Neutralization of Chlamydia trachomatis - Infection and Immunity

INFECTION AND IMMUNITY, Feb. 1993,

p.

Vol. 61, No. 2

498-503

0019-9567/93/020498-06$02.00/0 Copyright © 1993, American Society for Microbiology

Effects of Antibody Isotype and Host Cell Type on In Vitro Neutralization of Chlamydia trachomatis ELLENA M. PETERSON,* XUN CHENG, SUKUMAR PAL, AND LUIS M. DE LA MAZA

Department of Pathology, Medical Science I Building, Room D440, University of California, Irvine, Irvine, California 92717 Received 14 August 1992/Accepted 12 November 1992

Monoclonal antibodies (MAbs) E-4, E-21, and DIII A3, which recognize the same or similar overlapping peptides in the variable domain IV of the major outer membrane protein of Chlamydia trachomatis but differ in isotype, were used in a complement-independent (CI) in vitro neutralization assay. These MAbs had previously been shown to neutralize chlamydial infectivity in HeLa 229 cells in a complement-dependent assay. In this report, all three MAbs neutralized chlamydial infectivity in HaK cells in a CI assay. However, when HeLa cells were used as the host cell, MAb E-4 (immunoglobulin G2b [IgG2b]) and MAb DIII A3 (IgG2b) failed to neutralize infectivity, while MAb E-21 (IgGl) neutralized chlamydial infectivity. These findings are consistent with the proposal that because of the presence of Fc'yRIII receptors, HeLa cells facilitate infectivity and thus block neutralization through the uptake of an IgG2b-chlamydia complex. Since Fc-yRIII receptors do not bind or bind poorly to IgG1, neutralization of C. trachomatis by MAb E-21 in HeLa cells is also corroborative evidence for the role of Fc-yRIII receptors in this interaction. A fivefold enhancement of infectivity was seen when 10 and 1 ,ug of MAb E-4 per ml were tested in a CI assay with HeLa cells. In performing CI neutralization synergy studies in HeLa cells with MAbs E-4 and E-21, antagonism between MAbs E-4 and E-21 was observed at MAb E-4 concentrations of 10 and 1 ,ug/ml for all concentrations of MAb E-21 tested (10 to 0.1 ,ug/ml). When HaK cells were used in the same studies, no antagonism between the MAbs was found. In addition, when HeLa cells were used in a CI assay, polyclonal serum raised to a peptide representing variable domain IV of the major outer membrane protein inhibited the neutralizing ability of MAb E-21. The blocking of neutralization and the enhancement of infectivity by chlamydia-specific antibodies seen in this investigation with HeLa cells may have important clinical implications for developing preventive strategies for chlamydial infections. The authors hypothesized that the lack of neutralization seen with the HeLa cells could be due to the uptake of MAb A-20-bound elementary bodies by HeLa cells through the Fc-yRIII receptors. On the other hand, HaK cells, lacking the Fc-yRIII receptor, were able to reflect CI neutralization. These authors presented corroborating data in which Fab fragments of MAb A-20 were compared with unaltered MAb A-20 in an in vitro neutralization assay with the two cell lines. When the Fc fragment of the MAb was absent, the same neutralization results were obtained in both HeLa and HaK cells. It is known that IgG2b and IgG3 isotypes bind to Fc-yRIII receptors, while the IgGl isotype does not (18). Therefore, if the lack of neutralization seen with HeLa cells is due to the uptake and internalization of the antibody-C. trachomatis complex mediated by the FcyRIII receptor, the neutralizing ability of IgGl MAbs should not be affected by the expression of Fc-yRIII receptors by the host cell. In order to test this hypothesis, we used MAbs that represent both the IgG2b and IgGl isotypes and that we had previously characterized as neutralizing from our results with HeLa cells in a complement-dependent neutralization assay. We present evidence from the use of these MAbs in a CI neutralization system that supports the proposal of Su et al. (21) that FcyRIII receptors on HeLa cells may play a role in facilitating uptake of the antibody-C. trachomatis complex. Using the same CI neutralization system, we also demonstrated that immune polyclonal sera and MAbs, depending on the isotype and host cell employed, can actually enhance chlamydial infectivity and can block or mask the neutralizing ability of other MAbs.

