stability, immunogenicity and expression of foreign antigens in bacterial vaccine vectors

Stability, immunogenicity and expression of foreign antigens in bacterial vaccine vectors

L u c i a C f i r d e n a s a n d J o h n D. C l e m e n t s *

The use ~?/ attenuated strains o,1 Salmonel la as vaccine vectors frequently involves the introduction o['heterologous antigens on recombinant plasmids. To overcome the problem ~/'plasmid instability, we have integrated the gene that codes for a potential immunogen into the chromosome of a galE mutant o f Salmonel la t yph imur ium. Comparative in vi t ro and in vivo studies were conducted between the strain carrying the gene chromosomally integrated and an isogenic strain carrying the same gene on a multicopy plasmid. Levels o/'expression of the foreign antigen were significantly lower when the antigen was expressed from the chromosome than when it was expressed from the plasmid. The in vivo maintenance of the genes coding for antigen e,x'pression was determined on organisms recovered JJ'om spleen, liver and Pe)'er's patches o[ orally inoculated mice. By 24 h postinoculation, the mq/ority of tissue isolates Ji'om the plasmid-containing strain had lost the plasmid and the abilio' to o, nthesize the antigen. By contrast, 100% of the recovered cointegrate isolates retained the abili O' to express the antigen throughout the 21 days of the e.\'periment. Si,qnificantO', humoral and mucosal antibody levels against the antigen were greater when the antigen was expressed J?om the plasmid stabilized by the presence c~[ the antibiotic than when the antigen was expressed from the chromosome. These observations indicate that the most important event for the development of an immune response against a foreign antigen delivered by these vectors may be the initial amount g/antigen that primes the gut-associated lymphoid tissue and not persistence o/ the vector h7 tissues.

Keywords: Vaccine vectors; plasmid instability; chromosome; immunogenicity

I N T R O D U C T I O N

A number of attenuated mutants of Salmonella are candidates for use as vaccine carriers for virulence determinants of other organisms ~ 13. Studies in animal models have demonstrated that immunization with these heterologous vaccines is an effective and safe way to induce mucosal and serum antibodies against both Salmonella and the foreign antigen. The use of attenuated mutants of Salmonella as vaccine carriers implies the introduction of foreign genes, usually on recombinant plasmids, into the Salmonella vehicle. These plasmids generally carry antibiotic resistance markers to facilitate identification and to keep selective pressure in favour of plasmid retention. Although most of these plasmids have been shown to be retained in vitro, their stability in vivo is more critical and less certain. In general, we have found these plasmids to be unstable in vivo and also in ritro in the absence of a stabilizing antibiotic (Cardenas and Clements, unpublished results). Different strategies have

Department of Microbiology and Immunology, Tulane University School of Medicine, New Orleans, LA 70112, USA. *To whom correspondence should be addressed

0264~410X"93/02012~ 10 ~ 1993 Butterworth-Heinemann Ltd

126 Vaccine, Vol. 11, Issue 2, 1993

been proposed to address the issue of plasmid instability and to avoid the necessity for a stabilizing antibiotic. One method, reported by Nakayama et al. 14, employs Aasd mutants of Salmonella typhimurium and an Asd + expression-cloning vector carrying the spaA gene of Salmonella sobrinus. Since absence of the recombinant plasmid is lethal for the cells, the genes that code for the heterologous antigen are stably maintained both in vitro and in vivo. This combination of cloning vector and mutant host eliminates the need for antibiotics to stabilize the cloning vector and may be useful where expression of the antigen from a recombinant plasmid is desirable.

An alternative approach to obtain stable antigen expression is to integrate the foreign genes into the chromosome of the carrier strain15-17. Hone et al.l~ have developed one system whereby heterologous DNA may be recombined into the chromosome of Sahnonella. This system involves two steps: integration ofa hisOG deletion mutation into the chromosome, and replacement of the hisOG deletion by the complete hisOGD region and a segment ofheterologous DNA which encodes the antigen of interest. This strategy was used to integrate the genes encoding K88 fimbriae of Escherichia coli into the chromosome of a gale mutant of S. typhimurium. It was

Antigen stability

observed that after 54 generations of growth in vitro, 90% of the recombinant organisms tested were K88 + However, 18 days after immunization only 73% of the bacteria recovered from the tissues of inoculated mice were K88 +. The loss of expression of K88 may have been due to either undetectable point mutations or to a spontaneous deletion of the K88 gene 16. A similar study was carried out by Strugnell et al. 17 using the aroC gene as the site of integration for the heterologous gene and a poIA S. typhimurium strain as the recipient of the recombinant plasmid. Two heterologous antigens were integrated into the Salmonella chromosome using this system: the C fragment of tetanus toxin and the tpd gene from Treponema pallidum which encodes a lipoprotein product. Both antigens were expressed, albeit at lower levels than when carried on a plasmid. Results of stability and immunization studies with these recombinant strains are not available.

