Chlamydia trachomatis native major outer membrane protein induces partial protection in nonhuman primates: implication for a trachoma transmission-blocking vaccine.
ABSTRACT A vaccine is likely the most effective strategy for controlling human chlamydial infections. Recent studies have shown immunization with Chlamydia muridarum major outer membrane protein (MOMP) can induce significant protection against infection and disease in mice if its native trimeric structure is preserved (nMOMP). The objective of this study was to investigate the immunogenicity and vaccine efficacy of Chlamydia trachomatis nMOMP in a nonhuman primate trachoma model. Cynomolgus monkeys (Macaca fascicularis) were immunized systemically with nMOMP, and monkeys were challenged ocularly. Immunization induced high serum IgG and IgA ELISA Ab titers, with Abs displaying high strain-specific neutralizing activity. The PBMCs of immunized monkeys produced a broadly cross-reactive, Ag-specific IFN-gamma response equivalent to that induced by experimental infection. Immunized monkeys exhibited a significant decrease in infectious burden during the early peak shedding periods (days 3-14). However, at later time points, they exhibited no difference from control animals in either burden or duration of infection. Immunization had no effect on the progression of ocular disease. These results show that systemically administered nMOMP is highly immunogenic in nonhuman primates and elicits partially protective immunity against ocular chlamydial challenge. This is the first time a subunit vaccine has shown a significant reduction in ocular shedding in nonhuman primates. A partially protective vaccine, particularly one that reduces infectious burden after primary infection of children, could interrupt the natural trachoma reinfection cycle. This would have a beneficial effect on the transmission between children and sensitized adults which drives blinding inflammatory disease.
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ABSTRACT: The cervix and/or fallopian tubes of pigtailed macaques were experimentally infected with Chlamydia trachomatis. Their sera were collected at varying time points and screened for identification of immunodominant antigens using a whole-genome protein microarray. The effect of doxycycline treatment on the antibody response generated in these macaques was also investigated. Twenty-five female macaques were infected with C. trachomatis serovars D or E in the cervix and/or fallopian tubes. Bloods were collected at baseline and at various intervals after challenge. Serum samples were tested for antibodies using a C. trachomatis serovar D protein microarray. Twenty chlamydial antigens reacted with sera from at least 68% (17/25) of the macaques. In addition to some well-known chlamydial antigens, nine different proteins, not previously recognized as immunodominant, including four hypothetical proteins (CT005, CT066, CT360 and CT578), were identified. Monkeys infected in the fallopian tubes developed a more robust antibody response than animals inoculated in the cervix. Treatment with doxycycline significantly decreased Chlamydia-specific antibody levels. In summary, using protein microarray serum samples from experimentally infected pigtailed macaques were screened for immunodominant chlamydial antigens. These antigens can now be tested in animal models for their ability to protect and as markers of disease progression.Journal of Proteomics 05/2014; · 3.93 Impact Factor
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ABSTRACT: An efficacious vaccine is needed to control Chlamydia trachomatis infection. In the murine model of Chlamydia muridarum genital infection, multifunctional mucosal CD4T cells are the foundation for protective immunity, with antibody playing a secondary role. We previously identified four Chlamydia outer membrane proteins (PmpE, PmpF, PmpG and PmpH) as CD4T cell vaccine candidates using a dendritic cell-based immunoproteomic approach. We also demonstrated that these four polymorphic membrane proteins (Pmps) individually conferred protection as measured by accelerated clearance of Chlamydia infection in the C57BL/6 murine genital tract model. The major outer membrane protein, MOMP is also a well-studied protective vaccine antigen in this system. In the current study, we tested immunogenicity and protection of a multisubunit recombinant protein vaccine consisting of the four Pmps (PmpEFGH) with or without the major outer membrane protein (MOMP) formulated with a Th1 polarizing adjuvant in C57BL/6, Balb/c and C3H mice. We found that C57BL/6 mice vaccinated with PmpEFGH+MOMP elicited more robust cellular immune responses than mice immunized with individual protein antigens. Pmps elicited more variable cellular immune responses than MOMP among the three strains of mice. The combination vaccine accelerated clearance in the three strains of mice although at different rates. We conclude that the recombinant outer membrane protein combination constitutes a promising first generation Chlamydia vaccine construct that should provide broad immunogenicity in an outbred population.Vaccine 06/2014; · 3.49 Impact Factor