In vitro assays have been used in an attempt to define the surface of Chlamydia trachomatis that make this organism vulnerable to neutralization by antibodies. There are many variables in performing these assays, including the cell line, the use of centrifugation, the diluent, and the addition of a source of complement (14, 15, 21). Using a complement-dependent assay with HeLa 229 cells and centrifugation, we have developed a reproducible assay to determine the in vitro neutralization ability of monoclonal antibodies (MAbs) and polyclonal sera (5, 15, 16). However, other investigators who have not used centrifugation or a source of complement with HeLa cells have had difficulty identifying antibodies that neutralize the infectivity of C. trachomatis in vitro (25). An explanation for the lack of neutralization seen when HeLa cells are used in a complement-independent (CI) neutralization system was recently reported by Su et al. (21). Using a CI neutralization system which compared, as the host cell, human genital epithelial cells (HeLa 229) with Syrian hamster kidney cells (HaK), these investigators noted marked differences in the neutralization results between cell lines. With an immunoglobulin G3 (IgG3) MAb, A-20, directed at the variable domain (VD) I of the major outer membrane protein (MOMP) of C. trachomatis serovar A, they found that while neutralization of C. trachomatis infectivity occurred in HaK cells, no neutralization was seen with HeLa cells. In addition, they presented evidence for the presence of FcyRIII receptors on the surface of HeLa cells. areas on

*

Corresponding author. 498

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EFFECT OF MAb ISOTYPE ON NEUTRALIZATION OF CHLAMYDIAE

MATERIALS AND METHODS MAbs and polyclonal sera. Four MAbs raised in our laboratory, E-4, E-21, E-23, and L3-1, as well as MAb DIII A3, supplied by H. Caldwell (Rocky Mountain Laboratories, Hamilton, Mont.), were used in this study. The MAbs were purified from ascitic fluid with a protein A MAPS II column (Bio-Rad Laboratories, Richmond, Calif.). MAb-containing fractions were dialyzed against phosphate-buffered saline (PBS) and frozen at -70°C. The peptide (Immunodynamics, San Diego, CA) used in the mouse immunizations, CSATAIFD1TTLNPTIAGAGD VKASAEGQLG, corresponded to 30 amino acids of VD IV of serovar E (Bour) with an additional N-terminal C that was linked to keyhole limpet hemocyanin (KLH) (17). The peptide was stored dry at 4°C until solubilized in sterile PBS, at which time it was frozen at -70°C. Immunizations and characterization of the antisera obtained in response to this peptide have been described previously (5). Briefly, 4- to 6-week-old BALB/c mice (Simonsen Laboratories, Gilroy, Calif.) were injected intraperitoneally with 100 p,g of KLH-VD IV peptide in complete Freund's adjuvant. Animals were boosted intraperitoneally 14 days later with 100 ,ug of KLH-VD IV peptide in incomplete Freund's adjuvant. Control mice were injected with 100 ,ug of KLH by the same protocol as described for the mice immunized with the KLH-linked peptide. The sera used in this investigation were obtained on day 20 of the immunization protocol and frozen at -70°C. Neutralization assays. CI neutralization assays were performed as described by Su et al. (21) with some modifications. C. trachomatis serovar E (Bour) was used in all neutralization assays. The organism was raised and stored as described previously (16). Dilutions of MAbs made in 2-SPG (0.2 M sucrose, 0.02 M sodium phosphate [pH 7.2], 5 mM glutamic acid) were mixed with C. trachomatis serovar E (Bour) at a final concentration of 1.5 x 105 inclusion-forming units (IFU) per ml. Reactions were carried out in a volume of 0.6 ml. After the MAbs and organisms were mixed, the reaction tubes were incubated at 37°C for 45 min with rotation at 150 rpm. Subsequently, 0.14 ml was added to two shell vials, each containing monolayers of HeLa 229 or HaK cells which had been washed with 1 ml of 2-SPG. Inoculated vials were incubated at 37°C with rotation at 120 rpm for 2 h, after which 1 ml of Eagle's minimal essential medium with Earle's salts containing 10% fetal bovine serum, gentamicin (50 ,ug/ml), and cycloheximide (1 ,ug/ml) was added. After 48 h of incubation at 37°C, the monolayers were fixed with methanol and stained as described previously (15). Except where noted, the number of IFU in test mixtures was compared with the number in control mixtures which contained either MAb E-23 or anti-KLH serum. The percent inhibition of control IFU or the percentage of control IFU was calculated for each concentration of MAb or polyclonal sera assayed. Synergy studies. Synergy testing to assess antagonistic, indifferent, or synergistic effects among MAbs was conducted by testing both single concentrations of the MAbs alone and setting up a checkerboard of combinations of MAb concentrations. ELISA with peptides overlapping the VD IV. Overlapping peptides were synthesized by the method of Geysen et al. (7, 8) as recommended by the supplier of the epitope mapping kit (Cambridge Research Biochemicals, Cambridge, En-

