Induction of lymphocyte responsiveness by the outer membrane-peptidoglycan complex of rough strains of Brucella abortus

I"eterinary Immunology and Immunopathology, 26 ( 1990 ) 3 1 - 4 8

31

Elsevier Science Publishers B.V., A m s t e r d a m

Induction of lymphocyte responsiveness by the outer membrane-peptidoglycan complex of rough strains of Brucella abortus Roger Smith III, L. Garry Adams, Blair A. Sowa and Thomas A. Ficht Departments QfVeterinary Patholog3, and Veterinary Microbiolog3', Texas .4&M Untversity, College Station, TX 77843-4463 (U.S.A.) Texas Agriczdtural Experiment Station, College Station, TX 77843 (U.S.A.) (Accepted 7 December 1989 )

ABSTRACT Smith, R., Ill, Adams, L.G., Sowa, B.A. and Ficht, T.A., 1990. Induction of lymphocyte responsiveness by the outer membrane-peptidoglycan complex of rough strains of Brucella abortus. I "et. lmmunol. Immunopathol.. 26:31-48. The outer membrane-peptidoglycan complex ( OM-PG ) from rough strains of Brucella abortus was tested for its ability to induce lymphocyte responsiveness in cattle. Six groups of heifers were immunized with varying doses and administration schedules of rough OM-PG and assayed for responsiveness of their lymphocytes in proliferation assays in vitro. All OM-PG preparations were emulsified in a commercial adjuvant for administration. Two other groups of heifers were immunized with strain 19 vaccine or adjuvant alone. Three groups of heifers received two inoculations of OM-PG antigens from a naturally-occurring rough strain at a 57-day interval. The doses of OM-PG given in these three groups were 400/tg, 1200/~g, and 4000/tg at each inoculation. The frequency of cows that responded in lymphocyte proliferation assays increased with the dose of OM-PG given. Two groups received single inoculations of OM-PG, either 2400/tg or 8000 #g. Although there were responsive cows in these immunization groups, their frequency was lower than in the groups receiving the same total dose in two inoculations. A sixth group of cows was inoculated with OM-PG from a rough transposon mutant of B. abortus, and the frequency of responsive cows in this immunization group was comparable to that of responsive cows immunized with the same dose of OM-PG from the spontaneous rough mutant. In comparisons of cows inoculated with strain 19 to those inoculated with OM-PG preparations, differences were observed in the relative responsiveness of their lymphocytes to whole cells and OM-PG in the in vitro lymphocyte proliferation assays. These differences suggested that lymphocytes stimulated by strain 19 vaccination have different specificities than those stimulated by immunization with OM-PG of rough mutant strains orB. abortus.

INTRODUCTION

D e v e l o p m e n t of subunit vaccines that stimulate a protective i m m u n e response, but do not cross-react on standard serological tests is a goal of several laboratories interested in bovine brucellosis (Alausa et al., 1986 ). Candidates

0165-2427/90/$03.50

© 1 9 9 0 - - Elsevier Science Publishers B.V.

32

R. SMITH Ill ET ~L.

for such a vaccine are the outer membrane-peptidoglycan complex (OM-PG) and cell envelopes from rough mutants of Brucella abortus. Because of their deficiency of O-antigen, OM-PG or cell envelopes of rough strains would not induce antibodies detected by standard serological tests (Alausa et al., 1986 ). Previous studies in our laboratory and that of Winter demonstrated that OMPG or cell envelopes of B. abortus induced lymphocyte responsiveness in a substantial fraction of cattle (Winter et al., 1983; Winter et al., 1986; Winter and Rowe, 1988; Smith et al., 1990a). These studies used relatively small numbers of experimental animals and did not assess immunization variables such as dosage, administration schedule, and sources of the OM-PG or cell envelopes. In this report, we specifically address these issues in a large sampling of cows immunized with rough OM-PG. We provide evidence for a doseresponse effect and for a requirement for two immunizations to induce lymphocyte responsiveness. In addition, we report that two stable rough mutants are equivalent in the immunogenicity of their OM-PG. Finally, we provide preliminary evidence for a difference in the antigenic specificity of lymphocytes from cows immunized with strain 19 and those from cows immunized with rough OM-PG. MATERIALS AND METHODS

COWS The cows used as sources of lymphocytes were 1-2-year-old, cross-bred heifers. All cows were from a certified brucellosis-free herd in a class A state. None had been vaccinated with strain 19. Prior to entry into the experiment, all cows were sampled twice, and standard serological assays (Card test, rivanol, complement fixation, hemolysis-in-gel and ELISA) were performed to confirm that they had not been previously exposed to B. abortus (Alton et al., 1975; Heck et al., 1980; Ruckerbauer et al., 1981 ).

Bacteria B. abortus strains used in these studies were strain 2308 (Dr. B.L. Deyoe, U S D A / N A D C , Ames, IA), strain 19 (Coopers Animal Health, Kansas City, MO), and strain RB51 (Dr. G.G. Schurig, Virginia Tech, Blacksburg, VA). B. abortus strain 2308:Tn5 lacZ (designated strain M 106) is a rough mutant derived by Tn5 mutagenesis of strain 2308 (T.A. Ficht, unpublished results, 1989). Immunogen preparations Eight experimental groups of cows were used, including one group that was immunized with strain 19 (3.4× 108 cfu) and a second group immunized with adjuvant only. The adjuvant was a commercial preparation of monophosphoryl lipid A, cell wall skeleton, and trehalose dimycolate in squalane

B. ABORTUS OM-PG COMPLEXAND LYMPHOCYTE RESPONSIVENESS

33

TABLEI Summary of immunogens and experimental groups Experimental group 1 2 3 4 5 6 7 8

Immunogen

Adjuvant' Strain 19 RB 51 OM-PG RB 51 OM-PG RB 51 OM-PG RB 51 OM-PG RB 51 OM-PG M 106 OM-PG