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ABSTRACT: There is a need to implement a vaccine to protect against Chlamydia trachomatis infections. To test a new vaccine, mice were immunized with the Chlamydia muridarum native major outer membrane protein (nMOMP) solubilized with either amphipol A8-35 or the detergent Z3-14. OVA was used as a negative control, and mice were inoculated intranasally with C. muridarum as positive controls. Animals vaccinated with nMOMP mounted strong Chlamydia-specific humoral and cell-mediated immune responses. Mice vaccinated with nMOMP/A8-35 had a higher ratio of Abs to denatured elementary bodies (EB) over live EB, recognized more synthetic MOMP peptides and had higher neutralizing titers than sera from mice immunized with nMOMP/Z3-14. T cell lymphoproliferative responses and levels of IFN-γ were also higher in mice vaccinated with nMOMP/A8-35 than with nMOMP/Z3-14. Following immunization, animals were challenged intravaginally with C. muridarum. On the basis of the number of mice with positive vaginal cultures, length of vaginal shedding, total number of positive vaginal cultures, and number of Chlamydia inclusion forming units recovered, nMOMP/A8-35 elicited a more robust protection than nMOMP/Z3-14. By depleting T cells with Abs, we determined that CD4(+) and not CD8(+) T cells mediated the protection elicited by nMOMP/A8-35. Mice were subsequently mated, and based on the number of pregnant mice and number of embryos, animals that were vaccinated with nMOMP/A8-35 or nMOMP/Z3-14 had fertility rates equivalent to the positive control group immunized with live EB and the fertility controls. In conclusion, increased accessibility of epitopes in the nMOMP/A8-35 preparation may account for the very robust protection against infection and disease elicited by this vaccine.The Journal of Immunology 04/2014; · 5.36 Impact Factor
Chlamydia trachomatis Native Major Outer Membrane Protein
Induces Partial Protection in Nonhuman Primates: Implication
for a Trachoma Transmission-Blocking Vaccine1
Laszlo Kari,* William M. Whitmire,* Deborah D. Crane,* Nathalie Reveneau,2*
John H. Carlson,* Morgan M. Goheen,* Ellena M. Peterson,†Sukumar Pal,†
Luis M. de la Maza,†and Harlan D. Caldwell3*
A vaccine is likely the most effective strategy for controlling human chlamydial infections. Recent studies have shown immuni-
zation with Chlamydia muridarum major outer membrane protein (MOMP) can induce significant protection against infection and
disease in mice if its native trimeric structure is preserved (nMOMP). The objective of this study was to investigate the immu-
nogenicity and vaccine efficacy of Chlamydia trachomatis nMOMP in a nonhuman primate trachoma model. Cynomolgus monkeys
(Macaca fascicularis) were immunized systemically with nMOMP, and monkeys were challenged ocularly. Immunization induced
high serum IgG and IgA ELISA Ab titers, with Abs displaying high strain-specific neutralizing activity. The PBMCs of immunized
monkeys produced a broadly cross-reactive, Ag-specific IFN-? response equivalent to that induced by experimental infection.
Immunized monkeys exhibited a significant decrease in infectious burden during the early peak shedding periods (days 3–14).
However, at later time points, they exhibited no difference from control animals in either burden or duration of infection.
Immunization had no effect on the progression of ocular disease. These results show that systemically administered nMOMP is
highly immunogenic in nonhuman primates and elicits partially protective immunity against ocular chlamydial challenge. This is
the first time a subunit vaccine has shown a significant reduction in ocular shedding in nonhuman primates. A partially protective
vaccine, particularly one that reduces infectious burden after primary infection of children, could interrupt the natural trachoma
reinfection cycle. This would have a beneficial effect on the transmission between children and sensitized adults which drives
blinding inflammatory disease. The Journal of Immunology, 2009, 182: 8063–8070.
North America, but it continues to be hyperendemic in many of the
poorest areas of Africa, Asia, Australia, and the Middle East.
Trichiasis is a painful sequela of trachoma where, due to conjunc-
tival scarring caused by chlamydial infection, eyelashes turn in-
ward to touch the cornea. This condition leads to decreased visual
acuity, and eventually blindness, through corneal abrasion. The
World Health Organization has proposed a four-element approach
to eliminate trachoma as a cause of incident blindness, called the
SAFE strategy (2). The acronym stands for Surgery to correct
trichiasis, Antibiotics to treat infection, and Facial cleanliness and
Environmental improvements to reduce chlamydial transmission.
rachoma, a chronic ocular disease caused by Chlamydia
trachomatis, is a leading cause of preventable blindness
(1). The disease has largely disappeared from Europe and
In humans, infection is asymptomatic in many individuals; thus,
treating only those with clinical symptoms will not control the
spread of infection. A vaccination program would have greater
impact on decreasing the prevalence of blinding trachoma.
C. trachomatis is also the causative agent of the most common
bacterial sexually transmitted diseases and lymphogranuloma ve-
nereum. C. trachomatis strains are divided into 15 serogroups (se-
rovars), of which A–C cause trachoma, D–K cause genital STDs,
and L1–L3 cause lymphogranuloma venereum (3–6). Chlamydiae
are obligate intracellular parasites exhibiting a unique biphasic life
cycle. The infectious, but metabolically inactive, elementary bod-
ies (EB)4attach to cells and enter to form intracellular inclusions.
