requirement of the conformational stability of a salmonella ribosomal vaccine for its mouse...

FEMS Microbiology Immunology 76 (1991) 229-240 © 1991 Federation of European Microbiological Societies 0920-8534/91/$03.50 Published by Elsevier ADONIS 092085349100081H

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FEMSIM 00169

Requirement of the conformational stability of a Salmonella ribosomal vaccine for its mouse protection

Eiji Kita, Daisuke Oku, Fumiko Nishikawa, Masashi Emoto, Kiyoshi Yasui and Shuzo Kashiba

Department of Bacteriology, Nara Medical Unicersity, Kashihara City, Japan

Received 21 December 1990 Revision received 29 April 1991

Accepted 30 April 1991

Key words: Ribosomal vaccine; B-cell mitogen; Formaldehyde condensation; Salmonella typhimurium

1. SUMMARY

The 43-kDa non-O antigenic component isolated from the crude ribosomal fraction of Salmonella typhimurium [9] was further purified by affinity chromatography (43-kDa protein: 43- kDp). Immunization with 43-kDp did not induce complete mouse protection in CF1 mice to 500 LD50 of S. typhimurium, although it elicited a substantial IgG antibody response. The 43-kDp exhibited the mitogenicity to splenocytes (CF1 and C 3 H / H e J) and B cell-rich populations (CF1). Complexing 43-kDp with the compact ribosomes of Streptococcus pyogenes by formaldehyde (complex vaccine: CV) elicited both IgM and IgG antibodies to 43-kDp. CV induced a boosting effect to enhance IgG antibody response. More- over, CV generated delayed-type hypersensitivity to salmonella antigens and also conferred complete protection against 500 LDs0 challenge of S.

Correspondence to: E. Kita, Department of Bacteriology, Nara Medical University, 840, Shijyo-cho, Kashihara City, Nara 634, Japan.

typhimurium to CF1 mice. These abilities of CV were reduced or impaired by RNase digestion. CV was able to induce partial or complete protection in inbred mouse strains (C3H/HeN, C3H/HeJ , DBA/2 and A/J) . These data, in addition to other reports, suggest that conformational stability between ribosomes and contaminating substances such as 43-kDp or O-antigens might be required for the overall effects of the ribosomal vaccine.

2. INTRODUCTION

A number of studies on salmonella ribosomal vaccines have been reported since Venneman and Bigley [1] demonstrated the immunogenicity of a subcellular, ribonucleic acid preparation of Salmonella typhimurium. Many investigators indicated that the efficacy of salmonella ribosomal vaccines varies with mouse strains [2], and that contaminating lipopolysaccharide (LPS) exerted the major effect [3,4]. In addition, salmonella ribosomal vaccines can induce mouse protection comparable to that induced by immunization with

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a sublethal dose of viable virulent S. typhimurium [5,6] or with viable cells of attenuated strains [7].

Previous studies from our laboratory have shown that a subcellular, ribosome-rich extract of S. typhimurium contained the immunogenic non- O antigenic component of an estimated molecular mass of 43-kDa [8,9]. This crude antigen alone was not protective but its immunogenicity was enhanced when it was administered together with the purified homologous ribonucleic acid (RNA). Furthermore, this non-O antigenic component could induce delayed-type hypersensitivity (DTH) to salmonella antigens when it was injected into mice with homologous RNA. Eisenstein and Angerman [10] have postulated that ribosomal vaccines contain LPS plus an additional mitogenic substance closely associated with LPS ex- tracted by mild procedure. We postulated that phenol extraction might deprive the ribosomal fraction or LPS of this additional mitogenic substance [8]. This hypothesis is supported by the fact that LPS fortuitously associated with the ribosomal fraction could have been expected to be complexed with a B-cell mitogenic endotoxin protein [11] which can be easily solubilized in the phenol phase by the phenol-water extraction of TCA endotoxin [12].

