Factors affecting the immunogenicity of tetanus toxin fragment C expressed in Lactococcus lactis

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MICROBIOLOGY

ELSEVIER

FEMS Immunology and Medical Microbiology 14 (1996) 167-177

Factors affecting the immunogenicity of tetanus toxin fragment C expressed in Lmtococcus lack Pamela M. Norton

*, Henry

W.G. Brown, Jeremy M. Wells, Angela M. Macpherson, Peter W. Wilson, Richard W.F. Le Page

Department of Pathology, Uniuersiry of Cambridge, Tennis Court Road, Cambridge CB2 lQP, UK

Received 10 January 1996;revised 12 March 1996;accepted 12 March 1996

Abstract The relative immunogenicity of tetanus toxin fragment C (TTFC) has been determined in three different strains of inbred mice when expressed in Lacrococcu~ luctis as a membrane-anchored protein (strain UCP1054), as an intracellular protein (strain UCP1050), or as a secreted protein which is partly retained within the cell wall (strain UCP1052). Protection against toxin challenge (20 X LID,,) could be obtained without the induction of anti-lactococcal antibodies. When compared in terms of the dose of expressed tetanus toxin fragment C required to elicit protection against lethal challenge the membrane-anchored form was significantly (lo-20 fold) more immunogenic than the alternative forms of the protein. Keywords: L.acfococcus lactis;

Targeting antigen; Tetanus toxin fragment C; Bacterial vaccine vector; Vaccine

1. Introduction The development d recombinant bacteria as antigen delivery vehicles has so far focused predominantly on the use of live, attenuated strains of mycobacteria and salmonella [l-5]. The efficacy of these bacterial vectors as vaccines is believed to depend on their invasiveness, capacity to survive and multiply, and on the occurrence of adequate levels of antigen gene expression in viva. In contrast to thins approach, other experimental studies have shown that inert microparticles and

* Corresponding author. Present address: Institute for Animal Health, Compton, Nr. Newbury, Berkshire RG16 ONN,UK. Tel: +44 (1635) 578 411; Fax: +44 (1635) 577 237.

liposomes can serve as antigen delivery vehicles [6]. The results obtained in the course of experiments with microparticles and liposomes raise the possibility that non-colonising bacterial vectors induced to express high levels of antigen prior to inoculation might be similarly useful for antigen delivery. Such a system would represent a novel approach to the use of recombinant bacteria as vaccine vectors. If based on an innocuous organism suitable for oral inoculation this approach would enable paediatric, geriatric and immunosuppressed individuals to be immunised with exceptionally low levels of risk. Although a number of lactic acid bacteria might be exploited in this way our own interest centres on Zmtococcus Zuctis. This bacterium has a low innate antigenicity [7]. Under certain conditions it is nevertheless capable of eliciting protective level immune responses.

0928-8244/96/$15.00 Copyright 0 1996 Federation of European MicrobiologicalSocieties. Published by Elsevier Science B.V. PII SO928-8244(96)00028-4

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A regulated high-level gene-expression system has been developed for L. Zuctis [8,9] and used to overexpress tetanus toxin fragment C (TTFC). TTFC is a 52 kDa non-toxic papain cleavage product of the holotoxin (amino acids 865-1315) which has previously been shown to be a protective immunogen in mice and guinea pigs [ 10-121. The expression vectors described previously produce TTFC intracellularly in soluble form or as a secreted protein with some intracellular accretion [8,9]. As it is probable that antigen associated with the bacterial surface may be relatively more immunogenic by virtue of greater accessibility to the immune system [ 13-151 we have now constructed a vector which can be used to target heterologous proteins to the bacterial cell envelope. This vector allows antigens to be expressed as fusions to the C-terminal membrane anchoring domain of a lactococcal cell surface associated proteinase @rtP>. [ 161. The relative immunogenicity of the different forms of TTFC which our vectors make it possible to express in L. lactis has now been investigated by the subcutaneous inoculation of three different inbred strains of mice with different recombinant strains of L. lactis. For each L. lactis strain we have determined the dose of TTFC-expressor bacteria required to protect mice from lethal toxin challenge.

