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Exp. Eye Res. (1999) 69, 75–84Article No. exer.1999.0678, available online at http :}}www.idealibrary.com on
Monoclonal IgA Antibodies Protect Against
Acanthamoeba Keratitis
HENRY LEHER, FERNANDO ZARAGOZA, SHERINE TAHERZADEH,
HASSAN ALIZADEH JERRY Y. NIEDERKORN*
Department of Ophthalmology, University of Texas Southwestern Medical Center, Dallas,
TX 75235-9057, U.S.A.
(Received Columbia 12 November 1998 and accepted in revised form 23 February 1999)
Acanthamoeba keratitis is a rare, yet sight-threatening corneal infection. Ocular infection does not appearto induce protective immunity as repeated corneal infections occur in both humans and experimentalanimals. However, we have recently demonstrated that activation of the common mucosal immunesystem by oral immunization with Acanthamoeba antigens protects both Chinese hamsters and pigsagainst ocular infection with A. castellanii. Protection correlates closely with the appearance of anti-Acanthamoeba antibodies in the tears. To test the hypothesis that oral immunization induces specificprotective IgA antibodies, two monoclonal IgA antibodies specific for Acanthamoeba antigens weregenerated. Both antibodies detected epitopes on the surface of fixed Acanthamoeba trophozoites. Whendelivered intraperitoneally, one monoclonal antibody (14E4) was detected in stool and tear samples. Thisclone also protected naive animals against ocular challenge with Acanthamoeba trophozoites (43%infection rate compared to a 91% infection rate in animals receiving control IgA). In vitro functionalstudies showed that neither antibody induced encystment or directly killed Acanthamoeba trophozoites.However, both monoclonal anti-Acanthamoeba IgA antibodies produced a three-fold inhibition in theadherence of trophozoites to corneal epithelial cells in vitro. These data show that monoclonal anti-Acanthamoeba IgA antibodies can protect against Acanthamoeba keratitis and suggest that this occurs byinhibiting adhesion of the parasite to the corneal epithelium. # 1999 Academic Press
Key words : Acanthamoeba ; keratitis ; monoclonal IgA; tear ; adhesion.
1. Introduction
Corneal infections have caused millions of cases of
blindness (Adamis and Shein, 1994; Pavan-Langston,
1994). With many pathogens, adhesion of the parasite
to the corneal epithelium is a crucial first step in
pathogenesis (van Klink et al., 1993; Beving, Soong
and Ravdin, 1996; Hocini et al., 1997; Fluckiger,
Jones and Fischetti, 1998).
Acanthamoebae are ubiquitously distributed pro-
tozoal parasites known to cause the sight-threatening
corneal inflammatory disease Acanthamoeba keratitis
(Visvesvara and Stehr-Green, 1990). Pathogenesis
probably results from contaminated contact lenses
which bring the parasite into close proximity with the
cornea. Adhesion of Acanthamoeba trophozoites to the
corneal surface is known to be a critical first step in
pathogenesis (Niederkorn et al., 1992; van Klink et
al., 1993). In vitro analysis has shown a direct
relationship between the ability of the parasite to bind
to corneal epithelial cells and the ability of
Acanthamoebae to produce disease in animal models
(Niederkorn et al., 1992). Adhesion is species specific
in that trophozoites appear to bind to corneal epithelial
* To whom all correspondence should be addressed : J. Y.Niederkorn, Department of Ophthalmology, University of TexasSouthwestern Medical Center, 5323 Harry Hines Blvd., Dallas, Tx.75235-9057, U.S.A.
cells from only four species : human, pig, rabbit and
Chinese hamster (Niederkorn et al., 1992; Yang, Cao
and Panjwani, 1997). Following adherence, the
trophozoites elicit soluble cytolytic factors which allow
the parasite to invade the stroma (Leher et al., 1998c;
He et al., 1990). Parasite-derived collagenase pro-
duced inside the stroma is believed to contribute to the
pathology seen in this disease (He et al., 1990).
Although Acanthamoeba keratitis can be a chronic
infection, therapeutic modalities have greatly
improved in recent years.
Acanthamoebae are known to be resistant to nu-
merous topical chemotherapeutics and to several
components of the immune system (Alizadeh et al.,
1995; Alizadeh, Niederkorn and McCulley, 1996; van
Klink et al., 1997; Toney and Marciano-Cabral,
1998). Our laboratory has shown that repeated
intramuscular immunizations induced specific
Acanthamoeba-specific serum IgG and delayed-type
hypersensitivity responses, yet failed to protect against
ocular challenge with Acanthamoeba trophozoites
(Alizadeh et al., 1995; van Klink et al., 1997).
