Immunogenicity of subcellular fractions of Brucella abortus: Measurement by in vitro lymphocyte proliferative responses

Veterinary Immunology and Immunopathology, 25 (1990) 83-97 Elsevier Science Publishers B.V., Amsterdam - - Printed in The Netherlands

83

Immunogenicity of Subcellular Fractions of BruceUa abortus: Measurement by In Vitro Lymphocyte Proliferative Responses R O G E R S M I T H III,L. G A R R Y ADAMS, T H O M A S A L B E R T M. W U

A. FICHT, BLAIR A. S O W A and

Departments of Veterinary Pathology and Veterinary Microbiology, Texas A & M University and the Texas AgriculturalExperiment Station,CollegeStation, TX (U.S.A.) (Accepted 14 October 1989)

ABSTRACT Smith, R., III, Adams, L.G., Ficht, T.A. Sowa, B.A. and Wu, A.M., 1990. Immunogenicity of subcellular fractions of Brucella abortus: measurement by in vitro lymphocyte proliferative responses. Vet. Immunol. Immunopathol., 25: 83-97. Five groups of heifers were immunized with various subcellular fractions of BruceUa abortus and tested for their responsiveness in lymphocyte proliferative responses in vitro. The five subcellular fractions used as immunogens were: (1) a mixture of recombinant outer membrane proteins fused to Escherichia coli fl-galactosidase, (2) a mixture of outer membrane proteins BaomI, BaomIIB1, and BaomIII1, (3) a mixture of outer membrane proteins 7.5 kDa and 8.8 kDa, (4) a complex of smooth lipopolysaccharide and proteins, and (5) a complex of outer membranes and peptidoglycan (OM-PG complex) from a rough strain. All immunogens were emulsified in adjuvant and administered twice at a 61-day interval. Two other groups of cows were included; one immunized with strain 19 and the other with adjuvant only. Strain 19 and the rough OM-PG complex induced responsiveness in lymphocyte proliferation assays in a high percentage of immunized cows. The smooth lipopolysaccharide-proteincomplex induced responsiveness in fewer cows. The lowest frequencies of responding cows were found in groups that received either recombinant proteins or purified protein mixtures. Based on these results, we concluded: (1) cellular immunity, as measured by in vitro lymphocyte proliferative responses, can be induced with subcellular fractions of B. abortus and (2) the more complex the immunogen, the greater the frequency of responding cows.

INTRODUCTION

An important issue in bovine brucellosis is the development of subunit vaccines that stimulate a protective immune response, but do not cross-react on standard serological tests (Alausa et al., 1986). It is generally accepted that protective immune responses in cattle require contributions of both the cellular and humoral components of the immune system (Alausa et al., 1986; Collins 0165-2427/90/$03.50

© 1990 Elsevier Science Publishers B.V.

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and Campbell, 1982; Schultz, 1981; Winter, 1987). While it is relatively easy to assess the humoral response of cattle to immunization by using panels of the many serological assays developed for BruceUa abortus, there is no single assay, or panel of assays, that effectively measures cellular immune responses. Most laboratories now rely on in vitro lymphocyte proliferative assays as a measure of cellular immunity to B. abortus (Winter et al., 1983; Confer et al., 1987; Brooks-Alder and Splitter, 1988; Wilkinson et al., 1988; Smith et al., 1990a). To design an effective subunit vaccine for brucellosis, it will be necessary to assess the immunogenicity of candidate antigens in cattle. Two experimental approaches have been taken to address this question. Some investigators have used antigenic fractions to assess responsiveness of lymphocytes taken from cattle vaccinated with strain 19 (Baldwin and Winter, 1985; Baldwin et al., 1985; Brooks-Alder and Splitter, 1988). Others have immunized cattle with subcellular fractions of Brucella, then tested for in vitro lymphocyte reactivity (Winter et al., 1983; Winter and Rowe, 1988) and for protection against experimental challenge (Confer et al., 1987). A broad spectrum of antigens or antigenic combinations has been identified by these approaches. Among those Brucella antigens reported to induce lymphocyte reactivity in cattle are the major outer membrane proteins 2 and 3 (Winter et al., 1983; Winter and Rowe, 1988), peptidoglycan (Winter and Rowe, 1988), and salt-extractable proteins (Confer et al., 1987). Furthermore, antigens of high, medium, and low molecular weights were reported to be stimulatory for lymphocytes from strain 19vaccinated cows (Brooks-Alder and Splitter, 1988). A problem of interpretation arises in some of these experiments as to the role of Brucella lipopolysaccharide (LPS). Its presence in most fractions, regardless of the care with which they are prepared, has made it difficult to assess its contribution to lymphocyte reactivity. Especially troublesome is the possibility that proteins are covalently bonded to Brucella LPS (Perera et al., 1984 ). Recently, it was reported that LPS is not stimulatory for B. abortus-specific bovine lymphocytes (Brooks-Alder and Splitter, 1988). However, another report suggested a role for LPS, or proteins co-extracted with LPS, in the induction of bovine lymphocyte reactivity (Baldwin and Winter, 1985). We report the results of our testing of five combinations of subcellular antigens fcr their ability to induce lymphocyte proliferative responses in vitro. The fractions were prepared such that three were free of LPS, one included only rough LPS deficient in O-antigen, and the last was an enriched fraction of LPS and associated proteins. Some, but not all, cows were able to respond to each of these preparations. The frequency of responsive cows was greatest when the more complex antigenic preparations were given as immunogens.

