity and functionality of the purified MAbs were
tested in vitro using enzyme-linked immunosorbent assays, complement deposition, and opsonophagocytic
assays. We found that a MAb (MAb F598) that bound the best to nonacetylated or backbone epitopes on PNAG
had superior complement deposition and opsonophagocytic activity compared to two MAbs that bound
optimally to PNAG that was expressed with a native level (>90%) of N-acetyl groups (MAbs F628 and F630).
Protection of mice against lethality due to S. aureus strains Mn8 and Reynolds further showed that the
backbone-specific MAb had optimal protective efficacy compared with the acetate-specific MAbs. These results
provide evidence for the importance of epitope specificity in inducing the optimal protective antibody response
to PNAG and indicate that MAbs to the deacetylated form of PNAG could be immunotherapeutic agents for
preventing or treating staphylococcal infections.
Staphylococcus aureus continues to be a major pathogen for
both hospital- and community-acquired disease (2, 4, 8, 12, 36).
The rise in antibiotic resistance of S. aureus highlights the need
for alternative treatments and preventative measures to com-
bat this infectious agent (6, 15). There are several surface
proteins and carbohydrates currently under investigation as
targets for antibody-based immunotherapies (7, 9, 10, 32, 34).
One such staphylococcal surface carbohydrate, poly N-acetyl-
glucosamine (PNAG), also referred to as the polysaccharide
intercellular adhesin, has been shown to elicit opsonic antibod-
ies when used as a vaccine in goats and rabbits. In addition,
these polyclonal antibodies passively protect mice against S.
aureus bacteremia and renal infection as well as against lethal-
ity following a high-dose infection (17, 18, 20). Animal anti-
bodies to PNAG also mediate killing of S. epidermidis strains
that express this antigen (18), and these strains constitute a
significant proportion of clinical isolates (36).
A key feature of the immune response to PNAG is the
differing properties of antibodies with specificities for different
epitopes on this molecule. Recent work showed that antibodies
that bind well to PNAG with a native level (?90%) of acetate
substituents on the glucosamine monomers, but poorly to the
antigen when the majority of the acetates are chemically re-
moved (?15% residual acetylation), are inferior in opsonic
and protective properties compared to antibodies elicited
against the deacetylated form of PNAG (dPNAG) (18). The
latter antibodies bind comparably to the antigen regardless of
the level of acetylation; these epitopes are referred to as back-
bone epitopes. Epitope specificity with respect to PNAG has
also been studied using antibodies present in the sera of human
cystic fibrosis patients who were colonized with S. aureus by
comparing the opsonophagocytic activity of affinity-purified
antibodies that bound to native PNAG with that of affinity-
purified antibodies that bound to dPNAG (14). As with the
animal-derived antibodies, the human backbone-specific anti-
bodies were, in general, better able to mediate opsonophago-
cytic killing activity than antibodies that required the acetate
groups to be present to bind well to PNAG.
To pursue further the role of epitope specificity as an im-
portant property distinguishing protective from nonprotective
antibody to the PNAG antigen, we produced fully human
monoclonal antibodies (MAbs) to this antigen that had differ-
ent properties of binding to native PNAG and dPNAG and
characterized their immunologic and protective characteristics.
In addition, fully human MAbs are being developed as treat-
ments for infections by bacterial, viral, and fungal pathogens
(16, 19, 22, 38), and similar reagents are already in use for the
* Corresponding author. Mailing address: Channing Laboratory,
Brigham and Women’s Hospital, Harvard Medical School, 181 Long-
wood Ave., Boston, MA 02115. Phone: (617) 525-2269. Fax: (617)
525-2510. E-mail: firstname.lastname@example.org.
treatment of numerous inflammatory diseases (21) and tumors
(33). Fully human MAbs have been shown to have few side
effects and low immunogenicity when given to patients (13). In
light of these prior observations regarding immunity to staph-
ylococcal PNAG, we hypothesized that MAbs specific to the
backbone epitopes on PNAG would have superior S. aureus
killing activity compared to MAbs that require the acetate
substituents in order to bind well to PNAG. In this paper we
describe the production of immunoglobulin G2 (IgG2)-secret-
ing hybridomas as well as cell lines transfected with DNA to
produce V region-identical recombinant IgG1 MAbs reactive
with PNAG and dPNAG antigens. In addition, we compared
the properties of the IgG1 and IgG2 MAbs by use of in vitro
assays measuring complement deposition and opsonophago-
cytic killing and further studied the IgG1 MAbs by use of in
vivo protection studies of mice. Overall, we found the IgG1
MAb with specificity to the dPNAG antigen had the greatest
complement deposition and opsonic and protective activities
against S. aureus.