Moreover, repeated ocular infections do not induce
protective immunity (Alizadeh et al., 1995; van Klink
et al., 1997). These findings parallel the human
disease. Human environmental exposure to
Acanthamoebae is apparently common and results in
systemic immune responses against the parasite in
50–100% of the normal adult population (Wang and
0014–4835}99}01007510 $30.00}0 # 1999 Academic Press
76 H. LEHER ET AL.
Feldman, 1967; Cerva, 1989; Cursons et al., 1980).
Nonetheless,Acanthamoebakeratitis occurs in immuno-
competent individuals. This point is reinforced by the
recrudescent nature of this disease in humans. Infected
individuals may sustain this infection for months and
sometimes years before complete resolution of the
disease occurs (Garner, 1993; Lindquist, 1998).
Our laboratory has provided preliminary evidence
showing that activation of the common mucosal
immune system can provide protection against
Acanthamoeba keratitis (Alizadeh et al., 1995; Leher et
al., 1998a, 1998b). Pigs and Chinese hamsters orally
immunized with Acanthamoeba antigens in the pres-
ence of the mucosal adjuvant cholera toxin (CT) were
protected against ocular challenge with Acanthamoeba-
laden contact lenses (Alizadeh et al., 1995; Leher et
al., 1998a, 1998b). The orally immunized animals
produced IgA antibodies specific for Acanthamoeba
antigens (Ac-ag) which could be detected in mucosal
secretions, including tear samples (Leher et al., 1998a,
1998b). When examined functionally, IgA from orally
immunized animals inhibited the adhesion of tropho-
zoites to corneal epithelial cells in vitro (Leher et al.,
1998a, 1998b). These results strongly suggest that
anti-Acanthamoeba IgA antibodies are the mediators of
immunity against Acanthamoeba keratitis in orally
immunized animals. However, the most compelling
proof of this hypothesis is to protect susceptible animals
through the passive transfer of monoclonal IgA
antibodies specific for Acanthamoeba. Accordingly, we
developed two anti-Acanthamoeba IgA monoclonal
antibodies which recognized surface epitopes on A.
castellanii trophozoites. The capacity of these mono-
clonal antibodies to provide protection against
Acanthamoeba keratitis was tested in Chinese hamsters.
2. Materials and Methods
Animals
Chinese hamsters (Cricetulus griseus) were
purchased from Cytogen Research and Development,
Inc. (West Roxbury, MA, U.S.A.). Animals were used
when they were 4–12 weeks old. All animals were free
from corneal defects and abnormalities prior to
experiments and were handled in accordance with the
Association for Research in Vision and Ophthalmology
(ARVO) Resolution on the Use of Animals in Research.
Cell Lines
An ocular human isolate of Acanthamoeba castellanii
was acquired from American Type Culture Collection
(ATCC No. 30868, Rockville, MD, U.S.A.) and was
maintained in axenic culture at 35°C in peptone-
yeast-glucose media (PYG) as previously described
(Alizadeh et al., 1995). Complete minimal essential
medium (cMEM) is defined as follows: MEM (JRH
Biosciences, Lenexa, KS, U.S.A.) supplemented with
1% -glutamine (BioWhittaker, Walkersville, MD,
U.S.A.), 1% sodium pyruvate (BioWhittaker), 1%
MEM vitamins (BioWhittaker), and 1% penicillin,
streptomycin and fungizone (BioWhittaker) with 10%
fetal bovine serum (FBS; Hyclone Labs, Logan, UT,
U.S.A.). Chinese hamster corneal epithelial cells
(CHCE) were immortalized from a corneal explant
using the E6}E7 papilloma virus oncogene as pre-
viously described (Wilson et al., 1995). CHCE were
cultured in cMEM containing 500 µg ml−" geneticin
(Sigma, St Louis, MO, U.S.A.). Complete Dulbecco’s
modified Eagle’s medium (cDMEM) is defined as
follows: DMEM (JRH Biosciences) supplemented with
1% -glutamine (BioWhittaker), 1% sodium pyruvate
(BioWhittaker), 10 m HEPES (BioWhittaker), 1%
non-essential amino acids (BioWhittaker), and 1%
penicillin, streptomycin and fungizone (BioWhittaker)
with 15% FBS (Hyclone Labs, Logan, UT, U.S.A.).
Hybridoma cell lines were established and maintained
in cDMEM containing 0±1 m hypoxanthine, 0±4 n
aminopterin and 16 n thymidine (Sigma; HAT-
DMEM). An anti-trinitrophenol (TNP) IgA producing
hydridoma was obtained from ATCC (No. MOPC 315)
and was maintained in cDMEM. Production of anti-
TNP IgA antibodies were verified by enzyme linked
immunosorbent assay (ELISA) using microtiter plates
coated with TNP covalently linked to bovine serum
albumin (BSA) (TNP-BSA was kindly provided by Dr
Dorothy Yuan, University of Texas Southwestern
Medical Center, Dallas, TX, U.S.A.). Mouse anti-TNP
IgA gave an optical density score of 0±447³0±031 at
a 1:200 dilution compared to an irrelevant isotype
control antibody which gave an optical density of
0±086³0±0006.
