Journal of General Microbiology (1986),132, 1599-161 0. Printed in Great Britain
1599
Antigenic Specificity of Human Antibody to Chlamydia in Trachoma and
Lymphogranuloma Venereum
By M I C H A E L E . W A R D , ' * J O H N D . T R E H A R N E * A N D A L A N M U R R A Y '
Department of Microbiology, Southampton University Medical School, Southampton General
Hospital, Southampton SO9 4XY, U K
Sub-Department of Virology, Institute of Ophthalmology, Judd Street, London WCI SQS, UK
(Received 27 November 1985)
An understanding of the molecular basis of the humoral immune response to chlamydial
infections in man requires the identification of target antigens to which antibodies are directed.
The antigenic specificity of antibody from patients with lymphogranuloma venereum (LGV) or
trachoma was therefore assessed by Western blotting. Surface polypeptides were first identified
using purified chlamydial outer membrane complex as antigen. Antibodies in sera from patients
with LGV but not from control negative sera reacted with a wide range of chlamydial surface
polypeptides with molecular masses of 19,29,41, 5 8 , 6 3 and 65 kDa. The major component of
the antibody response detected by both immunoblotting and immunoprecipitation assay was
directed against the major outer membrane protein (MOMP). Antibody to MOMP was speciesspecific on Western blotting, whereas antibody to several other polypeptides recognized
common immunodeterminants on polypeptides of C. psittaci Cal-10 of equivalent molecular
mass. Immunologically C. psittaci Cal-10 was more closely related to LGV strains of C.
trachomatis than a guinea pig inclusion conjunctivitis strain of C. psittaci.
Trachoma sera collected from a village in southern Iran showed predominantly type-specific
antibody on micro-immunofluorescence to serotype A or B trachoma agents. These sera showed
a weak immune response to MOMP, a pronounced response to a polypeptide of 36 kDa and
much less widespread reactivity with other chlamydial polypeptides. The lack of an immune
response to SDS-stable immunodeterminants on MOMP might contribute to the susceptibility
of trachoma patients to repeated cycles of ocular infection with chlamydiae.
INTRODUCTION
Lymphogranuloma venereum (LGV) and trachoma are clinically and immunologically
distinct manifestations of human infection with Chlamydia trachornatis. In LGV, chlamydiae of
serotypes L1, L2 or L3 invade and multiply within lymph nodes draining the genital tract,
producing a strong, systemic antibody response reactive with multiple chlamydial serotypes.
Hyperendemic trachoma, by contrast, is a localized ocular infection due to chlamydiae of
serotypes A, B or C, which engender a weak type-specific antibody response in both tears and
sera. Multiple reinfection is the norm but eventually gives rise to protective immunity as shown
by the fact that trachoma is primarily a childhood disease (Schachter & Dawson, 1978).
Antibody to the serotype-specific immunodeterminants offers limited protection in man or
primate models to ocular challenge with organisms of homologous serotype (Monnickendam &
Pearce, 1983). However, severe disease was observed in subjects immunized experimentally
with whole trachoma organisms who subsequently became infected with organisms of
heterologous serotype. The implication was that repeated ocular exposure to cross-reactive
Abbreviations: EB, elementary body; LGV, lymphogranuloma venereum; micro-IF, micro-immunofluorescence; MOMP, major outer membrane protein.
0001-31 13
a 1986 SGM
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M. E. W A R D AND OTHERS
chlamydial antigens in a region of hyperendemic trachoma transmission gave rise to damaging
immune hypersensitization leading ultimately to severe scarring and blindness (Schachter &
Dawson, 1978). Empirical vaccination having failed, any further vaccine trials necessitate an
understanding of the contribution of individual chlamydial surface antigens to the human
immune response.