MATERIALS AND METHODS
Bacterial strains. S. aureus strains MN8 (capsular type 8 [CP8]), NCTC 10833
(ATCC 25904; CP untypable), Reynolds (CP5), and Newman (CP5) and S.
epidermidis strain M187 were obtained and propagated as previously described
(3). Methicillin-resistant S. aureus (MRSA) Panton-Valentine leukocidin (PVL)-
producing strains NRS 123 (also known as MW2 and USA400), NRS 192, and
NRS 193 were obtained from the repository of the Network on Antimicrobial
Resistance in Staphylococcus aureus, as was methicillin-susceptible, PVL-produc-
ing strain NRS 157. Strains were grown in tryptic soy broth (TSB) supplemented
with additional 1% glucose (TSBG) for opsonic killing assays, immunofluores-
cence studies, and protection studies.
Purification and chemical modification of PNAG. Purification of PNAG was
performed as previously described (17), using the culture supernatant of S.
aureus strain Mn8m grown in a chemically defined medium. To remove ?80% of
the N- and O-linked substituents from native PNAG, purified PNAG was dis-
solved at 0.5 mg/ml in 5 M NaOH and incubated for 18 h at 37°C with stirring.
The solution was neutralized to a final pH between 6 and 8 and dialyzed against
deionized water for 24 h and then freeze dried. The residual level of acetate
groups was determined by nuclear magnetic resonance spectroscopy as described
Hybridomas. Informed consent to take a blood sample from a patient 3 years
after an episode of S. aureus bacteremia and then isolate B cells for processing
for hybridoma production was obtained as stipulated by the Committee on
Clinical Investigation at the Beth Israel Deaconess Medical Center. Hybridomas
were created essentially as described previously (27, 28). In brief, B cells were
transformed with Epstein-Barr virus and then dispersed into multiple culture
wells and grown for several weeks. Supernatants were screened for the presence
of antibody that could bind to highly acetylated, native PNAG (75% to 100% N
acetylated) and/or to poorly acetylated PNAG (dPNAG; ?15% acetylated).
Cells in positive wells were then fused with the HMMA 2.5 cell line (27) to
generate stable, antibody-producing hybridomas, which were cloned for homo-
geneity by limiting dilution as described previously (26). Three MAb clones,
designated F598, F628, and F630, were obtained for further study.
Cloning of antibody-variable regions. RNA was extracted from ?6 ? 106cells
of each hybridoma by use of an RNeasy kit (QIAGEN Inc., Valencia, CA)
according to the manufacturer’s instructions. A 1-?g volume of total RNA was
reverse transcribed using a QIAGEN Omniscript kit. A 1-?l volume of cDNA
product was used as a template for PCRs. Each reaction consisted of 50 ?l of
PCR SuperMix High Fidelity (Invitrogen Corp., Carlsbad, CA), 100 pmol of each
primer (see Table 1), and 1 ?l of cDNA template. For PCR amplification ?30
cycles were used with the following protocol: 94°C for 30 s initially followed by
cycles of 94°C for 30 s, 65°C for 30 s, and 72°C for 1 min, with a final extension
at 72°C for 5 min. PCR products from at least three independent PCRs were
sequenced at least three times until a consensus sequence for the variable regions
could be determined. A consensus sequence was determined to be correct when
the sequencing results from at least three different PCRs were found to be
identical. Sequencing was done at the Harvard Medical School DNA core se-
quencing facility, and the resultant sequences were compared against known
germ line sequences using Ig BLAST on the NCBI database. The final sequences
have been deposited in GenBank.
Construction of IgG1 MAbs. The TCAE6 vector containing the human lambda
and human IgG1 constant region was used as previously described (26, 30).
Cloned heavy (H) chain V-region genes from the three hybridomas were di-
gested with SalI and NheI restriction enzymes and ligated into the TCAE6 vector
cut with the same enzymes. The ligation reaction mixture was then transformed
into competent Escherichia coli TOP10F cells (Invitrogen). Plasmids within in-
dividual clones were purified using a QIAGEN plasmid Miniprep kit and V-
region H chains digested and sequenced to ensure that the correct sequence had
been inserted into the vector. Cloned light (L) chains were digested with BglII
and AvrII restriction enzymes and ligated with the TCAE6 vector already con-
taining the matching H chain variable region and cut with the same enzymes.
Plasmids were transformed into E. coli, and then individual clones were isolated,
plasmids were obtained, and inserted DNA was sequenced to insure that the
correct L chain V-region sequence had been cloned. In the end, three constructs
were created containing the H and L chains from F598, F628, and F630, the three
initial MAbs. Each plasmid construct was transfected into CHO DHFR?/?cells
by use of Lipofectamine 2000 (Invitrogen) according to the manufacturer’s
instructions. Stably transfected cells were selected using geneticin (Invitrogen),
and clones were obtained by limiting dilution as described previously (26).