Collection of Chinese Hamster Mucosal Secretions
Tear secretion was induced by intraperitoneal (IP)
administration of 24 mg kg−" pilocarpine (Sigma).
Tears were collected 10 min after pilocarpine adminis-
tration using 2 µl micro-pipettes (Drummond Scien-
tific, Broomall, PA, U.S.A.). Samples were placed into
sterile microfuge tubes and snap frozen in liquid
nitrogen prior to storage at ®80°C. Stool samples
were collected and immediately placed into phosphate
buffered saline (PBS; pH¯7±2) containing 5% BSA
(Sigma) and a single protease inhibitor cocktail tablet
(Boehringer Mannheim, Indianapolis, IN, U.S.A.).
Stool samples were centrifuged for 5 minutes in an
Eppendorf microfuge (Brinkman Instruments Inc.,
Westbury, NY, U.S.A.) at 100 g, aliquoted, and stored
at ®80°C until used.
ELISA and Western Analyses
Ninety six well microtiter plates were coated with
10 µg ml−" aqueous proteinaceous Acanthamoeba
trophozoite extract (Ac-ag) overnight in 0±1 car-
bonate buffer (Sigma; pH 9±6). Plates were washed 4
times with PBS containing 0±01% Tween-20 (Sigma;
MONOCLONAL IGA AGAINST ACANTHAMOEBA 77
wash buffer), and blocked with 5% BSA in PBS
(blocking buffer) for 1 hr at 37°C. Subsequent anti-
bodies were diluted in blocking buffer. Concentrated
culture supernatant containing either monoclonal
anti-Acanthamoeba IgA antibodies or anti-TNP control
IgA antibodies were added undiluted, 1:10, 1:100 or
1:200. Mucosal fluids were diluted as follows: stool
samples were diluted 1:1. Tears were diluted from
1:100 to 1:800 using two-fold serial dilutions.
Samples were incubated for 1 hr at 37°C. Rabbit anti-
Chinese hamster IgA hyperimmune sera (Leher et al.,
1998a) was added (1:100) and the plates incubated
at 4°C for 2 hr. Plates were washed and 1:10000 goat
anti-rabbit IgG-horse radish peroxidase (HRP) (Ac-
curate, San Diego, CA, U.S.A.) was added. Plates were
developed by adding 1±0 m 2,2«-azinobis(3-ethyl-
benzthiazoline-6-sulfonic acid) (Sigma) containing
0±003% H#O#
and incubated for 30 min at room
temperature. After development, 0±1 ml of 10% SDS
(Sigma) was added per well prior to reading on a
microplate reader (Molecular Devices Corp.) at
405 nm. Naive tears produced an optical density of
0±418³0±008 for all dilutions (1:100 through
1:3200), suggesting a nonspecific uniform back-
ground. The mean optical density obtained from each
dilution of naive tears was subtracted from the same
dilutions of experimental tears. Likewise, naive stool
samples produced a background optical density of
0±305³0±016 which was subtracted from the optical
densities obtained from experiment samples.
Western blot analysis of culture supernatant was
carried out using conventional techniques. Briefly,
25 µg of protein were resolved on a 12% reducing
SDS-polyacrylamide (SDS-PAGE) gel using a BioRad
minigel apparatus (BioRad, Hercules, CA, U.S.A.). Gels
were transferred to Immobilon-P nylon membranes
(Millipore) using a BioRad minitransfer apparatus as
per the manufacturer’s recommendations. Blots were
blocked for 1 hr with 5% casein (Sigma) in PBS with
shaking prior to addition of rabbit anti-Chinese
hamster IgA immune sera diluted 1:100 in the same.
After 1 hr, membranes were washed again and
1:1000 goat anti-rabbit IgG-HRP in PBS containing
5% casein was added. The membrane was incubated
for 1 hr, washed, and developed using an ECL Western
blotting kit (Amersham, Buckinghamshire, U.K.) as
per the manufacturer’s instructions. Monomeric,
dimeric, and polymeric IgA were determined by
comparison with molecular weight standards and
comparison with a well-characterized control mono-
clonal anti-TNP IgA antibody (ATCC MOPC 315)
which is known to produce monomeric and polymeric
IgA (Weltzin et al., 1989).
Chinese Hamster Acanthamoeba Keratitis
Ocular infection with Acanthamoeba was achieved
by placing parasite-laden contact lenses onto scarified
eyes. Briefly, contact lenses were fashioned from
dialysis tubing (Spectrum Med. Inc., Houston, TX,
U.S.A.) using a 3 mm trephine. Lenses were sterilized
in a contact lens heat sterilizer prior to overnight
incubation in PYG containing 50% trophozoites and
50% cysts (3¬10' Acanthamoebae ml−"). Hamsters
were anesthetized by intramuscular injection of
10 mg kg−" ketamine (Ft. Dodge Laboratories Inc., Ft.