Dose

Number of cows

Day 0

Day 57

Total

3.4x 108 400/2g 1200/~g 4000/tg 2400 ltg 8000/tg 1200/lg

400 ~tg 1200~g 4000/2g 1200/lg

3.4× 108 800 Hg 2400#g 8000 ltg 2400/tg 8000/~g 2400/tg

23 24 23 22 21

24 22 23

~Adjuvant used was: 0.25 mg monophosphoryl lipid A, 0.25 mg cell wall skeleton. 0.25 mg trehalose dimycolate, 0.02 ml squalane, and 0.002 ml Tween80. All groups, except group 2 (strain 19 ), received the immunogen in adjuvant.

and Tween 80 (Ribi I m m u n o c h e m , Hamilton, MT ). All immunogens, other than strain 19, were emulsified in adjuvant, lyophilized, and stored at 4°C prior to immunization. The OM-PG immunogens were prepared by a modification of the Lutkenhaus procedure (Lutkenhaus, 1977; Sowa et al., 1983; Sowa, 1990). Brucella abortus strain RB51 or strain M 106 cells were 7-irradiated and centrifuged at 12 000 X g for 3 min, the supernatant was discarded, and the pellet was resuspended in at least three volumes of Lutkenhaus buffer ( 10 m M Tris-HC1, 5 m M EDTA, and 1 m M 2-mercaptoethanol) at 4°C. Chilled cells were immediately lysed by sonication using a microprobe (Braunsonic, B. Braun, Melsungen AG) delivering 100 W at 20 000 Hz for five repetitions, each consisting of 1.5 min of sonication followed by 0.5 min of cooling. Unbroken cells and debris were removed by centrifugation for 3 min at 12 000 × g, and the supernatant was then centrifuged at 134 0 0 0 × g for 25 min in an A-95 rotor (Airfuge ®, Beckman Instruments, Fullerton, CA). The pellet consisting of OM-PG complex was resuspended in Lutkenhaus buffer by mild sonication, and the centrifugation steps were repeated twice. The protein content of each OM-PG preparation was determined by the BCA assay (Pierce Chemical Co., Rockford, IL). Routinely, OM-PG preparations were analyzed for consistency by analytical sodium dodecylsulfate-polyacrylamide gel electrophoresis. The OM-PG was lyophilized prior to emulsification in adjuvant. Five groups of cows received varying doses of strain RB51 OM-PG, either in one dose or two (Table 1 ). A sixth group received strain M 106 OM-PG in two doses.

Peripheral blood mononuclear cell preparation Blood (12 ml) was collected from the cows in 1000 U heparin (Upjohn Co., Kansas City, MO). Peripheral blood mononuclear cells were isolated the

34

R. SMITH lIl ET AL.

following day by differential centrifugation of heparinized blood over ficollsodium metrizoate (Sigma Chemicals, St. Louis, MO), and resuspended in culture m e d i u m at 2 X l 0 6 per ml. Culture m e d i u m consisted of RPMI-1640 (Hazelton Research Products, St. Lenexa, KS) supplemented with sodium bicarbonate (Hazelton), non-essential amino acids (Hazelton), L-glutamine (Hazelton), HEPES buffer (Hazelton), sodium pyruvate (Hazelton), and 10% fetal bovine serum (HyClone Laboratories, Logan, UT), as previously described (Smith et al., 1990b; Smith et al., 1990c). In the assays reported here, however, 2-mercaptoethanol was not added to the culture medium. Preliminary experiments demonstrated that omission of 2-mercaptoethanol resuited in lower spontaneous uptake of thymidine, but did not alter responses to mitogens or antigens (Smith, unpublished, 1987 ).

Lymphocyte proliferation assay Peripheral blood mononuclear cells were assayed in 96-well round-bottom culture plates. Briefly, 2 X 105 peripheral blood mononuclear cells were cultured overnight prior to addition of antigen or mitogen. Two antigen preparations were used; 5 X 107 ),-irradiated B. abortus strain 2308 whole cells or 1 /Lg of strain RB51 OM-PG. Cultures were incubated for an additional 5 days at 37°C in a humidified atmosphere of 7.5% CO=. Four hours prior to the termination of culture, the cells were pulsed with 10/~1 of [ 3H ]-thymidine (ICN Biomedicals, Inc., Costa Mesa, CA) at 100 #Ci per ml. The cultures were harvested onto glass fiber paper and counted by liquid scintillation. The negative control was the thymidine incorporation by cells in cultures with m e d i u m alone, and the positive control was the incorporation by cells in cultures with 250 ng concanavalin A (Pharmacia Inc., Piscataway, NJ). Data are expressed as the net counts per minute (cpm), defined as the experimental minus the control values ( E - C ): E - C = cpm in cultures with a n t i g e n - c p m in cultures with m e d i u m

Experimental design and data analysis Cows were randomly assigned to one of eight experimental groups and immunized on day 0. Those cows that received two doses of immunogen were reimmunized on day 57. All cows were sampled, and proliferative assays performed, twice prior to immunization. Subsequent samples were taken and assays were performed at 14, 28, 42, 57, 70, 91, 119, 147, 182, 212,237, and 245 days after primary immunization. On each of the twelve sampling times after primary immunization, one-third of the cows were sampled. All cows were bred, and pregnant cows were inoculated conjunctivally with 0.8 X 107 cfu B. abortus strain 2308 at mid-gestation (245 days post-immunization). Samples for proliferative assays were collected at 21, 41, 69, 83, and 97 days post-challenge.

B. ABORTUS OM-PG COMPLEX AND LYMPHOCYTE RESPONSIVENESS

35

For purposes of data analysis, individual assays were judged as invalid or uninterpretable if the cpm in cultures with medium exceeded 10 000. Invalid assays represented approximately 10% of the total. Two criteria were established for inclusion of individual cows into the data analysis. First, the results of the preimmunization assay must have been negative (see Results for definition of positive and negative assays). Second, there must have been three valid assays after the primary immunization (in order to make comparisons in terms of frequency of positive responses ).