Cloned cells were screened by enzyme-linked immunosorbent assay (ELISA) to
identify those producing maximal levels of IgG1 for the native PNAG and
Production of MAbs. Transfected cells were weaned to serum-free medium
containing geneticin (CHO-SFMII medium; Invitrogen). To increase antibody
production, methotrexate replaced geneticin in the medium and further clonal
selection was undertaken as described previously (26). Bulk cultures were grown
in flasks in 800-ml volumes, and after 2 to 3 liters of culture was obtained, the pH
of the pooled supernatants was adjusted to 6.5 and then run over a protein G
column (Invitrogen) according to the manufacturer’s instructions except that the
running buffer was adjusted to pH 6.5 and, after elution of bound MAb in 0.1 M
glycine (pH 2.6), the eluate was neutralized using 1 M phosphate buffer (pH 6.4).
Fractions containing protein were pooled and dialyzed against phosphate-buff-
ered saline (PBS) (pH 6.5). MAb concentrations were determined by ELISA as
ELISAs. Immulon 4 HBX microtiter plates (Thermo Lab Systems, Franklin,
MA) were coated with 100 ?l of a previously determined optimal binding con-
centration of each antigen (0.6 ?g/ml for native PNAG or 3 ?g/ml for dPNAG)
dissolved in sensitizing buffer (40 mM phosphate buffer, 0.02% azide, pH 7.4)
and incubated overnight at 4°C. Plates were washed three times with PBS (0.0144
g/liter KH2PO4, 0.9 g/liter NaCl, 0.0795 g/liter Na2HPO4) containing 0.05%
Tween 20 (washing buffer) and blocked with 200 ?l of a 5% solution of skim milk
dissolved in PBS. Plates were again incubated overnight at 4°C. Plates were
TABLE 1. Primers used to clone hybridoma antibody
aFor the light chain primers, the Bgl II site is underlined and the starting ATG
is shown in bold. For the lambda constant primer, the Avr II site is underlined.
For the heavy chain primers, the SalI site is underlined and the starting ATG is
shown in bold. For the heavy chain constant primer, the Nhe1 site is underlined.
VOL. 74, 2006 HUMAN MAbS TO PNAG2743
washed, and purified MAbs diluted in 5% skim milk–0.05% Tween–PBS (dilu-
tion buffer) were added at various concentrations. Plates were then incubated for
1 h at 37°C and washed three times. A 100-?l volume of secondary antibody
(anti-human IgG whole molecule conjugated to alkaline phosphatase) was added
at a 1:1,000 dilution in the dilution buffer. Plates were incubated at 37ofor 1 h
and washed three times. A 100-?l volume of p-nitrophenyl phosphate at a
concentration of 1 mg/ml in substrate buffer (800 mg NaHCO3, 1.46 g Na2CO3,
10 mg MgCl, 20 mg Na3N in 500 ml water) was added, plates were incubated at
room temperature for 30 min, and then color development was read at 405 nm.
To quantify the concentration of MAb preparations, rows on ELISA plates were
first sensitized with anti-human IgG to which purified human IgG lambda
(Sigma) was added. The optical density at 450 nm (OD450) obtained was plotted
versus the concentration of the IgG standard to derive a regression equation
from which OD values of the test MAbs could be taken to calculate specific MAb
Binding competition experiments. ELISA plates coated with native PNAG as
described above were used to determine whether the MAbs competed for bind-
ing to related epitopes on this antigen. Constant amounts of the IgG2 form of
one MAb (a concentration resulting in an OD405reading of 1.0 under standard
ELISA conditions) were mixed with various amounts of the IgG1 form of a
competitor MAb, and the binding of the mixture to immobilized PNAG was
performed as for the direct ELISA described above. Secondary monoclonal
mouse anti-human IgG2 (Zymed) was then used to determine the ability of the
IgG1 form of a different MAb to compete for binding to the PNAG antigen.
Controls included uninhibited IgG2 MAb and PNAG-coated wells without pri-
mary MAb but probed with secondary antibody to determine only the back-
ground binding of the secondary antibody.
Immunofluorescence. Glass-bottom microwell plates (Mattek, MA) were
coated with 4% Celltak (BD Biosciences, MA) in 0.1 M NaHCO3and 20 mM
NaOH. Plates were coated for 30 min at room temperature and then washed
twice with distilled water. A 1-ml volume of a bacterial suspension freshly grown
in TSBG (?108CFU/ml) was added to the plates and allowed to adhere for 20
min at room temperature. After two washes with PBS, bacterial cells (S. aureus
Mn8m and S. aureus Mn8?ica) were fixed with 1% formaldehyde in PBS for 30
min at 4°C. Cells were washed twice with PBS and then blocked with PBS–1%
bovine serum albumin–2% normal rabbit serum for 30 min at 4°C with rocking.
The blocking solution was replaced with 1 ml of the primary antibody solution,
MAb F598 or F628 (2 ?g/ml) in PBS with 1% bovine serum albumin and 2%
normal rabbit serum, and allowed to incubate for 2 h at 4°C with rocking. After
four washes with PBS, 1 ml of a secondary anti-human IgG, Alexa 488 (Molec-
ular Probes, Corvallis, OR) (diluted 1:2,000), was added and the mixture was
incubated at 4°C for 1 h. The plates were then washed four times with PBS. The
plates were examined by phase-contrast and fluorescence microscopy. Images of
the same field viewed by the two different microscopic methods were acquired by
a camera, and images were processed by computer using the LSM 5 image
Complement deposition assay. Determinations of the deposition of the opso-
nically active C3 component were based on assays described previously (29).