Dodge, IA, U.S.A.) and a topical application of
proparacaine (Alcaine, Alcon, Ft. Worth, TX, U.S.A.).
Corneas were gently abraded (25% of surface) using a
sterile cotton swab. Parasite-laden lenses were placed
on the abraded corneas and eyes were closed with a
single suture (6.0 Ethilon, Johnson and Johnson,
Somerville, NJ, U.S.A.). The lenses were removed 7
days later and the infection scored on a scale of 0–5
based on the following parameters : corneal infil-
tration, corneal neovascularization, and corneal ul-
ceration. The pathology was recorded as follows: 0¯no pathology, 1¯!10% of the cornea involved, 2¯10–25%, 3¯25–50%, 4¯50–75%, and 5¯75–
100%. Any animal receiving a score of at least 1±0 for
any parameter was scored as infected.
Production of Chinese Hamster Anti-Acanthamoeba
IgA Monoclonal Antibodies
Hamsters were lightly anesthetized by methoxy-
flurane (Metofane, Mallinckrodt Veterinary Inc.,
Mundelein, IL, U.S.A.) inhalation. Animals received
0±25 ml of 0±1 sodium carbonate (Sigma; pH 9±6) by
gavage prior to oral administration of 10 µg of CT
mixed with 100 µg of Ac-CT in a total volume of
100 µl. All oral immunizations were performed by
gavage once per week for 4 weeks.
Those animals which demonstrated anti-Acantha-
moeba IgA in stool samples by ELISA and were
protected against ocular challenge with Acanthamoeba-
laden contact lenses were selected as B-cell donors.
Peyer’s patches collected from the orally immunized
hamsters were minced with scissors prior to filtration
through nylon mesh (Nitex, Tetko Inc., Kansas City,
MO, U.S.A.). The cells were washed 3¬ in cDMEM
and mixed with mouse myeloma cells (X63Ag8.653,
kindly provided by Dr Philip Thorpe, UT Southwestern
Medical Center, Dallas, TX, U.S.A.) at a ratio of 4:1.
The cells were centrifuged at 500 g and resuspended
in 0±5 ml of 50% polyethylene glycol with 10%
dimethylsulfoxide (Sigma). The fused cells were diluted
in serum-free cDMEM, centrifuged at 100 g, and
resuspended to 10' cells ml−" in cDMEM containing
0±5¬ hypoxanthine, aminopterin and thymidine
(HAT) media supplement (HAT-cDMEM). One hundred
microliters of the cellular suspension were placed into
96-well plates containing Chinese hamster peritoneal
exudate cells (feeder layers). Cultures were fed every 3
days with fresh HAT-cDMEM until colonies were
visible in the wells.
Supernatants collected from growth positive wells
were tested for the presence of anti-Acanthamoeba IgA
78 H. LEHER ET AL.
F. 1. Western analysis of anti-Acanthamoeba IgA monoclonal antibodies. Concentrated hybridoma culture supernatantswere resolved on 12% SDS-PAGE gels loaded at 25 µg lane−". The gels were transferred to nylon membranes and Westernanalysis performed using rabbit anti-Chinese hamster IgA hyperimmune sera. The secondary antibody was goat anti-rabbit IgG-HRP and the blot was developed by ECL Western blotting kit. The lanes contained the following concentrated culturesupernatants : Lane 1¯ anti-Acanthamoeba IgA (clone 14E4) ; Lane 2¯ anti-Acanthamoeba IgA (clone F6C3) ; Lane 3¯ anti-TNP mouse IgA (Control IgA) ; and Lane 4¯ concentrated fresh media (Media). Bracket A indicates region of dimeric andpolymeric forms of IgA and bracket B indicates monomeric IgA based on comparisons with molecular weight standards andcontrol anti-TNP IgA (Lane 3).
by ELISA as described above. Growth positive wells
producing anti-Acanthamoeba IgA were recultured into
three 96-well plates, and the process repeated, until
90% of the growth positive wells were also positive for
anti-Acanthamoeba IgA. Selected wells were subjected
to limiting dilution to isolate monoclonal lines. Two
cell lines, each originating from different primary
wells, were expanded (clones 14E4 and F6C3).
Hybridomas producing anti-Acanthamoeba or con-
trol anti-TNP IgA were cultured in cDMEM-HAT or
cDMEM respectively. Cultures were grown until the
media were exhausted. The cells were then removed
by centrifugation at 250 g (Jouan CR-412, Jouan,
Inc., Winchester, VA, U.S.A.). Culture supernatants
were concentrated to one-tenth the original volume
and dialyzed using a Filtron Ultrasette concentrator
(Pall-Filtron, Northborough, MA, U.S.A.) containing a
10 kDa exclusion membrane as per the manu-
facturer’s recommendations. The supernatants were
further concentrated on a 100 kDa Filtron (Pall-
Filtron) microcentrifugal concentrator prior to storage
at ®80°C.