Furthermore, Phillips, Eisenstein and Meissler [13] have speculated that LPS in complex with the ribosomes could be converted to a T-dependent form of the antigen, based on their study using formaldehyde-fixed S. typhimurium LPS to ribosomes of Brucella abortus and the endotoxin-resistant C 3 H / H e J mouse.

Our present study was undertaken to clarify the role of the non-O antigenic 43-kDa protein (43-kDp) component in the immunogenicity of salmonella ribosomal vaccines by using the same technique by which this molecule was cross-linked to heterologous ribosomes by formaldehyde condensation [14].

MATERIALS AND METHODS

3.1. Animals Conventionally-raised CF1 female mice of 7 -9

weeks old were used as previously reported [8].

Female mice of inbred strains, C 3 H / H e J , Balb/c, C57BL/6 (susceptible strains), and C 3 H / H e N , D B A / 2 and A / J (resistant strains) (purchased from Japan Clea, Tokyo) were used at the age of 10 weeks.

3.2. Organisms Salmonella typhimurium LT2 was used

throughout the study. The intraperitoneal (i.p.) LDs0 of this strain for CF1 mice is 500 colony- forming units (cfu) [8]. The i.p. LDs0 of this strain for inbred mice was determined by the method of Reed and Muench [15] after survival was measured over 30 days. The i.p. LDs0 values for inbred mouse strains were as follows: 2 × 105 cfu for D B A / 2 ; 1.5 × 106 for A / J ; 10 ~' for C 3 H / H e N ; 10 for C 3 H / H e J ; 20 for C57BL/6 and < 10 for Balb/c .

3.3. Purification of the acm~e component The subcellular fraction containing the 43-kDp

(designated 43-kDa fraction) was prepared as previously reported [9]. After SDS-polyacryl- amide gel electrophoresis (SDS-PAGE) using a standard tube gel apparatus, the particular por- tion corresponding to the 43-kDa band was sliced, and the protein in the 43-kDa band was recovered by the electrophoretic elution with the Maxyield-NP (50 V, 60 rain, Atto, Tokyo) using seamless cellulose membrane (Mr-cut off, 14000; Wako Pure Chemical Industries, Osaka). To re- move SDS, the electrophoretically-recovered sample was supplemented with 9 volumes of cooled methanol and was centrifuged at 10 000 × g for 20 rain at 0°C after it was kept at -20°C overnight. The sedimented sample was dissolved in saline, then dialyzed and its protein concentration was measured by Lowry's method [16].

Antiserum to the 43-kDa band was raised in rabbits and its y-globulin fraction was coupled to column matrix by the standard CNBr coupling method [17] as previously reported [18]. The 43- kDa fraction was applied to the affinity column and the protein binding for the antibody was eluted with glycine buffer (pH 4.0). Fractions eluted with glycine buffer were pooled, concen- trated and dialyzed by ultrafiltration, and used as the purified 43-kDa protein (43-kDp). The purity

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same dose. Fourteen days after the second vacci- nation, mice were bled to obtain serum to be tested for antibody titers (secondary response).

The remaining five mice in each group were tested for delayed footpad response (DFR) on day 14 of primary immunization, using formaldehyde-killed cells (FKC) of strain LT2 [7].

3. 9. Enzymatic digestion of CV To 2% CV or LT2-CRF solution in 50 mM

Tris. HC1 buffer (pH 7.2), ribonuclease I-A (RNase I-A, EC 2.7.7.16, Sigma) was added at a concentration of 2 /zg per ml and the mixture was incubated at 40°C for 3 h. Digestion was carried out under sterile conditions and 1 /50 volume of sterile zinc acetate solution (100 mM) was added to the mixture to inhibit nuclease activity. The digested samples were diluted with sterile HBSS to obtain several concentrations required for in- jections. Controls consisted of HBSS containing RNase I-A and zinc acetate. For tissue cultures, the digested samples were diluted with RPMI complete medium and controls consisted of RPMI complete medium containing RNase I-A and zinc acetate.