Inside cells, EBs transform into noninfectious, metabolically ac-
tive reticulate bodies which divide by binary fission and eventually
convert back to EBs (7). During the extracellular EB stage, Abs
present in genital tract or ocular secretions can inhibit infection
both in vivo and in tissue culture (8–10). However, the reticulate
bodies, residing within the intracellular inclusion, remain inacces-
sible to Abs. Resolution of infection at this stage requires a cell-
mediated immune response likely controlled by IFN-?-secreting
Th1 cells. Thus, an ideal C. trachomatis vaccine should induce
both local neutralizing Abs to prevent infection by EBs, and a Th1
response to limit infection once it is initiated. The intracellular
lifestyle of the bacteria, where they reside in a well-protected in-
clusion, makes the production of either an effective natural or an
artificial immune response difficult.
*Laboratory of Intracellular Parasites, Rocky Mountain Laboratories, National Insti-
tute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT
59840; and†Department of Pathology and Laboratory Medicine, University of Cal-
ifornia, Irvine, CA 92697
Received for publication December 31, 2008. Accepted for publication April 6, 2009.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by the Intramural Research Program of the National
Institute of Allergy and Infectious Diseases, National Institutes of Health, and by
Public Health Service Grant AI-32248 (to L.M.de la M.) from the National Institute
of Allergy and Infectious Diseases, National Institutes of Health.
2Current address: Sanofi Pasteur Ltd., Toronto, Ontario M2R 3T4, Canada.
3Address correspondence and reprint requests to Dr. Harlan D. Caldwell, Laboratory
of Intracellular Parasites, Rocky Mountain Laboratories, National Institute of Allergy
and Infectious Disease, National Institutes of Health, 903 South fourth Street, Ham-
ilton, MT 59840. E-mail address: firstname.lastname@example.org
4Abbreviations used in this paper: EB, elementary body; MOMP, major outer mem-
brane protein; nMOMP, native-MOMP; SPG, sucrose phosphate glutamate; IFU, in-
clusion-forming units; RT, room temperature; VD, variable domain.
The Journal of Immunology
Development of a vaccine against C. trachomatis is a high pri-
ority. Computer modeling has indicated that even a partially pro-
tective vaccine would substantially reduce infections worldwide
(11, 12). Efforts to create a vaccine have been unsuccessful to date.
In fact, humans vaccinated with killed EBs present more severe
disease than nonvaccinated individuals after naturally acquired in-
fection (13–15). This suggests that dead intact chlamydiae harbor
immunopathogenic components, thus arguing against the use of
either inactivated or live-attenuated vaccines. Hence the major ef-
fort in the development of a chlamydial vaccine has focused on
subunit immunogens capable of evoking protective immunity
without sensitization to damaging immunopathogenic Ags.
The major outer membrane protein (MOMP) is regarded as one
of the most promising subunit vaccine candidates. Highly immu-
nogenic and immunoaccessible, it elicits both neutralizing Abs and
T cell immunity (10, 16–21). MOMP is the dominant surface pro-
tein (contributing to 60% of the total protein mass in the outer
membrane) and consists of four variable domains interspersed be-
tween five constant domains (22, 23). The four variable domains
contain serovar-specific epitopes; the five constant domains are
highly conserved between the different serovars and contain sev-
eral conserved CD4 and CD8 T cell epitopes (24–26). MOMP has
been used in several vaccine studies, together with various adju-
vants and delivery systems. Still, attempts to induce protection
using MOMP, MOMP peptides, or plasmids expressing MOMP
yielded disappointing results, in both small animal models (27–32)
and cynomolgus monkeys (33, 34). These studies demonstrated
either no protection or limited protection against C. trachomatis
An important exception is the recent study by Pal et al. (35) that
showed systemic immunizations with MOMP purified in native
conformation (nMOMP) induced protection against genital chal-
lenge in the murine model. The protective immune response, as
measured by postchallenge infectious burden, duration of shed-
ding, and disease (infertility), was equal to that induced by exper-
imental infection. Currently, this remains the most successful at-
tempt of using a chlamydial subunit vaccine to mimic natural
immunity. Because of these very encouraging results, we have
extended these studies to nonhuman primates. Here we describe
the immunogenicity of nMOMP subunit vaccination and the re-
sulting partially protective immunity achieved in the nonhuman
primate ocular trachoma model.
Materials and Methods
C. trachomatis strains
C. trachomatis serovar A strain A2947 (A2497), serovar A strain
A/HAR-13 (A/HAR-13), serovar B strain B/TW-5/OT (B), serovar Ba
strain Ba/AP-2/OT (Ba), and serovar C strain C/TW-3/OT (C) were grown
in HeLa 229 cells with DMEM (Mediatech) containing 10% (v/v) FCS, 4.5
g/L glucose, 2 mM glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 55
M 2-ME, and 10 ?g/ml gentamicin. Density gradient-purified EBs were
stored in 0.2 M sucrose, 20 mM sodium phosphate and 5 mM glutamic acid
buffer (SPG) at ?80°C.