Passive Immunization of Chinese Hamsters with Anti-
Acanthamoeba IgA
Chinese hamsters received 2±0 mg (0±5 ml) of
concentrated monoclonal antibody culture super-
natant containing either anti-Acanthamoeba IgA 14E4
or F6C3, or anti-TNP IgA. The supernatants were
concentrated as described above and administered i.p.
twice per day for 7 days (i.e., 14 injections). The
animals received ocular Acanthamoeba infections as
described above 6 days after the last immunization.
MONOCLONAL IGA AGAINST ACANTHAMOEBA 79
Seven days after receiving Acanthamoeba-laden contact
lenses, the ocular infections were scored by two
independent, masked observers.
Adhesion Assay and Immunofluorescent Staining of
Trophozoites
Adhesion of Acanthamoeba trophozoites to CHCE in
vitro was carried out by radiolabeling 2¬10'
trophozoites ml−" overnight in 0±1 µCi $&S-
methionine}cysteine (New England Nuclear, Boston,
MA, U.S.A.). The labeled parasites were washed 3¬ in
HBSS (BioWhittaker) and resuspended (1¬10'
trophozoites ml−") in HBSS containing 1:400 or
1:800 of one of the following concentrated culture
supernatants : anti-Acanthamoeba IgA clone 14E4,
anti-Acanthamoeba IgA clone F6C3, or control mouse
anti-TNP IgA monoclonal antibody. Trophozoites were
mixed with IgA preparations (100 µl well−") and then
added to a 96-well plate containing confluent CHCE
monolayers. Cultures were incubated at 35°C for 45
minutes, and then washed 3¬ with the HBSS. One
hundred microliters of 10% SDS were added and the
plates were incubated 15 min at room temperature
prior to transferring the well’s contents to scintillation
vials. Counts were measured on a Beckman LS3801
scintillation counter (Beckman, Irvine, CA, U.S.A.). All
samples were tested in quadruplicate.
Immunofluorescent staining of trophozoites was
performed as follows. Trophozoites were washed 3¬with HBSS prior to a 1-hr fixation in ice-cold acetone
(EM Science, Darmstadt, Germany). The fixed tropho-
zoites were washed three times and resuspended to
1¬10' trophozoites ml−" in PBS containing 5% BSA
(blocking buffer; Sigma) for 1 hr. A 1:50 dilution of
concentrated culture supernatant from one of the
following hybridoma cell lines was added to 0±1 ml of
trophozoite suspension: anti-Acanthamoeba IgA clone
14E4; anti-Acanthamoeba IgA clone F6C3; or control
mouse anti-TNP IgA. Following incubation for 1 hr on
ice, a 1:50 dilution of rabbit anti-Chinese hamster IgA
was added. Trophozoites were washed and incubated
for 1 hr with goat anti-rabbit IgG-FITC (1:1,000;
Accurate). Trophozoites were washed and then
visualized with a Leica Diaplan microscope (Deerfield,
IL, U.S.A.) using a 40¬ epiflourescence objective.
Images were captured using a CCD camera (COHU,
San Diego, CA, U.S.A.) with an integrator}storer
(Colorado Video Inc., Boulder, CO, U.S.A.) and digitized
using a Data Translator DT3155 image-acquisition
card (Data Translation, Marlboro, MA, U.S.A.) on a
DELL pentium computer.
IgA-mediated Cytotoxicity Assay
Acanthamoeba castellanii trophozoites were washed
3¬ in HBSS and resuspended to 1¬10& tropho-
zoites ml−" in a 1:400 dilution of one of the following
concentrated culture supernatants : anti-Acanthamoeba
IgA clone 14E4, anti-Acanthamoeba IgA clone F6C3, or
control mouse anti-TNP IgA monoclonal antibody.
After 1, 4 or 8 hr, trophozoite suspensions were
diluted 1:2 in trypan blue and examined micro-
scopically for viability and encystment. All samples
were tested in triplicate and the average numbers of
viable trophozoites were reported with standard
deviation of the mean.
Statistical Analysis
Statistical analyses were carried out using a
Student’s t test.