3.10. Statistics All experiments were done three times, and

the levels of significance for the observed fre- quencies were determined by Fisher's test for 2 × 2 table or Student's t-test.

4. RESULTS

4.1. Immunogenicity of purified 43-kDp and CV Graded doses of 43-kDp were tested for pro-

tective activity in CF1 mice against 500 LDs0 of S. typhimurium LT2, and results are shown in Table 1. Mice were not completely protected with any doses of 43-kDp alone, although mean survival days were increased as compared with control mice at each immunizing dose. Thus, the complex vaccine (CV) was tested for its immunogenicity. As shown in Table 1, the protective capacity of 43-kDp was significantly enhanced by this procedure, and a single immunization with 50 ~g of CV (composed of 10/xg of 43-kDp and 40

Table l

Protective activity of 43-kDp to S. typhimurium LT2 in CFI mice

Dose a Survival rate (%) (p~g/mouse) 43-kDp CV Mixed vaccine

1 0 (9 .2 ) b ND ND 5 0 (10.6) 20 (22.3) 0 (13.8)

10 0 (13.2) ND ND 20 10 (14.5) ND ND 25 ND 70 (27.6) 5 (14.2) 50 15 (16.0) 100 15 (17.2)

100 15 (16.5) 100 25 (18.8) Control

(saline) 0 (6.8)

a Each group consisted of 10 mice immunized i.p. with indicated doses of 43-kDp, CV or mixed vaccine. Mice were challenged i.p. with 500 LDs0 of strain LT2 14 days after immunization, and kept for 30 days. Results are expressed as the mean survival rate (%) of three separate experiments. Immunization with 100 /xg of streptococcal ribosomes re- suited in only 10% mouse-survival rate.

b Numbers in parenthesis represent the mean survival clays of dead mice. Statistically not significant when survival rates were 20% or less by Fisher's test for 2 × 2 table.

/xg of streptococcal ribosomes) generated complete protection in CF1 mice against 500 LDs0 challenge. In contrast, the mixed vaccine used as a control induced a low degree of protection, though statistically significant, only at a dose of 100 /xg. However, higher doses of the mixed vaccine did not increase the magnitude of its protective activity, and streptococcal ribosomes alone were unable to confer mouse protection even at doses higher than 100 /xg/mouse (data not shown).

4.2. Immune responses after immunization with 43-kDp

The antibody responses in CF1 mice after immunization with 43-kDp are summarized in Table 2. The purified 43-kDp at doses higher than 5 /xg/mouse induced only a low degree of IgM response after either primary or secondary immunization. There was no significant boosting effect of antibody responses after the second injection of 43-kDp at any immunizing dose. In contrast, immunization with salmonella ribosomes (LT2- CRF) resulted in both IgM and IgG responses

of 43-kDp was assured by SDS-PAGE and im- munodiffusion test before it was used for further study.

3. 4. Cell preparation A single-cell suspension of splenocytes was

prepared following the conventional method, and was depleted of macrophages, activated lymphocytes, and other adherent cells by passage over Sephadex G-10 (Pharmacia Fine Chemicals, Upp- sala, Sweden) as described by Ly and Mishell [19]. The T-cell fraction was enriched by filtrating pooled cells through nylon wool columns [20], and the B-cell fraction was enriched by treatment of pooled cells with anti-mouse T-cell serum and complement as described previously [21]. The purity of each cell preparation was confirmed by selective responses to mitogens and also by im- munofluorescent antibody analysis [21]. Cell preparations with viability of higher than 98% were used for experiments.

3.5. Blastogenic responses Blastogenic responses of unseparated spleen

cells, enriched T- or B-cells, were done as previously described [21].

Mitogens employed for controls were LPS of S. typhimurium (Westphal type, Difco, Detroit, MI) and concanavalin A (Con A, Sigma, St. Louis, MO).