Six healthy adult male cynomolgus macaques (Macaca fascicularis) main-
tained in the nonhuman primate wing at Rocky Mountain Laboratories
(Hamilton, MT) and cared for under standard practices implemented by the
Rocky Mountain Veterinary Branch were used for all clinical procedures.
Once entered into experiments, animals were housed in single cages. All
clinical and handling procedures were reviewed and affirmed by the Ani-
mal Care and Use Committee at Rocky Mountain Laboratories, and work
was conducted in full compliance with the Guide for Care and Use of
Laboratory Animals, as well as all applicable federal laws and regulations.
The facilities are fully accredited by the American Association for Accred-
itation of Laboratory Animal Care.
nMOMP was prepared from A2497 as previously described by Pal et al.
(35). Three anesthetized macaques (ketamine hydrochloride, 1 mg/kg body
weight) received 200 ?g of nMOMP divided equally between s.c. injec-
tions on both sides of the shaved neck and i.m. injections into the shaved
right and left triceps. Injections contained a 3:7 ratio of CpG ODN-2395 (1
mg; Coley Pharmaceutical Group) and nMOMP to Montanide ISA 720
(Seppic) in a total volume of 1.4 ml/animal (0.35 ml/injection site). Three
other macaques received 200 ?g of OVA in a similar manner. Vaccinated
and control animals were boosted twice at 71 and 141 days after vaccina-
tion using the same routes and doses.
Challenge of vaccinated monkeys
All six macaques were challenged at 175 days after vaccination with 1 ?
104A2497 EBs in 20 ?l of SPG, placed under the protracted upper and
lower eyelids of each eye (2 ? 104EBs/ eye). After inoculation, the upper
lids of closed eyes were briefly rotated with sterile forceps to ensure com-
plete coverage of the inoculum.
Culture and disease evaluation
Three and 7 days after challenge, and weekly until clearance of disease,
swabs from each eye of every animal were cultured on HeLa 229 cells in
96-well microtiter plates. Sterile type 1 Calgiswabs (Puritan Medical Prod-
ucts) were pressed onto the inner surface of the upper and lower lid of each
eye of anesthetized animals and passed back and forth 8–10 times. Swabs
were placed in 2-ml microfuge tubes containing 0.5 ml of SPG and three
sterile glass beads, mixed on an Eppendorf Thermomixer (Brinkmann In-
struments) for 2 min at 1400 rpm and 4°C, and titered for recoverable
inclusion-forming units (IFUs).
Before each swab collection, monkeys were scored for hyperemia and
follicle formation on the upper conjunctival surfaces in both eyes, as de-
scribed by Taylor et al. (36). Hyperemia was scored as follows: 0, no
hyperemia; 1, mild hyperemia; and 2, severe hyperemia. Follicles were
scored as follows: 0, no follicles; 1, 1–3 follicles; 2, 4–10 follicles; 3, ?10
follicles; and 4, follicles too numerous to count. The scores recorded for the
upper conjunctival surfaces of both eyes were added for each animal and
termed the clinical response score. The highest possible score was 12.
Statistical analyses of the clinical response and recoverable IFUs were
nMOMP three times. The immunizations were a combination of i.m. and s.c. injections. Three other monkeys were immunized with OVA in a similar
manner. Approximately 1 mo after each immunization, blood and tear samples were collected to monitor the immune response. Thirty-four days after the
second boost, all animals were challenged with serovar A strain A2497. Chlamydial shedding and gross clinical response were monitored weekly after
Immunization and infectious challenge schedule for nMOMP-vaccinated monkeys. Three cynomolgus monkeys were immunized with
8064IMMUNIZATION OF NONHUMAN PRIMATES WITH nMOMP
done by the statistical language R and used the nonparametric Wilcoxon
rank sum test. Differences were considered significant at a value of
p ? 0.05.
Coomassie and immunoblot analysis
Purified MOMP was loaded onto 10% polyacrylamide gels to view via
Coomassie staining (500 ng/lane) or Western blot analysis (100 ng/lane).
To view MOMP trimers, samples were kept below 37°C, whereas MOMP
monomers were viewed by boiling the sample for 5 min. GelCode Blue
Stain Reagent (Thermo Scientific) was used to stain proteins according to
the manufacturer’s specifications. For immunoblot analysis, proteins were
transferred to 0.2-?m pore size nitrocellulose membranes (Bio-Rad) and
blocked at room temperature (RT) for 30 min. The membranes were rinsed
three times for 5 min in hybridization buffer (3% BSA, 1? PBS (pH 7.3),
0.05% Tween 20, 0.02% NaN3). Primary Abs were then added to aliquots
of hybridization buffer at a 1/1000 dilution and incubated at RT overnight.
The primary Ab solution was removed, and the membranes were rinsed
serovar A strain A2497-infected cells. The purity of the immunogen and its heat-labile trimeric state were evaluated by SDS-PAGE followed by Coomassie
staining or immunoblotting with or without boiling. A, Coomassie staining of nMOMP. Arrows on the left show trimeric and monomeric forms. B, Control
immunoblot of a serovar A-specific mAb (A-20). C, Immunoblot using the three immunized monkey sera. D, Immunoblot using pools of OVA and prebleed
controls. kD, Kilodaltons.