Statistical analysis For determination of a cutoff point to differentiate negative and positive samples, 467 samples were assayed on six separate dates prior to immunization. Of those, 430 were considered valid by the above criteria. As a cutoff point for discrimination of positive and negative samples, the mean plus two standard deviations was used. To compare experimental groups for frequency of responding individuals, chi-square analysis was used, with c~= (0.05/N), where N = t h e number of comparisons, to correct for multiple comparisons (Matthews and Farewell, 1988). For comparisons among the eight experimental groups, seven tests were made. thus setting a = 0.0071. RESULTS

Determination of positive and negative results In the six assays prior to immunization, 430 of the 467 assays were considered valid by the criteria described. The distribution of the proliferative responses of these unvaccinated cows is presented in Fig. 1. The mean value of the net response to strain 2308 whole cells ($2308 WC) was 972 (_+ 2950) cpm, and to strain RB51 OM-PG (RB51 OM-PG) 223 (_+ 1835) cpm. The cutoff values (mean plus two standard deviations) were 6871 for $2308 WC and 3893 for RB51 OM-PG. Using rounded values of 7000 and 4000 cpm as the cutoffs for negative responses, 18 assays (4.2%) were above the cutoff for one or the other in vitro antigen. Based on the criteria of at least one negative response prior to vaccination, four cows were eliminated from further consideration (all of which had no valid assays prior to immunization ). To determine whether the assay, as presented, would identify positive responses following an active experimental infection, cows were assayed at intervals after challenge (Table 2). In the group that received adjuvant alone (unimmunized cows), positive responses to $2308 WC ranged from 60 to 100% of cows tested in an individual assay. A lower frequency of positive responses (25-57%) was observed in response to RB51 OM-PG in this group. For all immunized cows, 80-98% of the cows responded to $2308 WC and 60-87% to RB51 OM-PG.

36

R. SMITH I I I ET AL.

A. 300

200'

"4 E Z

1oo

9

5

2

1

2

0 -10

-5

0

5

10

15

20

25

30

10

15

20

25

30

B. 300'

239

200'

z

100"

6 -10

-5

0

5

E--C (cpm x 103) Fig. 1. Results of 200 proliferative assays on lymphocytes from cattle unexposed to B. abortus. The frequency of assays is plotted for each interval of the net response ( E - C ) . The number above each bar indicates the number of assays in that interval. Panel (A), the antigen was $2308 WC. Panel (B), the antigen was RB51 OM-PG.

Reponses o f u n i m m u n i z e d cows and cows i m m u n i z e d with strain 19 All cows were r a n d o m l y assigned to o n e o f eight i m m u n i z a t i o n g r o u p s a n d tested for r e s p o n s i v e n e s s in l y m p h o c y t e p r o l i f e r a t i v e assays at twelve intervals after i m m u n i z a t i o n , with each cows b e i n g tested four times. In the g r o u p o f cows t h a t r e c e i v e d a d j u v a n t alone ( g r o u p 1), 16 o f 84

B, ABORTUSOM-PG COMPLEXAND LYMPHOCYTERESPONSIVENESS

37

TABLE 2 Frequency of positive responses to B. abortus strain 2308 WC and strain RB51 OM-PG in cattle experimentally challenged with B. abortus strain 2308 Days post-challenge In vitro antigen $2308 WC

In vitro antigen RB51 OM-PG

21

41

55

69

83

97

21

41

55

69

83

97

Unimmunized cows Number tested Number positive Percent positive

9 6 67

7 6 86

4 3 75

7 7 100

5 3 60

4 3 75

9 3 33

7 2 29

4 l 25

7 4 57

5 2 40

4 2 50

Immunized cows Number tested Number positive Percent positive

39 35 90

42 41 98

41 33 80

37 3l 84

30 29 97

37 30 81

39 34 87

42 35 83

41 34 86

37 32 86

30 18 60

37 24 65

All cows Number tested Number positive Percent positive

48 4l 85

49 47 96

45 36 80

44 38 86

35 32 91

41 33 80

48 37 77

49 37 76

45 35 78

44 36 82

35 20 57

41 26 63

(19.0%) in vitro assays were positive with $2308 WC as antigen, and two of 84 (2.3%) were positive with OM-PG as antigen (Tables 3 and 4). Of the twelve cows that gave positive responses to $2308 WC, only four responded in more than one assay (Table 3). For all of these four cows, the second positive assay was on day 212, an assay that tended to give high responses in all groups. Those cows receiving strain 19 as immunogen (group 2) responded in 50 of 83 (60.2%) assays with $2308 WC. Of the 24 cows in this group, one did not respond to $2308 WC in any assay (Table 3). Sixteen were considered responders to $2308 WC, in that they responded in at least two assays following immunization. Most of the positive responses were observed in the first assays after immunization, and persisted for the duration of the experiment. A different level of responsiveness was observed with RB51 OM-PG as the in vitro antigen. There were only 27 of 83 (32.5%) positive assays with RB51 OM-PG antigen. Overall, only seven cows immunized with strain 19 were considered responders to RB51 OM-PG in vitro (Table 4). In all assays, if a response was observed to RB51 OM-PG, the cow also responded to $2308 WC. The converse, however, was not true in this immunization group.

Responses of cows immunized with varying doses of strain RB51 OM-PG Three doses of RB51 OM-PG, over a one log range, were used for cows in groups 3, 4 and 5. For the assays using $2308 WC as antigen, there was an

38

R. SMITH III ET -XL.

TABLE 3 Frequency of positive responses to B. abortus strain 2308 WC in cattle immunized with strain 19 or rough O M - P G o f Brucella lmmunogen

N u m b e r of positive responses ~ ( percent ) None

[

2

versus strain 19

-~djuvant

I1 (47.8)

8 (34.8

4 17.4)

0 (0.0)

0 (0.0

na -~

na

23 (84)

Strain 19

1 (4.2)

7 t29.2

6 25.0)

9 (37.5)

1 (4.2

18.9" ( <0.001 )

na

24 (831

RB51 O M - P G ( 2 X 4 0 0 ug)

4 (17.4)

5 ~21.7

6 26.1)

7 (30.4)

l (4.3

12.2 (0.016)

(0.645/

23 (851

RB51 O M - P G ( x 1200.ug)