3.6. Fixation of 43-kDp to heterologous ribosomes Heterologous ribosomes were prepared from

Streptococcus pyogenes N-3A (Group A, type 14; isolated from a patient in our laboratory) following the method of Kita and Kashiba [22].

Complexes of 43-kDp and compact streptococcal ribosomes were made by a formaldehyde fixation procedure [23]. One volume of the 43-kDp solution (200 tzg/ml) and four volumes of the ribosome solution (800 izg/ml) were mixed, dialyzed against a mixing buffer containing 10 mM sodium phosphate, 10 mM magnesium acetate and 4% neutralized formaldehyde (pH 7.2) for 72 h at 20°C, and then against the same buffer without formaldehyde for at least 48 h at 4°C. Before use, the complexes were treated with 0.3% sterile formaldehyde at 4°C for 72 h, followed by

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repeated washing with sterile 10 mM sodium phosphate buffer (pH 7.2) containing 10 mM sodium acetate. The pelleted complexes sus- pended in sterile HBSS were used as the complex vaccine (CV). The sterility was confirmed by cul- turing CV in thioglycolate broth at 37°C for 72 h.

3. 7. Measurement of antibody titers Antibodies to the purified 43-kDp in mouse

serum were measured by enzyme-linked immunosorbent assay (ELISA) following the method of Rote et al. [24]. Goat anti-mouse u chain and 3' chain conjugated with horseradish peroxidase (Organo Teknika, Cappel Product, West Chester, PA) were used in the assay. Titers were determined by the extrapolation to background values of lines obtained by plotting the log~0 of the absorbance vs. the log10 of the serum dilution, following the method of Eisenstein et al. [25]. Titers of samples of unknown serum run on dif- ferent days were normalized by adjusting the absorbance of the high-titered serum developed by hyperimmunization of CF1 mice with LT2-CRF (standard serum) to minimize day to day varia- tion.

3.8. Immunization and challenge Groups of 10 mice were injected i.p. with 0.1

ml of graded doses of 43-kDp or CV. Fourteen days later mice were challenged i.p. with 500 LDs0 of S. typhimurium LT2. Controls received 0.1 ml of saline, 50 /xg of streptococcal ribosomes, or the mixed vaccine in which 43-kDp and streptococcal ribosomes were simply mixed at a ratio of 1:4 just before injection. Groups of 10 inbred mice were immunized i.p. with 50 Ixg of each vaccine, and were challenged i.p. 14 days later with 108 cfu for resistant strains or 10 z cfu for susceptible strains.

For studies of immune responses, groups of 15 CF1 mice were immunized i.p. with graded doses of each vaccine. After 14 days, blood (about 100 /xl) was obtained from each individual mouse (10 mice in each group) by bleeding from the retroor- bital venous plexus, and IgM and IgG antibodies to the 43-kDp were measured by ELISA (primary response). Animals were rested for 48 h and then revaccinated i.p. with the same vaccine in the

Table 2

Immune responses in CF1 mice after immunization with 43-kDp

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Vaccine Dose type (/*g/mouse)

Antibody response (logl0 ELISA titer) a

Primary response Secondary response

IgM IgG IgM IgG

DFR b (xO.I mm) to FKC

43-kDp

LT2-CRF

Saline

1 <2.0 c <2.0 <2.0 <2.0 2.7±0.2 5 2.24±0.11 <2.0 2.36±0.22 <2.0 2.6±0.3

10 2.76±0.13 <2.0 2.84±0.15 <2.0 2.8±0.4 20 2.90±0.16 <2.0 3.12±0.34 <2.0 3.1±0.3

5 2.92±0.11 2.24±0.15 3.96±0.2 4.82±0.36 11.4±2.2 3.45±0.17 3.52±0.23 4.57±0.32 5.33±0.52 13.2±1.8

<2.0 <2.0 <2.0 <2.0 2.6±0.8

a Groups of 10 mice were immunized i.p. with indicated doses of the desired vaccine, and after 14 days mice were bled to obtain serum (primary response). Two days later mice were revaccinated, and blood was obtained after 14 days (secondary response). Group titers were calculated as the geometric mean titers of 10 individual mice, Each assay was done in triplicate.