2. Materials and methods 2.1. Bacterial strains, plasmids

and growth media

E. coli recA + strain MC1022 [ 171 was used as the host for the pLET vectors and their derivatives.

Table 1 Features of recombinant

strain

Expression

ucP1000 UCP1049 UCP1050

14 (1996) 167-l 77

The recombinant L. Zactis strains used in this study are listed in Table 1. The lactococcal expression host strain UCPlOOO (Table 1) is a derivative of L. lactis strain MG1820 [18] which carries the lactose utilisation plasmid ~820 and plasmid pILPo1 containing the T7 RNA polymerase gene under control of the inducible lactococcal lac promoter [8]. L. Zactis was routinely cultured in Ml7 broth (Difco Laboratories Ltd.) or on Ml7 agar plates containing 0.5% glucose at 30°C or 37°C. E. coli strains were grown in Luria-Bertani (LB) broth or on LB agar plates. Erythromycin was added to Ml7 medium to a final concentration of 5 pg/ml. Chloramphenicol was used at final concentrations of 5 ,ug/ml and 15 pg/ml for L. lactis and E. coli respectively.

2.2. Recombinant

DNA techniques

PCR amplification of the TTFC gene and the C-terminal region of the gene encoding PrtP was performed under high fidelity conditions using Taq DNA polymerase (Cambio Ltd.) in a total volume of 100 ~1 [19]. Thirty cycles of PCR amplification were run in a thermal cycler (Techne model PHC3) under appropriate conditions for each set of primers. General molecular cloning techniques were carried out as previously described [20]. Restriction endonucleases, calf intestinal phosphatase and a DNA ligation kit (Amersham) were used according to the recommendations of the supplier. The methods used for the transformation of L. Zuctis and the preparation of plasmid DNA have been described previously [21].

L. lactis strains used in this study Key features

Relative amount of TTFC expressed

Reference

None pMIG1 pLET1 -l-l-FC

Expression host Non-expressor strain Intracellular over-expression of TIFC to 22% of total soluble protein

0 0 200

[81

UCP1052

pLET2-TTFC

Secretion vector accumulates soluble TTFC within the wall

40

[91

UCP1054

pLET4-TTFC

Expresses a membrane anchored TTFC-PrtP fusion protein

1

This work

vector

D31

P31

PamelaM. Norton et al. / FEMS Immunology and Medical Microbiology 14 (1996) 167-I 77

2.3. Cell fractionation and enzymatic wall associated proteins

release of cell

L. lactis was fractionated by homogenisation as described [8]. For smaller scale preparations of cell fractions zirconium beads (0.1 mm diameter) were used in a minibead beater (Biospec products) for 3 X 2 min cycles. Samples were cooled on ice for 1 min between each cycle. The zirconium beads were removed by filtration through a 5 pm filter (micropore) and the homogenate centrifuged at 13 000 X g for 15 min in a microfuge to remove the insoluble pellet. The membranes were separated from the soluble protein fraction by centrifugation at 150 000 X g for 1 h at 4°C. In order to release protein that might be attached to the cell wall induced cells were treated with lysostaphin and lysozyme to degrade the cell wall. Approximately 5 OD,,,, units of cells were washed three times in 2 ml of 20 mM Tris-HCl pH 7.5, 150 mM NaCl and finally resuspended in 500 ~1 of 20 mM Tris-HCl pH 7.5 10% (w/v> sucrose. 50 ~1 of distilled H,O containing 0.125 mg of lysostaphin and 0.5 mg of lysozyme were added to this suspension, and the mixture incubated at 37°C for 30 min. The cells were then pelleted by gentle centrifugation and the supematant containing the degraded cell wall material and cell wall associated proteins was decanted. The pellet of protoplasted cells was gently washed, lysed by the addition of 100 ~1 of SDSPAGE sample buffer, and boiled for 5 min before samples were loaded onto electrophoresis gels. Proteins were recovered from the supematant fractions by phenolic extraction, precipitated with ethanol, and analysed by SDS-PAGE.

