(~ INSTITUT PASTEUR/ELsEVIER Paris 1990
Res. z~icrobiol. 1990, 143,893-899
N E W A T T E N U A T E D DERIVATIVES O F VIBRIO C H O L E R A E
G.D.N. Pearson (l), V.J. DiRita (1), M.B. Goldberg (2), S.A. Boyko (2), S.B. C a l d e r w o o d (2) and J . J . Mekalanos (i)
(l) Department of Microbiology and Molecular Ger,e~ics, Harvard Medical School, Boston, MA 02115, and (2) Infectious Disease Unit, Massachusetts General Hospital, Boston, MA 02114 (USA)
Introduction. After more than a century of research on cholera, there is still no effective commercial vaccine. The disease remains a major health problem in the developing countries of Asia and Africa. Despite effective treatments, the rapidity of the disease still results in over 30,000 deaths per year in Bangladesh alone and perhaps over 200,000 worldwide (Holmgren et al., 1989). One approach to the development of an effective cholera vaccine is the construction of attenuated Vibrio cholerae mutants by specific deletion of genes encoding virulence factors. Deletion of the structural gene encoding the toxic A subunit of cholera toxin (ctxA) but not the immunogenic B subunit has yielded a number of derivatives that have been tested in human volunteers (Mekalanos et al., 1983; Kaper et al., 1984; Levine et al., 1988a,b; Herrington et al., 1988). While these strains were strongly immunogenic, they also induced a spectrum of side effects in volunteers that have limited their utility as vaccines. Although there exists one strain that appears less reactogenic than others (Levine et al., 1988b), the molecular definition of relevant properties causing its further attenuation is not straightforward because of the history of the parental strain used in its construction. Deletion mutations in either of two genes (tcpA and toxR) have been defined that are strongly attenuating (Taylor et al., 1987) but which also adversely affect imn,,~nogenicity (Herrington et al., 1988). Thus, additional defined mutations need to be identified which can be incorportated into ctx deleted vaccine strains to give full attenuation but still maintain immunogenicity. Live attenuated strains also suffer from the theoretical drawback of genetic instability in the environment where they may recombine with toxinogenic strains. The cholera toxin genes are located on a genetic element (Mekalanos, 1933; Goldberg and Mekalanos, 1985) that may be picked up by vaccine strains and then complement ctx deletions responsible for attenuation. In this report, we describe new attenuated derivatives of V. cholerae with mutations that cause intermediate reductions in intestinal colonization. Partial reduction in intestinal colonization may be one strategy for further attenuation of cholera live vaccines. In addition, some of these strains have properties that make them safer in terms of their likelihood to revert to toxinogenicity by re-acquisition of functional cholera toxin genes.
894
G.D.N. PEARSON
ET AL.
Materials and methods. Strains and plasmids. - Conditions for the growth and storage of V. cholerae strains have been reported (Kaper et al., 1984; Taylor et al., 1987). Construction of the recombinant suicide plasmids pGP52, pVJ22, and pVJ23 utilized plasmid pJM703.1 (Miller and Mekalanos, 1988) and will be described elsewhere (in preparation). The construction of other strains used in this study are described in the text or in cited references. Biochemical and genetic analysis. - Competition intestinal colonization assays, auto-agglutination selection, Southern blot hybridization and SDS-PAGE were done as described (Taylor et al., 1987).
Results. Characterization o f the ctx genetic element.