Immunized monkeys recognize both trimeric and monomeric MOMP by Western blot analysis. nMOMP was purified from C. trachomatis
serovar A EB (A2497). The serum Ab responses of the immunized mon-
keys were measured by ELISA using formalin-fixed EBs (A2497). A, Se-
rum IgG; B, serum IgA. ELISA titers were measured ?30 days after the
primary vaccination and the first and second boosts.
Kinetics of the serum ELISA IgG and IgA titers against
different trachoma serovars. The trachoma strain specificity of the serum
Ab response was evaluated by ELISA using EBs of four different strains.
A, Serum IgG; B, serum IgA. ELISA titers were measured 34 days after the
Titer and specificity of the serum ELISA Ab responses for
8065The Journal of Immunology
twice in wash buffer (3% BSA, 1? PBS (pH 7.3), 0.05% Tween 20) fol-
lowed by an extended 1-h wash. Secondary Ab (goat anti-monkey IgG-
HRP-conjugated or rabbit anti-mouse IgG-HRP-conjugated Ab; MP Bio-
medicals) was diluted 1/1000 in wash buffer, and membranes were
incubated at RT for an additional 2 h. Membranes were then washed three
times in 1? PBS (pH 7.3), 0.05% Tween 20; twice in 1? PBS; and once
in H2O. Finally, membranes were treated with Lumi-LightPLUSWestern
Blotting Substrate (Roche) following the manufacturer’s specifications,
and Ab-MOMP complexes were visualized via a Typhoon Variable Mode
Imager (GE Healthcare).
Levels of IL-4, IL-10, IL-12p70, IFN-?, and TNF-? were determined from
culture supernatants of nonhuman primate PBMCs stimulated with UV-
killed EBs of A2497, B and C. Blood was collected from each anesthetized
animal into a sterile heparinized cell preparation tube (BD Biosciences)
and processed according to the manufacturer’s instructions. After hypo-
tonic lysis of the remaining RBC, collected PBMCs were washed twice in
PBS, counted, and adjusted to 5 ? 106cells/ml in DMEM culture medium.
Two hundred microliters of each cell suspension were then placed into
flat-bottom microtiter plate wells in triplicate, and either 50 ?l of killed
EBs at a total protein concentration of 50 ?g/ml or 50 ?l of SPG were
added to test or control wells, respectively. Plates were rocked at 37°C for
1 h and then further incubated at 37°C for 72 h. After incubation, 130 ?l
of culture supernatant were collected from each well, placed into polypro-
pylene microtiter plates, sealed, frozen at ?20°C, and sent for multiplex
cytokine analysis at LINCO Diagnostic Services.
Serum and tear collection
Serum was obtained from venous blood, and tears were collected by ab-
sorbing onto autoclaved and dried 5- ? 20-mm Whatman No. 41 filter
strips. Each strip was placed under the lower eyelid and allowed to saturate
with tears. Saturated strips were then placed into microfuge tubes contain-
ing 0.2 ml of PBS, refrigerated overnight, and frozen until assayed.
Serum and tear IgG and IgA Ab titers for each animal were determined by
ELISA. Two-fold dilutions of serum or tears (100 ?l/well) were added to the
wells of Immulon 2 HB flat-bottom 96-well plates (ThermoLabsystems)
coated with formalin-fixed A2497, A/HAR-13, Ba, or C EBs (1 ?g of total
protein per well). After incubation at 37°C for 2 h, plates were washed
three times, and 100 ?l of biotin-conjugated goat anti-monkey IgA or IgG
(Rockland Immunochemicals; 1/60,000 and 1/5,000, respectively) was
added to appropriate wells for another 1 h of incubation at 37°C. Plates
were then washed, incubated with 100 ?l/well of streptavidin (1:5,000),
washed, and exposed to 100 ?l of p-nitrophenyl phosphate (Invitrogen) for
30 min in the dark at RT. Reactions were stopped with 50 ?l of 1 N NaOH
per well and analyzed on a plate reader at 405 nm.
In vitro serum neutralization assays
C. trachomatis neutralization assays were performed in HAK cells as pre-
viously described (37). Briefly, 1 ? 106EBs per ml were added to 2-fold
dilutions of preimmune and immune sera from vaccinated macaques and
incubated for 1 h at 37°C in microfuge tubes placed on a rotator. Two
hundred microliters of each dilution were then inoculated (in triplicate)
onto 4 ? 105HAK cells/well in a 24-well plate and incubated with rocking
for 2 h at 37°C. Inocula were then removed, and monolayers were fed with
DMEM containing 1 ?g/ml cycloheximide and incubated at 37°C for
for different trachoma serovars. The infection neutralization capability of
the serum Abs was measured in an in vitro neutralization assay in HAK
cells. A, The kinetics of the neutralization titers were measured ?30 days
after the primary vaccination and the first and second boosts using A2497.