In a more recent report 15, a chromosomal expression vector was devised using a modified version of the mini-transposon mini-Tnl0 carried on bacteriophage lambda. This vector possesses a transposase gene located outside the transposable portion of the transposon, which in itself carries a kanamycin resistance gene and a ~-IacZ fragment under the control of a lacUV5 promoter. The gene coding for a sporozoite protein (CS) from Plasmodium yoelii was cloned into this vector fused to the lacZ transcription signals. This construct was transformed into E. coil and plasmids from positive clones were introduced into a restriction-deficient S. typhimurium. In the absence of the lac repressor the transposase is expressed and the mini-Tnl0, along with the CS gene, transposes onto the chromosome. The last step in this system involves the transduction of the insertions into an attenuated Salmonella vaccine strain using a P22 lysate. The heterologous protein was expressed by the recombinant strain as detected by immunoblots of whole cell lysates using an anti-CS monoclonal antibody. Animal studies demonstrated that S. typhimurium recovered from spleens of orally infected mice 3 weeks postimmunization were still able to express the CS protein, although a small deletion in the CS gene was observed in one of the isolates recovered. Although immunization of mice with this vaccine did not confer protection against challenge with viable sporozoites and the antibody responses against CS were negligible, there was a significant anti-CS CTL response.

The purpose of the study reported here was to provide a direct comparison of antigen stability, expression and immunogenicity between organisms expressing a foreign gene from a plasmid vector and organisms expressing the same gene from the chromosome. Based on the system developed by Hone et al.16, we integrated the gene that codes for the B subunit of the heat-labile toxin (LT-B) of enterotoxigenic E. coli into the chromosome of S. typhimurium. This antigen was chosen based upon our extensive experience immunizing animals with attenuated mutants of Salmonella expressing this antigen from a recombinant plasmid z-5. We "report here the details of that construction and the results of a comparative study of in vitro and in vivo stability, expression and immunogenicity between an attenuated Salmonella strain carrying a gene for expression of LT-B on a plasmid vector and an isogenic strain carrying the same gene integrated into the chromosome. Additionally, we examined the intracellular distribution of this antigen between these strains.

in bacterial vaccine vectors: L. Cardenas and J.D. Clements

M A T E R I A L S A N D M E T H O D S

Bacterial strains and plasmids. E. coli JM83 (pJC217) F-O8061acZ AM15 A(lac-pro AB) ara rpsL is a K-12 derivative transformed with a 3.5 kilobase (kb) plasmid (pJC217) which carries a gene that codes for production of the B subunit from the heat-labile toxin (LT-B) of enterotoxigenic E. coil 4. E. coli HB101 F - mcrB mrr hsdS20 ( r B - m B ) fecal3 leuB6 ara-14 proA2 lacY1 9aIK2 xyl-5 mtl-l rpsL supE44 2- was obtained from Gibco BRL Life Technologies, Inc. (Gaithersburg, MD). S. typhimurium J706 9alE-H1 AhisOG and plasmid pADE17116'18 were kindly provided by David Hone (University of Maryland School of Medicine). Plasmid pADE171 is a pSCI01 derivative with a 6kb fragment containing DNA from 3 kb upstream of the his regulatory region to the start of the hisC gene. This plasmid codes for resistance to streptomycin and spectinomycin. S. typhimurium LB5010 9ale leu3121 ilv452 pro hsdLT hsdSA hsdSB rnetA22 metE551 trpD2 (Ref. 19) was obtained from L. Bullas (Loma Linda University, Loma Lilada, CA).

Media and growth conditions. Trypticase soy broth (TSB) was obtained from BBL Microbiology Systems (Cockeysville, MD). Solid media used were trypticase soy agar (TSA) (BBL) and minimal medium M9 containing per litre: 6g Na2HPO4, 3g KH2PO4, 0.5g NaC1, 1 g NH4C1 , 2g glucose, 0.11 g CaC12 and 0.25g MgSO4-7H2 O2°. Where indicated, ampicillin and streptomycin were added at 100/~gm1-1 and spectinomycin was added at 120#gml-1. X-Gal (5-bromo-4-chloro-3- indolyl-fl-D-galactoside) was added to TSA plates to a final concentration of 40 ~g ml- 1. All strains were grown under standard conditions except as noted.

Penicillin enrichment. Isolation of S. typhimurium transformants in which the plasmid conferring resistance to spectinomycin and streptomycin was lost was performed by the penicillin enrichment methods described by Carlton and Brown 21

Isolation of DNA. Plasmid DNA was isolated by the alkaline lysis procedure 22. Small-scale chromosomal DNA preparations for Southern hybridizations were performed as described 22.

Restriction endonuclease digestion. Restriction enzymes, ligase and reagents were obtained from Gibco BRL and used according to manufacturer's directions. Phosphoryl- ated PstI linkers were purchased from New England Biolabs (Beverly, MA).

Transformation. Transformations were carried out using standard procedures 22.

Electrophoresis. Agarose gel electrophoresis was performed on 0.9% slab gels in 0.04M Tris-0.2M sodium acetate-0.002M EDTA (pH 7.8) (TAE). Phase 2 DNA fragments generated by HindIII digestion and phage ~bX174 RF DNA fragments generated by HaeIII digestion were used as molecular weight standards. Sodium dodccyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed by the technique of Laemmli 2a. For SDS PAGE, Rainbow Markers (Amer- sham International, Amersham, UK) were used as molecular weight markers.