Several potentially important chlamydial surface antigens have already been identified. The
chlamydial major outer membrane protein (MOMP) functions as a porin (Chang et al., 1982;
Bavoil et al., 1984), comprising some 60% of the protein at the surface of the infectious
elementary bodies (EB). Serotype-, subspecies- and species-reactive immunodeterminants have
been identified on this protein with monoclonal antibodies (Stephens et al., 1982; Matikainen &
Terho, 1983)and account for much of the serological cross-reactivity between chlamydial strains
observed by micro-immunofluorescence (micro-IF) (Caldwell & Schachter, 1982). Some EB
surface proteins are cysteine-rich, forming disulphide-linked oligomers, which maintain the
essential structural rigidity of the EB in the absence of peptidoglycan (Hatch et al., 1984). A
number of other surface proteins of unknown function have also been identified (Wenman &
Lovett, 1982a, b). In addition, an endotoxic lipopolysaccharide (LPS) gives rise to complementfixing antibody reactive with C.psittaci, C.trachomatis and the LPS core of other Gram-negative
bacteria (Volkert & Matthiesen, 1956; Nurminen et al., 1984).
The contribution of these surface antigens to the human immune response can be identified
by immunoblotting or by immunoprecipitation. Newhall et al. (1982) used Western blotting in a
study of sera from patients with chlamydial genital tract infection. Antibody to a protein of
62 kDa was common in patients with infection but widespread reaction of other control sera
from non-chlamydia1 infected individuals was identified to other antigens including MOMP.
This paper describes the antigenic specificity of sera from patients with trachoma and LGV
investigated by Western blotting and by radioimmunoprecipitation.
METHODS
Chlamydiae. The C. trachomatis strains used were A/SA l/OT, B/TWS/OT, C/UW 1/OT, E/DK20/0N and
L1/440/LN. C. psittaci strains were meningopneumonitis Cal-10 and guinea pig inclusion conjunctivitis (GPIC)
A10. C. trachomatis strains SAI, TW5 and UWl were grown in BGMK cells (Hobson et al., 1982) in Dulbecco's
minimal essential medium in the presence of 1 pg cycloheximide ml-l. All other chlamydial strains were grown in
cycloheximide-treated McCoy cells, prepared and purified as previously detailed (Salari & Ward, 1981).
Chlamydia1 protein concentrations were determined by comparing the A z s o of samples dissolved in 0.1 M-NaOH
with a standard curve based on a known mass of bovine serum albumin (BSA).
Sera. Monoclonal antibodies were prepared by conventional procedures using purified C. trachomatis
L1/440/LN as antigen and hybridomas derived from Balb/c mouse spleens and NS1 cells. Sera from patients with
the typical clinical features of LGV and high antibody to LGV strains of C. trachomatis originated from the
laboratoriesof Dr R. Ballard in South Africa and Dr D. Mabey in The Gambia, West Africa, to whom we are most
grateful. Sera from patients with typical active trachoma (Tl, T2 or T3) and positive antibody to C. trachomatis
serotype A, B or Con micro-IF were from the collection of sera at the Institute of Ophthalmology, London. These
patients were from the village of Sar-rig in southern Iran, a region with hyperendemic blinding disease. Trachoma
agents were isolated from approximately half of the patients. Psittacosis sera were selected from patients with
atypical pneumonia responsive to tetracycline who demonstrated positive antibodies to a pool of C.psittaci strains
by micro-IF. Rabbit antiserum was from an animal hyperimmunized with C. trachomatis L2/434/Bu in Freund's
adjuvant (Difco) over several months (Ward & Murray, 1984). Samples of sera were thawed once, mixed with an
equal volume of glycerol as anti-freeze, then stored liquid at -20°C.
Serology. Micro-IF was done at the Institute of Ophthalmology, London, as previously described (Treharne et
al., 1977).
Polyacrylamide gel electrophoresis. Polyacrylamide slab gels, 0-75mm thick, comprised a running gel with a
linear gradient of 10 to 25% (w/v) acrylamide, a stacking gel of 5 % (w/v) acrylamide and the discontinuous buffer
system of Laemmli as previously described (Salari & Ward, 1981). Chlamydiae were derivatized by boiling for
5 min in dissociating buffer and electrophoresed for 16 h at 175 V and 4 "C at a starting current of 25 mA.
Molecular mass standards (Sigma) were myosin, p-galactosidase, phosphorylase b, catalase, actin, DNAase,
trypsinogen and myoglobin.