1 (4.5)

4 (18.2

8 36.4)

8 (36.4)

1 (4.5

20.0* ( <0.0011

1.l (0.894)

22 (79)

RB51 O M - P G (2×4000pg)

1 (4.8)

2

(9.5

7 33.3)

7 (33.31

4 (19.0)

23.8* ( < 0,001 )

4.8 (0.3081

21 (80)

RB51 O M - P G ( 1 x2400¢tg)

5 (20.8)

7 (29.2

9 37.51

3 (12.5)

0 ~0.0)

7.5 (0.058)

"t . _~ (0.126~

24 (90)

RB51 O M - P G ( 1 X 4000 l~g )

3 (13.6)

II (50,0)

4 18.2)

4 (18.2)

0 (0.0)

8.7 (0.034)

5.2 (0.267)

22 (81~

M 106 O M - P G ( 2 x 1200/~g)

0 (0.0)

6 (26.1)

7 30.4)

6 (26.1)

4 (17.4)

3.7 (0.448)

23 (87)

Total

26

51

4

Number o f cows assays )

versus adjuvant

50

3

7. e (P)

44

11

22.0* ( < 0.001 )

182 (669)

'Lymphocytes from each co,*. were assayed at four time points covering a period of 245 days after immunization, thus. 4 is the m a x i m u m n u m b e r of positive responses possible. -'Not applicable. *Difference is significant from the comparison group ( a = 0 . 0 5 / 7 = 0 . 0 0 7 1 ) .

increasing frequency of responses as the dose o f immunogen increased: 49.4% positive at two doses of 400 pg, 60.8% positive at two doses of 1200 #g, and 66.2% positive at two doses of 4000 #g (Table 3 ). Equivalent responses were observed when RB51 OM-PG was used as the antigen in proliferative assays: 47.1%, 55.7%, and 63.8% positive assays at the three increasing doses of immunogen (Table 4). Several o f the cows demonstrated responses after primary immunization, but others required a second immunization to respond in the proliferative assay. In all assays, responses to the two in vitro antigens were parallel; that is, proliferative responses to $2308 WC were paralleled by proliferative responses to RB51 OM-PG, and vice versa. At 400/tg doses of OM-PG, 14 of the 23 cows responsed to $2308 WC in at

B. ABORTUSOM-PG COMPLEX AND LYMPHOCYTERESPONSIVENESS

39

TABLE 4 Frequency of positive responses to B. abortus strain RB51 OM-PG in cattle immunized with strain 19 or rough OM-PG of Brucella Immunogen

Number of positive responses ~ ( Percent ) None

1

2

3

;(2 (P) 4

Number of cows ( assays )

versus adjuvant

versus strain 19

na 2

na

23 (84)

na

24 (83)

Adjuvant

21 (91.3)

2 (8.7)

0 (0.0)

0 (0.0)

0 (0.0)

Strain 19

9 (37.5)

8 (33.3)

2 (8.3)

5 (20.8)

0 (0.0)

RB51 OM-PG (2X400#g)

3 (13.0)

8 (34.8)

4 (17.4)

8 (34.8)

0 (0.0)

29.2* (<0.001

4.4 23 (0.221) (85)

RB51 OM-PG (2X 1200/tg)

1 (4.5)

4 (18.2)

11 (50.0)

6 (27.3)

0 (0.0)

36.1" ( <0.001

14.1" 22 (0.003) (79)

RB51 OM-PG (2 X4000/zg)

0 (0.0)

3 (14.3)

9 (42.9)

6 (28.6)

3 (14.3)

39.0* ( < 0.001

RB510M-PG (1X2400/tg)

4 (16.7)

10 (41.7)

6 (25.0)

4 (16.7)

0 (0.0)

27.0* ( <0.001

4.4 24 (0.221) (90)

RB510M-PG (1X4000/~g)

5 (22.7)

11 (50.0)

5 (22.7)

1 (4.5)

0 (0.0)

22.1" ( <0.001

5.7 22 (0.127) (81)

MI06 OM-PG (2X 12001~g)

1 (4.3)

6 (26.1)

8 (34.8)

6 (26.1)

2 (8.7)

36.2* (<0.001

12.4 23 (0.015) (87)

Total

44

52

45

36

5

15.2* (0.002

18.8" (<0.001)

21 (80)

182 (669)

~Lymphocytes from each cow were assayed at four time points covering a period of 245 days after immunization, thus, 4 is the maximum number of positive responses possible. -'Not applicable. *Difference is significant from the comparison group ( a = 0 . 0 5 / 7 =0.0071 ).

least two proliferative assays, and 12 o f 23 to RB51 OM-PG in at least two proliferative assays (Tables 3 and 4). That frequency increased to 17 of 22 responding to either antigen at the 1200/tg dose and 18 of 21 at the 4000/tg dose.

Comparison of one- and two-dose immunization schedules Two groups of cows were given a single dose of rough OM-PG immunogens (groups 6 and 7). The single doses were chosen to correspond to the total dose ofimmunogens delivered in two doses to groups 4 (2400/tg) and 5 (8000 /lg). At both doses, the frequency of cows responding to a single immunization was less than that to two doses. At the 2400 ~g dose, 37.8% o f the prolif-

40

R. SMITH IIl ET AL.

erative assays were positive in response to either $2308 WC or RB51 OM-PG (Tables 3 and 4 ). Responses to the higher dose of immunogens were approximately the same, with 37.8% of the assays positive with $2308 WC antigen, and 29.6% of the proliferative assays positive with RB51 OM-PG antigen. Of the 24 cows immunized with 2400/tg in one dose, twelve responded in more than one assay to $2308 WC, and ten in more than one assay to RB51 OMPG. This represented a drop in the frequency of responsive cows from 77.3% of cows given two doses to 50% for the combined dose, when $2308 WC were used as the in vitro antigen. When RB51 OM-PG were used as in vitro antigen, the frequency dropped from 77.3% responsive cows given two doses to 41.7% responsive cows given as single dose. These differences were not, however, statistically significant. At the highest total dose (8000/lg), the frequency of responsiveness to $2308 WC in vitro dropped from 85.7% for two doses to 36.4% for the combined dose, and to RB51 OM-PG from 85.7% to 27.3% (6 of 22). These differences were significant by chi-square analysis, with Z2= 12.7 (P=0.013, 4 df, o~=0.05) for $2308 WC and 17.0 (P=0.002, 4 df, a = 0 . 0 5 ) for RB51 OM-PG.