Characterization of the cloned ctx genes from several strains of V. cholerae (Kaper et al., 1984; Mekalanos, 1983; Goldberg and Mekalanos, 1986) has led to the proposal that the ctx genes reside on a 7-kb genetic element that has a structure similar to complex transposons (i.e., it is flanked by repeated sequences called RS sequences which are analogous to insertion sequences). We have characterized the RS sequence: associated with the ctx element in an assay designed to detect their transposition or site-specific recombination properties. The assay tests the ability of RS sequences to mediate integration of a conditionally replication-deficient plasmid into the chromosome of a strain of V. cholerae (2740-80) that lacks the entire ctx element. The plasmid pGP52 carries cloned RS sequences and can be mobilized by conjugation into strain 2740-80. As shown in table I, pGP52 could integrate into the 2740-80 chromosome at very high frequency. This integration event occurred at a specific site on the chromosome and at a frequency at least a million-fold higher than that of the control plasmid pVM55 (Miller and Mekalanos, 1988) bearing several hundred bases pairs of homology. The DNA on the 2740-80 chromosome where integration of pGP52 occurs was sequenced and compared to the sequences at the ends of several RS elements. The chromosome shares only a 17 nucleotide sequence (CCTAGTGCGCATTATGT) with the ends of RS1 which we have named ER2. Southern blot hybridization confirmed that the ER2 sequence is present at the ends of the ctx genetic element in several strains of V. cholerae. These data suggest that the ctx genetic element is a site-specific transposon that can integrate at high frequency into ER2 sequences on the V. cholerae chromosome.
TABLE I. - - Integration frequency of suicide plasmids pGP52 and pVMSS into various strains of V. eholerae. Recipient strain
Plasmid transferred
Transfer frequency
2740-80 2740-80 recA8 2740-80 2740-80 recA8
pGP52 pGP52 pVM55 pVM55
1.5 x 105 1.0 x 104 < 0.1 < 0.1
Integration was selectedafter conjugativetransfer of plasmids from E. coil as described (Miller and Melakanos, 1988). Transfer frequency is expressedas ampicillin-resistantrecipients per donor cell after 3 h of mating.
A TTENUA TED LIVE CHOLERA
VACCINES
895
We also tested the ability of pGP52 to integrate into strains of V. cholerae carrying the recA8 mutation (this represents a deletion in the recA gene constructed by recombinant methods). Integration was decreased 16-fold in the 2740-80 recA8 background (table I) and about 20-fold in the 0395-N1 recA8 background. Thus, the recA8 mutation may be capable of significantly reducing the probability of a non-toxinogenic vaccine strain aquiring the cholera toxin genes by either homologous recombination or site-specific recombination with environmental strains. Recently, Ketley et al. (1990) have reported that a recA mutant of V. cholerae vaccine strain CVDI03 was deficient in colonization of human volunteers. Because CVD103 is a derivative of strain 569B, a strain that has defects in a number of colonization properties (Baselski et al., 1978; 1979; see below), we examined the colonization properties of strain 0395-N1R, a recA8 derivative of strain 0395-N1. 0395-N1 is known to intestinally colonize mice and humans efficiently (Herrington et al., 1988; Taylor et al., 1987). In mixed infections in suckling mice, 0395-N1R was only outcompeted by about 5-10-fold by its parental RecA ÷ strain 0395-N1. This level of colonization defect is far less than was observed for mutants defective in tcpA or toxR, which are outcompeted about 500-1000-fold in suckling mice (Taylor et al., 1987). We conclude that recA alleles may have utility as stabilizing and attenuating mutations in some vaccine candidates like 0395-N1 without adversely affecting immunogenicity. Construction o f toxS deletion derivatives o f strain 0395-N1. The reduced colonization properties of strain 569B relative to other strains of V. cholerae might be explained by the fact that this strain has a deletion in a regulatory gene called toxS (Miller et al., 1989). The toxS gene stimulates the expression of toxRcontrolled genes of V. cholerae such as the gene for the major outer membrane protein OmpU. Because strain 569B is reduced in OmpU production, we sought to isolate toxS-defective mutants of strain 0395-N1 and then test them for OmpU production and colonization properties. We constructed a deletion mutation in the toxS gene of strain 0395-N1 by enriching for