Protein iodination. A single Iodobead (Pierce Chemical Co.) was incubated for 5 min at room temperature with a
reaction mixture of 100 pg protein A (Sigma), 1 mCi (37 MBq) Nal 251(Amersham) and 100 pl PBS (Dulbecco A,
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Oxoid). Unbound iodine was removed from labelled protein by gel filtration on Sephadex G25. Specific activity
was 2-4 pCi (pg protein)-'. Bolton and Hunter iodination was done as recommended in the accompanying
product leaflet from Amersham but using 100pg C. trachomatis L1/440/LN, 500 pCi (18.5 MBq) [1251]Boltonand
Hunter reagent in 100 pl PBS adjusted to pH 8-2.
Zmmunoblorting. Chlamydia1 antigens separated by SDS-PAGE were equilibrated for 1 h in 4 M-urea, 50 mMNaC1,2 mM-Na,EDTA, 0.1 mhl-dithiothreitol in 10 mM-Tris/HCI, pH 7.4. This procedure is reported to improve
the antigenicity of polypeptides separated on SDS-PAGE (Bowen er al., 1980). Antigens were electroblotted onto
nitrocellulose (0.45 pm; Schleicher & Schuell) at 50 V for 4 h at 4 "C in 20 mhl-Tris, 150mhl-glycine and 20% (v/v)
methanol in water. Molecular mass markers transferred to nitrocellulose were stained by the procedure of Dubois
& Rosen (1983), which prevents alteration in the size of the membrane, facilitating comparison with the parent
gel. Non-specific binding sites in the membrane were blocked overnight at 4 "C in 3% (wp) BSA (Sigma, Fraction
V, pH 7), 0.9% (w/v) NaCI, 0.05% (w/v) NaN3 in 10 mM-Tris/HCl, pH 7.4. After washing in gelatin-NP40 buffer
(Towbin et al., 1979)the membrane was cut into strips approximately 5 mm wide, and incubated overnight on ice
with 250 p1 test serum diluted 1 in 10 (trachoma sera) or 1 in 50 in gelatin-NP40 buffer. After washing in gelatinNP40, strips were incubated for 45 min on ice with 21Op1 [1251]proteinA equivalent to 2 x lo5 c.p.m. lZ51.
Unbound protein A was removed by successive washing in buffer consisting of sodium lauryl sarcosinate, 16-0g;
NaC1, 2343; Na,EDTA, 7.44g; gelatin, l o g ; NaN,, 2.0g; Tris, 24.23 g; water to 41, pH 7.4, until no further
radioactivity eluted. Autoradiograms of strips were prepared at - 70 "C using Kodak XAR5 film between
intensifying screens. For quantification, 251-labelled bands on nitrocellulose were located from the
autoradiogram, excised, and the radioactivity was measured in a gamma counter. Western blots of dilutions of
polyclonal antibody against constant antigen established that protein A binding was antibody-limited and thus
proportional to antibody present.
Radioimmunoprecipitationassay. Freshly radiolabelled chlamydiae were solubilized by incubation for 3 h at
40°C in 1 % (v/v) Empigen BB (Albright & Wilson) plus 0.1% (w/v) SDS in PBS, pH7.2. The extract was
centrifuged for 4 rnin at 12000g, 1 mM-phenylmethylsulphonyl fluoride (Sigma) was added and the supernatant
stored at 4 "C for a maximum of 1 month. A 2 p1 sample of the supernatant, 10 p1 of a 1 in 10 dilution of test serum
in PBS and 190p1 0.5% (v/v) Empigen BB and 0.1 % (w/v) SDS dissolved in PBS were mixed overnight at 4 "C.
Then 50 p1 of a 5% (w/v) suspension of protein A-Sepharose beads (Pharmacia) in PBS containing 1 % (w/v) BSA
and 0.05% (w/v) NaN, was added and mixed for 4 h at 4 "C. The beads were sedimented by centrifuging for 1 min
at 12000g and washed five times in Empigen BB/SDS mixture. Radiolabelled antigen bound via antibody to the
immobilized protein A was eluted by boiling for 5 min in 30 p1 dissociating buffer, centrifuged at 12000g for 4 min
and the radioactivity of the supernatant determined by gamma counting (typically 5000-12000 c.p.m. for immune
sera). The derivatized antigens were separated by SDS-PAGE and radioactivity due to individual polypeptides
were determined by autoradiography at - 70 "C as before.