2.4. Growth and induction strains

of recombinant

L. lactis

To induce expression of TTFC exponentially growing cells (OD,,, nm 0.3-0.6) were pelleted by centrifugation at 1500 X g for 7 min and resuspended in two volumes of Ml7 containing 0.5% lactose. The cultures were then incubated for 2 h at 30°C (in the case of UCP1052 and UCP1054) or at 37°C (strain UCP1050) before being centrifuged, washed and resuspended in ice-cold PBS to the required concentration. Cells were maintained on ice

169

for up to 30 min before being inoculated into mice. Aliquots of 1 X 10’ cells were taken from these suspensions and checked for the production of TTFC by Western blot analysis using polyclonal rabbit anti-TTFC antiserum. 2.5. Immunisation

and challenge

Female mice (6-8 weeks old) were inoculated subcutaneously (in the inguinal fold) on alternate weeks with doses of either 5 X 106, 5 X 107, or 5 X 10’ live, induced expressor strains of bacteria, or with non-expressor strains. Additional groups of mice were inoculated with purified recombinant TTFC (Boehringer). No adjuvant was used in any of the immunisations. Serum samples were taken seven days after each inoculation. The immunisations were repeated in three different inbred strains of mice: CBA, BALB/c and C57BL/6 (supplied by Harlan OLAC Ltd.). Immunised mice were challenged by subcutaneous inoculation of either 5 X , or 20 X LD,, of purified tetanus toxin, and humanely killed if any hind leg paralysis was observed. Mice which remained free of tetanic signs for 4 days were taken to be fully protected, since no animals displayed clinical signs of tetanus later than 4 days after challenge. 2.6. Immunoblotting

and ELISA

Total cell protein extracts from L. lactis and samples from the fractionation were separated by SDS-PAGE [22] and electroblotted onto nitrocellulose [23]. TTFC was detected by the use of a rabbit anti-TTFC antiserum and goat anti-rabbit immunoglobulins (Nordic Immunological Laboratories). The ELISA for analysis of immune sera followed the protocol described previously [8]. Isotype analysis of anti-TTFC antibodies was performed in essentially the same way except that a standard curve was generated using pure immunoglobulins derived from the following myeloma cell lines: MOPC 315 (IgA), MOPC 104E (IgM) and MOPC 22 (IgG) (Sigma). Appropriate anti-isotype horseradish peroxidase conjugated antibodies (Sigma Chemical Co.) were used to detect specific antibodies. To detect antibodies to L. lactis proteins by an ELISA, microtitre plates (NUNC Maxisorp) were first coated with 100 ~1 of a 1 mg/ml solution of

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lactococcal soluble proteins in carbonate buffer (pH 9.6) by incubating overnight at 4°C. The plates were then blocked for 1 h at 37°C in blocking buffer (phosphate buffered saline, pH 7.4 (PBS) containing 0.05% Tween 20,2% BSA; 200 ml per well), washed once with PBS/Tween and then incubated with a 1:lOO dilution of test serum (diluted in 1% BSA, PBS/Tween) for 2 h at 37°C. The plates were washed 4 times in PBS/Tween and then incubated with horseradish peroxidase-conjugated goat antimouse immunoglobulins (Dako Ltd.) for 1 h at 37°C. The plates were washed as before and finally in PBS before developing with TMB. The reaction was stopped after 5 min with 12.5% H,SO, and the OD read at 450-540 nm in a Titertek MC340 plate reader.

3. Results 3.1. Construction of an expression vector (pLET4) for targeting antigens to the cell envelope in L. lactis The C-terminal region of PrtP (nt 65 18 to 6913) was obtained by PCR amplification using purified plasmids of strain L. Zactis NCD0763 as template and sequenced to confirm its identity with reference to the published sequence [16]. To facilitate cloning the PCR primers were designed to incorporate BamHI and BgZII restriction sites at the 5’ ends of the sense and antisense primers respectively. The purified PCR fragment was cloned into the BamHI site of pLET2 [9] to generate pLET4. In pLET4 the reconstituted BamHI site lies between the signal leader and the inserted cell-wall spanning and membrane anchoring domain of PrtP (Fig. la). To construct a vector which would express TTFC as a fusion to the C-terminal region of PrtP a fragment encoding TTFC was cloned into the BamHI site of pLET4. The fragment encoding TTFC (amino acids 865 to 1315) was obtained from plasmid pSS1261 [24] by PCR using primers with restriction sites for BarnHI at their 5’ end. The resulting expression construct, designated pLETCTTFC was partially sequenced to ensure that the ITFC gene in pLET4-‘ITFC was in-frame with both the signal leader and wall-spanning domain (Fig. lb).