spontaneously occurring mutants deficient in TCP pili production by selecting for the non-autoagglutinating phenotype (Agg-) (Taylor et al., 1987). The strain isolated (Agg-2) appears to carry the same deletion in toxS that is carried by strain 569B and which apparently also occurred spontaneously in this strain's history (data not shown). When analysed by SDS-PAGE (fig. 1), outer membranes of Agg-2 appear similar in OmpU/OmpT ratio to those of strain 569B or to 0395 pVJ22 (the latter strain carries a null insertion mutation of pVJ22 into toxS). The defect in OmpU expression seen in strain Agg-2 and 569B was complemented by pVJ23 which carries an active copy of the toxS gene. Finally, strain M 13, a hypotoxinogenic mutant strain (Holmes et al., 1978), appears identical in OmpT/OmpU ratio and in its response to complementation by pVJ23 to that seen with its parent strain 569B. This result suggests that MI 3 is probably not completely defective in ToxR function or this strain would not make any OmpU even in the presence of pVJ23 (Miller and Mekalanos, 1988). It also suggests that the low reactogenicity of this strain (Woodward et ai., 1975) may in fact be due in part to its unusual outer membrane profile as well as its reduced toxin production phenotype (Holmes et al., 1978). Under optimal culture conditions, Agg-2 still expresses significant levels of TCP pili and cholera toxin B subunit, but never produces wild type levels of the OmpU protein. We reasoned that if the intestinal environment presents optimal signals for induction of toxR-regulated genes, then toxS mutants might be only mildly defective in colonization unless OmpU plays an essential role in this process. We analysed the colonization properties of Agg-2 by competition assay with 0395. We found that Agg-2 was about 16 times more efficient at intestinal colonization than strain JJM43 (carrying the toxR43 allele (Herrington et al., 1988; Taylor et al., 1987). Thus, Agg-2
896
G.D.N. PEARSON
. . . . .
ET AL.
~
2tJ~ I
i
O395
I
~-2
i
~gB
i
M13
FiG. I. - - SDS-PAGE analysis of total proteins produced by various V. cholerae strains. Strain background~ are indicated below. Lanes marked with plasmids represent derivatives carrying integrated versions of pVJ22 (causing a toxS null phenotype) or pVJ23 (complementing toxS mutations). The positions of the outer membrane proteins OmpT and OmpU are indicated togther with molecular weight standards.
is intermediate in its colonization properties between 0395-N1 (its highly immunogenic parent) and JJM43 (a poorly immunogenic toxR mutant derivative). These data suggest that Agg-2 is not highly defective in production of TCP pili and other colonization properties in vivo, but rather has a mild colonization defect perhaps due to reduced expression of OmpU. Construction o f irgA mutants o f strain 0395-N1. The irgA locus of V. cholerae encodes a 77-kDa outer membrane protein that is expressed best only when the organism is grown under iron limited conditions (Goldberg et al., 1990). Recently we have cloned the irgA gene and determined a portion of its DNA sequence corresponding to its 5' end. The deduced amino acid sequence encoded by irgA was found to be highly homologous to the FepA protein of Escherichia coli. A portion of this homology is shown in figure 2. Because FepA functions as the ferrienterochelin receptor in E. coli, it seems plausible that irgA encodes an outer membrane receptor for a V. cholerae siderophore and therefore may play an important role in iron optake in the intestine. Consistent with this conclusion, an irgA::TnphoA mutant (MBG40) of strain 0395 is about 10-fold reduced in intestinal colonization of suckling mice by a competition
A TTENUA TED LIVE CHOLERA
VACCINES
IrgA
132
Q A I E R I E V I R G P M S
FepA
139
E M I E R I E V I R G P A R
897
FIG. 2. - Region of homology between the peptide encoded by irgA and that encoded by FepA. The number of residues from the amino terminal end of each protein to the start of the alignment is indicated on the left.
assay (Goldberg et al., 1990). However, this irgA mutant is about 100-fold less virulent as assessed by LDs0, suggesting that irgA may play multiple roles in the pathogenesis of cholera. Thus, a mutation in irgA may be an excellent candidate for a second attenuating mutation in cholera live vaccine strains. In this regard, we have constructed a strain (SBC20) which carries a irgA mutation in the 0395-N1 background and are currently examining this mutant's colonization properties. Discussion.