Microtiter plates were coated, as described for the ELISA, with PNAG antigen.
After incubation with dilutions of the MAbs and washing, normal human sera
diluted 1:50 was used as a source of complement. Prior to use, this serum was
absorbed with three different S. aureus strains to remove endogenous antibody to
this microbe. Plates were incubated for 15 min at 37°C. After the plate was
washed, goat anti-human C3 (MP Biomedicals, Irvine, CA) at a dilution of
1:2,000 was added and, after 1 h of incubation at 37°C followed by three washes,
an anti-goat IgG-alkaline phosphate conjugate (Sigma) was added at a 1:2,000
dilution and the mixture was again incubated at 37°C for 1 h. Plates were
developed as in the ELISA except that OD values were read after 15 min of
Opsonophagocytic assays. The basic protocol has been previously described
(17). In brief, the target S. aureus strain was grown to midlogarithmic phase
(OD650of 0.4; ?1 ? 109CFU/ml) in TSBG and diluted 1:100 into RPMI
medium with 15% fetal bovine serum (HyClone, Logan, UT). Complement
(infant rabbit serum; Cedarlane Laboratories, Hornby, Ontario, Canada) (di-
luted 1:15) was absorbed for 1 h with S. aureus strain Mn8 (1 ml of diluted
complement per 1 ml of packed cells obtained from suspensions of an OD650of
1.0 prepared from cells grown on tryptic soy agar plates overnight at 37°C).
Polymorphonuclear neutrophils (PMNs) were separated from freshly drawn hu-
man blood by use of heparin-dextran (1:1 mix) and adjusted to a concentration
of 2 ? 107cells/ml before being added to the assay tubes. Components (MAb
dilution, target bacteria, complement source, PMNs) (100 ?l each) were added
together and incubated for 1.5 h at 37°C on a rack rotating end over end.
Bacterial enumeration was carried out by making serial 10-fold dilutions in TSB
with 0.05% Tween, and dilutions were plated on tryptic soy agar. After plates
were incubated overnight at 37°C, bacterial colonies were counted. Controls
included tubes containing only PMNs, complement, and bacteria; PMNs and
bacteria alone; and complement and bacteria alone. To rule out apparent de-
creases in bacterial CFU due to aggregation of the target bacteria by the MAbs,
a control containing bacteria, complement, and monoclonal antibodies but lack-
ing PMNs was also included. The percentage of reduction in CFU was calculated
using as a denominator a control containing complement, PMNs, bacteria, and
an irrelevant IgG myeloma protein (Sigma).
Animal models. For lethality studies, 4- to 6-week-old FVB female mice
(Taconic Farms, Hudson, NY) (?20 g) were injected intraperitoneally with
different amounts of the MAbs 4 h before bacterial challenge section. A purified
myeloma protein, human IgG1 lambda, was used as an antibody control (Sigma).
Bacteria were injected intraperitoneally at a challenge dose of 2.5 ? 108to 9.0 ?
108per mouse. Mouse survival was monitored for 5 days.
Statistics. Survival analysis and calculations of antibody binding and inhibiting
properties used Prism software for a Macintosh computer. To determine half-
maximal binding activities and epitope overlap of the MAbs, the log of the
concentration of the MAbs was plotted against the activity being measured
(defined as a percentage of maximal binding or inhibition); either linear or
nonlinear regression was used to generate a formula to calculate the reported
values. Microsoft Excel was used for regression analysis for antibody quantifi-
Nucleotide sequence accession numbers. The final sequences determined as
described above have been deposited in GenBank under accession numbers
DQ231549 to DQ231554.
PNAG- and dPNAG-specific hybridomas and monoclonal
antibodies. B cells from a patient who had recovered 3 years
previously from S. aureus bacteremia were used to produce
human hybridomas. Throughout the growth, cloning, and se-
lection processes, the antibodies secreted by the hybridomas
were screened for the ability to bind to the acetylated and/or
deacetylated PNAG. We chose three hybridomas for further
study and named them F598, F628, and F630 for their fusion
numbers. All three hybridomas were of the IgG2 lambda sub-
class. This is consistent with observations of a predominantly
IgG2 human immune response to polysaccharides. Due to the
known superiority of IgG1 antibodies to IgG2 antibodies for
both fixing complement and interacting with Fc receptors on
neutrophils, two qualities important in antibody-mediated
opsonophagocytosis and bacterial clearance, we chose to clone
the heavy and light chains of the original hybridomas and
create IgG1 equivalents. Table 1 describes the primers used to
clone the heavy and light chain of each hybridoma into the
TCAE6 vector, which contains both the human lambda and
human IgG1 constant regions. Recombinant TCAE6 con-
structs containing the heavy and light chains of each hybridoma
TABLE 2. Germline gene usage and changes from germ-line
nucleotide sequences of PNAG-specific hybridomas
or L chain
V gene usage
No. of amino acid
aFWK, framework region; CDR, complementarity-determining region.