Vaccine, Vol. 11, Issue 2, 1993 127

Ant igen stabi l i ty in bacter ia l vaccine vectors: L. C&rdenas and J.D. Clements

DNA hybridization. Plasmid and chromosomal DNA were digested with the appropriate restriction enzymes, separated by agarose gel electrophoresis, and transferred to nitrocellulose as described by Maniatis et al. 24. The LT-B-specific probe consisted of a 600bp fragment contained within the HindIII-EcoRI sites of plasmid pJC220 (Figure 1). The his-specific probe was a 900bp fragment obtained by digestion of plasmid pADE171 with PstI and XmaIII. The probe fragments were separated by electrophoresis in a ! % agarose gel in TAE, isolated, electroeluted, and labelled with 32p by nick

translation. DNA hybridization and autoradiography were performed by standard procedures 22.

Purification and analysis o f L T-B. LT-B was purified from E. coli JM83 (pJC217) and from S. typhimurium J1000 by agarose affinity chromatography as previously described 25. Purified LT-B was concentrated by ultra- filtration on Amicon PM-10 membrane filters and stored in TEAN at 4°C prior to analysis by SDS-PAGE on 15% polyacrylamide gels and Western blot. Western blots were performed by standard procedures 22 using

E H A P X E

pADE171 I l \ / I I

Pv E

pUC8

PHE S I i l l l LT-B

H Pv

pUC8

Remove internal Psd site with EcoRI digest. Rcligatc.

pJC220

Pv E S

LT-B

f

H Pv

pUC8 pUC8

Digest with PvuII and add Pstl linkers

p E S

I.nscrt inu) Psd si~c of pADE171

LT-B

H P

pJC220

E H A P E S,

LT-B

H P X

I

E

I pALCSO

i i i •

LT-B probe his probe

I Integrate LT-B gcnc into the chromosome by homologous re.comblnation.

H A P H S E P X I

J Chromosome ~--~--~, LT-B ~ l [ Cl'u-omosom¢ ] JI000

Figure 1 Integration of the gene coding for production of LT-B into the chromosome of S. typhimurium. The LT-B gene was cloned upstream of the his promoter on a low copy number plasmid (pADE171) that carries hisOGD from S. typhimurium. This plasmid was used to transform a hisOG-deleted strain (J706). The LT-B gene was integrated into the chromosome of strain J706 by homologous recombination to yield strain J1000. Restriction enzyme code: A (Aatll), E (EcoRI), H (Hindlll), P (Pstl), Pv (Pvull), S (Sstl), X (Xmalll). Drawing is not to scale


Antigen stability

monospecific goat antiserum directed against LT-B and rabbit antiserum directed against goat IgG con- jugated to horseradish peroxidase (Sigma Chemicai Co., St Louis, MO). Blots were developed using the IBI Enzygraphic T M Web (International Biotechnologies, Inc., New Haven, CT).

Protein determinations. Protein determinations were made by the method of Lowry et al. 26.

Analysis of in vitro expression and stability. Analysis of expression of LT-B from different strains was performed by ELISA as previously described 4. Bacterial samples were suspended in PBS-Tween and lysed by successive freeze and thaw. Expression was quantified using purified LT-B as a standard and is expressed in terms of specific activity as ng LT-B (based on ELISA) per mg total protein. In order to compare the in vitro stability of expression of LT-B between the cointegrate and the plasmid-containing strain, organisms were grown in 20 ml of TSB and subcultured into fresh medium every 18 h for 5 consecutive days. The plasmid containing strain S. typhimurium LC21 (this study) was grown in either the presence or absence of ampicillin, the stabilizing antibiotic. Bacterial lysates were prepared and assayed for expression as described above. Dilutions from the overnight cultures were differentially plated onto either TSA or TSA containing 100pgm1-1 of ampicillin and 25 individual colonies from each strain were restreaked and tested for expression of LT-B by ELISA.

Animals. Female Balb/c mice 6-10 weeks of age were purchased from Charles River. Mice received water and food ad lib and did not receive treatment with antibiotics.

Colonization, invasion and persistence in mouse tissues and in vivo stability. Three groups containing 12 to 15 mice each were inoculated intragastrically with 10 l° c.f.u. from log phase cultures of one of three S. typhimurium strains: J1000 (carrying the LT-B gene integrated in the chromosome), LC21 (carrying the LT-B gene on the multicopy plasmid pUCS) grown in the presence of ampicillin, and LC21 grown without stabilizing antibiotic. On days 1, 3, 6, 14 and 21 postinoculation, three to four mice from each group were killed. Samples from blood, Peyer's patches, spleen and liver were removed under aseptic conditions. After mincing the soft tissues, all samples were cultured in Selenite broth (Difco) at 37°C overnight. Cultures containing samples from animals inoculated with S. typhimurium J1000 were diluted and plated on TSA. Cultures containing samples from animals inoculated with S. typhimurium LC21 were diluted and plated on TSA and TSA supplemented with 100 fig ml 1 of ampicillin. Twenty-five colonies from each of the positive cultures were picked and tested for expression of LT-B by ELISA in order to determine the in vivo stability of the LT-B gene in each of the three strains.