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MATERIALS A N D M E T H O D S Cow$

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 strains 2308 and 1119.3 (Dr. B.L. Deyoe, USDA/NADC, Ames, IA), strain 19 (Coopers Animal Health, Kansas City, MO), and strain RBS1 (Dr. G.G. Schurig, Virginia Tech, Blacksburg, VA ). Immunogen preparations Seven experimental groups of cows were used, including one group that was immunized with strain 19 (5 × l0 s 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 squalene and Tween 80 (Ribi Immunochem, Hamilton, MT). All imlnunogens, except strain 19, were emulsified in adjuvant, lyophilized, and stored at 4 ° C prior to immunization. Strain 19 was resuspended and administered according to manufacturer's recommendations. Immunogens consisting of B. abortus peptides fused to E. colifl-galactosidase were selected from a ~t-gtll library, and are referred to collectively as fusion proteins (Ficht et al., 1988). Briefly, random size fragments ofB. abortus (strain 2308 and strain 19) DNA with an average length of 1-2 kb were generated by partial digestion with restriction enzymes, cloned into ~t-gtll, and grown in E. coli Y1090 cells. Expression of the fusion proteins was induced with isopropyl thio-fl-D-galactopyranoside and detected with monoclonal antibodies or monospecific rabbit antiserum to B. abortus outer membrane proteins BaomI, BaomIIB1, BaomIII1, and a 7.5 kDa protein (Holman et al., 1983 ). Selected clones expressing these B. abortus peptides were used to obtain immunogens, which were purified by passing the cell lysates over a fl-galactosida~ affinity column. Bound immunogens were eluted at low pH, dialyzed, mixed, and lyophilized prior to emulsification in adjuvant. The dose given to the cows consisted of 30/tg of each of the four peptides. Two doses were given at a 61-day interval. Immunogens consisting of purified B. abortus proteins were prepared by elution from electrophoretic gels (Ficht et al., 1988). Briefly, B. abortus strain

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RB51 or strain 2308 cells were disrupted by repeated sonication in hypotonic buffer. The complex of outer membranes and peptidoglycan (OM-PG complex) was separated from the cytosol and cytoplasmic vesicles by differential centrifugation. Proteins contained in the OM-PG complex of B. abortus were solubilized by boiling in 5% (w/v) sodium dodecyl sulfate and subjected to polyacrylamide gel electrophoresis (Laemmli, 1970). The gels were lightly stained with Coomassie blue, and the relevant bands were carefully excised. The proteins were then electroeluted from the gel, dialyzed, mixed and lyophilized prior to emulsification in adjuvant. All preparations were tested for purity by electrophoresis. The immunogen consisting of a mixture of BaomI, BaomIIB1, and BaomIII1 (Baom proteins) was prepared from strain RB51 cells, and the immunogen consisting of a mixture of 7.5 kDa and 8.8 kDa (7.5/ 8.8 kDa proteins) proteins from strain 2308 cells. Using these strain combinations, such that B. abortus LPS did not migrate in the region from which the proteins were excised, permitted preparation of protein immunogens in which LPS contamination was undetectable by Limulus assay. The dose given to the cows consisted of 30/~g of each peptide. Two doses were given at a 61-day interval. Immunogens consisting of enriched B. abortus lipopolysaccharide complexed with proteins were prepared by fractionation procedures previously described and are referred to as smooth lipopolysaccharide (sLPS-protein complex) (Wu et al., 1987). Briefly, strain 1119.3 cell envelopes, prepared by repeated sonication, were extracted with hot phenol and the LPS precipitated twice in methanol and dissolved in distilled water. The fraction used as an immunogen in these experiments, f5b, contained approximately 17% protein, by the Lowry phenol method using bovine serum albumin as standard (Lowry et al., 1951 ). The LPS fraction was lyophilized prior to emulsification in adjuvant. The dose given to the cows consisted of 30/~g, given twice at a 61-day interval. Immunogens consisting of B. abortus OM-PG complex were prepared from strain RB51, as described above. The OM-PG complex was lyophilized prior to emulsification in adjuvant. The dose given to the cows consisted of sufficient OM-PG complex to contain 400/~g of protein by the BCA assay (Pierce Chemical Co., Rockford, IL), given twice at a 61-day interval.

