Clin. exp. Immunol. (1978) 33, 57-64.

Cell-mediated immune responses in owl monkeys (Aotus trivirgatus) with trachoma to soluble antigens of Chlamydia trachomatis D. L. SACKS, W. J. TODD & A. B. MACDONALD Department of Microbi ology, Harvard University School of Public Health, Boston, Massachusetts, USA

(Received 6 February 1978)

SUMMARY

The first temporal study of the cell-mediated immune responses (CMI) following ocular infections with Chlamydia trachomatis is presented. We examined the CMI of owl monkeys infected with trachoma to soluble antigens of C. trachomatis by leucocyte migration inhibition (LIF) and delayed hypersensitivity skin testing. Delayed hypersensitivity of a systemic nature developed after a local eye infection in owl monkeys; clearance of inclusions from conjunctival cells coincided with the onset of this response. The association of eye secretion and circulating antibodies with recovery from primary infection was not so striking. Both cellular and humoral immune responses persisted for at least 2 months, at which time all test animals were completely resistant to re-infection. The elicitation of cell-mediated immune reactions with solubilized chlamydial antigens may permit the isolation of specific antigens involved in the generation of protective immunity in the owl monkey model. INTRODUCTION The owl monkey (Aotms trivirgattis) has been used as a model for trachoma infection in man (Bell & Fraser, 1969). Owl monkeys are highly susceptible to experimentally induced infection, producing acute purulent conjunctivitis with numerous inclusions in conjunctival epithelial cells. Complete recovery generally occurs within 3 weeks, whereafter monkeys remain immune to rechallenge with both homologous and heterologous strains of Chlamydia trac-homatis (Murray et al., 1971; Fraser et at., 1975). The mechanisms of immunity in oxxl monkeys have heretofore been investigated exclusively in terms of eye secretion and serum antibodies. These antibodies develop as a consequence of infection and accompany the disappearance of inclusions from infected conjunctival cells (Murray et at., 1971). The presence of eye secretion antibodies at the time of challenge has correlated 'A-ell with resistance to reinfection (McComb, Peters & Fraser 1971; Fraser et al., 1975). Passive transfer of ovl monkey sera containing anti-chlamydial antibodies did not render recipient monkeys immune (Orenstein, Mull & Thompson, 1973). Neutralization studies have demonstrated that eve secretion and plasma from human patients with active trachoma can neutralize trachoma infectivity in ovl monkey eyes (Nichols et al., 1973), and that the immunoglobulin fraction is responsible for this neutralization (Barenfanger & MacDonald, 1974). However, the role of neutralizing antibodies in actual protection from trachoma infection remains in doubt because: (a) the children who served as sources of neutralizing antibody were not themselves Present address: Department of Anatomy, Colorado State Unix ersity, F4ort Collins, Colorado, USA. Correspondence: Dr A. B. MacDonald, Department of Microbiology, Harv ard School of Public Health, Boston, Massa-

chusetts 02115, USA.

0099-9104/78/0700-0057$02.00 () 1978 Blackwell Scientific Publications

57

58

D. L. Sacks, W. J. Todd C A. B. MacDonald

protected (Nichols et aL, 1973); (b) the mechanisms of neutralization are unknown-the role of complement is undefined; and (c) eye secretions from owl monkeys with trachoma may lack neutralizing activity, as evidenced by the failure of trachoma-positive eye secretions to delay the appearance of inclusions in conjunctival epithelial cells (McComb, personal communication), whereas positive human tears delayed infectivity for 5 days. Given the intracellular nature of the pathogen, the investigation of trachoma infections in terms of cell-mediated immune responses in both humans and animal models has remained of considerable interest. Kuo et al. (1971) have demonstrated that delayed hypersensitivity in guinea-pigs to specific antigens of C. trachomatis can be induced by inoculation with killed organisms. Similar results have been reported in studies of active or healed trachoma cases who were challenged intradermally with antigen (Bietti et al., 1967; Ghione, Werner & Grulle, 1974; Grayston et al., 1960). In addition, uninfected individuals who had received killed trachoma vaccine intramuscularly also demonstrated a positive cell-mediated response as assayed by blast transformation with specific antigen (Soldati et al., 1971). The present study describes the development and persistence of delayed hypersensitivity to solubilized chlamydial antigens after an ocular infection in owl monkeys. The temporal nature of the study permits evaluation of the role of cell-mediated immunity in recovery from and resistance to trachoma infections in the owl monkey model. In addition, the use of soluble test antigens provides a basis for the identification and purification of the major antigens involved in the developmental cycle of C. trachomatis, and in particular those specific antigens which may be responsible for the generation ofa protective host response.