2744KELLY-QUINTOS ET AL.INFECT. IMMUN.
were transformed into CHO cells for MAb synthesis and pu-
The DNA V-region germ line genes used and mutational
changes from consensus germ line genes that arose in the
variable-region genetic elements to form each of the H and L
chain V regions in the three MAbs are shown in Table 2 and
Table 3. Sequences are deposited in GenBank under accession
numbers DQ231549 to DQ231554. We found that common
germ line genes were used to produce these three antibodies,
and all three of the L chains used the same germ line IGLV4-69
and IGLJ-3 genes, but differences due to somatic mutation
were found in the final sequences. All three heavy chains con-
tained the same germ line IGHD gene (IGHD3-22). The F598
and F628 H-chain germ line gene sequences matched in every
case except for the heavy chain IGHJ gene, and the F598 and
F630 germ line gene sequences matched in every case except
for the heavy chain IGHV gene sequence. This result agrees
with previous observations indicating that a tightly restricted
use of germ line genes occurs in the human immune response
to carbohydrate antigens (37). Although the germ line genes
used to make the different MAbs were highly related in their
sequences, none of the three H or three L chains had identical
amino acid sequences among the three hybridomas studied.
Comparative antigen binding and epitope specificities of the
human MAbs. ELISAs were used to determine the compara-
tive strength of antigen binding of each purified MAb by eval-
uating binding to both the acetylated and deacetylated forms of
PNAG. As shown in Fig. 1A, all of the MAbs bound to native
PNAG, with clear differences in the amounts of each MAb
needed to achieve maximal binding to this antigen. Determi-
nations of the amount of each MAb needed to achieve 50%
maximal binding in the ELISA showed that MAb F598 was
TABLE 3. Results of CDR3 analysis
H or L
Size of CDR3
(no. of amino
D regionJ region
FIG. 1. ELISA reactivities of IgG1 (A and B) and IgG2 (C and D) MAbs to PNAG (A and C) and dPNAG (B and D) antigens. (A) Binding
of isotype-switched IgG1 MAbs F598, F628, and F630 to native PNAG. (B) Binding of IgG1 MAbs to dPNAG antigen. (C) Binding of IgG2 MAbs
to PNAG antigen. (D) Binding of IgG2 MAbs to dPNAG antigen. The irrelevant antibody is a human IgG1 antibody specific for P. aeruginosa
VOL. 74, 2006 HUMAN MAbS TO PNAG2745
significantly superior to MAb F628 in antigen binding, and
both of these MAbs were greatly superior to MAb F630 in
antigen binding (Table 4). Of note, there was little difference
between the results for binding of the IgG1 and IgG2 forms of
each MAb to PNAG (Fig. 1A and C). We also found that MAb
F598 bound to dPNAG better than the other two MAbs (Fig.
1B and D), and the low level of binding of MAbs F628 and
F630 to dPNAG precluded calculation of a half-maximal bind-
ing level with any biologic significance. Apparent differences in
the final OD readings obtained for the IgG1 (Fig. 1B) and
IgG2 (Fig. 1D) forms of the MAbs to dPNAG likely were due
to reduced overall binding of the MAbs to this form of the
PNAG antigen and use of a secondary antibody, directed to
the whole IgG molecule, which likely had different reactivity to
IgG1 and IgG2. MAbs F598 and F628 were used in immuno-
fluorescence studies to compare binding to wild-type and ica-
deleted S. aureus strain MN8. Results shown in Fig. 2 depict
the strong binding of both MAbs to the wild-type strain but not
to the ica-deleted strain. All three MAbs were also tested
against a panel of antigens in an ELISA and found to be
nonreactive against alginate from Pseudomonas aeruginosa
strain PAO 581 (24), the P. aeruginosa LPS O antigens from
serogroups 02 and 06 (25), and the PS/A antigen of Bacteroides
fragilis (23) (data not shown).
Determination of epitope overlap by competitive binding
assays using IgG1 forms of the MAbs to compete for binding to
native PNAG of the IgG2 form of the MAbs (held at a con-
centration that would give rise to an OD405of ?1.0 in a
standard ELISA) revealed that MAbs F598 and F628 had an
apparent partial overlap in epitope specificity. Inhibition of the
binding of 72 ng/ml of the IgG2 form of MAb F598 by the IgG1
TABLE 4. Calculation of amount of each MAb needed to achieve
50% maximal binding to PNAG in an ELISA
aDetermined by nonlinear regression of data for IgG1 MAb binding to PNAG
(as shown in figure 1A) by use of a sigmoidal curve-fitting function for a variable
slope and plotting the log10concentration of the MAb versus percent maximal
FIG. 2. Immunofluorescence study of binding of MAbs F598 and F628 to wild-type (WT) and ?ica S. aureus strain Mn8, showing specificity
of binding to the PNAG-producing parental strain.