Peripheral blood mononuclear 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 following day by differential centrifugation of heparinized blood over ficoll-sodium metrizoate (Sigma Chemicals, St. Louis, MO), and resuspended in culture medium at 2X l0 s per ml. Culture medium consisted of RPMI-1640 (Hazelton Research Products, St. Lenexa, KS) supplemented with sodium bicarbonate (Hazelton), non-essential amino acids (Hazelton), 1-glutamine

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87

(Hazelton ), H E P E S buffer (Hazelton), sodium pyruvate (Hazelton), 2-mercaptoethanol (Sigma), and 10% fetal bovine serum (HyClone Laboratories, Logan, UT), as previously described (Smith et al.,1990a; Smith et al.,1990b). Lymphocyte proliferation assay Peripheral blood mononuclear cells were assayed in 96-well round-bottom culture plates. Briefly, 2 X 10s peripheral blood mononuclear cells were cultured overnight prior to addition of 5 × 107 y-irradiated B. abortus strain 2308 whole cells, as antigen. Cultures were incubated for an additional 5 days at 37 °C in a humidified atmosphere of 7.5% C02. Four hours prior to the termination of culture, the cells were pulsed with 10 #1 of [3H]-thymidine (ICN Biomedicals, 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 medium alone, and the positive control was the incorporation by cells in cultures with 250 ng concanavalin A (Pharmacia, 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 B. abortus- cpm in cultures with medium.

Experimental design and data analysis Cows were randomly assigned to one of seven experimental groups and immunized on day 0 (Table 1). Those cows that received two doses of immunogen were re-immunized on day 61. Thirty days prior to immunization, approximately halfof the cows were sampled, and on the day of primary immunization, allcows were sampled and proliferativeassays performed. Subsequent samples were taken and assays were performed at 40, 61, 99, 133, 157, and 189 days after primary immunization. On each of the six sampling times afterprimary immunization, only half of the cows were sampled, with even-numbered cows alternating with odd-numbered cows. All cows were bred and inoculated conjunctivally with 107 cfu B. abortus strain 2308 at mid-gestation (215 days postimmunization). Samples for proliferative assays were collected at 27, 56, 85, and 117 days post-challenge. For purposes of data analysis, individual assays were judged as invalid, or uninterpretable, if the thymidine-uptake in cultures with medium exceeded 10 000 cpm. Invalid assays represented approximately 15% of the total.Two criteriawere established for inclusion of individual cows in 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 afterthe primary immunization (in order to make comparisons in terms of frequency of positive responses).

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TABLE 1

Summary of immunogens and experimental groups Immunogen Adjuvant Strain 19 Fusion proteins BaomI BaomIIB1 BaomIII1 7.5 kDa sLPS-protein complex

Rough OM-PG complex Purified proteins 7.5 kDa 8.8 kDa Purified proteins BaomI BaomIIB1 BaomIII1