Hindlll

T7p

SL

II

TTFC . . . . .1.: . . . . . . . . . . . . . . . . . .

PrtP anchor

T

Fig. 1. (A) Plasmid PLET4 a lactococcal vector for the cell wall targetting of proteins. The coding sequence of the chloramphenico1 (cat) resistance gene is depicted as an arrow on which the direction of transcription is indicated. Ori: replicon functions; T: terminator; p T7: promoter; SL: usp45 signal leader sequence; PrtP. (B) Schematic representation of expression cassette in PLET4-TTFC. Coding sequences are boxed and labelled accordingly. SL: usp45 signal leader; TTFC: tetanus toxin fragment C gene; Prt anchor: lactococcal proteinase c terminal anchoring domain. The T7 bacteriophage promoter sequence (T7p) and terminator (T) are indicated.

3.2. Cellular location and estimation of the relative quantity of TTFC-PrtP fusion protein in L. lactis UCPI 054 The pLET4-TTFC expression vector was transferred to the L. Zactis expression host strain UCPlOOO by electroporation to generate expression strain UCP1054 harbouring pLET4-TTFC (Table 1). When strain UCP1054 was grown and induced as described above (see Section 2) a fusion protein of the correct size was recognised by anti-TTFC antibodies (Fig. 2). Immunoblotting experiments with sub-cellular fractions of induced cells showed that the fusion protein was not detectable in the soluble cytoplasmic fraction and was associated with the membrane and cell wall fractions of the cell envelope. No other proteins were detected in the Western blot suggesting that the fusion protein was not running anomalously under these conditions. Likewise all material

PamelaM. Norton et al. / FEMS Immunology and Medical Microbiology

A

B

12345676

M

1

s c

lTFC

14 (1996) 167-177

171

In order to compare the relative levels of TTFC expression in the expression strains Western blots of total cell extracts (from 1 X lo9 induced cells) were analysed with antisera to TTFC. Targeting the antigen to the cell envelope appeared to substantially reduce the amount of antigen expressed (Table 1). To investigate whether the fusion protein was covalently anchored to the cell wall, induced expressor cells were digested with lysostaphin and lysozyme and then separated from the material released into the supematant by centrifugation (see Section 2). TTFC could be not be detected in this supernatant (results not shown). It appeared that all the TTFC produced in strain UCP1054 was anchored in the cell membrane not in the cell wall. When Western blots of varying dilutions of total cell extracts were compared it was estimated that strains UCP1052 and UCP1054 contained approximately 20% and 0.5% respectively of the TTFC produced by strain UCP1050 2 h after induction (Fig. 2b and Table 1). 3.3. The lTFC-expression strains of L. lactis could induce the formation of anti-TTFC serum antibodies: the responsiveness of inbred strains of mice difSered

Fig. 2. (A) Immunoblotting with total cell protein extracts from the three different TTFC expression strains UCP1050, UCP1052 and UCP1054. Bacteria were grown to an OD,, of 0.5 and expression of ‘lTFC induced for 2 h. Proteins from 5 X lo* bacteria were immunoblotted with rabbit anti-TTFC sera. Lane 1: 100 ng purified recombinant TTFC (Boehringer) UCP1054, lanes 2: UCP1054, lanes 3-5: 1:20, 1:lO and 1:5 dilutions of UCP1052 respectively, lanes 6-9: 1:200, l:lOO, 1:50 dilutions of UCP1050 respectively. (B) Expression of TTFC in L. la&. Immunoblotting with sub-cellular fractions of induced cells harbouring the expression plasmid PLET4-TTFC (expression strain UCP1054). M: membrane fraction, I: insoluble fraction; S: soluble fraction. C: total cell extracts from the control strain UCP1049; TTFC: purified recombinant TTFC. Sizes of the prestained marker proteins are shown at the right.

appeared to enter the gel. These results demonstrated that the TTFC-PrtP fusion protein was intimately associated with the purified cell membrane and the crude cell wall fraction (comprising peptidoglycan, other components of the cell wall, and attached cell membrane). However, this TTFC appeared not to protrude outside the pelptidoglycan, since it could not be detected on the surface of intact expressor cells by immunogold or immunoflourescent labelling techniques.

