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Analysis of the Original Antigenic Sin Antibody Response to the Major Outer
Membrane Protein of Chlamydia trachomatis
Jody D. Berry, Rosanna W. Peeling,
and Robert C. Brunham
Department of Medical Microbiology, University of Manitoba,
Winnipeg, and Laboratory Centre for Disease Control, Winnipeg,
Canada
The anamnestic antibody response to the Chlamydia trachomatis major outer membrane
protein (MOMP) was evaluated in mice after priming with serovar C and boosting either with
the homologous serovar or with heterologous serovars (A, H, K, and B). Microimmunofluorescence antibody responses demonstrated that boosting with heterologous serovars
strongly recalled antibody to serovar C, typical of an original antigenic sin (OAS) response.
Boosting with serovars antigenically related to serovar C (A, H, and K) recalled antibody to
the variable domain 1 (VD1) peptide of the MOMP of serovar C as determined by a pinpeptide ELISA. Complete amino acid substitution analysis of the VD1 peptide epitope of the
MOMP showed that the original antigenic sin response to each boosting serovar contained
antibodies with unique patterns of VD1 peptide recognition. The data suggest that antigenically related C. trachomatis serovars differentially recruit B cell lineages from a heterogeneous
memory B cell pool that had been induced by priming with the original serovar and thus
account for the OAS antibody response.
Shortly after the development of the microimmunofluoresence (MIF) antibody assay that uses Chlamydia trachomatis
elementary bodies (EBs) as antigen, Wang and Grayston [1, 2]
confirmed the strain diversification of the organism and noted
the type specificity of antibody responses during C. trachomatis
infection. They also recognized that reinfection with a C. trachomatis serologic variant (serovar) different from the original
infecting serovar recalled antibodies to the priming serovar, a
phenomenon termed original antigenic sin (OAS) [1, 3, 4]. This
serologic phenomenon was initially observed in seroepidemiologic studies of influenza A virus infection [5, 6] and is
believed to be a common feature of infectious diseases that
elicit strain-specific immune responses due to antigenic
variation.
The immunologic basis for OAS remains incompletely defined, although at least four observations are relevant to the
phenomenon. First, OAS is a property of antigen-specific memory B lymphocytes. B lymphocytes undergo affinity maturation
and are selectively recruited by antigen into the memory compartment [7]. Affinity-matured antibody molecules are encoded
by variable region genes that contain either beneficial somatic
mutations or novel high-affinity minicassette combinations or
Received 29 January 1998; revised 19 August 1998.
Financial support: Medical Research Council of Canada (GR13301) and
Canadian Bacterial Diseases Network Centers of Excellence Program.
J.D.B. was supported in part by a University of Manitoba Graduate
Fellowship.
Reprints or correspondence: Dr. R. C. Brunham, Dept. of Medical
Microbiology, University of Manitoba, 543–730 William Ave., Winnipeg,
R3E 0W3, Canada ([email protected]).
The Journal of Infectious Diseases 1999; 179:180–6
q 1999 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/99/7901-0023$02.00
both [8]. Somatic mutation of a germline variable region gene
can alter antigen-binding specificity and is thus speculated to
contribute to OAS [9]. For example, a haptenic analogue of a
priming antigen was demonstrated to recall antibody encoded
by somatically mutated but not by germline variable region
genes [10]. Second, memory B lymphocyte responses to a given
antigen are dominant over naive B cell responses [11], with the
activation differences between memory and naive B cells due
in part to differential effects of the FcgRII receptor (IgG receptor) on the B cell surface [12]. Preexisting antibody inhibits
activation of naive B cells, because coengagement of the immunoglobulin and Fc receptor on the B cell surface leads to
an activation signal in memory B cells but not in naive B cells
[12]. Thus, in the presence of anti-hapten antibody, memory B
cells are preferentially activated in vivo by a structurally related
haptenic analogue of the priming hapten [13, 14]. Third, OAS
is a property of structurally related antigenic determinants. It
has been estimated that to elicit an OAS response, a boosting
antigen can differ by no more than 33%–42% in amino acid
sequence from a priming protein antigen [15]. Last, it has been
demonstrated with hybridoma technology that boosting with
a structurally variant antigen recalls only a portion of the B
cell population that participated in the initial B cell response
[14]. In aggregate, these observations suggest that OAS may
result from the fact that primary B cell responses to a given
antigen are diversified through somatic mutation and affinity
selection, thus creating a pool of heterogeneous memory B cells.
Some of these memory B cells exhibit improved reactivity for
structural variants of the priming antigen, and certain lineages
within the pool can be differentially expanded by exposure to
a related antigen.