Antibody responses. Three groups containing seven mice each were inoculated orally with 10 l° c.f.u, harvested from log phase cultures of one of the S. typhimurium strains as described above. A second dose of the same strain was administered on day 4 post primary immunization and booster doses were given on days 21 and 25 post primary immunization. Animals were killed 2 weeks after the last boost. The time points for the boosters and

in bacterial vaccine vectors: L. Cardenas and J.D. Clements

killing of the animals were chosen because extensive studies in this and other laboratories have indicated that following this regimen of immunization, initial levels of both serum and mucosal antibody peak between 3 and 4 weeks post primary immunization 2 5,7,14.16,2'7--29. Serum and mucosal samples obtained and processed as previously described 5 were assayed for the presence of serum immunoglobulin G (IgG) and mucosal immunoglobulin A (IgA) directed against the LPS of the immunizing strain and against LT-B by ELISA. LPS was prepared by the method of Sutherland and Wilkinson 3°.

Intracellular distribution of L T-B. Cellular fractiona- tions of S. typhimurium strains J1000 and LC21 were prepared by harvesting 35 ml of overnight culture in 0.2 M Tris, pH 8.0, containing 1 M sucrose to 20 mg cells (wet weight) per ml of buffer. EDTA was added to a final concentration of 5 mM and, after a 10min incubation at room temperature, lysozyme was added to 200 pg ml-1 The bacterial mixture was diluted 1:2 with water, incubated for 30min at 37~C, and centrifuged at 50009 for 15 min. The supernatant with the periplasmic contents was collected and saved. The pellet was washed once in 0.2M Tris, pH 8.0, 0.5 M sucrose, resuspended in 0.2M Tris, pH 8.0, and subjected to repeated freeze and thaw. The lysed cells were centrifuged at 900009, to separate the cytoplasm and the membrane fractions. All fractions were dialysed against 0.02M Tris, 0.01 M MgCI2, pH 8.0 (Ref. 31). Alkaline phosphatase (AP) and glucose-6- phosphate dehydrogenase (G-6-P) activities were determined in each fraction as previously described 32. One unit of AP is defined as the amount required to hydrolyse 1.0 pM of p-nitrophenyl phosphate to p-nitrophenol and inorganic phosphate per rain at room temperature 33. One unit of G-6-P is defined as the amount of enzyme in a l ml sample that in a 3 ml assay at room temperature increases the absorbance of the solution by 0.001 at 340 nm in 1 min 32. The amount of LT-B in each fraction was determined by ELISA as described above.

R E S U L T S

Construction of plasmid pALC50 Plasmid pJC217 carries the gene that codes for LT-B

cloned as a 0.72 kb fragment in the HindIII site of the polylinker region of plasmid pUC84. There is an internal EcoRI site in this fragment located upstream of the initiation codon for LT-B (Figure 1). Plasmid pADEI71, the shuttle vector used for these studies, has a single Pstl site adjacent to the hisOGD region. This region provides the homology for the recombinational event. In order to eliminate the PstI site present in the pUC8 polylinker region of plasmid pJC217, the plasmid was cut with EcoRI, isolated by electroelution following agarose gel electrophoresis, and religated. The new plasmid, designated pJC220, was approximately 250 base pairs smaller than pJC217 and lacked the entire polylinker region from pUC8, including the unwanted PstI site.

E. coli strain HB101 was transformed with pJC220 and ampicillin-resistant transformants were selected and tested for production of LT-B by ELISA. All the selected colonies were positive. There was no quantitative difference in the amount of antigen produced, when compared with the same strain transformed with the original plasmid pJC217. Plasmid pJC220 was then isolated and cut with Pvull and the reaction products


Antigen stabil ity in bacterial vaccine vectors: L. C&rdenas and J.D. Clements

were separated on a 0.9% agarose gel. Two fragments, sizes 2.3 and 0.8 kb, were obtained. The smaller fragment, containing the LT-B gene and the lac promoter and operator from pUC8, was excised from the gel, electroeluted, and ethanol-precipitated. It was necessary to include the lac promoter and operator region as LT-B has no promoter or operator of its own 34. The 0.8 kb fragment was then flanked with PstI linkers and ligated into plasmid pADE171, which carries the hisOGD region from Salmonella and the genes encoding resistance to streptomycin and spectinomycin. The ligation mixture was used to transform E. coli HB101. The presence of the lac promoter and operator in this construct permitted selection on the basis of titration of the lac repressor in HB101, i.e. selection of blue colonies on X-Gal containing media. The resultant construct was designated pALC50.

1 R 3 4 5 6

2 3 - W 9 .4 -

6 .6 -

Integration of the gene coding for production of LT-B into the chromosome of S. typhimurium

Plasmid pALC50 was used to transform the host- specificity mutant S. typhimurium LB5010 prior to transformation of S. typhimurium J706 which carries a deletion in the hisOG region 16. This strategy allowed the LT-B gene to integrate into the chromosome due to recombination between the his and adjacent regions of the plasmid and homologous regions on the chromosome. Selected spectinomycin-resistant transformants were grown to log phase and aliquots of these cultures were plated on minimal medium agar plates to isolate His + revertants. Following penicillin enrichment, spectinomycin-sensitive, His + revertants, having lost the shuttle plasmid, were selected and screened for the expression of LT-B by ELISA. These revertants were obtained at a frequency of approximately 1 in 102. One such spectinomycin- sensitive, His +, LT-B-producing isolate, designated J 1000, was selected for further study (Fi#ure 1).