RESULTS
Antigenic specijkity of LG V sera
Western blotting was used to determine the reactivity of 14LGV sera with C. trachomatis
L1/440/LN. Specificity of the blotting procedure was controlled using 6 human sera negative for
chlamydial antibody by both micro-IF and ELISA (Fig. la). Control sera showed no false
positive reactions with any of the separated chlamydial polypeptides. LGV sera reacted with a
wide range of chlamydial polypeptides including a 17 kDa polypeptide, a 29 kDa doublet, a
60 kDa doublet (1 1/14 sera), a 65 kDa polypeptide (8/14 sera) and polypeptides of approximately
128and 150 kDa. Antibody to polypeptides of 38 and 41 kDa in the MOMP region of the gel was
present in all LGV sera and accounted for around a third of total [ 251]proteinA binding to the
nitrocellulose. Certain sera reacted with both these polypeptides (nos 2, 5, 16 and 18) whilst
others reacted preferentially with the upper (nos 3 and 13) or lower (nos 4, 14 and 17) molecular
mass polypeptide, confirming that they were immunologically distinct.
The identity of chlamydial surface antigens was confirmed using Western blots of selected
sera against chlamydial outer membrane complex purified by the method of Caldwell et af.
(1981). Strong antibody responses were observed in the MOMP (40 kDa) region of the gel and to
polypeptides with approximate molecular masses of 58 and 63 kDa (Fig. 1b). Weaker antibody
responses were detected to polypeptides of 30, 50 and 75 kDa.
The reactivity of the same LGV sera on radioimmunoprecipitation with the same organism
was then determined. Purified C . trachomatis L1/440/ON was radiolabelled at free amino groups
with [1251]Boltonand Hunter reagent and solubilized in Empigen BB. When 5 x lo5 c.p.m. of
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Fig. 1. (a) Sera from patients with LGV infection and control negative sera lacking antibody to chlamydiae (*) were reacted by immunoblotting with separated
polypeptides of an LGV strain of C. frachomatis (L1/440/LN). Antibody to a range of polypeptides was identified in LGV sera, with MOMP at approximately
40 kDa, a major antigen. Negative control sera did not react with any chlamydial polypeptides, confirming the specificity of the blotting procedures. ( h )Chlamydia]
surface antigens were identified by the Western blot procedure using purified chlamydial outer membrane complex as antigen. The approximate molecular masses
(kDa) from SDS-PAGE of the antigens detected are indicated on the right-hand side of this and subsequent figures.
t 4
Q\
0
c
Antibody to chlamydial polypeptides
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Fig. 2. Radioimmunoprecipitation assay of antibody to an LGV strain of C.trachomatis in sera from
patients with LGV and from negative controls (*). C. trachomatis L1/440/LN was radiolabelled with
[12sI]Boltonand Hunter reagent and solubilized in Empigen BB. Sera were incubated with the extract,
the antigen-antibody complexes formed were bound to protein A-Sepharose beads, and the radioactive
antigen in the complex was identified by SDS-PAGE followed by autoradiography. The dominant
antibody response detected was to polypeptides in the MOMP region of the gel.
solubilized antigen was incubated with test serum, 2-3 % of the radioactivity became associated
with the protein A immunoadsorbent in the case of LGV sera and 0.18% for control sera. The
251-labelledantigens in the immune complexes were separated by SDS-PAGE and located by
autoradiography (Fig. 2).
Antibody to MOMP was found in all LGV sera tested corresponding to 50-88% of the radiolabelled antigen (mean 67%) detected, with typical activity around 250 c.p.m. after background
subtraction. A t least two antigenic polypeptides were detected in the MOMP region of the gel,
but were poorly resolved. Weak antibody to polypeptides of 62 kDa and 29 kDa was detected in
6 and 10 of the 12 sera investigated, accounting for 10% and 14% respectively of the
radioactivity due to bound antigen. No significant antibody response was detected in control
negative sera.