2746 KELLY-QUINTOS ET AL.INFECT. IMMUN.
form of this MAb revealed that it took 1.8 ?g/ml (95% confi-
dence interval, 1.2 to 2.6 ?g/ml) of the homologous MAb to
reduce the binding by 50%. Similarly, it took 1.1 ?g MAb
F598/ml (95% confidence interval, 0.48 to 2.5 ?g MAb F598/
ml) to reduce binding by 50% of 600 ng MAb F628/ml to
PNAG. We could not determine whether MAb F628 could
reduce the binding of MAb F598 to PNAG, as the greater
amount of binding of the latter MAb needed to achieve half-
maximal binding (?4.8 times for half-maximal binding; Table
4) precluded adding sufficient amounts of MAb F628 as a
competitor to achieve significant inhibition of binding of MAb
F598. Neither MAb F598 nor F628 inhibited ?50% of the
binding of 1.2 ?g MAb F630/ml when up to 10 ?g/ml compet-
itor MAb was used, suggesting there was minimal to no overlap
in epitope specificity for the native PNAG antigen between
MAb F630 and the other two MAbs.
Complement deposition activity of MAbs. As deposition of
opsonically active fragments of the third component of com-
plement (C3) often correlates with in vitro phagocytic killing
and in vivo protection against gram-positive pathogens, we
measured complement deposition onto immobilized PNAG
antigen by both the IgG1 and IgG2 forms of each MAb. As
seen in Fig. 3, the IgG1 form of the antibody was able to
deposit the C3 component of the complement cascade to a
greater extent than the IgG2 forms of the same antibody.
Interestingly, there was a modest but clear difference for MAb
F598, whereas a larger difference was seen with MAbs F628
and F630. Although changing MAbs F628 and F630 to the
IgG1 form did increase complement binding ability over that
achieved by the IgG2 form, this manipulation did not increase
the overall complement deposition to the level achieved with
either the IgG1 or IgG2 forms of MAb F598.
Opsonophagocytic activity of MAbs. The functional activity
of the MAbs was also measured in vitro by utilizing an op-
sonophagocytosis assay. As seen in Fig. 4, MAb F598 in both
the IgG1 and IgG2 isotypes had the most killing activity com-
pared to the MAbs F628 and F630. In agreement with the
complement deposition data, there was some difference in
opsonophagocytic activity between the IgG1 and IgG2 forms of
MAb F598: the IgG1 form killed 25% to 30% more bacterial
CFU than did the IgG2 form at comparable MAb concentra-
tions. More notably, changing MAb F628 from the IgG2 form
to the IgG1 form resulted in a 60% increase in opsonophago-
cytic killing at the highest concentration tested (25 ?g MAb/
ml) whereas at the lower concentrations of MAb F628 the
increased killing by the IgG1 isotype resulted in 15% to 20%
more CFU killed. Changing MAb F630 to the IgG1 isotype
had only a minimal effect, with only low levels of opsonic killing
activity achieved with either the IgG1 or IgG2 isotype of MAb
F630. Further opsonic assays found that MAb F598 mediated
killing of all S. aureus strains tested, including four PVL-
producing clinical isolates (three of which were also MRSA
strains), as well as one S. epidermidis strain (Fig. 5).
Protective activity of MAbs. Because of the overall superi-
ority of the IgG1 form of the antibodies in mediating comple-
ment deposition and opsonic killing, we chose to evaluate this
subclass for the in vivo protection studies. A lethality model
was used in which a single dose of an MAb was given 4 h prior
to bacterial challenge. As seen in Fig. 6, 600 ?g of either MAb
F598 or MAb F628 could significantly protect mice from chal-
FIG. 3. Relative deposition onto purified PNAG of the third com-
ponent of complement (C3) by IgG1 (filled symbols) and IgG2 (open
symbols) MAbs. C3 deposition was measured by ELISA with a goat
anti-human C3. The MAb to alginate is an irrelevant human IgG1
antibody specific for the P. aeruginosa alginate capsular antigen.
FIG. 4. Opsonophagocytic activity of IgG1 and IgG2 MAbs. Target
strain is S. aureus Mn8. Bars represent means; error bars represent the
standard errors of the means for three to eight replicates. Percent
reduction in CFU was calculated compared to controls containing
complement, PMNs, bacteria, and IgG control antibodies.
VOL. 74, 2006 HUMAN MAbS TO PNAG 2747
lenge with S. aureus strain Reynolds, a CP5 strain. When the
amount of MAb passively administered was reduced to 300 ?g
per mouse, the F598 MAb continued to significantly protect
the mice; however, MAb F628 was no longer providing signif-
icant protection (P ? 0.05). We found with S. aureus strain
MN8 (CP8) that somewhat lower doses of the MAbs could be
used in passive protection studies. When we administered 400
?g of MAbs F598 and F628, we found that both MAbs medi-
ated significant protection at this dose, but at a dose of 200
?g/mouse, only MAb F598 was protective (Fig. 7). A 300-?g
volume of MAb F630 had no protective activity against chal-
lenge with S. aureus strain Mn8 (data not shown). Overall,
MAb F598, which bound the best to dPNAG and had the
optimal complement deposition and opsonic activity, was also
the most protective MAb in these studies.