Two control strains were also constructed. One control strain, designated LC21, was obtained by transformation of S. typhimurium J706 with plasmid pJC220. Strain LC21 is used as an isogenic strain expressing LT-B from a gene on a multicopy plasmid. A second control strain was constructed by transforming J706 with plasmid pADE 171 without the inserted LT-B gene and selecting for histidine prototrophy and sensitivity to spectinomycin. This strain, designated J500, was used as an LT-B negative control for all subsequent experiments.

Southern blot analysis Integration of the LT-B gene into the his locus of the

chromosome of S. typhimurium was confirmed by Southern blot analysis (Figure 2). The 600bp LT-B specific probe was shown to hybridize with plasmid pJC220 (lane 1 ), with plasmid pALC50 (lane 3), and with chromosomal DNA from S. typhimurium Jl000 (lane 4). The LT-B specific probe did not hybridize with plasmid pADE171 (lane 2) or with chromosomal DNA from S. typhimurium J500 (lane 5). There is a single SstI site within the LT-B gene; consequently, two different size fragments (4 kb and 23 kb) were observed to hybridize with the LT-B probe. The difference in intensity between these bands is attributed to the fact that the recognition site for SstI is close to the 5' end of the gene. The darker autoradiographic band reflects the region of greatest homology with the probe. The his-specific probe hybridized strongly to the 23 kb fragment from chromosomal DNA of J1000 (lane 6), indicating that the LT-B gene

2 . 3 "

2.0-

Figure 2 Southern blot analysis of integration of the LT-B gene into the chromosome of S. typhimuriurn J1000. Chromosomal and plasmid DNAs were digested with restriction endonucleases, electrophoresed, transferred to nitrocellulose, and hybridized to either a 32P-labelled LT-B-specific or his-specific probe. Lane 1: LT-B plasmid pJC220 digested with EcoRI; lane 2: shuttle plasmid pADE171 digested with Pstl; lane 3: LT-B plasmid pALC50 digested with Sstl; lane 4: chromosomal DNA from S. typhimurium J1000 digested with Sstl; lane 5: chromosomal DNA from S. typhimurium J500 digested with Sstl; lane 6: chromosomal DNA from S. typhimurium J1000 digested with Sstl. Plasmid and chromosomal DNAs in lanes 1-5 were hybridized to the LT-B specific probe; lane 6 contains chromosomal DNA from S. typhimurium J1000 hybridized to the his-specific probe. Size markers (in kilobases) are on the left

had been integrated just upstream of the his operon on the chromosome of S. typhimurium J706. The his-specific probe hybridized with the his region of plasmid pALC50, but not with a 23 kb band in S. typhimurium J500 (not shown). Based upon these findings, the integration of the LT-B gene into the chromosome of S. typhimurium J 1000 was mapped as shown at the bottom of Fi(jure 1.

SDS-PAGE/Western blot Since the gene that codes for production of LT is

not normally carried on the chromosome of wild type strains of E. coli, it was possible that LT-B produced by S. typhimurium Jl000 would be structurally or immunologically different than LT-B produced by strains with a plasmid-coded gene. LT-B was purified from S. typhimurium J 1000 by agarose affinity chromatography and analysed by Western blot following SDS-PAGE. As shown in Figure 3, LT-B purified from S. typhimurium J1000 (lane 1) was indistinguishable from LT-B purified from an E. coli strain expressing LT-B from plasmid pJC217 (lane 2). Preparations from both S. typhimurium


1 2

Antigen stability

46-

30-

21-

14-

Figure 3 Western blot analysis of LT-B. LT-B was purified from S. typhimurium J1000 and E. coli JM83 (pJC217) by agarose affinity chromatography and analysed by Western blot following SDS~PAGE. Samples containing l ~ 2 0 0 n g of protein were boiled for 5min prior to electrophoresis. Preparations from both S. typhimurium J1000 (lane 1, 150ng) and E. coli JM83 (pJC217) (lane 2,2O0ng) contained a band corresponding to monomeric LT-B that was recognized immunologically by antiserum directed against LT-B. Molecular weight markers (in kDa) are on the left

J1000 and E. coli JM83(pJC217) contained only a single band, corresponding to monomeric LT-B, and only that band was recognized immunologically by antiserum directed against LT-B.

Stability and expression in vitro In this experiment, stability and the levels of in vitro

expression of LT-B between the chromosomally integrated strain (Jl000) and an isogenic strain carrying the gene on a plasmid (LC21) were examined. The levels of expression of LT-B from these strains were compared by ELISA. S. typhimurium LC21 grown in the presence of ampicillin produced approximately 20 times more LT-B than did S. typhimurium J1000. Even in the presence of the stabilizing antibiotic, the plasmid-bearing strain exhibited significant plasmid loss ( > 8 0 % of the recovered isolates were ampicillin-sensitive at all time points after 24h). As shown in Table 1, LT-B was consistently expressed by both the cointegrate strain (Jl000) and the plasmid-containing strain grown in the presence of the stabilizing antibiotic throughout the 5 days of the experiment. In contrast, expression by the plasmid-containing strain was reduced to negligible levels following the first subculture when the stabilizing antibiotic was omitted from the growth medium. That this reduction in expression was due to plasmid loss was confirmed by differential plating of LC21 on medium with and without ampicillin (not shown). This analysis

in bacterial vaccine vectors: L. C~rdenas and J.D. Clemems

revealed that by the end of the first 24 h growth period, < 1% of LC21 recovered from the culture still contained the plasmid. These findings demonstrate that antigen expression is more stable in vitro when the gene is integrated into the chromosome than when the gene is carried on a plasmid in the absence of a stabilizing antibiotic. It should also be noted that even in the presence of the stabilizing antibiotic, only 20% of the isolates retain the recombinant plasmid.