Zhao et al. [16] and Fan and Stephens [17] were the first to
JID 1999;179 (January)
OAS Antibodies to C. trachomatis MOMP
initiate molecular studies of the OAS response engendered by
C. trachomatis. They demonstrated that the OAS response is
strongly directed to variable domain (VD) 1 of the major outer
membrane protein (MOMP) of the priming and challenge C.
trachomatis serovars and that the recall response to the original
serovar is correlated with the extent of sequence sharing between the two serovars [16]. They also demonstrated that OAS
antibody responses are associated with the development of
cross-neutralizing antibodies to both the priming and challenge
strains [17]. Nara [18] suggested that OAS provides cross-strain
protection against antigenic variants of a pathogen by enriching
host antibody responses to broadly neutralizing determinants.
Thus, the OAS response to C. trachomatis could also represent
a host defense mechanism against MOMP antigenic variation.
We have been interested in understanding OAS to determine
if it can be exploited in vaccination against the known multiple
serovars of C. trachomatis. Previously, we demonstrated that
the B cell response to a neutralizing epitope in VD1 of C.
trachomatis MOMP is diverse at the genetic level [19]. Among
eight different hybridomas recognizing the same nominal epitope, four different antibody variable gene assemblages were
identified. Fine-specificity analysis using systematic amino acid
substitutions within the peptide epitope recognized by these
monoclonal antibodies demonstrated that different B cell clones
had distinct antibody-binding patterns. As an explanation for
the OAS response to C. trachomatis MOMP, we queried
whether different B cell subsets are differentially recruited by
MOMP variants out of the diverse memory B cell pool. The
present study was undertaken to evaluate this possibility. The
polyclonal antibody recall response was compared in mice
primed with serovar C EBs and boosted with either serovar C
or other chlamydial serovars. On the basis of previous observations that B cell pleiotropy in monoclonal antibody binding
to an epitope found in the VD1 of MOMP can be discerned
by fine-specificity epitope mapping, for the present study we
assume that the immunochemical characteristics of the polyclonal antibody response will be predictive of differential activation of different B cell clones. As such, polyclonal antibody
fine-specificity epitope mapping patterns are used as a proxy
for individual B cell receptor specificity activated during the
secondary antibody response.
181
mL of an equal mixture of C. trachomatis serovar C EBs (containing
5 3 10 5 inclusion-forming units [ifu]) in sucrose-phosphate-glutamate (SPG), pH 7.4, and complete Freund’s adjuvant. Mice received an identical EB immunization in incomplete Freund’s adjuvant 14 days later. By 6 months, antibody titers had substantially
declined from peak values (data not shown), and mice were divided
into groups of 4 and boosted intraperitoneally with homologous
or heterologous (serovars A, H, K, or B) EBs in SPG without
adjuvant. Blood samples were obtained from mice before boosting
and 7 days after boosting. Sera of mice from the same immunization
group were pooled for analysis. Twenty naive mice were divided
into groups of 4 and intraperitoneally immunized with 200 mL of
a 1:1 mixture of C. trachomatis EBs (containing 5 3 10 5 ifu in SPG).
This was done to measure the primary IgG antibody response at
7 days in naive mice as control for primed mice. In all cases, the
7-day serum IgG titer to serovar C VD1 among naive mice was
!1/100 (data not shown).
MIF assay. Mouse sera were diluted 1/8 in PBS and assayed
in 2-fold dilutions for IgG antibody against purified formalinized
EBs of C. trachomatis serovars A, B, C/J, F/G, H, and K in the
MIF assay [2]. All sera were titrated to end point. Antibody titers
were expressed as the reciprocal of serum dilutions yielding distinct
fluorescence of C. trachomatis EBs.
Synthesis of pin-peptides. Pin-peptides were synthesized on polyethylene rods by using a commercially available kit (Cambridge
Research Biochemicals, Cambridge, UK) as described previously
[21, 22]. Two sets of peptides were used. The first set consisted of
8-mer peptides based on the serovar C MOMP VD1 sequence,
DVAGLQND. The second set represented 7-mer peptides based
on the serovar C MOMP VD1 peptide epitope VAGLQND, in
which each residue was serially replaced with each of the 19 alternative amino acids.