b Groups of 5 mice were immunized i.p. with indicated doses of the desired vaccine, and after 14days DFR was done using FKC of strain LT2 as an elicitin.

c Data were obtained from three separate experiments and results are expressed as the mean ± SD for three trials. Statistically not significant when ELISA liters were 2.10 or less, and when DFR was 3.8 or less by Student's t-test.

after a single injection. Furthermore, LT2-CRF afforded significant boosting effect, enhancing both IgM and IgG antibody to 43-kDp after a second injection.

Furthermore, DTH to salmonella FKC, as determined by DFR, was induced only by LT2-CRF, but not by 43-kDp (Table 2).

4.3. Immune responses induced by CV The antibody responses in CF1 mice after im-

munization with CV are summarized in Table 3. CV at doses of 5-100/xg/mouse stimulated both IgM and IgG antibody responses to 43-kDp in primary immunization. In this procedure, CV at a dose of 5 / ,g (1 ~,g of 43-kDp and 4 /zg of

Table 3

Immune responses in CF1 mice after immunization with CV a

Vaccine type Dose (/xg/mouse)

Antibody responses (Iogl0 ELISA titer)

Primary response Secondary response

IgM IgG lgM IgG

DFR (×0.1 mm) to FKC

CV 5 2.52 ± 0.23 25 2.74 ± 0.42 50 3.24 ± 0.23

100 3.68 ± 0.21

Mixed vaccine 5 < 2.0 (uncomplexed form) 25 2.30 + 0.23

50 2.82±0.16 100 3.36 ± 0.34

Saline < 2.0

2.66±0.05 2.98+0.17 3.44±0.28 3.82±0.43

<2.0 <2.0 <2.0 <2.0

<2.0

3.34±0.16 3.83±0.09 4.42±0.21 4.86±0.37

<2.0 2.51±0.27 2.96±0.33 3.45±0.31

<2.0

4.09±0.23 4.28±0.16 4.78±0.49 5.56±0.37

<2.0 <2.0 <2.0 <2,0

<2.0

8.6±0.7 9.7±1.2

11.6±1.4 11.8±2.2

2.5±0.7 2.8±0.4 2.7±0.3 2.8±0.2

2.6±0.3

a See footnote Table 2.

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streptococcal ribosomes) was able to induce de- tectable levels of IgM and IgG antibody to 43- kDp, while immunization with 100 p,g of the mixed vaccine containing 20 tx g of 43-kDp and 80 Ixg of streptococcal ribosomes did not evoke IgG antibody responses. Furthermore, CV was able to induce the boosting effect to enhance IgM and IgG antibody responses to 43-kDp after secondary immunization. In contrast, the mixed vaccine, at doses of 5-100 /xg/mouse, evoked no significant boosting effect to enhance IgM antibody response after secondary immunization.

In addition, immunization with CV induced DTH to salmonella FKC, while the mixed vaccine did not generate DTH at any dose.

4.4. Blastogenic responses induced by 43-kDp and CV

As shown in Fig. 1, 43-kDp stimulated [3H]TdR uptake by unfractionated splenocytes of CF1 mice

% ×

20

E EL U

C ~0 ~g

E

~a

-r.

0 10 -3 10 -2 10 -1 1 10 102 iJg lml

C o n c e n t r a t i o n s o f 43 k D p

Fig. 1. Mitogenicity to splenocytes of 43-kDp. Each point represents the mean of nine cultures. The bar represents the s tandard error, o , CF1 splenocytes; o, C 3 H / H e J splenocytes.

to a significant extent in the dose range of 1-100 txg/ml, with a maximum response obtained at 10 txg/ml. This activity was not attributable to endotoxin which might be contaminating 43-kDp preparation, since splenocytes of C3H/HeJ mice also responded significantly to this antigen.