B, The trachoma strain specificity of these neutralizing titers was measured
34 days after the second boost.
Titer and specificity of the serum neutralizing Ab responses
Table I. Cytokine secretion profiles of PBMCs from nMOMP-immunized monkeys pulsed with different trachoma serovars
MonkeyImmunogen SerovarTNF-? (pg/ml)IL-4 (pg/ml) IL-10 (pg/ml)IL-12 (pg/ml)IFN-? (pg/ml)
a?, ?3 pg/ml.
8066IMMUNIZATION OF NONHUMAN PRIMATES WITH nMOMP
36–42 h. After methanol fixation, monolayers were stained with a Chlamy-
dia-specific mAb (A-20) and counted for IFUs. Percent specific neutral-
ization for each dilution was calculated as [(preimmune IFUs ? immune
IFUs)/preimmune IFUs] ? 100.
Immunized monkey sera recognizes both trimeric and
monomeric MOMP under Western blot analysis
A schematic diagram showing the immunization and challenge
schedule of cynomolgus monkeys is shown in Fig. 1. Initially, we
analyzed the serum Ab response by Western blotting under con-
ditions that detect both trimeric nMOMP (65 kDa) and monomeric
MOMP (42 kDa). Coomassie staining revealed a strong trimeric
nMOMP band and a less abundant 42-kDa monomeric MOMP
band. No other protein bands were detected. After boiling, only the
monomeric form of MOMP was found (Fig. 2A). Western blots
were then performed using sera from immunized and control an-
imals. Sera from all three nMOMP-immunized monkeys reacted
with both trimeric and monomeric MOMP (Fig. 2C), whereas sera
from both the OVA-immunized animals and prebleed controls
were negative (Fig. 2D). A serovar A-specific mAb (A-20) was
used as a positive control. The A-20 Ab recognized both trimeric
and monomeric MOMP (Fig. 2B).
Kinetics and specificity of the serum ELISA Ab responses
against different trachoma serovars
The serum and tear Ab responses of the immunized monkeys were
measured by ELISA using formalin-fixed A2497 EBs. Low serum
IgG and IgA titers were detected post-primary immunization, but
reasonably high titers were measured after the first and second
boosts (Fig. 3). Tear IgG Abs were of very low titer, and tear IgA
Abs were not detectable (data not shown).
The trachoma strain specificity of the serum Ab response was
also evaluated by ELISA using EBs of four different trachoma
strains as coating Ags. For both IgG and IgA, the highest titers
were measured against the serovar A strains (A2497 and A/HAR-
13), followed by the serovar Ba and C strains (Fig. 4).
Titer and specificity of the serum neutralizing Ab responses
against different trachoma serovars
We next assayed the serum of immunized monkeys for neutraliz-
ing Abs. The sera of all three immunized monkeys revealed high
neutralizing titers following immunization (1/20,000–1/100,000;
Fig. 5A). The trachoma strain specificity of these neutralizing titers
was also evaluated. Surprisingly, and unlike the ELISA Ab titers,
these experiments showed highly specific neutralizing activity
against strain A2497 (Fig. 5B), the strain from which nMOMP was
prepared. Neutralizing titers against the other three trachoma
strains, including the other serovar A strain (A/HAR-13), were
relatively low (1/2400–1/4000) compared with the homotypic
Cytokine secretion profiles of PBMC from nMOMP-immunized
monkeys pulsed with different trachoma serovars
The Ag-specific cytokine-mediated immune response and its se-
rovar specificity were evaluated by profiling the Th1/Th2 cytokine
response of PBMCs. The cytokine profile showed that IFN-?, a
Th1 cytokine, was consistently induced after being pulsed with Ag
(Table I). The data also revealed that, unlike the serum ELISA and
primates. The images are examples of uninfected and
infected upper conjunctiva 4 wk postchallenge. The in-
fected conjunctiva exhibits severe hyperemia, edema,
and large follicles.
Follicular conjunctivitis in nonhuman
lenge of nonhuman primates. The nMOMP and OVA-
immunized nonhuman primates were ocularly chal-
lenged with strain A2497. Chlamydial shedding was
evaluated at weekly intervals to monitor the course of
infection. The nMOMP-immunized animals shed sig-
nificantly fewer organisms during the first 2 wk post-
challenge (p ? 0.002–0.026, Wilcoxon rank sum test).
Chlamydial shedding after ocular chal-
8067The Journal of Immunology
neutralizing titers, IFN-? induction was heterotypic, not dependent
on the serovar used for stimulating the PBMCs.