Stability in vivo The stability and the in vivo maintenance of the LT-B

gene between S. typhimurium Jl000 and the plasmid- containing strain S. typhimurium LC21 were examined next. S. typhimurium LC21 was grown in either the absence or in the presence of ampicillin as above. The ability of each strain to invade and persist in mouse tissues, maintain the genes coding for expression of the heterologous antigen, and to evoke serum and mucosal antibody responses against the heterologous antigen and against the lipopolysaccharide of the parent strain were examined.

As shown in Table 2, there was evidence for invasion and persistence in Peyer's patches, livers, and spleens of animals throughout the 21 days of the study. S. typhimurium J1000, S. typhimurium LC21 and S. typhimurium LC21 + A m p colonized the various tissues to approximately the same extent. Organisms were not recovered from the blood during this study.

The in vivo maintenance of the LT-B gene was determined on organisms recovered from spleen, liver and Peyer's patches of the orally inoculated mice. Up to 25 bacterial isolates were examined from each tissue sample obtained during the invasion and persistence study. Organisms were cultured overnight and examined by ELISA for the ability to express LT-B. As shown in Table 3, the ability to express LT-B was stably maintained in vivo by all colonies tested from the cointegrate strain, S. typhimurium J l000, throughout the 21 days of the study. In contrast, expression of LT-B from colonies recovered from animals inoculated with either S. typhimurium LC21 or S. typhimurium LC21 +Amp was markedly reduced. By day 21, none of the S. typhimurium LC2I or S. typhimurium LC21 + Amp isolates retained the ability to express LT-B. From these experiments it was evident that strain S. typhimurium Jl000 could persist in tissues and maintain the ability to express LT-B from the chromosomal integration site, while strain LC2I grown in the absence of antibiotic rapidly lost the plasmid in vivo and, consequently, the ability to express LT-B. When strain LC21 was grown in the presence of the stabilizing antibiotic the plasmid was retained in vivo for a longer period of time. However,

Table 1 Stability and expression of LT-B in vitro

Specific activity LT-B (ng mg x)

Hours J 1000 LC21 LC21 + Amp

24 1.6 1.7 54 48 2.3 ND 36 72 2.1 ND 48 96 2.5 ND 28

120 3.0 ND 36

ND, none detected

Vaccine, Voi. 11, Issue 2, 1993 131

Antigen stability in bacterial vaccine vectors: L. C~rdenas and J.D. Clements

Table 2 Invasion and persistence in mouse tissues

Strain and tissue type 1

No. of specimens culture positive/no, tested on day:

3 6 14 21

Peyer's patches 2/3 J1000 Liver 0/3

Spleen 0/3

Peyer's patches 0/3 LC21 Liver 0/'3

Spleen 0//3

~"Peyer's patches 3//3 LC21+Amp ,~ Liver 0//3

( Spleen 0/3

3/3 0/3 3/3 3/3 0/3 2/3 1/3 0/3 0/3 0/3 3/3 2/3

2/3 3/3 3/3 3/3 0/3 2/3 0/3 1/3 0/3 2/3 1/3 0/3

3/3 X 2/3 3/4 0/3 X 2/3 1/4 3/3 X 2/3 2/4

X, not done

Table 3 Expression of LT-B by isolates recovered from mouse tissues

Strain and tissue type 1

No. of specimens LT-B positive/no, tested on day:

3 6 14 21

~" Peyer's patches 25/25 J1000 ~ Liver

L Spleen

~ Peyer's patches LC21 ,~ Liver

L Spleen

~" Peyer's patches 6/25 LC21+Amp ,] Liver

L Spleen

25,,'25 25/25 25/25 25/25 25/25

25,/25 25/25

3/22 0/25 0/25 0/25 0/25 0/25 0/25 0/25

5/'25 X 5/25 0,/25 X 2/25 0//21

7/25 X 0/25 0/'25

X, not done

Table 4 Humoral response following immunization with the cointegrate and plasmid-containing strains

J1000 LC21 LC21 4- Amp

Anti-LT-BIgG (,ugml 1) 0 0 444-18 Anti-LT-B IgA(ngml 1) 0 0 2749_+1220 Anti-LPS IgG (,ugml 1) 16_+6 9+_2 19_+4 Anti-LPS IgA (ngml ~) 1770_+701 1326_+607 82_+64

by day 21 none of the recovered colonies retained the plasmid or the ability to express LT-B.