To evaluate whether 43-kDp can selectively stimulate certain lymphocyte populations, the enriched T- or B-cell fraction was cultured with 1 tzg/ml or 10 p.g/ml of 43-kDp for 72 h. Stimula- tion of B-cells with 10/xg./ml of 43-kDp yielded significant levels of blastogenesis, comparable to the response induced by the same dose of LPS (Fig. 2). These findings suggest that the purified 43-kDp preferentially stimulates mouse B-cell- rich populations.

On the other hand, CV and LT2-CRF (5 /xg/ml) exhibited mitogenic activity only to unfractionated splenocytes (CV: 22 250 + 3 718 cpm, P<0.001; LT2-CRF: 26583_+3718 cpm, P < 0.001) but not to enriched lymphocytes, whereas the mixed vaccine (5 >g/ml) stimulated incorporation of [3H]TdR into B-cells (15118_+2793 cpm, P < 0.001) as well as unfractionated splenocytes (10459 +_ 1 217 cpm, P < 0.001).

Furthermore, RNase-digestion of CV and LT2-CRF reduced their mitogenic activity to unfractionated splenocytes by 50% as determined by the incorporation of [3H]TdR, and only digested CV exhibited B-cell mitogenicity (14 289 + 3127 cpm, P<0.001). In contrast, RNase-digestion did not affect the mitogenicity of the mixed vaccine to B-cells and unfractionated splenocytes.

4.5. Effect of RNase digestion on the immunogenicity of CV

Comparison of the immunogenicity in CF1 mice between the undigested and RNase-digested CVs (50 txg/mouse) is shown in Table 4. Digestion of CV with RNase I-A resulted in the impairment of its capacity to elicit IgG antibody response to 43-kDp after primary and secondary immunization. Primary and secondary IgM antibody responses were also reduced after digestion of CV with RNase. Similar results were obtained when LT2-CRF was digested by RNase I-A, but immune responses induced by the mixed vaccine

6O

×

so

+1

E ~ q0 u T C

E ~ 3 0

-~ 2 0

"tJ b-

Unfract io- Enr iched Enr iched nated T-cel l B-cel l

s p l e n o c y t e s p o p u l a t i o n p o p u l a t i o n

Fig. 2. Blastogenic responses to 43-kDp of splenic cell populations. The column and bar represent the mean and standard error of nice cultures, rm, 1 ,ag/ml of 43-kDp; I , 10 txg/ml

of 43-kDp; N], 10/xg/ml of LPS; [], 2 p 4 / m l of Con A.

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did not change after RNase-digestion. Further- more, the ability of CV as well as LT2-CRF to induce DTH to salmonella FKC was also abro- gated by nuclease digestion. These changes were closely related to the reduction in mouse protection afforded by the digested CV or LT2-CRF, but nuclease digestion did not alter the immunogenicity of the mixed vaccine.

4.6. Protection by CV in inbred mouse strains Among resistant strains, C3H/HeN and A / J

mice were almost completely protected by CV, whereas highly-susceptible strains (C57BL/6 and Balb/c) were not protected (Table 5). Protection conferred by CV in these mice correlated well with their responses to the native LT2-CRF. Fur- thermore, CV was immunogenic even for the highly-susceptible C3H/HeJ mice to the same extent as for the resistant DBA/2 mice. Both strains were also protected to a lesser degree, though significantly, by LT2-CRF. However, the mixed vaccine did not protect mice of any strain tested. Thus, CV appears to be immunogenic in the mouse strains, regardless of the susceptibility

Table 4

Effect of RNase digestion of CV on its immunogenicity

Vaccine RNase Survival ~ Antibody responses (logi0 ELISA titer) b DFR to

type digestion rate (%) Primary response Secondary response FKC c (50/~g) (500 LDso) ( × 0.1 ram)

IgM IgG IgM IgG

CV '