Dose -

Administration schedule day 0 and 61

Number of cows

day 0 and 61

13 17 21

400 #g 30/~g each

day 0 and 61 day 0 and 61 day 0 and 61

13 9 18

30/zg each

day 0 and 61

15

5 × l0 s 30/~g each

30/~g

day 0

Statistical analysis For determination of a cutoff point to differentiate negative and positive samples, it was not possible to use the mean and standard deviation, because these statistics are not resistant to the effects of extreme values and because the distribution of responses was non-Gaussian. Instead, the median and the fourth-spread (similar, but not identical, to the interquartile difference) were used to identify "outliers." The formula for identifying outliers is the value of the upper fourth plus 1.5 times the fourth-spread (Emerson and Strenio, 1983 ). To compare experimental groups for frequency of responding individuals and to compare the frequency of positive responses to the outcome of bacterial cultures, chi-square analysis was used, with ~ = (0.05/N), where N = number of comparisons, to correct for multiple comparisons (Matthews and Farewell, 1988). For comparisons among the seven experimental groups, six tests were performed, thus setting ~ = 0.0083. RESULTS

Determination of positive and negative results In the assays prior to immunization, 200 were considered valid by the criteria described. The distribution of the proliferative responses of these unvaccinated cows is presented in Fig. 1. The median value of the net response was 1520 cpm, with 20 082 cpm being determined as the upper level for negative re-

IMMUNOGENICITY OF SUBCELLULAR FRACTIONS OF BRUCELLA ABORTUS

-'7--

94

100

8OOOO

8O ,~ ~

89

6OO(3O

60 2O000

2O

-10

0

10

20

30

40

50

60

70

80

90

100

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. Inset: box plot of the same data. The extent of the box represents the upper and lower fourths of the distribution, and the line inside the box represents the median. Fifty percent of the 200 assays fall within the box, The cutoff for positive and negative responses is represented by the vertical bars, and all outliers are represented as individual points. TABLE 2 FrequencyofpositiveresponsestoB. abortusstrain 2308in cattleexperimentallychallengedwith B. abortusstrain 2308 Days post-challenge

27 Unimmunized cows Number tested Number positive Percent positive Immunized cows Number tested

Number positive Percent positive All cows Number tested Number positive Percent positive

56

85

117

6 1 16.7

7 5 71.4

5 4 80.0

40

20

32

25

6 15.0

17 85.0

30 93.8

22 84.3

49 6 12.2

26 18 69.2

39 35 89.7

30 26 80.2

9 0 0.0

sponses. U s i n g a r o u n d e d value o f 20 000 c p m as t h e c u t o f f for negative responses, 20 a s s a y s ( 1 0 % ) were a b o v e t h e cutoff. B a s e d o n t h e criteria o f at least one negative r e s p o n s e p r i o r to v a c c i n a t i o n , 45 cows were e l i m i n a t e d f r o m f u r t h e r c o n s i d e r a t i o n (25 for w h i c h t h e r e were no valid assays, a n d 20 for which t h e r e were s p u r i o u s l y high r e s p o n s e s ) . T o d e t e r m i n e w h e t h e r t h e assay, as p r e s e n t e d , w o u l d identify positive re-

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sponses following an active experimental infection, cows were assayed at intervals after challenge. The distribution of proliferative responses in assays after experimental challenge is presented in Table 2. In the adjuvant group (unimmunized cows), five of seven (71.4%) cows responded at 85 days postchallenge, and four of five (80%) at 117 days post-challenge. For all immunized cows, 93.8% of the cows responded at 85 days, and 84.3% at 117 days. Taken together, approximately 80-90% of all experimentally infected cows responded positively in the proliferative assay by 12-17 weeks.

Responses of individual cows to immunization with subceUular fractions of B. abortus All cows were randomly assigned to one of seven immunization groups and tested for responsiveness in lymphocyte proliferative assays at six intervals after immunization, with each cow being tested three times. In the group of cows that received adjuvant only, five of 51 (9.8%) assays were positive. These five positive assays represented five different cows, so that none of these cows responded in two of the three assays (Table 3). The 9.8% responsiveness is TABLE3 Frequency of positive responses to B. abortus strain 2308 in cattle immunized with subcellular fractions of BruceUa Immunogen

Adjuvant Strain 19 Fusion proteins 7.5/8.8 kDa proteins Baom proteins sLPS-protein complex Rough OM-PG complex

Total

Number of positive responses (percent)

Z2 (P)

None

1

2

3

versus adjuvant

12 (70.6) 2 (15.4) 11 (52.4) 8 (44.4) 5 (33.3) 3 (23.1) 2 (22.2)

5 (29.4) 0 (0.0) 8 (38.1) 8 (44.4) 8 (53.3) 7 (53.8) 1 (11.1)

0 (0.0) 5 (38.5) 2 (9.5) 2 (11.1) 2 (13.3) 1 (7.7) 2 (22.2)

0 (0.0) 6 (46.1) 0 (0.0) 0 (0.0) 0 (0.0) 2 (15.4) 4 (44.4)

43

37

14

na a 23.0* (<0.001) 2.3 (0.317) 3.6 (0.165) 5.6 (0.061) 8.3 (0.040) 14.5" (0.002)

12

na = not applicable *Difference is significant from comparison group (~ = 0.05/6 = 0.0083 ).