3. Results
Monoclonal Anti-Acanthamoeba IgA Antibodies
Previous studies in our laboratory have shown that
oral immunization using CT and Ac-ag protected
against Acanthamoeba keratitis (Alizadeh et al., 1995;
Leher et al., 1998a, 1998b). Protection correlated
with the appearance of anti-Acanthamoeba IgA in the
tears (Leher et al., 1998a, 1998b). If tear IgA
antibodies were the protective entity induced by oral
immunization, then passive transfer of monoclonal
IgA antibodies specific for Acanthamoeba antigens
should protect naive hamsters from Acanthamoeba
keratitis. Accordingly, two monoclonal anti-
F. 2. Monoclonal anti-Acanthamoeba IgA antibodies arespecific for Acanthamoeba antigen. ELISA plates were coatedwith 10 µg ml−" Ac-ag overnight. Concentrated monoclonalantibody culture supernatants were diluted 1:10, 1:100, or1:200 and added to the wells. Rabbit anti-Chinese hamsterIgA was added as a second step, and after incubation, wasfollowed with goat anti-rabbit IgA-HRP. The ELISA plateswere developed and read at 405 nm. Data are the mean oftriplicate wells and are reported as mean optical density³..*P!0±0007 by Student’s t test. These results are from onerepresentative experiment. This experiment was performedthree times with similar results. *, Media Control ; V, IgAControl ; D, 14E4 Anti-Acanthamoeba IgA; -, F6C3 Anti-Acanthamoeba IgA.
80 H. LEHER ET AL.
F. 3. Anti-Acanthamoeba IgA monoclonal antibodies bind to fixed trophozoites. Trophozoites were fixed in ice-cold acetoneand incubated with either rabbit anti-Acanthamoeba hyperimmune sera (Hyperimmune sera), mouse anti-TNP IgA (ControlIgA), or anti-Acanthamoeba IgA (14E4 or F6C3). The cells were washed and incubated with rabbit anti-Chinese hamster IgAprior to exposure to goat anti-rabbit IgG-FITC. Photographs are representative of at least ten high-power fields per sample.
Acanthamoeba IgA antibodies (14E4 and F6C3) were
generated. Limiting dilution was performed twice to
insure that each selected cell line was monoclonal.
Because monomeric IgA is not readily transported to
the mucosal surface (Hexham, Carayannopoulos and
Capra, 1997), concentrated culture supernatants were
examined by Western analysis to determine whether
the monoclonal antibodies were produced as mono-
meric or polymeric IgA. Each clone produced mono-
meric, dimeric, and polymeric forms of the antibody
(Fig. 1). The mouse control IgA hybridoma also
produced monomeric and polymeric forms of antibody;
however staining on the Western blot was weaker
than the hamster IgA antibodies due to the limited
capacity of the rabbit anti-hamster IgA antibody to
cross-react with mouse IgA (Fig. 1). Analysis by ELISA
showed that both anti-Acanthamoeba antibodies bound
to Acanthamoeba antigens while anti-trinitrophenol
(TNP) control IgA antibodies did not (Fig. 2). More-
over, both anti-Acanthamoeba monoclonal antibodies
bound to epitopes on fixed trophozoites, while anti-
TNP IgA control antibodies did not (Fig. 3).
Effects of Passively Transferred Anti-Acanthamoeba
IgA Against Acanthamoeba Keratitis
To examine the ability of the monoclonal IgA
antibodies to protect against Acanthamoeba keratitis,
the following antibody preparations were administered
to three separate groups of hamsters : (1) anti-
Acanthamoeba IgA (clone 14E4), (2) anti-Acanthamoeba
IgA (clone F6C3), or (3) mouse anti-TNP IgA (control
IgA). Each animal received 2±0 mg of concentrated
antibody culture supernatant i.p. twice per day. The
MONOCLONAL IGA AGAINST ACANTHAMOEBA 81
F. 4. Passively transferred anti-Acanthamoeba IgAappears in mucosal secretions. Hamsters were passivelyimmunized with (1) anti-Acanthamoeba IgA clone 14E4, (2)anti-Acanthamoeba IgA clone F6C3, or (3) mouse anti-TNPcontrol IgA. Two-milligrams of antibody preparation wereadministered twice daily for 7 days. (A) Stool (n¯4) and (B)tear samples (n¯7) were collected on day 5 and assayed forthe presence of anti-Acanthamoeba IgA by ELISA. Back-ground optical densities produced by naive stools or tearsranged from 0±305³0±016 to 0±418³0±008 and weresubtracted from the respective experimental samples. Eachsample was tested separately. Optical densities for eachgroup were combined and reported as mean opticaldensity³.. of the mean. Statistics were carried out usingStudent’s t test. *P%0±03 compared to animals receivingcontrol IgA. These data are from a typical experiment whichwas performed two times with similar results. *, 14E4 Anti-Acanthamoeba IgA; V, F6C3 Anti-Acanthamoeba IgA; D,Control IgA.
injections were administered for 7 consecutive days
and the animals were challenged with ocular
Acanthamoeba infections on day 6 of the immunization
protocol.