The ability of expression strains UCP1050, UCP1052 and UCP1054 to induce serum antibodies to TTFC was tested in three inbred strains of mice. Three doses of either 5 X 106, 5 X 10’ or 5 X lo8 live, induced bacteria were inoculated subcutaneously (without adjuvant) on alternate weeks, and serum samples taken seven days after each inoculation; anti-toxoid serum antibody levels were then determined by ELISA. All the recombinant bacterial strains proved to be capable of inducing toxoid specific antibodies, but the total number of bacteria required to elicit these antibodies varied with the strain of bacteria (Fig. 3). This indicated that both the amount of TTFC and the form of the antigen influenced its immunogenicity (see below). For example, 2 doses of 5 X lo6 cells of UCP1050 (which contained the most TTFC) (Fig. 3, Group 12) induced antibody responses to TTFC, whereas no such responses were elicited by this dose of cells of strains UCP1052 or UCP1054 (Fig. 3, Groups 3 and 9) Similarly, a single dose of 5 X IO’ UCP1050 or 5 X 10’ UCP1052 induced toxoid specific antibodies (Fig. 3, Groups 11 and 1) but 2 doses of 5 X lo8

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PamelaM. Norton et al. / FEMS Immunology and Medical Microbiology

UCP1054 were required (Fig. 3, Group 7) to elicit any anti-TTFC antibodies. No antibodies to TTFC were detected in the sera of mice inoculated with the control strain UCP1049 (Table 1) or PBS alone. Since protection to tetanus is correlated with the formation of adequate levels of anti-toxin IgG, the classes (IgM, IgG, IgA) of serum anti-toxoid antibodies elicited in C57BL/6, CBA and BALB/c mice were investigated. In C57BL/6 mice a single subcutaneous dose of 5 X 10’ UCP1050 or two doses of 5 X lo8 UCP1052 or UCP1054 elicited serum IgG. When inocula of 5 X lo6 bacteria of UCP1050 and UCP1052 were used anti-toxoid IgG was elicited after two and three doses respectively. However, three doses of 5 X lo6 UCP1054 failed to elicit anti-toxoid immunoglobulin. Clearly all the recombinant strains of L. Zuctis were able to elicit IgG anti-toxoid antibodies, but the dose required to achieve this differed for each strain. When compared in terms of the total number of recombinant bacteria required to elicit IgG, antitoxoid strain UCP1050 (which produced the largest quantity of antigen) was the most effective immunogen. CBA (H-2k> and BALB/c (H-2d) mice were less responsive to the recombinant immunogens than C57BL/6 (H-2b) mice (Table 2). Tenfold higher doses of UCP1050, and up to one hundred-fold

14 (19%) 167-177

higher doses of UCP1052, were needed to induce the formation of anti-toxoid IgG in these strains of mice. In sharp contrast, the dose of strain UCP1054 required to induce anti-toxoid IgG was the same in all three strains of mice. Anti-toxoid serum IgA remained at background levels in all mice. 3.4. The form in which the antigen influenced its immunogenicity

was expressed

As the different recombinant bacterial strains produced differing quantities of antigen it was expected that the numbers of bacteria of the different strains required to elicit protective levels of IgG would vary. But if the forms of the antigen expressed in the different strains had all been equally immunogenic then the differences in the bacterial doses required to elicit protective levels of IgG would simply have reflected the relative total amount of antigen they contained. However, the results (Table 2) show that on the basis of the relative amounts of antigen given, one of the forms of the antigen was much more immunogenic than the others. In all three strains of mice the amount of the membrane-anchored form of the antigen required to elicit IgG anti-toxoid antibody responses was approximately 13-20-fold less than the amount of intracellular antigen. When the

t

UCPI 052

Fig. 3. Anti-tetanus toxoid serum strains expressing TTFC. Groups and 12 received 5 X lo6 bacteria titre is recorded as the OD,s,_,,, third bar, dose 2; and fourth bar,

UCP1049

UCPlOM

UCP1050

PBS

r7TFC

antibody titre in C57BL/6 mice following subcutaneous inoculatior with recombinant Lactococcus lactis 1, 4, 7 and 10 received 5 X 10s bacteria. Groups 2, 5, 8 and 11 received 5 X lo7 bacteria. Groups 3, 6, 9 Groups 13 and 14 received PBS or 10 pg recombinant ‘lTFC (Boehringer) in PBS respectively. Antibody at a 1:lOO dilution of serum. Bars as follows (from left to right): first bar, pre; second (solid) bar, dose 1; dose 3. * = P < 0.001 (Student’s t-test).