Cross-reactivity between LG V sera and C . psittaci
To determine whether LGV antibody to C . trachornatis polypeptides would identify genusspecific immunodeterminants on comparable C. psittaci polypeptides, the Western blots were
repeated using C. psittaci Cal-10 and GPIC A 10 as antigens. C .psittaci MOMP failed to transfer
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M . E . W A R D A N D OTHERS
Fig. 3. Western blot of sera from patients with LGV or psittacosis reacting with polypeptides of C.
psittaci Cal-10. At high concentrations of antigen, as here, MOMP failed to transfer to nitrocellulose.
Antibodies to a wide range of other polypeptides were detected, suggesting that these antigens carry
determinants common to both Cal-10 and LGV strains of C. truchomatis. At lower antigen
concentrations (not shown) MOMP successfully transferred but did not react with the LGV sera,
confirming the lack of genus-specific immunodeterminants.
to nitrocellulose at a loading of 400 pg protein per slab gel but transferred at a loading of 100 pg.
LGV antibody failed to react with successfully transferred C. psittaci MOMP of either strain but
reacted with a wide range of C.psittaci polypeptides, particularly in the range 58 to 65 kDa (Figs
3 and 4). Strain Cal-10 antigens showed more extensive cross-reactivity with LGV antibody
than the GPIC strain, suggesting that this organism is more closely related to LGV strains of C.
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Fig. 4. As for Fig. 3 with C. psittaci GPIC A 10 as antigen. This strain showed less reactivity with LGV
antibody than did C.psitraci Cal- 10, supporting the concept of considerable antigenic heterogeneity
within C. psittaci.
trachomatis. Clearly these polypeptides carry genus-specific immunodeterminants common to
both chlamydial species. When four sera from patients with psittacosis were reacted on the same
gels with C . psittaci antigens a similar pattern of reactivity to the LGV sera was observed. No
antibody to C . psittaci MOMP was detected, suggesting that these organisms may be
antigenically heterogeneous, but antibody was again detected to polypeptides in the range 58 to
65 kDa (Figs 3 and 4).
Antigenic spec8city of trachoma sera
Sera from trachoma patients from a region with hyperendemic blinding trachoma in southern
Iran were characterized by micro-IF. The sera were of low titre and were usually serotypeDownloaded from www.microbiologyresearch.org by
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Fig. 5 . Composite figure based on three comparable gels showing the reactivity by Western blot of trachoma and other immune sera with trachoma antigens:
C. truchomatis SAl, serotype A ( a ) ;TW5, serotype B (b, c). Most trachoma sera were type-specificon micro-IF. The type specificity and serum identification number
are indicated at the top of the figure. A few trachoma sera showed broader reactivity, with specificity for serotypes A, B and C. Three LGV sera were included for
comparative purposes as well as a monoclonal antibody (M) to MOMP and a rabbit hyperimmune serum (R)to C. trachomatis L2. Negative control sera confirmed
the specificity of the blots but have been omitted for brevity. For a description of the gels see main text.
Antibody to chlamydial polypeptides
1607
specific for trachoma agents of serotype A or B. A few more broadly reactive sera showed
specificity for serotypes A, B and C or A and C alone. The antigenic specificity of these sera was
then determined using Western blots to C. trachomatis serotypes A (Fig. 5 a), B (Figs 5 b and 5 c)
and C (not shown).
Sera repeatedly analysed by Western blot gave remarkably reproducible patterns of reactivity
with chlamydial polypeptides as shown by serum 57, the only serum common to all three blots
comprising Fig. 5. Thus it was possible to compare Western blots made on separate occasions.