Comparison of rough mutants as sources of OM-PG A newly derived rough mutant of strain 2308, designated strain M 106, was compared to strain RB51 as a source of OM-PG for immunization. The intermediate dose of 1200/~g, given twice, was chosen as the dose for comparison. The response to immunization with strain M 106 OM-PG was almost identical to the response to strain RB51 OM-PG (Tables 3 and 4). In 87 proliferative assays using $2308 WC as antigen, there were 54 (62.1%) positive responses, and 17 of the 23 cows in this group responded in more than one assay (Table 3). In assays using RB51 OM-PG as antigen, there were 48 (55.2%) positive responses, and 16 of these cows responded in at least two assays (Table 4 ). The data for all groups are summarized in Tables 3 and 4. By chi-square analysis, all immunization groups were significantly different ( P < 0.0071) from the adjuvant control group in the frequency of their responses to RB51 OM-PG as the in vitro antigen. When $2308 WC were used as antigen in vitro, neither group that received a single immunization was significantly different when a was set at 0.0071 to accommodate multiple comparisons (see Materials and Methods). In addition, the group that received the low dose in the two-immunization schedule (400/~g) was not significantly different from the adjuvant group in their in vitro responses to $2308 WC. The lower levels of significance with $2308 WC as the in vitro antigen was due to the higher frequency of positive assays in the adjuvant group, most of which occurred in a single assay on day 212. When the groups immunized with different doses or sources of rough OM-

B. ABORTUSOM-PG COMPLEXAND LYMPHOCYTE RESPONSIVENESS

41

PG were compared to the strain 19 group in responses to $2308 WC, there were no significant differences (Table 3 ). In responses in vitro to RB51 OMPG, however, there were significant differences in the frequency of responding cows between the strain 19 group and the groups immunized with the intermediate ( P = 0.003) and high ( P < 0.001 ) doses of RB51 OM-PG (Table 4).

Comparison of strain 2308 WC and strain RB51 OM-PG as in vitro antigens In cows immunized with varying doses and schedules of OM-PG from rough mutant strains, the proliferative responses to $2308 WC and to RB51 OMPG in vitro were approximately equivalent. In cows immunized with strain 19, however, there was a remarkable disparity in the magnitude and frequency (Tables 3 and 4) of positive responses to the different antigens in vitro. As noted previously, the disparity in frequency of responses was significant for two groups using a stringent test of significance. To compare this difference quantitatively, the magnitude of the response to the two in vitro antigens was plotted as a scatter plot, with each point representing the value TABLE 5 Correlation between magnitude ofreponses to $2308 WC and RB51 OM-PG complex after immunization and before experimental challenge Experimental group ~

Least squares regression line

r

df

Slope (standard error)

Intercept (standard error )

Strain 19

0.272 (0.019)

- 1120 (846)

0.846

81

RB51 OM-PG (2x400#g)

0.576 (0.027)

1790 (1486)

0.920

83

RB51 OM-PG ( 2 × 1200/tg)

1.011 (0.060)

-5216 (3183)

0.886

77

RB51 OM-PG (2 × 4000/~g)

0.766 (0.043)

- 339 ( 3468 )

0.896

78

RBSI OM-PG ( 1 × 2400/tg)

0.446 (0.034)

1151 (1087)

0.809

88

RB51 OM-PG ( 1 × 8000/tg)

0.604 (0.037)

- 314 (1504)

0.881

79

M 106 OM-PG (2)< 1200/tg)

0.722 (0.037)

- 1233 (2300)

0.915

85

~Experimental group 1 (adjuvant) is not included because the responses to both antigens were below the threshold values that define a positive response.

42

R. SMITH III ET .kL.

obtained from a single cow in a single assay. The correlation coefficient and regression line were obtained for each immunization group (Table 5). The correlation coefficients for the magnitude of the response to the two antigens were high in all groups ( 0 . 8 0 9 - 0 . 9 2 0 ) . The slopes of the regression lines, however, differed significantly in all groups, with the strain 19 group being lowest at 0.272. All OM-PG immunization groups were higher, ranging from 0.446 (2400pg in one dose) to 1.011 (2400 pg in two doses). Although there was no clear dose-response effect, those immunization groups with the highest frequency of responsive cows (groups 4, 5, and 9) had the greatest slopes ( 1.011, 0.766, and 0.722, respectively). After experimental challenge with viable strain 2308 organisms, a smaller number of proliferative assays was performed. The relative reactivity to $2308 WC and RB51 OM-PG in most experimental groups did not change substantially after exposure to the replicating organism (Table 6). The groups that received OM-PG immunogens had regression slopes ranging from 0.600 to 0.855, with the difference from the pre-challenge values being relatively small. In the two groups that received single immunizations, the slope increased to the range of those groups that had received two immunizations. The group T~BLE 6

Correlation between magnitude of responses to $2308 WC and RB5I O M - P G complex after experimental challenge Experimental group

Least squares regression line Slope ( standard error)

r

df

Intercept ( standard error)

-~djuvant

0.246 ( 0.047 )

385 ( 1386 )

0.666

34

Strain 19

0.365 (0.050)

-3611 (3264)

0.815

27

RB51 O M - P G ( 2 × 400 l£g)

0.600 ( 0.073 )

0.823

32

RBSI O M - P G ( 2 x 1200pg)

0.811 (0.055)

0.934

32

RB51 O M - P G ( 2 x 4000 pg)

0.732 (0.054)

-319 ( 6425 )

0.935

26

RB51 O M - P G (1 × 2400 pg)

0.708 (0.102)

245 (6408)