UCP1052

UCP1054

BALB/c

BALB/c

3 20 zoo 0.6 6 40 0.015 0.15 1

n/d O/4 4/4 O/4 O/4 4/4 n/d n/d n/d

n/d 3/3 3/3 O/3 O/3 3/3 O/3 n/d 3/3

are shown in column E. The levels of protection

n/d 4/4 4/4 O/4 O/4 4/4 n/d n/d n/d

3/3 3/3 O/3 O/3 3/3 O/3 O/3 3/3

4/4 n/d 214 4/4 n/d n/d n/d

n/d

2;4

to

of three)

following (20 X LD,,)

(up to a maximum

Survivors/total toxin challenge G

3/3 n/d 3/3 3/3 O/3 O/3 3/3

following (5 X LD,, )

n/d 3;3

Survivors/total toxin challenge F

dose and the number of inoculations

The relative amounts of ‘lTFC produced by the dose of bacteria required to elicit the formation of IgG anti-toxoid antibodies tetanus toxin challenge after three varying doses of the different bacterial strains are shown in columns F and G. No Ig * indicates that no anti-toxoid immunoglobulin was detected after three doses.

ucP1050

BALB/c

NoIg’ 2 2 NoIg* No@* 2 NoIg* No&* 2

UCP1054

CBA

5x106 5x107 5x10s 5x106 5x107 5x108 5x106 5x107 5x10s

UCP1052

CBA

3 20 200 0.6 6 40 0.015 0.15 1

UCP1050

CBA

NoIg* 2 2 NoIg’ IgM only 2 No&* IgM only 2

uCP1054

C57BL/6

5x106 5x107 5x108 5x106 5x107 5x10s 5x106 5x107 5x108

UCP1052

C57BL/6

2 20 100 0.6 4 40 0.015 0.15

5x106 5x107 5x108 5x106 5x107 5x10s 5x106 5x107 5x 108

UCP1050

C57BL/6

2 2 1 3 2 2 NoIg* 3 2

C

B

A

Relative amount of ITFC given in (D) E

L. la& strains, bacterial

Doses required to elicit anti-TTFC IgG D

Dose

Bacterial inoculum

Mouse strain

Table 2 Relationship between the quantity of antigen produced by the different recombinant required to induce protective levels of anti-toxoid IgG

$ B >

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PamelaM. Norton et al. / FEMS Immunology and Medical Microbiology

antigen was only loosely associated with the cell envelope (as in strain UCP1052) comparable increases in immunogenicity were not observed. 3.5. Serum antibodies elicited by TTFC expressed in all cellular compartments are protective In order to determine whether the presence of toxoid specific antibodies correlated with protection immunised animals were challenged with purified toxin. The three strains of mice were inoculated as before with different doses of bacteria and challenged with 5 X LD,, of purified toxin two weeks after the third dose. The results (Table 2) showed that all three bacterial strains were able to elicit immune responses which protected mice against 5 X LD,, of tetanus toxin. These results, shown in Table 2, indicated that primary IgM anti-toxoid responses were not protective; adequate levels of anti-toxoid IgG were obligatory. In concordance with the IgG anti-toxoid ELISA titres elicited the membrane bound form of the antigen was generally more protective. In the groups for which the most complete comparisons were obtained (the CBA and BALB/c mice) protection mirrored the observed IgG antibody responses. In order to assess the potential duration of protection the anti-toxoid serum antibody titres were followed over several months in a separate experiment. The IgG antibody titres remained high for more than six months and the survival rates following tetanus toxin challenge were the same as those shown in Table 2 (results not shown).