Pin-peptide ELISA. Serial dilutions of pooled antisera from
each group of mice were tested on the immobilized peptides by
ELISA as described earlier [19]. Results are expressed as reciprocal
values of the highest dilution that yielded a signal more than five
times the background optical density value for an irrelevant syn-
Materials and Methods
C. trachomatis strains and immunization methods.
Female
BALB/c mice were obtained from Jackson Laboratory (Bar
Harbor, ME) at 5–6 weeks of age. Renografin gradient–purified
C. trachomatis EBs used in these experiments include the following: A (G17/OT), B (TW5/0T), C (TW3/0T), H (UW43/Cx), and
K(UW31/Cx). Serovars C, A, H, and K belong to the C serogroup,
and serovar B to the B serogroup; figure 1 shows the primary amino
acid sequence for these serovars in the VD1 region of the MOMP
[20]. All strains were grown in HeLa 229 cell monolayers. A group
of 20 mice were primed by immunizing intraperitoneally with 200
Figure 1. Primary sequence of C. trachomatis MOMP variable domain (VD) 1 from C. trachomatis serovars used in study. Serovars of
C serogroup constitute sequence variants in VD1 region. B serogroup
has substantially different VD1 peptide sequences. Underlined sequence
corresponds to pin-peptide epitope used to evaluate sera in pin-peptide
ELISA. * indicates deletion; – indicates sequence homology. Nos. indicate amino acid positions of VD1 in primary structure of whole
MOMP protein based on reference [20].
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Berry et al.
thetic peptide evaluated under the same assay conditions. For the
amino acid substitution analysis, a dilution of 1/800 of immune
sera in 2% bovine serum albumin in PBS and Tween 20 (0.05%)
was used except for the serovar B–boosted group, which was done
at a 1/400 dilution. For fine-specificity mapping, the results were
expressed as percentage of the optical density value relative to the
parental peptide. In the present study, we defined an amino acid
as important for antibody recognition if its substitution in the
parent peptide reduced antibody binding by 180%. If four or more
amino acid substitutions at that position in the parent peptide
reduced antibody binding to <20%, that position was arbitrarily
defined as critical. This method allowed an estimation of the finespecificity epitope mapping pattern for a given polyclonal sera.
Similar definitions were used in a previous study to define the fine
specificity of monoclonal antibody binding to a MOMP epitope
[19].
Results
MIF antibody assay. Six months after priming with serovar
C, the mice had IgG antibody titers of between 128 and 512
against serovar C and between 32 and 512 against serovar B
in the MIF assay. The MIF response against serovars A, H,
F/G, and K ranged from 0 to 128, consistent with their antigenic
status as junior serovars [1]. Unexpected variation in the preboost titers was observed among all groups of mice, despite
priming in an identical fashion. Variation may have been due
to differences in decay rates of circulating antibody titers to
individual epitopes found on the whole organism as a result of
differences in antigen depot or other undefined variations in
the vaccination process. After homologous boosting, mice
showed an 8-fold increase in antibody titer against serovar C,
JID 1999;179 (January)
a 6-fold increase to serovar B, and 0- to 4-fold increases to
serovars A, H, F/G, and K. Boosting with heterologous serovars brought about an 8-fold increase in antibody titer against
both serovar C and the boosting serovar, accompanied by lesser
increases against other serovars (figure 2). This strong recall of
antibody to serovar C by heterologous boosting is the signature
of an OAS response. The results of the MIF assay also demonstrated that boosting with serovars more closely related to
serovar C increased antibody cross-reactivity more than did
boosting with serovar B. For this analysis, we compared postboost MIF titers to serovars A, H, F/G, and K; all 16 reactions
among serogroup C–boosted mice generated antibody titers
11/32 to those serovars, compared with 0 of 4 reactions among
serovar B–boosted mice (P ! .01 ; Fisher’s exact test, two-tailed).
MOMP VD1–specific serologic responses.
To evaluate
whether the differences in the MIF patterns could be correlated
with differences in responses to the serovar C VD1 peptide
epitope, sera were tested with the pin-peptide ELISA [19].
Whereas preliminary experiments demonstrated that naive mice
did not develop measurable serovar C MOMP VD1 antibody
responses 7 days after immunization (data not shown), homologous boosting of primed mice demonstrated a typical secondary antibody response to the serovar C VD1 peptide, with
titers rising ∼25 fold, from 1/200 to 1/5000, within 7 days (figure
3). Heterologous boosting (with serovars A, H, and K) also
resulted in recall antibody responses to the serovar C VD1
peptide epitope. As expected, boosting with serovar B did not
recall antibodies to the serovar C VD1 peptide, since its MOMP
differs substantially in amino acid sequence in the VD1 region
(see figure 1). These results confirm that the OAS response is
Figure 2. Microimmunofluorescence antibody patterns of pre- and post-boost sera using C. trachomatis elementary bodies for indicated serovars.