Serotype-specifictrachoma sera reacted with a reduced range of polypeptides compared with
more broadly cross-reactive trachoma or LGV sera. The sera reacting in Fig. 5 a with a serotype
A trachoma agent gave virtually identical results when C. trachomatis UW 1, serotype C was
used as antigen (data not shown). Traces of antibody were detectable in B-specific sera only, to
MOMP of serotype A or C (Fig. 5a) and were detected in more significant amounts in broadly
cross-reactive sera, including three LGV sera, a rabbit hyperimmune serum to serotype L2 and
one (no. 59) of two trachoma sera with reactivity for serotypes A, B and C. An interesting feature
of blots with serotype A or C antigen was that all trachoma sera reacted dominantly with a
polypeptide of 36 kDa. Antibody to this polypeptide was also present in cross-reactive sera and
in blots directed against serotype B antigen (Figs 5 b and 5c) but was of less importance. Three
serotype B-specific sera were the only trachoma sera showing strong reactivity with the
homologous MOMP, the position of which was defined by monoclonal antibody (track M in Fig.
Sc). A strong antibody response was also detected to polypeptides of around 63 kDa (e.g. sera 43,
53 and 57 in Figs 5a and 5b).
DISCUSSION
In active trachoma, lymphoid follicles with germinal centres develop in the conjunctiva,
giving rise to local, tear sIgA antibody. This antibody is probably important in short-lived
immunity after ocular chlamydial infection (Monnickendam & Pearce, 1983) but was not
available in adequate amounts for determination of its antigenic specificity. However,
migration of immunologically committed plasma blasts from the conjunctival site gives rise to
circulating antibody, which was the subject of this study. In man, tear and serum antibody in
trachoma is of similar specificity (Treharne et al., 1978). In the guinea pig model of trachoma,
both serum and tear antibodies correlate equally well with resistance to ocular challenge (Malaty
et al., 1981). Thus the antigenic specificity of serum antibody in trachoma is likely to be similar
to that of local tear antibody and can be related to protective immunity.
Micro-IF testing of the sera studied here showed that the trachoma sera were, in general,
narrowly serotype specific whereas the LGV sera reacted with a much broader range of the 15
C . trachomatis serotypes. This serological difference was matched by the range of polypeptides
with which the sera reacted on Western blotting. Trachoma sera were characterized by a weak
immune response to MOMP, despite the presence of an important serotype-specific
determinant on this protein, but a strong reaction with a 36 kDa polypeptide, and the virtual
absence of reactivity to polypeptides in the range 43 to 58 kDa. By contrast, in LGV sera the
dominant immune response was to MOMP. Could the lack of an immune response to critical
determinants on MOMP be an explanation for the susceptibility of children in hyperendemic
areas to repeated bouts of damaging trachoma infection? To answer this it is necessary to
consider the effects which immunoblotting might have on chlamydial antigens.
Chlamydia1 surface proteins are acidic and heavily cross-linked by S-S bridges. Thus
complete solubilization of chlamydial EB, necessary for immunoblotting, requires strongly
anionic, denaturing detergents like SDS, together with a reducing agent such as mercaptoethanol (Hatch et al., 1981; Bavoil et al., 1984). Such procedures are incompatible with the
preservation of secondary and tertiary protein structure, which must be maintained if
conformation-dependent epitopes are to be retained. Thus the major reason for the failure of
monoclonal antibodies to human IgG to react on Western blotting was the disruption of
secondary structure by mercaptoethanol (Thorpe et al., 1984). Antibodies in broadly crossreactive sera like the LGV sera binding to conformation-independent determinants would mask
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M. E . W A R D A N D OTHERS
the destruction of conformation-dependent epitopes on Western blotting unless the latter were
immunodominant. As MOMP failed to blot under non-reducing conditions it was necessary to
renature the separated antigens by removing the SDS with 6 M-urea before electrophoretic
transfer (Bowen et al., 1980). This method had beneficial effects on chlamydial antigenicity
because two of five monoclonal antibodies with specificity for the MOMP of C. trachornatis
serotypes B, D, E, L1 and L2 failed to react in Western blots unless it was used (data not shown).
Nevertheless, these urea-treated antigens should not be regarded as equivalent to the native
proteins.