Number of cows (assays) versus strain 19 na na 20.9* (<0.001) 18.9" (<0.001) 16.5" (<0.001) 11.8" (0.008) 2.2 (0.532)

17 (51) 13 (39) 21 (63) 18 (54) 15 (45) 13 (39) 9 (27) 106

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essentiallyidenticalto the frequency of falsepositive reactions observed prior to immunization (10.0%). Those cows receivingstrain 19 as immunogen responded in 28 of 39 (71.8%) assays. Of the 13 cows in this group, two did not respond in any assay (Table 3). Eleven were considered responders to immunization, in that they responded in at least two of the three assays following immunization. Most of the responses were observed in the firstassays after immunization, and persisted for the duration of the experiment. In those cows receivingtwo doses of fusion proteins,twelve of 63 (19.0%) assays were positive.Only two of 21 cows responded in more than one assay, with eight others responding in a single assay (Table 3). Of the two cows that responded in more than one assay, both responded on day 61, at the time of secondary immunization. One responded again at 157 days, but failed to respond at 187 days. For the other cow, the pattern was reversed, in that the responded at 187, but not 157 days after immunization. Twelve of 54 (22.2%) assays were positive in cows that were immunized with the mixture of the low molecular weight proteins.Eight cows did not respond to the 7.5/8.8k D a proteins,eight responded in one assay, and two responded twice (Table 3). Both cows that responded in two assays did so only after secondary immunization on day 61. Cows immunized with the mixture of three B a o m proteins responded in twelve of 45 (26.7%) proliferativeassays. In this group, five cows did not respond, eight responded in a single assay, and two responded in two assays (Table 3). In this group, all but one of the positive responses were observed after secondary immunization. Smooth L P S from strain 2308, containing 17% protein, induced responses in 15 of 39 (38.5%) assays on lymphocytes from 13 cows. Three cows responded in two or three assays, and seven in a singleassay (Table 3). Only three cows did not respond to sLPS-protein complex. Only two positive assays were observed before the secondary immunization at day 61. The group of cows immunized with rough O M - P G complex demonstrated positive responses in 17 of 27 (63.0%) assays. Six of these cows responded in two or three assays, and one cow responded in a single assay (Table 3). T w o cows did not respond to the rough O M - P G complex. Three of eight cows responded before secondary immunization, with other cows responding only after secondary inoculation. The data for all groups are summarized in Table 3. By chi-square analysis, there was no difference between the frequency of responsiveness and groups of cows immunized with adjuvant, fusion proteins,7.5/8.8kDa proteins,B a o m proteins, or sLPS-protein complex ( P > 0.0083). Both the strain 19 and the rough O M - P G complex groups were significantlydifferentfrom the adjuvant group (P < 0.0083). In comparison to the positive control group (strain 19), the group immunized with rough O M - P G complex was not significantlydifferent in frequency of responsive cows.

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DISCUSSION

Two conclusions can be drawn from data presented in this report: (1) cellular immunity, as measured by in vitro lymphocyte proliferative responses, can be induced wl~h subcellular fractions of B. abortus and (2) the more complex the immunogenic preparation, the greater the frequency of responding COWS.