MATERIALS AND METHODS Preparation of test antigen. A serotype B strain of C. trachomatis was propagated in 60Co-irradiated monolayers of BHK21 cells grown in enriched medium containing 10% foetal calf serum (McComb & Puzniak, 1974). For preparation of soluble antigens, 3-6 x 107 BHK21 cells were infected with between 1 and 10 inclusion-forming units of chlamydiae per host cell. The process of infection was enhanced by centrifugation of the chlamydiae for 1 hr at 1000 g onto the BHK21 monolayers. 40A4 hr after inoculation, the tissue culture medium containing detached cells was decanted and centrifuged at 2000 g for 20 min. The pellets were combined with the remaining monolayers, which were harvested by scraping into 0-1 M glycine buffer, pH 10-0, containing 1-0 M urea. The cells were homogenized and extracted with Triton X-100, 1-5% final concentration, followed by stirring for 1 hr at 35°C. The solution was centrifuged at 25,000 g for 30 min. The supernatant containing the soluble antigen was dialysed overnight against phosphate-buffered saline (PBS), 0-02 M phosphate, pH 7-2, 0 54 M NaCl. Identical preparations ofsoluble antigen were used in both LIF assays and skin tests. Owl monkeys. Nine monkeys with no previous history of trachoma were obtained by the New England Primate Research Center. Seven were infected by conjunctival inoculation and two were used as uninfected controls. Trachoma inoculum. A crude yolk sac preparation of C. trachomatis organisms serotype B (TRIC/B/SAU/HAR-36/OT), diluted in phosphate plus sucrose and glutamate buffer (PGS, 0-01 M phosphate, pH 7 0, 0-21 M sucrose, 0 0049 M potassium glutamate), was used to infect owl monkey eyes by previously described methods (Murray et al., 1971). The infectious dose was determined by titration of the inoculum in baby hamster kidney cell (BHK21) monolayers (Rota, unpublished). 2 months after the initial infection, the monkeys were again inoculated in each eye with an identically prepared inoculum. Determination of infectivity. Owl monkey conjunctival scrapings were placed on a microscope slide and processed as previously described (Murray et al., 1971; Nichols et al., 1967). Inclusions were detected by immunofluorescence with human LGV antiserum, which was conjugated with fluorescein isothiocyanate. Antibody titres ofeye secretions and plasma. Eye secretions were collected with heat-sterilized Weck-cel sponges, as previously described (McComb & Nichols, 1969). Plasma was prepared from blood collected from the femoral veins. Two-fold dilutions of eye secretions or plasma were tested for antibody to serotype B (HAR-36) antigens by indirect immunofluorescence, using conjugated rabbit antisera to owl monkey immunoglobulin, as previously described (Nichols & McComb, 1962). Leucocyte migration inhibition assay (LIF). The capillary tube technique adapted by Bendixen & S0berg (1969) for peripheral blood leucocytes was used. Monkeys were bled into 3 0 ml heparinized syringes and the cells allowed to sediment in 0 5 ml of 6% dextran (mol. wt. 2,000,000) for 1 hr at 37°C. The leucocyte-rich plasma was removed and centrifuged at 220 g for 10 min. The supernatant plasma was removed, the cells resuspended and washed in Hanks's balanced salt solution three times. After the third wash, the cells were resuspended in HEPES-buffered TC 199 medium (Grand Island Biological, New York), containing 10% foetal calf serum (Microbiological Associates, Maryland) and 220 u each of penicillin and streptomycin (Grand Island Biological, New York) to give a leucocyte concentration of at least 1-5 x 10' cells per ml. Sterile siliconized 20 pm capillary tubes were filled with the cell suspensions, the ends sealed with sterile clay and the tubes centrifuged at 220 g for 5 min. The capillary tubes were cut at the cell-fluid interface and immediately placed in migration plate culture chambers (Microbiological Associates, Maryland). The chambers were filled with approximately 0-5 ml of