gland). Enzyme-linked immunosorbent assays (ELISAs) performed with the pins containing the overlapping

were

499

TABLE 1. Characteristics of the MAbs used in the neutralization assays MAb

Isotype

MOMP region recognized

E-4 DIII A3 E-21 E-23a L3-1

IgG2b IgG2b IgGl IgGl IgGl

VD IV VD IV VD IV

TLNPTIA LNPTIAG TLNPTIA -

VD III

AEFPLDIT

Peptide

recognized

Neutralization of serovar E

Yes Yes Yes No No

a MAb E-23 recognizes the serovar E MOMP in a Western blot but does not recognize overlapping hexameric peptides representing serovar E VD IV. The location of the epitope on the MOMP has not been established.

peptides, as described previously (16). MAbs were tested on at least two different sets of peptide pins made to the VD IV region and with two control peptides, PLAQ and GLAQ, that appeared to be irrelevant to the VD IV sequence. Any reaction having an optical density (OD) of more than 0.2 units above the average of the two ODs obtained with these two control peptides was considered positive. Isotyping. Antisera and MAbs were isotyped as described by Simon et al. (20) with the following modifications. The wells of a 96-well microtiter plate were incubated overnight at 4°C with 0.1 ml of either KLH (10 ,ug/ml) or the VD IV peptide (10 ,g/ml) in 50 mM Tris, pH 7.5, containing 0.15 M NaCl. Coated plates were washed three times with PBS0.05% Tween, and 0.1 ml of twofold serial dilutions of antisera were incubated for 1 h at 37°C. From this point, a mouse isotyping kit (Bio-Rad) was used. Microtiter plates were read with a Bio-Rad Micro 450 plate reader. RESULTS Neutralization assays with HaK and HeLa cells. A profile of the MAbs used in the neutralization studies is shown in Table 1. Four of the MAbs, E-4, E-23, E-21, and DIII A3, were used in a CI in vitro neutralization assay in which HaK cells were compared with HeLa cells. In these assays, MAb E-23 was used as the control MAb because it recognizes the MOMP of serovar E in a Western immunoblot assay but does not neutralize the infectivity of this serovar. The other three MAbs recognize a similar region in the VD IV of the MOMP and represent both the IgGl and IgG2b isotypes (11, 12, 16). The results of the CI in vitro neutralization assay with both HeLa and HaK cells are shown in Fig. 1. All three MAbs, regardless of isotype, were able to neutralize the infectivity of C. trachomatis to approximately the same extent when tested in HaK cells. In contrast, when HeLa cells were used, neutralization was only seen with MAb E-21, which is of the IgGl isotype, but not with either of the two IgG2b MAbs used. In fact, at the higher concentrations of these two IgG2b MAbs, there was a four- to fivefold increase in the number of IFU over the control (Table 2). Testing combinations of MAbs for neutralization. As stated above, when HeLa cells were used in the CI neutralization system, an increase in infectivity was observed which could go as high as 500% of the control value when the two IgG2b MAbs, E-4 and DIII A3, were used at 10 to 1 ,ug/ml (Table 2). In order to see whether an IgG2b MAb could actually have an antagonistic effect on a neutralizing IgGl MAb, a checkerboard titration of MAbs was performed in which MAb E-4 at concentrations of 10, 1, and 0.1 ,ug/ml was tested against the same concentrations of MAb E-21. As shown in Table 2, when HaK cells were used, the MAbs were indifferent to one