Previous findings have shown PNAG is an effective antigenic
target for protecting animals against S. aureus and S. epider-
midis infection via elicitation of polyclonal antibodies. Further-
more, there was a superiority in opsonic and protective prop-
erties of antibodies that bound well to the backbone epitopes
on dPNAG compared with antibodies requiring acetate sub-
stituents to bind well to PNAG (18). In this study we confirmed
and extended these findings by showing that a fully human
MAb with good binding activity to the backbone epitopes on
the dPNAG antigen had better in vitro opsonic killing activity
and was more protective in vivo than two other MAbs that
bound less well to both native PNAG and dPNAG. Impor-
tantly, we found that the better binding of MAb F598 to PNAG
and dPNAG was correlated with superior C3 deposition, op-
sonic killing, and protection, indicating the interrelatedness of
these properties for protective antibodies for the PNAG anti-
gen. These in vitro correlates of protective immunity can be
used in experiments to help identify improved antibody-based
therapeutics and potentially find additive or synergistic com-
binations of antibodies to target PNAG-expressing pathogens.
Of note, we did not find any additive or synergistic activities
when we combined the three MAbs studied here in all possible
combinations and evaluated them in opsonic killing assays (C.
Kelly-Quintos, unpublished observation).
To improve the biologic properties of the initial IgG2 MAbs,
we switched isotypes for the cloned V regions to produce
human IgG1 MAbs. Interestingly, switching the F598 MAb
from IgG2 to the IgG1 subclass gave a modest improvement in
C3 deposition, with a concomitant 25% to 30% increase in
opsonic killing activity. The F628 IgG2 MAb had a good im-
provement in both of these characteristics when switched to
the IgG1 isotype, but significantly increased killing was
achieved only with the highest concentration of MAb tested
(25 ?g/ml). Of note, the IgG2 isotype of MAb F598 showed
two to three times more opsonic killing activity than the IgG1
form of MAb F628 at concentrations of 3 to 12.5 ?g/ml, con-
sistent with the increased level of C3 deposition achieved by
the IgG2 MAb F598. This result suggests MAb F598 may
recognize a densely packed epitope on the PNAG molecule, as
it has been shown that IgG2 antibodies have increased com-
FIG. 5. Opsonophagocytic killing of several staphylococcal strains
with MAb F598 at 12 ?g/ml. Bars represent means; error bars repre-
sent the standard errors of the means. S. aureus strains Mn8, Newman,
10833, and Reynolds and S. epidermidis strain M187 represent methi-
cillin-sensitive clinical and laboratory isolates. Strain 123 (also desig-
nated MW2 or USA400) is an MRSA strain carrying the PVL genes
whose genome has been sequenced. Strains 192 and 193 represent
MRSA, PVL-positive isolates from community-acquired infections.
Strain 157 is a methicillin-sensitive, PVL-positive S. aureus isolate from
a patient with lethal necrotizing pneumonia.
FIG. 6. Protection against S. aureus strain Reynolds with IgG1 MAbs
(12 mice per group). (A) A 600-?g volume of each MAb was adminis-
tered 4 h before bacterial challenge. (B) A 300-?g volume of each MAb
was administered 4 h before bacterial challenge. Challenge dose, 2.5 ?
2748 KELLY-QUINTOS ET AL.INFECT. IMMUN.
plement deposition and opsonic activity when epitope density
is high, as is commonly found on carbohydrate molecules (1).
Although changing the F630 MAb from the IgG2 to the IgG1
form also increased complement deposition activity, this in-
crease translated into only marginally better opsonophagocytic
activity. Overall, converting the least opsonic F630 MAb into
the IgG1 form did not improve the level of complement-fixing
activity to that of MAbs F598 or F628, indicating a close rela-
tionship between the ability of these MAbs to deposit C3 onto
the PNAG antigen and to mediate opsonic killing.
Even though it appeared that MAbs F598 and F628 had some
partial epitope overlap, as revealed by competition binding ex-
PNAG and dPNAG antigens was likely a factor in its superior
opsonic and protective properties. However, studies using human
polyclonal antibodies produced in response to infection showed
no effect of affinity on opsonic activity when comparing antibodies
with specificity for dPNAG to those that bound optimally to
native PNAG (14). This suggests that good binding to dPNAG,
and not overall binding to native PNAG, is an important factor
for the most opsonic and protective antibodies to S. aureus
PNAG. Furthermore, when using diphtheria toxoid-conju-
gated PNAG or dPNAG antigens as active vaccines in ani-
mals, the native PNAG conjugate vaccine induced relatively
low opsonic titers compared to the dPNAG conjugate vaccine,
and all of the opsonic killing activity elicited by the native
PNAG conjugate vaccine was inhibited by purified dPNAG
antigen (18). Thus, a consistent picture emerges showing that
even when there are antibodies present that bind better to
native PNAG versus dPNAG, the opsonically active ones are
those that bind to dPNAG. The findings reported here for the
relative antigen-binding and immune-effector activities of
MAbs F598 and F628 are consistent with this overall conclu-
Also, consistent with previous findings using polyclonal anti-
bodies (18), the dPNAG-binding MAb F598 had superior in vivo
protective activity. The lowest dose of MAb F598 that we found
was needed to achieve significant protection in the mice was ?10
mg/kg of body weight, which is quite comparable to the dose of
the humanized MAb Palivizumab given clinically to infants for
prophylaxis against respiratory syncytial virus infection. Palivi-
zumab is administered to infants at a dose of 15 mg/kg (11).