Serum immunoglobulin G (IgG) and mucosal immunoglobulin A (IgA) responses against LT-B and against the LPS of the parent S. typhimurium strain C5 were then examined. Anti-LT-B and anti-LPS values were determined by ELISA. As shown in Table 4, there was no anti-LT-B response from animals inoculated with either J1000 or with LC21 (grown without the stabilizing antibiotic). In contrast, there were significant anti-LT-B serum IgG and mucosal IgA responses in animals inoculated with LC21 grown in the presence of ampicillin. All animals responded with serum and mucosal antibodies directed against LPS, a reflection of the abilities of all three of these strains to invade and persist to approximately the same extent (Table 2).

lntraeellular distribution of LT-B It is not clear what influence cellular location will have

on the subsequent immunological response when antigens are presented to the secretory immune system by these

attenuated Salmonella. The flagellin studies reported by Newton et al. 35 were initiated, in part, to examine the immune response to surface antigens in this system. Therefore, it was important to determine the intracellular location of LT-B produced by strains with the gene chromosomally integrated. The gene that codes for LT is normally carried on a large, single-copy plasmid and not on the chromosome. Moreover, LT-B is transcribed from the LT-A promoter and has no promoter of its own 34. In these constructs, LT-B is expressed under control of the lac promoter. The intracellular distribution of LT-B between the plasmid-containing strain S. typhimurium LC21 and the cointegrate strain S. typhimurium J1000 was examined following overnight growth. The amount of LT-B in each cellular fraction was determined by ELISA and compared with the distribution of alkaline phosphatase and glucose-6-phosphate dehydrogenase as markers for efficiency of cell fractionation. As seen in Table 5, the majority of LT-B was found in the periplasmic fraction of both strains, irrespective of the location of the gene coding for this antigen. Alkaline phosphatase and glucose-6-phosphate dehydrogenase partitioned as expected.

D I S C U S S I O N

The use of attenuated Salmonella strains as vaccine vectors for heteroiogous antigens has been extensively studied. These mutants are able to establish a limited infection in the host, and during the natural course of this innocuous infection the bacteria deliver a series of


Antigen stability in bacterial vaccine vectors: L. C&rdenas and J.D. Clements

Table 5 Distribution of two marker enzymes and LT-B in Salmonella

Distribution in fraction (%)

Periplasm Cytoplasm Membranes

AP G-6-P LT-B AP G-6-P LT-B AP G-6-P LT-B

J1000 81 31 86 17 64 17 3 5 ND LC21 + Amp 92 19 67 3 72 20 5 9 13

ND, none detected

antigens directly to the B and T lymphocytes present in the gut-associated lymphoid tissue (GALT). It has been well established that immunization with these bivalent vaccines is an effective and safe way to elicit the production of serum and mucosal antibodies against both the Salmonella carrier and the foreign antigen.

One potential problem concerning the use of these vaccines in humans is that most of the candidate hybrid strains carry the heterologous DNA on multicopy recombinant plasmids. Recombinant plasmids generally carry antibiotic resistance markers to facilitate identification and to keep selective pressure in favour of plasmid retention. Several authors have reported the instability of such plasmids both in vivo and in vi tro 12'15'36 3 8

Preliminary studies in our laboratory indicated that within 24h of immunization > 9 9 % of organisms recovered from the tissues of animals that had been inoculated with a plasmid-carrying strain had lost the plasmid and, consequently, the ability to synthesize the foreign antigen. Strategies such as those employing conditionally lethal plasmids to maintain the heterologous antigens 14'39 do not address this problem since only those bacteria that retain the plasmid can be recovered.

One means of overcoming the problem of spontaneous plasmid loss is to introduce the cloned gene into the chromosome of the carrier. In the study reported here, we describe the integration of the gene coding for the B subunit of the heat-labile toxin (LT-B) from E. coli into the chromosome of a 9ale mutant of S. typhimurium. This was achieved by homologous recombination between the chromosome of a histidine-deleted mutant of Salmonella and a plasmid that carried the deleted region and the foreign gene. Integration of the gene into the His locus of the chromosome of S. typhimurium was confirmed by Southern blot analysis using an LT-B specific probe and a histidine- specific probe. We next compared the stability and levels of expression of LT-B between the cointegrate strain and the plasmid-carrying strain in vitro. Our data clearly indicated that the initial levels of expression of LT-B by the strain carrying the LT-B gene on the chromosome (J1000) and the plasmid-bearing strain grown in the absence of antibiotic were similar. However, the plasmid-containing strain (LC21) loses the ability to express the heterologous antigen very quickly (i.e. within 24 h) in the absence of a stabilizing antibiotic, whereas the cointegrate strain continues to maintain and express the antigen. The amount of LT-B produced by the cointegrate strain Jl000 was approximately 20-fold less than that produced by the strain carrying the multicopy plasmid when the antibiotic was supplied in the growth medium (LC21+Amp). The antigen was expressed from the chromosomal locus in a continuous and stable manner throughout the 5 days of the in vitro experiment

performed here (Table 1). The in vitro stability of the LT-B cointegrate reported here is in contrast to the findings of Hone et al. 16 in that their K-88 cointegrates were less stable. After 54 generations in vitro, 10% of their isolates had lost the ability to express antigen from the chromosomally integrated genes.