0.746

38

RB51 O M - P G ( 1X 8000//g)

0.855 (0.065)

- 11433 (5343)

0.921

31

M106 O M - P G ( 2 × 1200pg)

0.811 (0.113)

-6121 (11375)

0.814

26

4571 ( 7259 ) -6496 (5040)

B. ABORTUSOM-PG COMPLEX AND LYMPHOCYTE RESPONSIVENESS

43

that was i m m u n i z e d with strain 19 demonstrated a slight increase in its slope, from 0.272 to 0.365, which was still much less than any slopes of the groups i m m u n i z e d with OM-PG preparations. Finally, after experimental challenge, the group that received adjuvant alone had a slope (0.246) resembling that of the strain 19 group. DISCUSSION

The major observations reported here are: ( 1 ) cellular immunity, as measured by in vitro lymphocyte proliferative responses, can be induced with OMPG of rough strains of B. abortus, (2) there is a dose-response effect over the one log range used in this experiment, (3) a single dose is much less effective at inducing cellular responsiveness than two doses, (4) two strains of rough mutants were equivalent in their ability to induce cellular responses, and ( 5 ) the specificity of lymphocytes responding to immunization with OM-PG differs from those responding to strain 19 vaccination. The demonstration of a dose-response effect, the apparent requirement for two immunizations, and the interchangeability of two differently derived rough mutants provide significant additional information for the design of subcellular vaccines. In previous studies, we identified OM-PG preparations as being approximately equivalent to strain 19 in inducing lymphocyte responsiveness (Smith et al., 1990a). Other immunogens used in that experiment (purified outer membrane proteins, lipopolysaccharide-protein complex, and recombinant outer membrane proteins fused to fl-galactosidase ) did not stimulate lymphocyte responsiveness in as high a frequency of cows as strain 19 or OM-PG. This report extends our previous results by examining several critical parameters to optimize the conditions for successful induction of lymphocyte responsiveness using Brucella OM-PG. Because of the efficacy of the OM-PG preparations to induce lymphocyte responsiveness, the precise nature of the OM-PG immunogen is an important issue. In the modified Lutkenhaus procedure used to isolate these immunogens, sonication sufficient to lyse Gram-negative bacteria, without prior lysozyme treatment, breaks the cytoplasmic membrane into small vesicles that do not readily sediment during centrifugation, while the OM-PG is easily sedimented and free of cytoplasmic membrane (Sarvas, 1985). Five observations regarding our immunogens strongly suggest that these are complexes of the Brucella outer membrane and peptidoglycan (Sowa, 1990 ). First, a single bilayer was observed on electron microscopy of OM-PG. Second, the previously described group 2 and 3 proteins with strong affinity to peptidoglycan were consistently observed in these preparations. Third, Brucella lipopolysaccharide partitioned with the OM-PG preparations. Fourth, the OM-PG preparation behaved as a single band having a buoyant density of 1.22 g/ml, that increased to 1.26 g / m l after lysozyme treatment. The preparations contained

44

R. SMITH III ET AL.

no cystoplasmic membrane band ( 1.15 g/ml). Finally, biotin-labeled surface proteins were isolated with these OM-PG preparations. The first observation in the present report, that lymphocyte responsiveness can be induced by immunization with OM-PG from rough mutants of B. abortus, confirms our previous studies (Smith et al., 1990a) and those of Winter (Winter et al., 1983; Winter et al., 1986; Winter and Rowe, 1988). Winter and Rowe immunized eleven cows with cell envelopes, outer membrane proteins, or peptidoglycan from B. abortus strain 2308 and measured proliferative responses and dermal hypersensitivity reactions to group 2 (porin) and group 3 outer membrane proteins (Winter and Rowe, 1988). Both measures of cellular immunity detected responses in seven of the eleven cows in this experiment. The conclusion of this, and a second experiment comparing vaccination with either cell envelopes or peptidoglycan, both in adjuvant. was that the cell-mediated immune response induced by these immunogens was indistinguishable in duration and magnitude. In our previous work, responsiveness to OM-PG of strain RB51 was detected in six of nine cows immunized twice with 400 pg. The frequency of responsiveness to OM-PG was greater than that to purified outer membrane proteins, to recombinant outer membrane proteins fused to E. coli fl-galactosidase, or to a preparation of lipopolysaccharide-protein complex from a smooth strain. Only responsiveness to strain 19 vaccination was greater than that to rough OM-PG. The experiments presented in this report were designed to extend these preliminary observations. The design of this experiment addressed three specific questions: the effect of dosage, the necessity for secondary immunization, and the effect of alternate rough mutants. Three doses of OM-PG, at half-log intervals, were chosen to test the effect of dosage. Responsiveness in at least two proliferative assays was used as a criterion for a positive response to immunization. At the lowest immunization dose, 60.1% ( 14 of 23 ) of the cows responded in proliferative assays to $2308 WC and 52.1% ( 12 of 23) to RB51 OM-PG. This was comparable to the 66.7% (6 of 9) responsiveness to $2308 WC observed in our previous experiment using the same dose of RB51 OM-PG. At higher doses of OM-PG used for immunization, the frequency of responsiveness increased to 77.3% ( 17 of 22) at 1200/tg and to 85.7% ( 18 of 21 ) at 4000/zg. At these higher doses, there was no difference in the frequency of cows responding to $2308 WC or RB51 OM-PG. The second question we addressed was the requirement for two doses of immunogens. The two higher doses of OM-PG were used for this purpose. administering the total dose of immunogen in one inoculation rather than two. In both cases, the frequency of responses was lower for groups given a single dose. At the total dose of 2400 #g, the differences were not significant. But at the higher dose, the difference in frequency of responsive cows was

