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: email@example.com.
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
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.