I4 (I 996) 167-I 77

We have shown previously that inoculation of L. lactis into mice elicits serum antibodies which cross-react with antigens present in commensal and food-grade lactic acid bacteria [7]. The appearance of these antibodies was also investigated in these experiments in order to determine whether the modes of expression of the heterologous immunogen had any

Al

Bl

3

2

2

3

4

45

5

678

6

7

a

3.6. Protection to toxin challenge could be obtained without induction of antibodies to L. lactis The serum antibody response to lactococcal protein antigens was measured at those bacterial doses which protected against a tetanus toxin challenge. In all three strains of mice the subcutaneous inoculation of two or three doses of 5 X 10’ bacteria raised the basal level of anti-lactoccal serum IgM antibodies but did not elicit IgG anti-lactoccal antibodies. A dose of 5 X lo* (or more) lactococci was required to elicit anti-lactococcal IgG responses (results not shown).

Fig. 4. Induction of serum antibodies to LAB following subcutaneous inoculation of L. lactis MG1820. Imunoblot of total cell lysates from a range of food grade and commensal LAB incubated with sera from C57BL/6 mice inoculated with three doses of 5 X 10s (A), 5 X lo7 (B) or 5 X lo6 (CT) UCP1049. Isolates 117 and 6 are recent isolates from murine ileum and are probably Lactobacillus murinus. Lane 1: isolate 117; lane 2, isolate 6; lane 3, Lmtobacillus bulgaricus 1110; lane 4, Streptococcus thermophilus 1523; lane 5, Lactobacillus casei; lane 6, Lactococcus lactis MG1820, lane 7, Lactobacillus bulgaricus 1135; lane 8, Lactobacillus acidophilus.

PamelaM. Norton et al./ FEMS Immunology and Medical Microbiology

influence on the appearance of these antibodies, or on the patterns of antibody formation observed. Western blot analysis confirmed that a dose of 5 X 10’ expressor bacteria was required to raise strong antibody responses to L. lactis or other lactic acid bacteria (LAB) (Fig. 4,); however, the expression of TTFC in the different cellular locations did not significantly affect the extent or pattern of the responses to lactic acid bacterial proteins following subcutaneous inoculation, since the anti-lactococcal responses of mice immunised with the UCP1049 control strain were similar to those seen after inoculation with the expressor strains. 4. Discussion This paper considers whether targeting TTFC to different cellular locations in L. luctis has an overriding effect on its immunogenicity in three different strains of inbred mice. To determine this we have used three different recombinant strains of L. Zuctis, each of which expresses TTFC in a different cellular compartment. Two of the expression plasmids (pLET1 and pLET2) present in these strains have previously been described [8,9]. Plasmid pLET1 (in strain UCP1050) allows the production of intracellular, soluble TTFC in L. lactis, and pLET2 (in strain UCP1052) permits secretion of TTFC into the growth medium. The rate of production of ‘ITFC in UCP1052 exceeds the diffusion rate of the protein through the cell wall, resulting in the accumulation of soluble protein which is loosely associated with (but not anchored to> the peptidoglycan of the cell wall. For the purposes of the present study we have constructed an additional expression vector pLET4 which has allowed us to express TTFC fused to the C-terminal region of a cell surface associated lactococcal proteinase (PrtP) in L. Zuctis strain UCP1054. The lTFC-PrtP fusion protein is anchored in the cell membrane, and cannot be detected in the soluble cytoplasmic fraction or in enzymatically released cell wall material. Although it was anticipated that this construct might result in surface distribution of TTFC epitopes these were undetectable by immunogold or immunofluorescent labelling techniques. Hence the TTFC in UCP1054 is presumably largely buried within or below the peptidoglycan.