To insure that the antibodies were present at
mucosal sites, stool and tear samples were collected 1
day before infection and examined for the presence of
anti-Acanthamoeba IgA by ELISA. Anti-Acanthamoeba
IgA from clone 14E4 was present in both stool and
tear samples in significant quantities compared to
samples taken from naive animals or animals receiving
control IgA [Fig. 4(A) and (B)]. Surprisingly, tear
F. 5. Anti-Acanthamoeba IgA protects againstAcanthamoeba keratitis. Hamsters were passively immunizedwith (1) anti-Acanthamoeba IgA clone 14E4, (2) anti-Acanthamoeba IgA clone F6C3, or (3) mouse anti-TNPcontrol IgA. Two-milligrams of antibody preparation wereadministered twice daily for 7 days. On day 6, the animalswere infected with Acanthamoeba-laden contact lenses. Thecontact lenses were removed 7 days later and infection rateswere scored. The data show the infection rates for eachgroup. The infected}total number of animals and severityscores for each group were as follows: Control IgA (10}11animals were infected; combined severity score of 1±42);14E4 (6}14 animals were infected; combined severity scoreof 0±36); and F6C3 (15}17 animals were infected; combinedseverity of 1±55).
samples collected from animals given anti-
Acanthamoeba IgA clone F6C3 showed only a trace
amount of anti-Acanthamoeba IgA, which was not
significantly higher than control animals. No anti-
Acanthamoeba IgA was found in stool samples from
animals receiving clone F6C3 [Fig. 4(A) and (B)].
Seven days after infection with Acanthamoeba-laden
contact lenses, the clinical disease of the animals was
scored. A compilation of two experiments showed the
infection rate of Chinese hamsters receiving anti-
Acanthamoeba IgA clone 14E4 was 42±8% compared to
animals given control IgA (90±0%) or those receiving
anti-Acanthamoeba IgA clone F6C3 (88±2%; Fig. 5).
Function of Anti-Acanthamoeba IgA
As shown above, anti-Acanthamoeba IgA protected
over 40% of the naive Chinese hamsters against
ocular challenge with Acanthamoeba trophozoites. We
next sought to examine the functional characteristics
of the monoclonal antibodies in vitro. Anti-
Acanthamoeba IgA antibody might function to prevent
corneal disease by either killing the trophozoites,
inducing their encystment, or preventing their ad-
herence to the corneal epithelium. Accordingly, the
82 H. LEHER ET AL.
F. 6. The effects of anti-Acanthamoeba IgA monoclonalantibody on trophozoite viability. Trophozoites wereincubated with anti-Acanthamoeba IgA monoclonal antibody(clone 14E4 or F6C3), anti-TNP mouse IgA, or HBSS (Media)for 1, 4 or 8 hr. At each time point the parasites werecollected and examined microscopically for viability bytrypan blue staining. Viability was not significantly differentat any time point when compared to trophozoites treatedwith control IgA or media alone. The data are from arepresentative experiment which was repeated once withsimilar results. ,, Media; 7, Control IgA; , 14E4 Anti-Acanthamoeba IgA; , F6C3 Anti-Acanthamoeba IgA.
anti-Acanthamoeba IgA monoclonal antibodies were
examined for their effect on parasite viability and
adherence to corneal epithelial cells in vitro. The
results showed that neither of the anti-Acanthamoeba
IgA antibodies killed trophozoites (Fig. 6) or induced
their encystment in vitro (data not shown). However,
as shown in Fig. 7, both anti-Acanthamoeba IgA
monoclonal antibodies inhibited binding of tropho-
zoites to Chinese hamster corneal epithelial cells
(CHCE). Nearly three-fold fewer trophozoites incubated
in anti-Acanthamoeba IgA bound to CHCE than
parasites preincubated in either medium or control
anti-TNP IgA.
4. Discussion
Herpes simplex virus keratitis and trachoma are
responsible for millions of cases of blindness (Adamis
and Shein 1994; Pavan-Langston, 1994). Both can
result in devastating immune-mediated corneal de-
struction. Like most mucosal parasites, adherence to
epithelial surfaces is the first step in the infectious
cascade (Adamis and Shein, 1994; Pavan-Langston,
1994). In numerous parasitic infections, disrupting
this first step in the infectious process has a profound
effect on pathogenesis. Numerous studies have shown
that viral, bacterial and amoebic infections can be
diminished or prevented by specific IgA antibodies
which inhibit adhesion between the pathogen and the
F. 7. Anti-Acanthamoeba IgA monoclonal antibodyinhibits adhesion of trophozoites to corneal epithelial cells invitro. Radiolabelled trophozoites were incubated with eitheranti-Acanthamoeba IgA (clone 14E4 or F6C3), anti-TNPmouse IgA (control IgA), or media. The parasites wereplaced into 96-well plates containing confluent CHCEmonolayers. After 45 minutes incubation the cells werewashed and the remaining counts measured. Adherenttrophozoites are represented as mean counts per minute(CPM)³.. Data are the mean of 4 wells per sample andstatistical analyses were carried out using Student’s t test. *The number of trophozoites preincubated in anti-Acanthamoeba IgA were significantly different from all othergroups (P%0±0031). The data are from a representativeexperiment which was repeated two times with similarresults.
affected host mucosal surface (Beving et al., 1996;
Hocini et al., 1997; Fluckiger et al., 1998; Leher et al.,
1998a, 1998b).