B..4BORTUS OM-PG COMPLEX AND LYMPHOCYTE RESPONSIVENESS

45

significantly higher when the same dose was administered in two doses rather than in one ( P = 0.013 for $2308 WC and P = 0.002 for RB51 OM-PG). The last question that was addressed in this experiment was the effectiveness of OM-PG from an alternate rough mutant for induction of cellular responsiveness. A transposon mutant of strain 2308, designated strain M 106, was chosen for this purpose. The particular advantages of using a transposon mutant as a vaccine are the stability of the mutation imparted by transposon insertion and the ability to identify the mutation by cloning and sequencing of nucleotides flanking the transposon. In these experiments, the transposon mutant was as effective as strain RB51 for the induction of cellular responsiveness. When $2308 WC were used to assay responsiveness in vitro, 73.9% ( 17 to 23 ) cows immunized with strain M 106 OM-PG responded, compared to 77.3% of cows immunized with the same dose of RB51 OM-PG. When RB51 OM-PG were used to assay responsiveness, 69.6% ( 16 of 23 ) cows immunized with strain M106 OM-PG responded, compared to 77.3% of cows immunized with the same dose of strain RB51 OM-PG. The slightly lower frequencies of responsiveness in cows immunized with strain M 106 OM-PG were not statistically significant. For all cows in this experiment, we tested their responsiveness to two antigen preparations in vitro. The first, $2308 WC, was used to test their responsiveness to antigens present in a prototype virulent strain of B. abortus. The second, RB51 OM-PG, was used to test responsiveness to the immunizing agent. In the six experimental groups that received varying doses and schedules of strain RB51 or strain M106 OM-PG immunogens, there were only slight differences in the frequency of cows responding to either antigen in vitro. However, there was a difference in the frequency of responsiveness in cows immunized with strain 19, with fewer cows responding to RB51 OMPG than to the $2308 WC preparations in vitro. The differences in the frequency of responses was also reflected by differences in the overall magnitude of the response to the two antigen preparations. The discrepancy in the patterns of in vitro reactivity to these two antigen preparations between cows immunized with strain 19 and those immunized with OM-PG strongly suggests a difference in the specificity of the lymphocyte response in these experimental groups. There are three important differences between the strain 19 and the OMPG immunogens. The most obvious is that strain 19 is a replicating antigen, whereas the OM-PG preparations are not. The lowest dose of 400/~g represents the amount of OM-PG in approximately 3 × 109 whole cells, which was the recommended adult dose at the time the immunogens were being prepared for a preliminary immunization trial (United States Department of Agriculture, 1984). Because the degree of in vivo replication of strain 19 after vaccination is not known, it was not possible to make reasonable adjustments to approximate a replicating antigen. Instead, two doses were given in three

46

R. S M I T H Ill ET AL.

groups of cows, and a potent adjuvant was chosen for the non-replicating immunogens to augment the response. In the period between preparation for our preliminary experiment and the one reported here, the official recommended dose for adult vaccination was reduced from 3 X 109 cfu to 3 × 108 cfu strain 19 (the dose used in group 2). Therefore, the cows inoculated with OM-PG received approximately a 1-2 log excess of OM-PG over those cows vaccinated with strain 19, further compensating for the in vivo replication of strain 19. Whether the difference in dosage or capacity to replicate, or the related difference that the OM-PG immunogens were emulsified in adjuvant, could alter the specificity of the response is unknown. Two observations make dosage an unlikely reason for the differences in specificity. First, in vivo replication of strain 19 could have easily made up for the difference in the initial dose OM-PG in strain 19 and in the isolated preparations. Second, there appeared to be no shift in the relative response to $2308 WC and RB51 OM-PG with increasing doses of isolated OM-PG. Interestingly, it is unlikely that strain 19 is unique in not inducing strong responses to RB51 OM-PG antigens. After experimental challenge with strain 2308, the u n i m m u n i z e d cows (those receiving adjuvant alone) responded to $2308 WC at high frequencies, reaching 75-100% at 41-69 days (Table 2 ). On the other hand, fewer of the same cows responded to RB51 OM-PG, reaching 25-57% for the same time period. Thus, the differences in reactivity to the RB51 OM-PG preparations can not be attributed to something unique to strain 19. A second difference between the two immunogens is the presence of smooth lipopolysaccharide (sLPS) in strain 19 and its deficiency in the OM-PG from strain RB51 and strain M l06. There is evidence that sLPS may alter i m m u n e regulation in two possible ways. Splitter and Everlith have reported that Brucelia sLPS increased the expression of class II major histocompatibility complex glycoproteins on the surface of bovine macrophages (Splitter and Everlith, 1989). The net effect of sLPS, however, was to suppress lymphocyte activation in the presence of interferon-7 (Splitter and Everlith, 1989). No such suppressive effect was observed with rough LPS, such as that present in our OM-PG immunogens. Whether sLPS can alter the specificity of the response is not addressed by their experiments. A second possible contribution of sLPS relates to the important contribution of B lymphocytes as antigenpresenting cells in primary i m m u n e responses (Ron and Sprent, 1987; Janeway et al., 1989). Because the predominant serologic response to B. abortus is to the O-antigen of the sLPS (Alausa et al., 1986), there may be a preferential uptake and processing by B lymphocytes of T cell-stimulating antigens associated with the sLPS. The absence of such uptake and processing of antigens associated with rough LPS might alter the specificity of the T-lymphocyte response. The third difference between the two immunogens is the presence of cyto-

B. ABORTUSOM-PG COMPLEX AND LYMPHOCYTE RESPONSIVENESS

47

sol, cytoplasmic membranes, and periplasmic proteins in strain 19, but not in the OM-PG immunogens. Vaccination with strain 19 whole cells may have stimulated a preferential expansion of lymphocyte clones responding to antigens not represented in the RB51 OM-PG. In so, then fewer clones would be present to react with RB51 OM-PG in vitro. We have reported that oligoclonal T-cell lines from unimmunized cows experimentally inoculated with strain 2308 reacted with cytoplasmic antigens, as well as to OM-PG antigens (Smith, 1990). Some of the obvious candidates for cytoplasmic antigens are heat shock proteins that have been demonstrated to stimulate T lymphocytes in other bacterial and parasitic infections (Young et al., 1989). It has been hypothesized that expression of heat shock proteins, especially the hsp 70 family, is up-regulated as part of the process of adapting to life in an infected host, and that such flexible genetic regulation may be important for understanding the differential immune response to replicating and non-replicating bacteria. Nothing is yet known about heat shock proteins in Brucella. Regardless of the mechanisms, lymphocytes of different specificities were stimulated by immunization with strain 19 or OM-PG. Clearly, the form in which Brucella antigens are presented to the host is important in determining the specificity and the magnitude of the cellular response. Future efforts must be directed toward a better understanding of the spectrum of Brucella antigens that stimulate lymphocyte reactivity and their roles in the induction of protective immunity. ACKNOWLEDGEMENTS