CMI to soluble chlamydial antigens

59

either control medium or medium containing soluble antigen diluted 1/100 or 1/1000. Each chamber was sealed with a cover-slip and the plates were incubated at 370C in 5% CO2 for 18 hr. The migration areas were then determined with a projection microscope and measured by planimetry. Each test was done in triplicate. Percentage migration inhibition was calculated by the following formula: average migrations of cells in antigen x 100 ~~= ppercentage migration; average migration of cells with no antigens percentage inhibition = 100 -percentage migration Tests were considered positive ifthe average inhibition was greater than 15%. Skin testing. Owl monkeys were skin-tested on their shaved abdomens by intradermal inoculation of 0 1 ml of PBS alone, or 0 1 ml of soluble antigen diluted 1/10, 1/100 or 1/1000 in PBS. A positive response was considered to consist of at least a 3 0 mm induration at the test site 48 hr after injection of the test antigens. There was no evidence of any immediate reactions following antigen administration.

RESULTS

Quantification ofinfection Each eye of the seven infected monkeys received 2-4 x 106 inclusion-forming units, as determined by titration of the inoculum in cell culture. 5 days later, conjunctival scrapings from each infected monkey showed inclusions (Table 1). Except for two monkeys (Nos 6 and 7), the inclusion counts declined by day 9. By day 14, few inclusions were present, and in two monkeys they were entirely absent. By day 21 no microbiological evidence of trachoma infection could be detected in any monkey. Re-inoculation of the seven test animals occurred 2 months after the primary infection. In these animals, no inclusions could be found up to 2 weeks after inoculation. Control monkeys which were not inoculated remained negative for inclusions throughout the duration of the study. Antibody response The plasma and eye secretions of all nine monkeys had anti-chlamydial antibody titres of 1/10 or less than 1/10 on day 0 (Table 2). A rise in the antibody titre of eye secretions reached a maximum by day 21, declining slightly thereafter. Relative to other infected animals, monkey No. 4 experienced only a small increase in the eye secretion antibody titre. The peak of circulating titre generally appeared 1 week earlier, at day 14, and remained steady or declined only slightly up to 4 weeks later. Neither control animal demonstrated a rise in eye secretion antibody. However, one control monkey (No. 8) did experience a significant increase in circulating titre by day 21. TABLE 1. Inclusion counts of owl monkeys inoculated with 2-4 x 106 inclusion-forming units of C. trachomatis expressed as inclusions per 1000 conjunctival cells

Day following first inoculation: Owl monkey No. 1 2 3 4 5 6 7 Geometric mean*

Day following second inoculation:

5

9

14

21 33 54

0

5

9

14

21-8 26-1 1-8 1-5 7-6 5-8 4-1 7-1

2-2 6-4 1-4 05 1-4 6-6 8-7

09 0 04 0 0-7 0-6 04 03

0 0 0 0

0 0 0 0

0 0 0 0

0

0

0

0 0 0

0 0 0

0 0 0

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

0 0 0 0 0 0 0 0

3.9

*

Excluding monkey No. 4.

D. L. Sacks, W. 9. Todd & A. B. MacDonald

60

TABLE 2. Anti-chlamydial antibody response* in owl monkeys infected with C. trachomatis and uninfected controls

Monkey No.

Infected 1 2 3 4 5 6 7 Geometric Meant Uninfected 8 9

Eye secretions on day:

Plasma on day:

0

5

9

14

21

33

Cell-mediated immune responses in owl monkeys (Aotus trivirgatus) with trachoma to soluble antigens of Chlamydia trachomatis.

Clin. exp. Immunol. (1978) 33, 57-64. Cell-mediated immune responses in owl monkeys (Aotus trivirgatus) with trachoma to soluble antigens of Chlamydi...
1MB Sizes 0 Downloads 0 Views