500

INFECT. IMMUN.

PETERSON ET AL.

-1

Pu

0 z

-0-- E4-HaK E4-HeLa -

E

0 C.)

IL 0

6

z

0

-

m

/

410

z Iz

Lu

C.) LU

IL

.01

.1

1

1 0

MAb ug/ml FIG. 1. In vitro neutralization results with three different MAbs in two cell lines. All results were compared with those obtained when MAb E-23 was used as the control. Results are expressed as percent inhibition of control IFU. All experimental data points represent the average of at least two separate determinations. Error bars represent 1 standard deviation.

another when tested in combination for their ability to neutralize the infectivity of C. trachomatis in a CI assay. That is, the presence of MAb E-4 did not interfere with the neutralizing ability of MAb E-21. This is in contrast to the results obtained with HeLa cells. As shown in Table 2, there was antagonism between MAb E-4 and MAb E-21. The results in this table are expressed as percentages of the control values in order to illustrate the marked enhancement seen when MAb E-4 was assayed with HeLa cells. Whereas only 4 and 26% of control (no MAb) IFU were obtained when MAb E-21 was tested alone at concentrations of 10 and 1 ,ug/ml, respectively, both values rose to more than 500% of the control value when 10 and 1 ,ug of MAb E-4 per ml was added to the same concentrations of MAb E-21. Therefore, MAb E-4 at concentrations of 10 and 1 pg/ml is antagonistic to all concentrations of MAb E-21 tested. Effect of polyclonal immune sera on a neutralizing MAb. Since an MAb of the IgG2b isotype could affect the neutralizing ability of an IgGl MAb, we next wanted to examine the neutralizing ability of MAb E-21 when assayed in combination with a monospecific polyclonal immune serum. Antiserum to the VD IV region of serovar E was raised by linking the peptide to KLH, and as a control, antiserum was raised to KLH alone. The isotype content of the resulting antisera was determined by using both KLH and VD IV peptide as TABLE 2. Synergy testing resultsa % of control IFU at MAb E-4 concn

Cells

HeLa

HaK

(~lg/ml):

E-21 MAb(,ug/ml) concn 0

10

1

0.1

0 10 1 0.1

100 4 26 73

590

525

578 s

237 22

449

517 46Q

152

0 10 1 0.1

100 12 76 100

41 19 25 22

55 4 59 42

131 23 53 71

427

i16

a MAb E-21 (IgGl) and MAb E-4 (IgG2b) were tested alone and in various combinations. The no-MAb control IFU value was set at 100%. Values indicating antagonism between the MAbs are underlined.