Values are shown as prechallenge/postchallenge reciprocal end-point titers. Boldface type indicates antibody responses >1/512 7 days after boosting.
Underlines indicate responses to boosting serovar.
JID 1999;179 (January)
OAS Antibodies to C. trachomatis MOMP
in part directed to the VD1 region of the MOMP and that
VD1-specific OAS responses occur among serologically related
strains [16, 17].
Fine specificity of OAS antibody recognition of serovar C VD1
as determined by complete amino acid replacement analysis.
Variations in whole EB reactivity seen in the MIF assay following boosting with C. trachomatis suggested qualitative differences in the ability of MOMP antigenic variants to recall
antibody from the pool of memory B cells. We speculated that
determination of the fine specificity of the serovar C VD1 peptide–specific antibodies may further demonstrate qualitative
differences in recall at the single epitope level. We therefore
performed the pin-peptide ELISA with serially substituted
peptides.
Among animals primed with serovar C, the pre-boost critical
amino acid residue pattern with the serovar C VD1 peptide
epitope was -AGLxN- for all groups of mice (figure 4). Homologous boosting altered the pattern of fine specificity of
antibody recognition to -GLQN-. Boosting with heterologous
serovars also altered the antibody recognition pattern but in
distinctively different ways. Boosting with serovar A altered the
antibody recognition pattern to -GLxxD-, serovar K to -GL-,
and serovar H to -AGL-. Boosting with serovar B did not alter
the pattern of critical residues compared with the prechallenge
sera, consistent with the absence of recall of serovar C VD1
antibodies.
Figure 3. Pre- (open bars) and 7-day postchallenge (solid bars) antibody reactivity with pin-peptides representing serovar C variable
domain 1 linear epitope, DVAGLQND. Log10 of reciprocal of highest
dilution giving 15-fold increase in OD units over background is shown
on y axis. Error bars are too small to be accurately represented. Results
are from single representative experiment; 3 additional assays gave
similar results.
183
Differences in fine specificity as observed in the amino acid
replacement analysis of serovar C VD1 peptide were remarkably concordant with the patterns observed in the MIF assay,
in that less stringent requirements for specific amino acids
within the epitope region were correlated with broader MIF
cross-reactivity. More quantitatively, amino acid replacement
analysis demonstrated that the total number of intolerant
amino acid residue substitutions decreased after boosting with
heterologous serovars within the C serogroup. For example,
homologous boosting generated antibodies that exhibited a
total of 40 intolerant amino acid residue substitutions in the
peptide epitope sequence. Heterologous boosting with related
serovars consistently produced antibodies with fewer intolerant
substitutions. Sera from serovar A boosting had 29 intolerant
substitutions, while sera from boosting with serovars K and H
each had a total of 23 and 22 intolerant substitutions, respectively. Collectively, the data show that OAS antibodies recalled
by heterologous boosting are qualitatively different from antibodies recalled by homologous boosting.
Discussion
We evaluated the effect of sequential exposure to C. trachomatis antigenic variants on antibody responses. We demonstrated that OAS antibodies to serovar C EBs are readily
detectable by the MIF assay from sera of mice primed with
serovar C and boosted with serovars A, K, H, or B. Homologous boosting primarily recalled serovar C–specific antibodies,
whereas heterologous boosting with serovars in the C serogroup
recalled antibodies with increased reactivity to multiple serovars, including the serovar used for boosting.
The serologic characteristics observed in the MIF assay were
corroborated by the pin-peptide assay. Thus, serogroup C
MOMP variants such as A, K, and H recalled serovar C VD1
peptide–specific antibodies. Recall of these antibodies was dependent on the extent of sequence sharing among the serovars,
since serovar C VD1 antibodies were not recalled by boosting
with serovar B, which shares little sequence identity in the VD1
region with serovar C.
The fine specificity of epitope recognition by antibody reflected the nature of the recalled VD1-specific antibody repertoire. Homologous sera (serovar C–primed and –boosted)
exhibited a different VD1 fine-specificity epitope mapping
pattern than was found in sera before boosting, changing from
-AGLxN- before boosting to -GLQN- after boosting. The
MOMP C VD1–specific OAS antibody recalled by VD1 sequence variants of serovars A, K, and H had unique patterns
of fine specificity that differed from the fine-specificity pattern
produced by homologous boosting (see figure 4). The qualitative difference in the antibody recognition pattern to the same
nominal epitope suggests that different variable region genes
are selectively used in the OAS antibody response, despite quantitatively similar responses to serovar C VD1 as detected in the
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Berry et al.