Mixed vaccine

LT2-CRF

Saline

- 100 3.32+0.37 3.68_+0.33 4.51_+0.28 5.01_+0.45 13.4-+2.3 (P < 0.01) d (P < 0.01) (P < 0.01) (P < 0.005)

+ 15 3.02-+0.18 < 2.0 2.94-+0.23 < 2.0 3.6_+ 1.8

- 25 2.96-+0.31 < 2.0 3.12_+0.28 < 2.0 3.1 _+0.8 + 10 2.88_+0.25 <2.0 2.98-+0.31 <2.0 2.5_+0.5

- 100 3.46-+0.12 3.55+_0.27 4.55_+0.26 5.43_+0.3l 13.6_+ 1.9 (P < 0.05) d (P < 0.05) (P < 0.01) (P < 0.01) (P < 0.01) (P < 0.05)

+ 25 2.52_+0.23 < 2.0 2.78_+0.35 < 2.0 3.8_+ 1.3

0 < 2.0 < 2.0 < 2.0 < 2.0 2.5 _+ 0.3

Data were obtained from three separate experiments and results are expressed as the mean + SD for three trials. a Each group consisted of l0 mice immunized i.p. with 50 p,g of the desired vaccine, and after 14 days they were challenged i.p.

with 500 LDs0 of strain LT2. b Each group consisted of 10 mice. See footnote Table 2. c Each group consisted of 5 mice. See footnote Table 2. d Statistical comparison was examined between RNase-digested and undigested vaccines.

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Table 5

Protection conferred by CV in inbred mouse strains

Vaccine a Survival rates (%) b

type C 3 H / H e N C 3 H / H e J D B A / 2 (50 ~g)

A / J Ba lb / c C57BL/6

43-kDp 15 0 10 15 0 0 CV 95 80 85 100 0 0

(P < 0.01) (P < 0.01) (P < 0.01) (P < 0.01) Mixed vaccine 20 10 20 20 0 0 LT2-CRF 90 35 45 100 0 0 Streptococcal r ibosomes 0 0 0 0 0 0 Saline 0 0 0 0 0 0

Each group consisted of 10 mice immunized i.p. with 50 /zg of the desired vaccine, and after 14 days they were challenged i.p. with 10 s cfu (to resistant mouse strains) or 102 cfu (to susceptible mouse strains) of S. typhimurium LT2. Survival rate was determined at 30 days postchallenge~

h Data were obtained from two separate experiments and results are presented as the mean of two experiments. Statistical comparison was made between the CV-immunized and mixed vaccine-immunized groups by Fisher 's test for 2 × 2 table. Survival rate of each group was statistically not significant as compared with the saline-treated group, when survival rates were 20% or less.

to S. typhimurium, that would respond to LT2- CRF.

5. DISCUSSION

To elucidate the role of non-ribosomal antigens in the immunogenicity of ribosomal vaccines, Phillips and Rimler [26] immunized chick- ens with formaldehyde-fixed complexes of LPS from Pasteurella multocida and ribosomes from Brucella abortus or Aspergillus fumigatus, and they have shown that these complexes protected animals against virulent P. multocida, in which methylated bovine albumin, purified RNA and chicken liver ribosomes did not substitute for ribosomes of these two bacteria. This may indicate that only microbial ribosomes, even though from unrelated organisms, can serve as immuno- dulators, provided that their conformational stability is preserved. Thus, in the present study the purified 43-kDp was cross-linked with formaldehyde to streptococcal compact ribosomes to clarify the role of 43-kDp in the immunogenicity of the ribosomal vaccine of S. typhimurium.

Despite the fact that formaldehyde is a univa- lent aldehyde, formaldehyde treatment has been used as a method for fixing the particles. Studies

with model systems have indicated that amide and amino groups can form methylene condensation products in the presence of formaldehyde [14]. Formaldehyde treatment of ribosomes was found to induce the formation of extensive inter- molecular or intramolecular p ro te in-pro te in cross-links [23]. However, there is also a report [27] showing that an RNA-protein cross-linking reaction does occur. Thus, it can be speculated that cross-linking methylene bridges between amide or amino groups of 43-kDp and adenyl or guanidyl groups of RNA or amide or amino groups of ribosomal proteins can be formed.