In our studies aimed at developing a subcellular vaccine for brucellosis, the first goal was to analyze immunogenicity using doses that reflected the relative abundance of the component in the whole Brucella cell. Thus, the doses of immunogens in this report were chosen based on their approximate representation in the strain 19 live vaccine. For example, OM-PG represents about 10% of the dry weight of a whole cell. The 400/~g dose represents the amount of OM-PG in approximately 3 X 109 whole cells, which was the recommended adult dose at the time the immunogens were being prepared (United States Department of Agriculture, 1984). Similarly, each of the major outer membrane proteins represents 5-10% of the total protein content of the OM-PG, and the 30 /zg doses were chosen to reflect that proportion (Sowa, unpublished observations). 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, and a potent adjuvant was chosen for the non-replicating immunogens to augment the response. During preparation for this experiment, the official recommended dose for adult vaccination was reduced from 3 × 109 cfu to 5 X 108 cfu strain 19 (the dose used in group 2). Therefore, the cows inoculated with subcellular immunogens received approximately a six-fold excess of the subcellular component over those cows vaccinated with strain 19, further compensating for the in vivo replication of strain 19. Thus, the doses we chose are not necessarily the optimal doses for successful immunization, and it was not feasible to scan this number of potential immunogens and test for a dose response in a single experiment. Subsequent experiments have been designed to test for optimal dose and immunization schedule of the most successful immunogen, OM-PG, determined in this experiment. Responsiveness was measured in one or more cows in almost all groups of immunized cows, although a hierarchy of immunogenic fractions could be discerned. Combinations of outer membrane protein antigens induced responses in only a few cows; in each of these three groups, only two cows (9-13%) were considered responders ( > two positive assays), with eight others being questionable (one positive assay). A higher frequency of responding cows was observed using sLPS-protein complex as the immunogen, with three (23%) responders and seven (54%) questionable, although this group was not significantly different from the previous groups. The highest frequency of responsiveness was observed in groups immunized with either strain 19 or rough

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O M - P G complex. Eleven of 13 (85%) cows responded to strain 19, and six of nine (66.7%) to rough O M - P G complex. The lower responsiveness in the rough O M - P G complex group may be attributable to the requirement for secondary immunization in some cows, thus delaying the onset of detectable responses. Most cows receiving subcellular fractions of B. abortus did not respond until after secondary immunization. The exceptions to this generalizationwere three of the six responding cows immunized with rough O M - P G complex. Future experiments have been designed to test the requirement for secondary immunization for induction of lymphocyte reactivity to rough O M - P G complex (Smith et al.,1990c). Winter and colleagues have used various preparations of outer membranes, cellenvelopes, outer membrane proteins, and peptidoglycan to immunize cattle (Winter et al.,1983; Winter et al.,1986; Winter and Rowe, 1988). Immunization of four cows with proteins extracted from the outer membrane of rough strain 45/20 induced responses that could be measured by in vitro lymphocyte proliferativeassays (Winter et al.,1983). In a larger group of animals, similar responses could be observed in cows immunized with outer membrane proteins, outer membranes, or with whole cells.The responses measured in this lattergroup of animals, however, were secondary responses in that allhad been calfhood vaccinated with strain 19 (Winter et al.,1983). Winter and Rowe (1988) immunized cattle with cellenvelopes, outer membrane proteins, or peptidoglycan from B. abortus strain 2308 and measured proliferative responses and dermal hypersensitivity reactions to group 2 (potin) and group 3 outer membrane proteins. Proliferative responses were detected in one of two cows immunized with cellenvelopes, in two of three cows immunized with outer membrane proteins, in three of three cows immunized with peptidoglycan and adjuvant, and in one of three cows immunized with peptidoglycan alone. Dermal hypersensitivity tests did not correlate with in vitro lymphocyte proliferativeresponses in three of the heifers,but the overall results for the experimental groups were comparable. The conclusion of this and a second experiment comparing vaccination with either cell envelopes or peptidoglycan was that the cell-mediated immune response was indistinguishable in duration and magnitude for all of these immunogens. Confer et al., (1987), studied the response of cattle to salt extractable proteins (CSP) originally described by Tabatabai and Deyoe (Tabatabai and Deyoe, 1984a,b). In their experiments, cattlewere either vaccinated with C S P in Freund's complete adjuvant or with derivatized C S P (dCSP). Derivatized C S P was prepared by chemical modification with dodecanoyl anhydride (Tabatabai and Deyoe, 1984b). The immunization protocol included two inoculations at 6 week intervals in Freund's complete adjuvant, with some cows receiving the same immunogen both times and others receiving one inoculation of each. Lymphocyte proliferative assays were performed on five cows from each group at 2-week intervals starting 4 weeks after the initialvaccination.