the antigens. As shown in Fig. 2, the control KLH antiserum, as expected, did not react with the VD IV peptide. The predominant isotype in both the KLH antiserum and an anti-KLH-VD IV antiserum was IgGl, followed by IgG2b and IgG3. For in vitro neutralization assays, anti-KLH polyclonal serum was used as a control for the polyclonal anti-KLH-VD IV serum. The polyclonal serum was tested alone and in combination with MAbs for neutralization of serovar E in both HeLa and HaK cells. MAb E-21 was added to the polyclonal serum to see whether this IgGl MAb could neutralize infectivity in HeLa cells in the presence of the polyclonal immune VD IV serum. MAb L3-1 (20 ,ug/ml) served as the control MAb because it is also of the IgGl isotype yet does not neutralize or recognize serovar E (Table 1). The results shown in Fig. 3 are for a 10-2 dilution of the polyclonal antisera. Similar results, with greater neutralization in HaK cells and no neutralization in HeLa cells, were also obtained for a 10-1 dilution of the polyclonal sera. While MAb E-21 was able to neutralize serovar E better than polyclonal serum when tested with HaK cells, with HeLa cells, the ability of MAb E-21 to neutralize infectivity was totally abrogated in the presence of the polyclonal VD IV immune serum directed at C. trachomatis and attenuated in the presence of the anti-KLH control polyclonal serum. DISCUSSION In this study, MAbs of different isotypes that recognize the same peptide within the VD IV region of the MOMP yielded different neutralization results depending on the cell line used. An MAb of the IgGl isotype was able to neutralize the infectivity of C. trachomatis in both HaK and HeLa cells in an in vitro CI neutralization system. In contrast, while the neutralizing ability of an IgG2b MAb could be clearly demonstrated in HaK cells, the opposite finding appeared when HeLa cells were used in the CI in vitro neutralization system. Here, instead of neutralization, enhancement of infectivity was seen with MAbs of the IgG2b isotype, depending on the concentration used. Therefore, when HeLa cells are used in the CI system, the results are dependent not only on the epitope recognized but also on the isotype of the antibody. Su et al. (21) reported that while neutralization was


VOL. 61, 1993

--0KLH-VD

IV

ANTISERA

vs

VD

IV

-U*-

IgGi

IgG2a

-A-

IgG2b

-0-

IgG3

IgM 6

°

1.0

501

KLH-VD IV ANTISERA vs KLH 2.o0-

1.0

0.0

4

KLH

6

8

10

12

ANTISERA DILUTION

ANTISERA DILUTION

ANTISERA vs KLH

KLH ANTISERA

vs

VD IV PEPTIDE

2.0 2.0 -

0

c0

1.0 6I

1.0 -

6

8

10

12

ANTISERA DILUTION

o.o 4

6 10 ANTISERA DILUTION

12

FIG. 2. Isotype pattern of KLH-VD IV and KLH antisera tested against KLH or VD IV alone. The dilution numbers 4 to 12 correspond to twofold dilutions from 1:400 to 1:102,400.

observed when Fab fragments of MAb A-20 (IgG3) were tested in a CI in vitro neutralization assay with HeLa cells, they could not attain neutralization with native MAb A-20. However, neutralization with MAb A-20 was achieved in HaK cells with both the intact MAb and the Fab fragments of this antibody. These authors also reported evidence for

100

ANTI-KLH & MAb E-21 ANTI-KLH-VD IV

-

El

ANTI-KLH-VD IV & MAb ANT

I-z

I-KH-VI)

IV & MAb

13-1 E.21

80

0

LC 60

z

0

E 40 z

z W

20

0

HELA

HAK

CELL LINE

FIG. 3. Results of in vitro assays with polyclonal sera raised to KLH and KLH-VD IV to which a nonneutralizing MAb, 13-1, and a neutralizing MAb, E-21, were added. All results are expressed as percent inhibition of control IFU obtained when serovar E was incubated with anti-KLH serum mixed with MAb 13-1 (20 ,ug/ml) before being used to infect both HaK and HeLa cells. Error bars represent 1 standard deviation for two separate experiments.