The simplest explanation for the lack of antibody to MOMP is that the serotype-specific
immunodeterminant on MOMP recognized by trachoma sera is conformation-dependent and
thus destroyed by Western blotting. This does not explain why certain serotype B-specific
trachoma sera showed strong reactivity for MOMP of homologous serotype and weak reactivity
with MOMP of serotypes A and C. Perhaps the serotype B-specific determinant on MOMP is
more stable than its counterpart on serotypes A or C. Nevertheless, the lack of antibody
response in the trachoma patients studied here to the SDS-stable immunodeterminants on
MOMP is of interest because it might be associated with their evident lack of immunity to
recurrent infection. In support of this concept, antibody to MOMP is known to neutralize
chlamydial replication in vitro (Caldwell & Perry, 1982), probably by blocking the movement of
nutrients through the porin formed by this protein (Bavoil et al., 1984). The critical
immunodeterminants involved have not been identified.
In LGV sera antibody to MOMP failed to react with C. psittaci, confirming the species
specificity of MOMP. This antibody was directed against two closely adjacent but
immunologically distinct polypeptides with a difference of approximately 2 kDa (Salari &
Ward, 1981). It is unclear whether the lower molecular mass polypeptide in this doublet was
unrelated to MOMP or was a distinct form of the protein resulting either from incomplete
reduction of the S-S bridged oligomers described by Newhall & Jones (1983) or from incomplete
processing leaving the membrane insertion signal peptide still attached. Translation in vitro of a
15 kbp insert of chlamydial genomic DNA in Escherichia coli yielded two similar,
immunologically related bands identified as MOMP (Allan et al., 1984).
Western blotting of LGV sera against purified chlamydial outer membrane revealed, in
addition to MOMP, surface polypeptides of approximately 19,30,49,58,63 and 74 kDa. The 19
and 74 kDa polypeptides may be identical to the chlamydial surface polypeptides of 17 and
74 kDa which have recently been cloned (Wenman & Lovett, 1982a; Kaul& Wenman, 1985). If
so, this study suggests that these surface polypeptides were quantitatively of minor importance
in the immune response to LGV (Fig. 1a). The 30 kDa surface polypeptide may be equivalent to
the 29kDa polypeptide described by Newhall et al. (1982). The minor surface antigen of
approximately 49 kDa has not previously been described. The surface polypeptides between 58
and 65 kDa, antibody to which was genus-specific and occurred in most chlamydial infected
patients, probably overlap with the polypeptides of 60-62 kDa which Newhall et al. (1982)
found in the sera of patients with chlamydial genital tract infection. Antibody to LPS would not
have been detected in this study due to its greater electrophoretic mobility and its failure to bind
to nitrocellulose in the presence of detergent.
A major point of difference between this study and that of Newhall et al. (1982) lies in the
reactivity of chlamydial polypeptides with control sera. Newhall and colleagues detected
antibody to MOMP and the 29 kDa polypeptide in all control sera from subjects presumed, on
epidemiological grounds, not to have experienced chlamydial infection. Low levels of antibody
to porins on other bacteria might cross-react with chlamydial MOMP given the conserved
nature of the amino acid sequences of porins (Hofstra & Dankert, 1979) at least amongst
Enterobacteriaceaespecies. If so, these antibodies must be of low avidity. In this study conditions
of Western blotting were stringently established so that antibodies in immune sera were readily
detected, whereas no non-specific immunoglobulin binding was detected in the sera of control
subjects selected for their absence of chlamydial antibody (see Fig. 1). In 20 randomly selected
blood donor sera traces of antibody to the polypeptide of 62 kDa were detected in three sera only,
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of which two sera had chlamydial antibody detectable by ELISA. No antibody was detected to
MOMP or the polypeptide of 29 kDa.
A difficulty is that many of the chlamydial polypeptides antigenic in man identified by this
study are of unknown function. Once information on this becomes available the importance of
specific immune responses identified here should become more apparent. The techniques used
here are being applied in conjunction with clinical and epidemiologicalobservation of trachoma
in an African community to determine any protective role in man of antibody to defined
chlamydial antigens and whether such antibody plays a role in the immunopathology of blinding
trachoma.
We are grateful to Dr R. Ballard in South Africa and Dr D. Mabey in The Gambia for samples of sera from
patients with LGV. We thank the Medical Research Council for their financial support.
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T. M. & LOVETT,M. A.
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BAVOIL,
P., OHLIN,A. & SCHACHTER,
J. (1984). Role of
disulphide bonding in outer membrane structure and
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