Ocular challenge of nonhuman primates immunized with
At 34 days after the third immunization, the nMOMP- and OVA-
immunized nonhuman primates were ocularly challenged with
strain A2497. Chlamydial shedding and gross clinical pathology
were evaluated at regular intervals to monitor the course of infec-
tion. Gross clinical pathology was scored based on hyperemia and
follicle formation on the upper conjunctiva. Fig. 6 shows examples
of an uninfected naive upper conjunctiva before challenge and an
infected conjunctiva 4 wk postchallenge. The infected conjunctiva
exhibits the maximum clinical score of 12, presenting with severe
hyperemia, edema, and multiple large follicles.
The nMOMP-immunized animals shed significantly less organ-
isms during the first 2 wk postchallenge (p ? 0.002–0.026), with
98% (70-fold) reduction at the peak shedding period observed on
day 7 (Fig. 7). The total infectious burden (total bacterial shedding
during the entire experiment) for these nMOMP-immunized ani-
mals was reduced by 94% (18-fold). However, the duration of
bacterial shedding did not differ significantly from that in the con-
trol animals. Interestingly, monkey 139 had a delayed peak shed-
ding period (wk 4 instead of wk 1) that corresponded to the highest
serum ELISA IgG and neutralizing titers and the highest IFN-?
response. Furthermore, and surprisingly, the high nMOMP neu-
tralizing titers, strong IFN-? response, and reduced chlamydial
shedding in the immunized animals did not manifest in a signifi-
cant difference in gross clinical pathology between the two groups
We have shown that nMOMP is highly immunogenic in nonhuman
primates. Systemic immunization elicited high levels of serovar-
specific serum IgG and IgA Abs, but very low levels of tear IgG
and undetectable levels of tear IgA Abs, as measured by ELISA.
Serum from vaccinated monkeys was shown to contain high strain-
specific neutralizing Abs. Conversely, immunization induced a
broad trachoma strain cross-reactive IFN-? response. This immune
response resulted in highly significant protection against homo-
typic ocular challenge, reducing the infectious burden ?70-fold
over the first 2 wk postchallenge. However, protection was re-
stricted to early time periods postchallenge, with minimal differ-
ences observed between vaccinated and control monkeys in either
infectious burden or duration at later time points. Surprisingly,
these marked early differences in organism burden in the conjunc-
tival epithelia did not reduce the severity of ocular disease. Nev-
ertheless, this is the first time a subunit vaccine has shown a sig-
nificant reduction in ocular shedding in nonhuman primates.
Although two previous studies described partial protection in non-
human primates after subunit vaccination (33, 38), this protection
was limited to a transient decrease in clinical response with no
significant reduction observed in shedding.
A major and perhaps important finding of this work was the
high strain-specific neutralizing titers generated after immuni-
zation with nMOMP. We believe that the native trimeric struc-
ture of MOMP could be the reason for achieving such high
strain-specific neutralizing titers. Neither this high titer nor
strain specificity was found by ELISA when using purified forma-
lin-fixed EBs as Ag. Serum ELISA Ab titers showed virtually
identical titers against the two serovar A strains (A2497 and
ocularly challenged with strain A2497. Gross clinical pathology was evaluated at weekly intervals to monitor the course of infection. The clinical response
score was determined based on the hyperemia and follicular formation of the upper conjunctiva of both eyes. 0, No disease; 12, maximum disease. There
was no significant difference in the gross clinical response between the two groups.
Gross clinical pathology after ocular challenge of nonhuman primates. The nMOMP- and OVA-immunized nonhuman primates were
8068IMMUNIZATION OF NONHUMAN PRIMATES WITH nMOMP
AHAR-13), with lower but measurable titers against the heterol-
ogous Ba and C trachoma serovars. The significance of this finding
is unclear, but it is consistent with previous findings that protective
immunity against C. trachomatis ocular infection is serovar spe-
cific, with little to no cross-protection against different serovars
(39–41). Indirectly, these findings implicate serovar-specific neu-
tralizing Abs in ocular immunity. The exquisite degree of strain
specificity found in serum neutralizing Abs of nMOMP-immu-
nized monkeys was unexpected and unpredicted, as the MOMPs of
strains A2497 and AHAR-13 differ by only four amino acids (42).
Two of these differences are located in MOMP variable domains
(VD; aa 80 and 153 in VDs I and II, respectively), and the other
two in constant regions. According to the two-dimensional model
of MOMP (43), VD I is the latching loop for the trimers, and that
loop should be critical for trimer formation. Also, recombinant
phage clones expressing MOMP antigenic determinants revealed
that protective serotype-specific mAbs recognized epitopes in VD
I and II (26). Although speculative, these findings argue the four
amino acid substitutions, either independently or collectively, may
change the structural properties of trimeric nMOMP. These sub-
stitutions could thus generate immunodominant determinants rec-
ognized by highly efficient infection-blocking neutralizing Abs.
Immunization with nMOMP resulted in an Ag-specific produc-
tion of IFN-? by PBMCs. The response was broadly cross-reac-
tive, given that different trachoma strains were equally effective in
its induction. IFN-? is thought to play an important role in resolv-
ing C. trachomatis infection; however, our findings indicate that it
is not sufficient to significantly alter the course of ocular infection.