the presence of Fc-yRIII receptors on the surface of HeLa cells but did not find them on HaK cells. Therefore, they postulated, as an explanation for their results, that entry was

facilitated by MAbs binding to C. trachomatis and that subsequently the organism was internalized via the interaction of the Fc-yRIII receptor and the MAb-C. trachomatis complex. If this hypothesis is correct, since FcyRIII receptors do not bind IgGl antibodies, one would not expect a neutralizing MAb of the IgGl isotype to behave differently in an in vitro neutralization assay with cells that do express FcyRIII receptors (HeLa) from those that do not (HaK cells). The experimental data in this report support the proposal of these investigators. We have previously assayed the MAbs and polyclonal sera used in this report in an in vitro neutralization assay in which guinea pig serum was used as a source of complement and centrifugation was used in the infection step (5, 15, 16). In addition, all reactions were performed in PBS rather than the 2-SPG that was used here. Under those conditions, the results with HaK and HeLa cells were essentially the same. This is in contrast to the findings we have now obtained with a different in vitro neutralization assay. The reasons for the observed discrepancies between the two systems when HeLa cells and antibodies of the IgG2b isotype are used is not known at this time. Peeling and Brunham (14), using yet another variation of a neutralization assay in which guinea pig serum was used but centrifugation was omitted, also obtained neutralization with a C. trachomatis-specific IgG2b MAb, UM4. The role that centrifugation and complement play in neutralization assays and how they may affect the interactions of the antibody-chlamydia complex with Fc receptors remain to be established. As has been discussed by Pearce (13), centrifugation may promote a different entry

502

INFECT. IMMUN.

PETERSON ET AL.

mechanism(s) for chlamydiae which may affect the trafficking and thus the fate of the organism once inside the cell. This could provide a partial explanation for the difference in results that we observed for the neutralization assays that we performed with and without centrifugation. Which in vitro system better reflects the in vivo interaction of antibodies directed to C. trachomatis is still unclear. While several entry mechanisms have been described for chlamydiae, in vivo this complex interaction between host and parasite may be quite different from what we are measuring in vitro (24). In vivo, neutralization of C. trachomatis will most likely occur at mucosal sites. IgA may also play a role in neutralization of chlamydial infectivity, and how this relates to what we are measuring in vitro with IgG antibodies remains to be established (4). However, until we have a better understanding of the in vivo host-chlamydia interactions, we will need to draw our conclusions about neutralizing epitopes from reproducible in vitro neutralization assays. We have shown, with the CI neutralization assay in HeLa cells, that there is an up to fivefold enhancement of infectivity by the IgG2b MAbs E-4 and DIII A3. Antibody enhancement of viral infectivity has been described for several systems (3, 23); more recently, this enhancement has been reported with human immunodeficiency virus type 1 (HIV-1) (9, 19, 22, 23). This enhancement of viral infection ranges from twofold to as high as 10,000-fold with the dengue viruses, depending on the virus strain, host cell, and antibody source (3, 23). Recently, Mady et al. (10), working with dengue virus, have shown that other cell receptors, P2microglobulin, CD15, and CD33, in the presence of antibody directed at both the pathogen and the cell receptor, showed enhancement of infectivity. From their findings, the authors theorize that the cell receptors act mainly to focus the virus on the cell surface, facilitating binding to the receptor. The viral systems of antibody-mediated enhancement have many similarities to what we observed with chlamydiae. It has been reported for HIV-1 that IgG-F(ab')2 antibodies did not enhance infection, whereas polyclonal immune serum did enhance infection. In addition, work by several investigators with different cell lines has shown that, depending on the neutralization system used, MAbs to Fc-yRI and Fc-yRIII blocked enhancement of infection (9, 22). Immune sera from HIV-1-infected patients at high concentrations neutralized but at low concentrations (high dilutions) enhanced infectivity. Using polyclonal mouse serum raised to the KLH-VD IV peptide in HeLa cells in a CI neutralization assay, we found no neutralization over that in control anti-KLH serum at a 10-1 dilution and observed a threefold enhancement of infectivity at a 10-2 dilution. It is interesting that Takeda et al. (22) found a twofold increase in HIV-1 infectivity, similar to our findings with C. trachomatis, at a 10-3 dilution of polyclonal human serum from HIV-1-infected individuals, while at lower dilutions neutralization was observed. Thus, there are many interesting parallel findings between the in vitro assays with viruses and C. trachomatis. It has been thought that FcyRIII receptors are restricted in their distribution to macrophages, natural killer cells, myeloid precursor cells, and a neutrophil cell line (18). However, the finding by Su et al. of these receptors on a human epithelial cell line (21) brings up an important question. In vivo, do cells permissive for a C. trachomatis infection employ an entry mechanism(s) that enhances infection, as was seen with the HeLa cells used in this report? From our knowledge to date, FcyRIII receptors have not been found on epithelial cells in vivo. However, if we draw an analogy