JID 1999;179 (January)
Figure 4. Fine-specificity mapping of polyclonal sera from groups of mice immunized with different prime-boost regimens. Sera were reacted
with parent peptide (VAGLQND) and individual amino acid substitution analogues. Analogues for each amino acid position are in alphabetical
order based on single-letter code for amino acids. Values represent binding of analogue as % of parental peptide. Nonbinding substitutions (!20%
of parent peptide binding) are in bold. Critical binding motifs and total number of intolerant substitutions for given serum are shown, where x
stands for any amino acid. Optical density values substantially 1100% for substituted peptide analogues presumably represent heteroclitic antibody
binding.
JID 1999;179 (January)
OAS Antibodies to C. trachomatis MOMP
pin-peptide ELISA. We implicate the existence of memory B
cells with a diverse pool of affinity-selected variable region genes
to serovar C VD1 to explain the unique phenotype of OAS
antibody generated by heterologous boosting.
Previous experiments provide information relevant to OAS
and suggest a partial understanding of the antibody variable
region genotypes used in response to serovar C VD1. We previously identified several hybridomas that recognized serovar
C VD1 MOMP, including a hybridoma (monoclonal antibody
[MAb] C1.6) that produced antibody capable of neutralizing
infectivity of both serovar C and A in cell culture [19]. Remarkably, MAb C1.6 had a fine-specificity epitope mapping
pattern (-GLxxD-) identical to that observed with sera from
serovar C–primed, serovar A–boosted mice in the present study.
Thus, the serovar C–primed, serovar A–boosted OAS antibody
response may preferentially activate B cells with MAb C1.6–like
variable region genes. We suggest that similar B cell lineages
also exist that are capable of selective activation by serovars K
and H. We plan to test this speculation directly by generating
hybridomas from mice undergoing an OAS response to the
MOMP VD1 epitope.
OAS antibodies may be relevant to C. trachomatis immunobiology and could provide early immunity to reinfection
compared with the susceptibility of naive individuals. Indeed,
this speculation is supported by the ability of C. trachomatis
OAS antibody to neutralize both priming and heterologous
challenge strains in vitro [17] and by the cross-protective immunity engendered between serovars A and C as observed in
the monkey eye infection model [23]. The OAS phenomenon
may also be relevant to the protracted period of time that appears to be required for induction of C. trachomatis immunity.
For instance, it seems to take ∼10 years to acquire protective
immunity to C. trachomatis among commercial sex workers
[24]. A similar time frame also characterizes acquisition of immunity to childhood trachoma [25]. Given that C. trachomatis
infections are antigenically variant, it may be that immunity is
built on the OAS phenomenon and requires in part the generation of a diversified memory B cell pool. If so, the order in
which antigenic variants prime the host may be important. C.
trachomatis serovars such as B and C are classified as senior
serovars because they induce antibodies more broadly crossreactive than do junior serovars [1]. Thus, priming with a senior
serovar and subsequent boosting by a junior serovar may best
generate cross-protective antibodies. An identical phenomenon
of slow acquisition of immunity has also been observed with
Plasmodium falciparum infection [26]. Children develop extremely strain-specific antibodies to P. falciparum–infected
erythrocytes, whereas adults have broadly cross-reactive antibodies. When antibodies from adults are immunoaffinitypurified from P. falciparum–infected erythrocytes and evaluated
for type or cross-reactivity, they are found to exhibit broad
cross-reactivity. This directly shows that individual antibody
molecules of adults are qualitatively different and more cross-
185
reactive than is antibody from children. The differences in antibody reactivity between children and adults are thought to
explain in part the susceptibility of children and resistance (partial) of adults to P. falciparum infection.
OAS could have important implications for vaccine-induced
immunity. In fact, the immunologic capacity of variant antigens such as the MOMP to induce an OAS response may be
exploitable in a vaccination strategy. Our results suggest that
sequential immunization with a limited number of antigenic
variants should engender an OAS response that leads to the
accumulation of cross-protective antibodies. Sequential immunization rather than the simultaneous delivery [27] of antigenically variant MOMPs can avoid the problem of antigenic
competition that occurs when structurally related antigens are
coadministered [28]. Indeed, sequential vaccination with heterologous antigens has been noted to be a largely unexplored
area of vaccinology [29]. The concept of OAS has broad significance for vaccination not only against C. trachomatis but
also against other antigenically variant pathogens, such as rotavirus [30, 31] and human immunodeficiency virus [32], for
which cocktail-type formats are being contemplated.
Acknowledgment
We thank E. Dillon for expert technical assistance.
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