These studies were supported by the Texas Agricultural Experiment Station projects 6194, 6847, and 5320, by USDA cooperative agreement 58-519B-0880, and by USDA grants 86-CRSR-2-2833, 84-CRSR-2-2503, 85-CRSR-22607, and 86-CRSR-2-2806. We thank David Locke and Drs. T.R. Simpson and R.P. Crawford for their care of research animals, and Betty Rosenbaum, Doris Hunter, Bruce Crooker, Jan Patterson, and Sidney Sherwood for their expert technical assistance.

REFERENCES Alausa, O.K., Corbel, M.J., Elberg, S.S., Gargani, G., Gubina, E.A., Shi-Lang, L., Plommet, M., Schliesser, T. and Yadava, V.K., 1986. Report of the Joint F A O / W H O Expert Committee on Brucellosis, 6. World Health Organization, Geneva, 132 pp. Alton, G.G., Jones, Z.M. and Pietz, D.E., 1975. Laboratory Techniques in Brucellosis. World Health Organization, Geneva, 163 pp. Heck, F.C., Williams, J.D., Pruett, J., Sanders, R. and Zink, D.L., 1980. Enzyme-linked immunosorbent assay for detecting antibodies to Brucella abortus in bovine milk and serum. Am. J. Vet. Res., 41: 2082-2084.

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Janeway, C.A., Jr., Ron, J. and Katz, M.E., 1989. The B cell is the initiating antigen-presenting cell in peripheral lymph nodes. J. lmmunol., 138:1051-1055. Lutkenhaus, J.F., 1977. Role of a major outer membrane protein in Escherichia coli. J. Bacteriol., 131: 631-637. Matthews, D.E. and Farewell, V.T., 1988. Using and Understanding Medical Statistics, 2. S. Karger AG, New York p. 178. Ron, Y. and Sprent, J., 1987. T cell priming in vivo: a major role for B cells in presenting antigen to T cells in lymph nodes. J. Immunol., 138: 2848-2856. Ruckerbauer, G.M., Stemshorn, G.M. and Nielsen, K.H., 1981. An hemolysis-in-gel test for anti-Brucella antibody in cattle serum. Adv. Exp. Biol. Med., 137: 782-783. Sarvas, M., 1985. Membrane fractionation methods. In: T.K. Korhonen, E.A. Dawes and P.H. M~ikel/i (Editors), Enterobacterial Surface Antigens: Methods for Molecular Characterization. Elsevier Science Publishers Ltd., Cambridge, pp. l 11-122. Smith, R., III, 1990. T lymphocyte-mediated mechanisms of acquired protective immunity against brucellosis in cattle. In: Adams, L.G. (Editor), Advances in Brucellosis Research: An International Symposium. Texas A&M University Press, College Station, TX, in press. Smith, R., III, Adams, L.G., Ficht, T.A., Sowa, B.A. and Wu, A.M., 1990a. Immunogenicity of subcellular fractions of Brucella abortus: measurement by in vitro lymphocyte proliferative assays. Vet. Immunol. Immunopathol., 25: 83-97. Smith, R., lit, Kapatsa, J.C., Rosenbaum, B.A. and Adams, L.G., 1990b. Bovine T-lymphocyte lines reactive with Brucella abortus. Am. J. Vet. Res., 4:512-517. Smith, R., III, Kapatsa, J.C., Sherwood, S.J., Ficht, T.A., Templeton, J.W. and Adams, L.G., 1990c. Differential reactivity of bovine lymphocytes to species of Brucella. Am. J. Vet. Res., 4:518-523. Sowa, B.A., 1990. Membrane proteins of Brucella sp. In: Adams, L.G. (Editor), Advances in Brucellosis Research: An International Symposium. Texas A&M University Press, College Station, TX, in press. Sowa, B.A., Moor, D. and Ippen-Ihler. K., 1983. Physiology of F-pilin synthesis and utilization. J. Bacteriol., 153: 962-968. Splitter, G.A. and Everlith, K.M., 1989. Brucella abortus regulates bovine macrophages-T cell interaction by major histocompatibility complex class II and interleukin-1 expression. Infect. Immun., 57:1151-1157. United States Department of Agriculture, 1984. Brucellosis eradication. Uniform methods and rules. Animal and Plant Health Inspection Service, Washington, DC, p. 10. Winter, A.J. and Rowe, G.E., 1988. Comparative immune responses to native cell envelope antigens and the hot sodium dodecyl sulfate insoluble fraction (PG) of Brucella abortus in cattle and mice. Vet. Immunol. Immunopathol., 18:149-163. Winter, A.J., Verstreate, D.R.. Hall, C.E., Jacobson, R.H., Castleman, W.L., Meredith, M.P. and McLaughlin, C.A., 1983. Immune response to porin in cattle immunized with whole cell, outer membrane, and outer membrane protein antigens of Brucella abortus combined with trehalose dimycolate and muramyl dipeptide adjuvants. Infect. Immun., 42:1159-1167. Winter, A.J., Hall, C.E., Jacobson, R.H., Verstreate, D.R., Meredith. M.P. and Castleman, W.L., 1986. Effect of pregnancy on the immune response of cattle to a Brucella vaccine. J. Reprod. Immunol., 9: 313-325. Young, D., Lathigra, R. and Mehlert, A., 1989. Stress-induced proteins as antigens in infectious diseases. In: Pardue, M.L., Feramisco, J.R. and Lidquist, S. (Editors), Stress-Induced Proteins. Alan R, Liss, New York, pp. 275-285.