from major histocompatibility complex (MHC) class II expression studies in rodents, we cannot rule out the fact that, under altered host states, receptors traditionally thought to be restricted to cells of the immune system may be expressed by otherwise silent cells (1, 2). As an example, epithelial cells of the rat gut express MHC class II antigens during parasitic infections, whole-body gamma interferon infusion, and acute infectious colitis; in a normal state, these cells do not express these antigens (2). El-Asrar et al. (6) have reported the presence of HLA-DR on superficial conjunctival epithelial cell layers in trachomatous conjunctivitis. By double staining, they were able to show that epithelial cells expressing HLA antigens also contained chlamydial antigens. As illustrated by the synergy studies, neutralizing antibodies can be blocked by chlamydia-specific IgG2b antibodies that facilitate the entry of the pathogen into host cells. This could have important implications for clinical infections as well as for future vaccine development. Isotyping results with polyclonal serum to the VD IV region of the MOMP showed that the antiserum, although it had a predominance of IgGl antibodies specific for the VD IV peptide, also contained large quantities of other IgG isotypes, IgG2b and IgG3, that have the potential to bind to Fc receptors. This antiserum neutralized the infectivity of C. trachomatis when tested in HaK cells in the CI neutralization system presented here as well as in HeLa cells in a complement-dependent system (5). However, the same antiserum, when tested in HeLa cells in a CI assay, was not able to neutralize C. trachomatis infectivity and even masked the neutralizing ability of an IgGl MAb. Further in vivo and in vitro work, perhaps with cell lines derived from the genital tract or conjunctival surface, to determine the interactions of different neutralizing MAbs will need to be undertaken to see whether the potential for antagonism among antibodies directed to C. trachomatis can be demonstrated. Whether the enhancement of infectivity of chlamydiae can be mediated with any antibody that binds to the surface of chlamydiae or whether it occurs only with antibodies directed to specific epitopes needs to be addressed. If there is antibody specificity to the enhancement, then it will be essential for future vaccine development to map and identify specific immunogenic epitopes that elicit enhancing and/or blocking antibodies.

ACKNOWLEDGMENTS This work was supported by Public Health Service grants Al30499 and AI-26807 from the National Institute of Allergy and

Infectious Diseases. We thank H. Caldwell for his helpful comments on the manuscript.

REFERENCES 1. Barclay, A. N., and D. W. Mason. 1982. Induction of Ia antigen in rat epithelial cells and gut epithelium by immunological stimuli. J. Exp. Med. 156:1665-1676. 2. Bland, P. W., and C. V. Whiting. 1992. Induction of MHC class II gene products in rat intestinal epithelium during graft-versushost disease and effects on the immune function of the epithe-

lium. Immunology 75:366-371. 3. Brandt, W. E., J. M. McCowan, F. H. Top, Jr., W. H. Bancroft, and P. K. Russell. 1979. Effect of passage history on Dengue-2 virus replication in subpopulations of human leukocytes. Infect. Immun. 26:534-541. 4. Brunham, R., R. Peeling, I. Maclean, J. McDowell, K. Persson, and S. Osser. 1987. Postabortal Chlamydia trachomatis salpingitis: correlating risk with antigen-specific serological responses

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