The washing with NHaCI often used for the purification of ribosomes yielded smaller subunit particles [28] and also the stability of intact ribosomes in the compact state during storage was not assured. In contrast, CV was considered to be able to maintain the conformational stability, compatible with intact ribosomes, since the formaldehyde fixation procedure was known to prevent degradation of ribosomes [23,29]. In the analysis with SDS-PAGE, almost no protein could be detected in gels of undigested CV and the digestion with RNase I-A released most of the protein including 43-kDp from CV molecules (data not shown). Amons and M611er [23] showed that rRNA regions resistant to nuclease digestion

of the ribosomes in the compact state are protected against formaldehyde treatment and that RNA fragments obtained by nuclease digestion are derived from RNA regions fully accessible to formaldehyde. This seems to imply that 43-kDp in CV molecules is fixed to rRNA regions sensitive to nuclease digestion.

The observation in the present study that CV induced IgG antibody and gave anamnestic responses of both IgM and IgG isotypes indicates that bacterial ribosomes could have adjuvant effect. We also observed that nuclease digestion of CV resulted in not only decreasing IgM antibody responses but also in decreasing its capacity to induce DTH to FKC of S. typhimurium. In addition, digested CV failed to evoke IgG antibody responses. These changes were consistent with the results obtained from the RNase-digested LT2-CRF. Hence, these findings would indicate that rRNA regions sensitive to nuclease digestion are required for the immunogenicity of CV, and also that in this complex, B-cell mitogenicity of 43-kDp might not be expressed, probably due to concealing the reactive sites of 43-kDp to B-cells with rRNA regions cross-linked to it. The latter concept can be explained by the assumption that nuclease digestion impaired the conformational stability of CV, thereby allowing B-cell mitogenic 43-kDp to react with B-cells. This assumption can be deduced from the experimental fact that digested CV stimulated B-cells more than did undigested samples. Since CV did not directly stimulate T- and B-cells, another type of splenic cells, probably macrophages, would be required for the expression of the mitogenicity to unfractionated splenocytes of CV and LT2-CRF. Butler et al. [30] reported that nuclease digestion of salmonella ribosomal preparations destroyed their ability to stimulate the production of immuno-enhancing factors in serum of treated mice. Another report showed that bacterial RNA contributes to the serum interferon-inducer activity [31]. These reports implicate the important role of RNA in producing the helper factor(s) including inter- leukins, as previously reported [32]. Thus, the responsiveness of macrophages to stimulation by R N A could be another clue as to whether splenocytes can respond to ribosomal preparations. This

237

could also explain in part the variety of effective- ness of salmonella ribosomal vaccines among mouse strains [33].

Finally, CV conferred high protection to C 3 H / H e J mice as well as to resistant mouse strains (A / J , D B A / 2 and C3 H /H eN ) . Since none of these strains were protected by the mixed vaccine, formaldehyde fixation can induce the conformational stability of the vaccine, thereby increasing its immunogenicity even in mice which would be protected by the native LT2-CRF only at a low degree or would not be protected by the mixed vaccine. Therefore, protection induced by CV was usually higher than, or comparable to that induced by LT2-CRF. This could be explained by the assumption that CV might be conformationally more stable than the native LT2-CRF in vivo, although further studies are necessary.

The present study suggests that nonribosomal B-cell mitogenic contaminants such as 43-kDp or LPS [13] can be protective antigens when they are presented as part of a macromolecular complex with compact ribosomes, and that the conformational stability of ribosomes could be required to play a modulating role in the overall immunogenicity of salmonella ribosomal vaccines.

A C K N O W L E D G E M E N T

We thank Ms. I. Tanikawa for her excellent secretarial assistance.

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requirement of the conformational stability of a salmonella ribosomal vaccine for its mouse...

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