Possible explanations for this finding are that systemic cellular
immunity was ineffective mucosally or perhaps the levels of IFN-?
generated by systemic immunization were simply insufficient. A
more accurate role for IFN-? in ocular immunity against chlamyd-
ial infection may require, like neutralizing Abs, strategies capable
of targeting local ocular immune responses. Typically, C. tracho-
matis infections generate primarily homotypic immunity, provid-
ing less protection against heterotypic challenges (39–41). Our
findings suggest protective immunity is not solely reliant on the
cytokine-mediated immune response; homotypic neutralizing Abs
could also be important factors. Due to the limited number of
primates available for experiments, we were unable to challenge
nMOMP-vaccinated animals with strains other than A2497. Het-
erotypic challenge using other serovars could better define the role
of IFN-? in achieving the protection observed in our experiments.
Similarly, heterotypic challenge with A/HAR-13 would help char-
acterize the in vivo function of the strain specificity of the neu-
tralizing serum Abs.
The immunity induced by systemic immunization with nMOMP
was equivalent to or even better than the immunity induced by
experimental infection in nonhuman primates. In a previous ex-
periment, monkeys were infected with A2497 twice, and immunity
was evaluated 3 wk later (unpublished data). The serum ELISA
IgG and IgA titers and the IFN-? response of PBMCs were com-
parable with that induced by nMOMP immunization. However,
serum neutralizing titers were ?10-fold lower (4,000–8,000 vs
32,000–76,800) than those measured after nMOMP immunization.
Because the nMOMP immunizations induced chlamydial-specific
IFN-? and serum neutralizing Ab responses that correlated with a
significant reduction in the level of early bacterial shedding, the
lack of impact on gross pathology postchallenge was puzzling. In
mice, systemic immunization with nMOMP induced protective im-
munity comparable with that induced by infection with live bac-
teria. Discrepancies between the results of the murine and nonhu-
man primate experiments underscore the importance of using the
nonhuman primate trachoma model in preclinical studies. Aside
from the inherent differences between host and chlamydial species,
these discrepancies likely result from differences in infection sites.
Both the ocular conjunctiva and the upper female genital tract are
mucosal sites; however, the ocular mucosa is probably more re-
gionally isolated. Lacking a practical genital model in nonhuman
primates, we were unable to investigate this further. Another in-
teresting aspect of the partial protection induced by nMOMP im-
munization in nonhuman primates is that it does not differ much
from the protection induced by experimental infection. A single
experimental infection in primates also induces only partial pro-
tection, with a 1- to 2-log decrease in shedding and limited reduc-
tion in pathology (41, 44). This suggests that an ideal vaccine of
nonhuman primates and humans would need to produce immunity
that actually exceeds that of natural infection. At present, the un-
derlying mechanisms for this disparity remain unclear, but it is
certain that a better understanding of human immunity and C. tra-
chomatis virulence factors capable of altering natural and vaccine-
mediated immunity are needed. Vaccines that can provide effective
protection of the eye will probably require immunization strategies
that target regional ocular immune induction sites.
Computer modeling studies have predicted even a partially ef-
ficacious vaccine would have a significant effect on decreasing
chlamydial transmission (11, 12). Considerably reducing chlamyd-
ial shedding throughout communities could successfully interrupt
the trachoma reinfection transmission cycle. This approach would
in fact be similar to that of mass antibiotic treatment, which re-
duces transmission by temporarily reducing the total infectious
burden in a community. However, the effect of mass antibiotic
treatments is unsustainable, and treatment must be repeated as in-
fections return over time (45–48). In contrast, a vaccine providing
long-lasting partial protection need be administered only once, or
at least less frequently, to achieve a sustainable effect on transmis-
sion. In our study, systemic immunization with nMOMP reduced
the total infectious burden by 94% (18-fold). Thus, a community-
wide nMOMP immunization could significantly impact the fight
against blinding trachoma by interrupting the reinfection cycle.
Admittedly, this vaccination campaign could face significant lo-
gistical difficulties, such as the high production cost of the im-
munogen and the delivery and storage of the vaccine in rural Af-
rican villages. This might be overcome by expressing nMOMP in
a surrogate system and combining it with other childhood vac-
cines. Nevertheless, it is foreseeable that nMOMP vaccination in
combination with antibiotic treatment could be the most effective
way to eliminate trachoma.
We thank the Rocky Mountain Veterinary Branch of Rocky Mountain
Laboratories/National Institute of Allergy and Infectious Diseases; Stephen
Porcella, Daniel Bozinov, and Craig Martens for help with statistical anal-
ysis; Anita Mora for assistance in graphic art; Kelly Matteson for manu-
script formatting; and Kena Swanson for critical review of the manuscript.
The authors have no financial conflict of interest.
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8070IMMUNIZATION OF NONHUMAN PRIMATES WITH nMOMP