14 (1996) 167-177

175

Although expression of TTFC in UCP1054 is driven by the T7 promoter (as in the other expression strains) the amount of TTFC detected in the cells of UCP1054 was only about l/40 of that detected in the UCP1052 secretor strain, and l/200 of that detected in strain UCP1050 (Fig. 2 and Table 1). Immunoblotting of lysostaphin-released cell wall material from UCP1054 ruled out the possibility that this observed lower level of ‘ITFC expression was simply due to its removal from the soluble pool of TTFC by attachment to insoluble peptidoglycan. The restricted amount of antigen formed by strain UCP1054 is presumed to be due either to a toxic effect of TTFC when it is anchored in the membrane as a PrtP fusion protein, or to rapid intracellular degradation of this hybrid protein. Further studies are required with other proteins and with the anchoring domains of other Gram-positive bacterial surface proteins in order to determine whether this is a TTFC-specific effect or a general consequence of using the PrtF protein for the cell surface display of antigens and epitopes. The relative immunogenicity of the TTFC produced by the three different lactococcal TTFC expression strains was tested by subcutaneous inoculation of mice with varying doses of live recombinant bacteria. The dose of L. Zuctis required to elicit protective antibody responses in mice appeared to be primarily dependent on the amount of antigen produced by the different expression strains. Strain UCP1050, which produced the highest levels of TTFC (up to 20% of soluble intracellular protein) was able to induce protective IgG antibodies at a tenfold lower dose than either strain UCP1052 or UCP1054. When the results of immunising three inbred strains of mice with strain UCP1054 are compared to those obtained by immunising with the other strains it appears that, when only a restricted amount of TTFC is expressed, protective antibodies are more readily elicited when the TTFC is anchored into the cell membrane. Approximately 13-20-fold lower amounts of the membrane-anchored form of TTFC were needed to elicit anti-toxoid IgG than when the TTFC was expressed in a soluble form (Table 2). This suggests that the form in which the antigen is associated with the cell affects its immunogenicity, or that the proportional increase in the quantity of

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bacterial-derived substances mediates critical effects. Here the most likely explanations of the enhanced antigenicity of TTFC observed in strain UCP1054 are either that fusion of TTFC to PrtP, with its concomitant anchorage in the plasma membrane, exerts an adjuvant effect, or that the proportionally greater quantities of bacterial cell mass given in the UCP1054 immunisations potentiated the responses to TTFC. Also of interest is the finding that a protective dose of UCP1050 (5 X lo7 bacteria), which elicits high level antibody responses to TTFC, does not concomitantly elicit significant antibody responses to the antigens of L. lactis. This demonstrates that, in the L. lactis system, an immune response directed against the recombinant expressor cells is not required in order to obtain antibody responses to the expressed antigen. Thus, as with microparticles, an immunologically silent antigen carrier system can act as an effective delivery vehicle. This may be helpful in reducing the risk of inducing an antibacterial response which may inhibit the boosting of responses to expressed heterologous antigens, an effect which has been seen in Gram-negative bacterial antigen delivery systems [25,26]. The technique reported here of pre-loading bacteria with heterologous antigen means that the lactococcal system is somewhat more analogous to microparticles and to liposomes than to other recombinant bacterial vaccine systems. It also avoids the problems associated with a lack of plasmid stability in vivo seen, for example, with recombinant Salmonella given by mucosal routes. In the case of the Salmonella system the relative importance of sustained low levels of antigen synthesis versus high initial antigen dose has been investigated. The clear finding that emerged was that it was the quantity of antigen present in the bacteria at the time of inoculation which was critical in determining whether or not an immune response was induced following oral inoculation of the vaccine strains of these bacteria [27]. Our findings that the lactococcal expression strains studied here differ in their parental immunogenicity, and in particular that minimal quantities of membrane-anchored TTFC can elicit protective antitetanus responses raises the question as to whether comparable differences exist in the mucosal immunogenic&y of these strains. Experiments are now

in progress to answer this question. The findings reported here provide useful clues to the further development of L. Zactis as a vaccine vector. They also represent the first recorded use of Gram-positive recombinant bacteria to elicit immunity to tetanus.

Acknowledgements The authors are grateful for the financial support of the Ministry of Agriculture, Fisheries and Food (P.N. and H.W.G.B.); BBSRC (J.M.W.); Merck, Sharpe and Dohme, and the Isaac Newton Trust (R.W.F.LeP.). Rabbit anti-‘ITFC antibodies were a generous gift of Dr. J. Halpern. Tetanus toxoid and tetanus toxin were kindly provided by Dr. P. Knight. We thank Dr. L. Steidler for providing us with a method for releasing cell wall anchored proteins in L.lactis.

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