The in vivo stability of the genes coding for antigen expression was determined on organisms recovered from spleen, liver and Peyer's patches of orally inoculated mice. By 24h postinoculation, the majority of tissue isolates from the plasmid-containing strain had lost the plasmid and the ability to synthesize the antigen. By contrast, 100% of the cointegrate isolates maintained the genes coding for expression of the antigen throughout the 21 days of the experiment. These findings are also different from those reported by Hone et al. 16 in that by day 18 post primary immunization only 73% of their recovered isolates retained the ability to express the inserted gene. Since Hone and co-workers used the same system of chromosomal integration employed in this study, the variations in gene stability and antigen expression are not the result of differences in integration sites or vectors used. The observed differences may have resulted from differences in promoters, nucleotide sequence or insert length, since the Hone et al.16 construct contained more than 6kb of inserted DNA from the K88ab operon under control of the pBR322 tetracycline promoter. It is also possible that expression of a foreign gene in the membrane (K88 pill) may contribute to instability when compared with expression of LT-B, which is not transported beyond the periplasmic space.

Significantly, both humoral and mucosal antibody levels against the antigen were greater when the antigen was expressed from the plasmid stabilized by the presence of the antibiotic than when the antigen was expressed from the chromosome. A possible explanation for these findings is that the lower levels of LT-B produced by S. typhimurium J1000 are not sufficient to induce a humoral response. S. typhimurium J1000 produces approximately 20-fold less LT-B than does strain LC21 grown in the presence of the stabilizing antibiotic. The observed differences in immunological responses could, therefore, be a function of antigen dose. One strategy to confirm this theory would be to increase the amounts of antigen expressed from the chromosome by either expressing the antigen under the control of a stronger promoter or by integrating the gene encoding the foreign antigen at multiple sites in the chromosome.

It is also important to determine the effectiveness of this system of immunization in an animal model that will permit challenge studies. Since the known strains of enterotoxigenic E. coli are not pathogenic for mice, it is not possible to use this animal model to determine whether or not this bivalent vaccine provides protection


Ant igen stabi l i ty in bac ter ia l vacc ine vectors: L. Ca rdenas and J.D. C lemen ts

against challenge with virulent organisms from which the foreign antigen (LT-B) was originally isolated. In general, the lack of relevant animal models has hindered the final assessment of all of the candidate Salmonella vector-based vaccines developed to date. In a number of studies, vaccination and challenge experiments have been performed via intraperitoneal or intravenous injection 1'4° Since this route of inoculation deviates from the natural route of infection used by mucosal pathogens, the conclusions concerning efficacy and protection deduced from this kind of experiment may not apply to natural challenge by the virulent organisms. Furthermore, when the end point of the challenge experiments is death, as is the case in the mouse protection assays, it is not possible to determine the individual contributions of mucosal, serum and cell-mediated immune responses to animal survival.

The expression of LT-B from a chromosomal locus did not interfere with the ability of the Salmonella vaccine carrier to colonize, invade and persist in mouse tissues, as evidenced by the fact that all three strains tested were recovered from tissues of inoculated animals throughout the 21 days of the experiment. The humoral response to LPS is also a reflection of the ability of the strains to initiate an effective immunization. An intriguing observa- tion was the approximately 20-fold reduction of mucosal anti-LPS IgA in animals inoculated with strain LC21 grown in the presence of the stabilizing antibiotic, when compared with strain J1000 or strain LC21 grown without the stabilizing antibiotic. The reason for the observed reduction in anti-LPS response is not clear. LT-B is known to be a highly antigenic molecule. It is possible that the introduction of large amounts of this T-dependent antigen into the mucosal lymphoid tissues diminishes the subsequent antibody response to the polysaccharide antigen LPS. It is also possible that the exaggerated immune response to LT-B is a function of the amount of LT-B produced or that the highly immunogenic nature of the protein influences the immunological and mitogenic properties of LPS.

There are a number of points to consider regarding the observed instability of the plasmid expressing the recombinant antigen when the strains are grown in the presence or absence of the stabilizing antibiotic. It should be noted that the studies reported here were performed with a single recombinant antigen (LT-B) on a derivative of a pUC plasmid with a pMB1 origin of replication 41. The relative instability of these plasmids has been widely noted by a number of investigators performing similar studies12.14.~.36 3s. However, other plasmids derived from different replicons (i.e. ColE1 ) have also been found to be unstable in these systems. Salas-Vidal et al. 3s, for instance, observed a 70% plasmid loss in strains recovered from the tissues of infected animals, even when the rop gene was introduced to stabilize the plasmid. It has been noted that overproduction of many recombinant antigens is toxic to the bacterial host. Whether this plays a role in the observed instability of these constructs is not known. Another consideration is the influence, if any, of the virulence-associated plasmid 42 46 on stability of the recombinant plasmids.

The study reported here provides important information for the use of attenuated bacteria as carriers of heterologous antigens. By integrating the gene coding for the heterologous antigen into the chromosome of the carrier, antigen expression can be stably maintained without the

requirement for stabilizing antibiotics. However, the mucosal and serum antibody responses observed indicate that the most important event for the development of an immune response against a foreign antigen delivered by these vectors may be the initial amount of antigen that primes the GALT, and not persistence of the vaccine strain in the tissues.

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

This work was supported by Public Health Service Grant AI28835 from the National Institute of Allergy and Infectious Diseases.

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