The importance of parasite adhesion has been
demonstrated in the pathogenesis of Acanthamoeba
keratitis. Yang et al. (1997) have shown that a
136 kDa mannose binding lectin mediates adhesion of
Acanthamoeba trophozoites to corneal epithelial cells.
As stated earlier, adherence is critical as trophozoites
can only induce disease in species in which the
parasite can bind to the cornea (Niederkorn et al.,
1992). IgA antibodies, the sentinels of humoral
defense at mucosal surfaces, often act by interfering
with adhesion of pathogens to mucosal epithelium.
Animal models of Acanthamoeba keratitis have pro-
vided a reproducible model in which to examine the
role of mucosal immunity in the form of IgA antibodies
against corneal parasitic challenges.
The present study showed that passive transfer of
anti-Acanthamoeba IgA monoclonal antibodies pro-
MONOCLONAL IGA AGAINST ACANTHAMOEBA 83
tected a significant percentage of naive Chinese
hamsters against ocular infection with Acanthamoeba
trophozoites. Both antibodies (14E4 and F6C3) recog-
nized epitopes from proteinaceous trophozoite extracts
and on the surface of the trophozoites. Furthermore,
the antibodies were shown to be functional by their
ability to inhibit adhesion of trophozoites to corneal
epithelial cells in vitro. Only one of the two monoclonal
antibodies (14E4) was protective upon passive transfer
into naive Chinese hamsters. Curiously, 14E4 was
present at the mucosal surface while the other anti-
Acanthamoeba IgA, F6C3, was not. One likely ex-
planation is that 14E4 was able to bind to the
polyimmunoglobulin receptor, the receptor protein
required for translocation of polymeric IgA onto a
mucosal site (Hexham et al., 1998). Because both
antibodies were the result of a fusion procedure which
produced cells containing chromosomes from each cell
type, it is possible that a recombination event mutated
F6C3 such that it could no longer bind to the
polyimmunoglobulin receptor. Myeloma cells, the
fusion partner used to create these hybridomas, are
known to actively recombine exogenously introduced
deoxyribonucleic acid (Fell et al., 1989). A recom-
bination event may lead to loss, addition, or re-
placement of amino acids which could subsequently
impede attachment of polymeric IgA to the poly-
immunoglobulin receptor. Nevertheless, one antibody
(14E4) successfully passed onto the mucosal surface
and was protective against Acanthamoeba keratitis.
These data demonstrate that multimeric IgA anti-
bodies delivered i.p. can appear at mucosal surfaces
including the cornea. Masinick et al. (1997) have also
shown that functional IgA can be induced to appear at
the corneal surface. Direct experiments suggesting a
mechanism whereby IgA functions at the corneal
surface have not been conducted; however, it seems
likely that the IgA antibodies are present in association
with the mucinous tear layer (Hazlett, Wells and Berk,
1981; Wells and Hazlett, 1985). IgA immobilized in
this fashion would be available to recognize infectious
agents, incarcerate them on the corneal surface, and
render them susceptible for removal by the shear
forces of the blink reflex. Such a mechanism of
protection is advantageous because nonspecific inflam-
matory cells such as neutrophils would not respond.
Thus, injury to bystander tissues, produced by non-
specific inflammation, would be averted.
Collateral damage inflicted by inflammatory
responses are responsible for much of the blinding
pathology associated with such diseases as HSV-
keratitis and trachoma (Adamis and Shein, 1994;
Pavan-Langston, 1994). Because IgA antibodies are
not strongly associated with inflammatory responses,
immunization procedures which stimulate IgA anti-
bodies provide a different form of protection than
conventional immunization. A growing body of evi-
dence has demonstrated the efficacy and protective
ability of IgA antibodies against such diverse
pathogens as the influenza virus, Herpes simplex virus,
plague and leprosy (Ramaprasad et al., 1997; Eyles et
al., 1998; Higaki et al., 1998; Gallichan and
Rosenthal, 1998). This study extends these obser-
vations by directly demonstrating the protective effects
of IgA against an ocular pathogen. This report
demonstrates the ability of IgA to prevent infection by
a corneal pathogen. These results support the feasi-
bility of mucosal vaccines for other, more common
corneal pathogens.
Acknowledgements
We would like to thank Rolf Brekken for his excellenttechnical advice concerning hybridoma cells. We also thankDr W. Matthew Petroll for his advice and assistance on theimage capturing computers used for immunofluorescence.This work was supported in part by Grants EYO9756 andT32-AIO7520 from the National Institutes of Health,Bethesda, Maryland, and an unrestricted grant from theResearch to Prevent Blindness, New York, NY, U.S.A. DrNiederkorn is a Research to Prevent Blindness SeniorScientific Investigator.
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