Similarly, a humanized MAb to S. aureus clumping factor A was
used at 10 and 30 mg/kg to protect rabbits against consequences
of S. aureus endocarditis (7) and this MAb has been evaluated in
humans infused with doses of 2, 5, 10, or 20 mg/kg, with linear
dose-response kinetics found for the final maximal serum con-
centration (31). These results further indicate that our MAbs are
effective in mice at doses comparable to those likely to be used in
humans. Thus, while it may be possible to improve the affinity or
other properties of MAb F598 to make it more potent, based on
the in vivo protection studies of mice, it is already in the potency
treating infections in humans.
A possible explanation for the molecular basis of the superior
activity of antibodies that bind well to the backbone epitopes on
PNAG was recently described (35). Synthesis of PNAG is depen-
dent on the protein products encoded by the genes in the inter-
cellular adhesin (ica) locus (5). The protein encoded by the icaB
gene is responsible for deacetylation of PNAG. It was found that
in-frame deletion of the icaB gene in the ica locus of both S.
aureus (N. Cerca, K. K. Jefferson, D. B. Pier, T. Maira-Litran, C.
Kelly-Quintos, D. A. Goldmann, J. Azeredo, and G. B. Pier,
unpublished data) and S. epidermidis (35) (where PNAG was
referred to as PIA) resulted in production of only fully acetylated
PNAG, and this form of PNAG was not retained on the cell
surface. Instead, fully acetylated PNAG was all released into the
PNAG was retained on the cell surface and the overall level of
acetylation was lower than that of the PNAG produced in the
absence of functional IcaB (35). Thus, some amount of deacety-
lation is required for surface retention of PNAG. Further work
showed that overexpression of the IcaB protein in a wild-type
strain of S. aureus resulted in greater surface retention of PNAG
and enhanced opsonophagocytic killing of dPNAG-specific anti-
bodies without any effect on the opsonic killing activity of anti-
bodies specific to native PNAG (N. Cerca, K. K. Jefferson, D. B.
Pier, T. Maira-Litran, C. Kelly-Quintos, D. A. Goldmann, J.
Azeredo, and G. B. Pier, unpublished data). These results indi-
cate that dPNAG-specific antibodies may have an advantage
over antibodies that require the presence of the acetate groups
on PNAG to bind well to this antigen, based on the superior
cell surface association of partially deacetylated PNAG. In
FIG. 7. Protection against S. aureus MN8 lethal infection with IgG1
MAbs to PNAG (eight mice per group). (A) A 400-?g volume of each
MAb was administered 4 h before challenge. Strain Mn8 challenge
dose, 5 ? 108. (B) A 200-?g volume of each MAb was administered 4 h
before challenge. Strain Mn8 challenge dose, 9 ? 108; ?, P value less
than 0.05 compared to IgG1 control MAb.
VOL. 74, 2006HUMAN MAbS TO PNAG 2749
addition, the reduction in acetylation that accompanies cell sur-
face binding of PNAG would reduce the density of acetate-de-
pendent epitopes and concomitant binding of antibodies requir-
ing acetate for maximal binding, resulting in a less effective
antibody, as was shown here.
Overall, the findings in this study complement previous re-
sults regarding the importance of epitope specificity in the
human immune response to S. aureus PNAG. The results fur-
ther support the conclusion that antibodies that bind well to
the backbone of PNAG are superior in their ability to kill and
protect against S. aureus infection compared with antibodies
that require the acetate groups for maximal binding to PNAG.
In addition, the fully human MAb to staphylococcal dPNAG
has excellent in vitro properties that translate into high levels
of in vivo protective efficacy. These are key preclinical findings
supporting further development of this reagent as an immu-
notherapeutic for prophylaxis and possibly for adjunctive treat-
ment of S. aureus infection.
This work was supported by Public Health Service grant AI-48917
from the National Institute of Allergy and Infectious Diseases.
We thank David Kuhrt for technical expertise in the development of
hybridomas and the Network on Antimicrobial Resistance in Staphy-
lococcus aureus for provision of indicated strains.
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Editor: J. N. Weiser
2750KELLY-QUINTOS ET AL. INFECT. IMMUN.