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Only at week 4 (prior to the booster inoculation) were the responses significantly greater in the vaccinated cattle than in the control group. At other time points, there were sporadic increases in one group or another, but no consistent increase in lymphocyte reactivity. Comparisons of our results to those of Winter and Confer are difficult. Strains used as the sources of our immunogens, methods of isolation, and adjuvants were different in all cases. In addition, our protein preparations were more limited than those of Winter. The group 2 outer membrane proteins of Winter consist of a collection of proteins in the range of 37-42 kDa, whereas, in this report, the fusion proteins and purified proteins excised from gels represented a single species of the group 2 proteins, BaomIIB1. The CSP and dCSP proteins of Tabatabai are not comparable to any of the immunogens in our experiments. Despite the differences in these studies, a common theme is the ability of at least some individual animals to respond to the subcellular immunogen used. In our studies, the more complex immunogens stimulated responsiveness in a larger percentage of cattle. One might speculate that the increased immunogenicity of rough OM-PG complex over other subcellular fractions may be attributable either to the increased number of antigens present or to the preferential presentation of proteins antigens covalently bound to peptidoglycan or LPS in the envelope preparation (Perera et al., 1984; Gomez-Miguel and Moriyon, 1986; Sowa, submitted, 1989). In support of the latter hypothesis, Splitter and Everlith (1989) have demonstrated that Brucella sLPS increased the expression of class II major histocompatibility complex glycoproteins on the surface of bovine macrophages. Although the net effect of sLPS 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 rough OM-PG complex. In these experiments, the effect of rough LPS from Brucella on class II glycoprotein expression was not tested. In addition, the observation that B lymphocytes are the primary antigen-presenting cell in a primary immune response may be relevant (Ron and Sprent, 1987; Janeway et al., 1989). Because the major serologic response is to the Oantigen (Alausa et al., 1986 ), the preferential binding of the O-antigen of sLPS by B lymphocytes might favor the processing and presentation of LPS-associated antigens. Regardless of the mechanisms by which the rough OM-PG complex induced greater responsiveness in bovine lymphocytes, these studies have two potential implications for the design of new subunit vaccines for brucellosis. First, based on our studies and those of others, the presence of only a few antigenic moieties in a vaccine may not be sufficient to stimulate cellular immunity in a majority of cattle. Second, the form in which the immunogen is presented may have a major effect on its success in stimulating cellular immunity. For example, the presence or absence of LPS may augment the effectiveness of macrophage pro-

IMMUNOGENICITYOF SUBCELLULARFRACTIONSOF BRUCELLA ABORTUS

95

cessing and presentation of other antigenic determinants by inducing higher levels of class II expression. These two tentative conclusions are not necessarily mutually exclusive, and further immunization studies m a y clarify the relative contributions of the n u m b e r of epitopes and their form to inducing lymphocyte activation. ACKNOWLEDGEMENTS These studies were supported by the Texas Agricultural Experiments Station projects 6194, 6847, and 5320, by U S D A cooperative agreement 58-519B0-880, and by U S D A grants 86-CRSR-2-2833, 84-CRSR-2-2503, 85-CRSR-22607, and 86-CRSR-2-2806. W e 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, Joyce Kapatsa, Sidney Sherwood, and Pare O'Rear 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 techniquesin brucellosis,World Health Organization, Geneva, 163 pp. Baldwin, C.L. and Winter, A.J., 1985. Blastogenic response of bovine lymphocytes to BruceUa abortus lipopolysaccharide.Infect.Immun., 47: 570-572. Baldwin, C.L., Verstreate,D.R. and Winter, A.J., 1985. Immune response of cattleto Brucella abortus outer membrane proteins measured by lymphocyte blastogenesis.Vet. Immunol. Immunopathol., 9: 383-396. Brooks-Alder, B. and Splitter,G.A., 1988. Determination of bovine lymphocyte responses to extracted proteins of Brucella abortus by using protein immunoblotting. Infect.Immun., 56: 2581-2586, Collins,F.M. and Campbell, S.G., 1982. Immunity to intracellularbacteria.Vet. Immunol. Immunopathol., 3: 5-66. Confer, A.W., Tabatabai, L.B., Deyoe, B.L., Oltjen,S.L.,Hall, S.M., Oltjen,J.W., Morton, R.J., Fulnechek, D.L., Smith, R.E. and Smith, R.A., 1987. Vaccination of cattlewith chemically modified and unmodified salt-extractableproteins from BruceUa abortus. Vet. Microbiol.,15: 325-339. Emerson, J.D. and Stranio,J., 1983. Boxplots and batch comparison. In: D.C., Hoaglin, F. Mostellerand J.W. Tukey (Editors),Understanding Robust and Exploratory Data Analysis.John Wiley, New York, NY. pp. 58-96. Ficht, T.A., Bearden, S.W., Sowa, B.A. and Adams, L.G., 1988. A 36-kilodaltonBrucella abortus cellenvelope protein in encoded by repeated sequences closelylinked in the genomic DNA. Infect.Immun., 56: 2036-2046. Gomez-Miguel, M.J. and Moriyon, I.,1986. Demonstration of a peptidoglycan-linkedlipoprotein and characterizationof its trypsin fragment in the outer membrane of BruceUa spp. Infect. Immun., 53: 678-684. Heck, F.C., Williams, J.D., Pruett, J., Sanders, R. and Zink, D.L., 1980. Enzyme-linked immu-

96

R. SMITH III ET AL.

nosorbent assay for detecting antibodies to BruceUa abortus in bovine milk and serum. Am. J. Vet. Res., 41: 2082-2084. Holman, P.J., Adams, L.G., Hunter, D.M., Heck, F.C., Nielsen, K.H. and Wagner, G.G., 1983. Derivation of monoclonal antibodies against Brucella abortus antigens. Vet. Immunol. Immunopathol., 4: 603-614. 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. Immunol., 138:1051-1055. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227: 680-685. Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem., 193" 265-273. Matthews, D.E. and Farewell, V.T., 1988. Using and Understanding Medical Statistics, 2 Karger, New York NY, 228 pp. Perera, V.Y., Winter, A.J. and Ganem, B., 1984. Evidence for covalent binding of native haptenprotein complexes to smooth lipopolysaccharide of Brucella abortus. FEMS Microbiol. Lett., 21: 263-266. 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 antiBrucella antibody in cattle serum. Adv. Exp. Med. Biol., 137: 782-783. Schultz, R.D., 1981. The role of cell-mediated immunity in infectious diseases of cattle. Adv. Exp. Biol. Med., 137: 57-90. Smith, R., III, Kapatsa, J.C., Rosenbaum, B.A. and Adams, L.G., 1990a. Bovine T-lymphocyte lines reactive with BruceUa abortus. Am. J. Vet. Res., (in press). Smith, R., III, Kapatsa, J.C., Sherwood, S.J., Ficht, T.A., Templeton, J.W. and Adams, L.G., 1990b. Differential reactivity of bovine lymphocytes to species of Brucella. Am. J. Vet. Res., (in press ). Smith, R., III, Adams, L.G., Sowa, B.A. and Ficht, T.A., 1990c. Induction of lymphocyte responsiveness by the outer membrane-peptidoglycan complex of rough strains of BruceUa abortus. Vet. Immunol. Immunopathol., 26: in press. 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. Tabatabai, L.B., and Deyoe, B.L., 1984a. Characterization of salt-extractable protein antigens from BruceUa abortus by crossed immunoelectrophoresis and isoelectric focusing. Vet. Microbiol., 9: 549-560. Tabatabai, L.B. and Deyoe, B.L., 1984b. Biochemical and biological properties of soluble protein preparations from BruceUa abortus. Dev. Biol. Stand., 56: 199-211. United States Department of Agriculture, 1984. Brucellosis eradication. Uniform methods and rules. United States Government Printing Office, Washington, DC, 107 pp. Wilkinson, R., Cargill, C. and Lee, K., 1988. Humoral and cell-mediated immune responses in non-pregnant heifers following infection and vaccination with BruceUa abortus. Vet. Immunol. Immunopathol., 18: 379-383. Winter, A.J., 1987. Discussions on BruceUa abortus. Ann. Inst. Pasteur (Microbiol), 138: 135137. Winter, A.J. and Rinse, G.E., 1988. Comparative immune responses to native cell envelope antigens and the hot sodium dodecyl sulfate insoluble fraction (PG) ofBrucella 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.,

IMMUNOGENICITYOF SUBCELLULARFRACTIONSOFBRUCELLAABORTUS

97

1986. Effect of pregnancy on the immune response of cattle to a Brucella vaccine. J. Reprod. Immunol., 9: 313-325. Wu, A.M., AdRms, L.G. and Pugh, R., 1987. Immunochemical and partial chemical characterization of fractions of membrane-bound smooth lipopolysaccharide-protein complex from Brucella abortus. Mol. Cell. Biochem., 75: 93-102.