Three new live, attenuated cholera vaccine derivatives have been proposed based on the preliminary work presented here (table II). The three new attenuated strains noted here are all derivatives of 0395-N1. Of interest is the fact that 0395-N1, as a live attenuated cholera vaccine is relatively low in reactogenicity and high in immunogenicity in human volunteers. For example, at a dose of 108 0395-N1 organisms, only 1 of 13 (8 07o)volunteers experienced scorable diarrhoea compared to 6/46 (13 070)for recepients of CVDI03 at the same dose (Herrington et aL, 1988; Levine et al., 1988b). These same volunteers averaged geometric mean vibriocidal titres of 8273 and 1260 for 0395-N1 versus CVDI03, respectively (Herrington et ai., 1988; Levine et al., 1988b). While 0395-N1 appeared slightly more reactogenic than CVDI03 in categories other than diarrhoea (e.g. fevers above 37.8°C), it is clearly a highly immunogenic vaccine with relatively low reactogenicity that is suitable for further investigation, particularly if additional attenuating mutations can be found.
TABLE II.
--
New
proposed cholera live vaccines.
Strain
Properties
0395-NIR
Derivative of strain 0395-N1 (Mekalanos et al., 1983); deleted for the recA gene; reduced in intestinal colonization about 5-10 fold; reduced in homologous and RS mediated site-specific recombination. Derivative of strain 0395-N1 (Makalanos et al., 1983); deleted for the toxS gene; reduced in intestinal colonization about 10-fold; altered in the expression of OmpU. Derivative of strain 0395-N1 (Mekalanos et al., 1983); carries an insertional inactivated irgA gene; reduced in intestinal colonization about 10-fold; presumably altered in iron uptake.
Agg-2 SBC20
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G.D.N. PEARSON
ET AL.
All three new mutants proposed here show about 10-fold reduced colonization when compared with their parental strain 0395-N1. We believe that stepwise reduction in colonization capacity is the best way to accurately examine the relationship between reaetogenicity and immunogenicity in volunteers. The three new mutations examined (recA, toxS and irgA) each reduce colonization by presumably different mechanisms (growth rate reduction, regulatory alteration, iron uptake defect, respectively). We anticipate that a series of strains can be produced by sequentially adding these mutations to the 0395-N 1 background, thus providing a continuum of vaccines for study in different human age groups or geographical populations. In this way, an immunogenic but non-reactogenic vaccine can be identified in volunteer studies in North America and then be tested in volunteers from a cholera endemic population. If such a vaccine fails to colonize the endemic volunteers, it would be a relatively simple matter to move one mutation back in the series to a better colonizing strain that presumably would overcome the immune, genetic or physiological factors responsible for poor colonization of the vaccine in endemic volunteers. Such a cognitive approach is not possible for strains like CVD103 and its derivatives (Levine et aL, 1988b) because its parent strain 569B starts out as a poor colonizing strain with many undefined mutant phenotypes (Miller and Melakanos, 1988; Ketley et al., 1990; Baselski et aL, 1978, 1979; Miller et aL, 1989). The difference in reactogenicity of 569B versus 0395 derivatives has been proposed to be related to the production of a shiga-like toxin (O'Brien et al., 1985). This now seems less likely for two reasons. First, the production of shiga-like toxin by 0395 has not been detected reproducibly in our hands. Second, we have cloned DNA fragments from 0395 showing detectable homology to E. coil sit-1 gene probes, and when they were sequenced we found no significant homology to the sit-1 coding sequence. It might finally be emphasized that 0395-NIR, the derivative that carries a deletion in the recA, is not only reduced in colonization properties but is also about 20-fold reduced in its ability to integrate pGP52 into its chromosome. The relative resistance to RS-mediated site-specific recombination and homologous recombination that 0395-NIR shows should make it less likely to revert in a cholera endemic area by picking up wild type cholera toxin genes through conjugation, transduction or transformation. KEY-WORDS: Vibrio cholerae, Cholera, Live vaccine; Attenuated mutants, Intestinal colonization.
References.
BASELSKI,V.S., MEDINA,R.A. & PARKER,C.D. (1978), Isolation and phen0typic characterization of virulence deficient mutants of Vibrio cholerae. Infect. lmmun., 22, 181188. BASELSKI,V.S., MEDINA,R.A. & PARKER,C.D. (1979), In vivo and in vitro characterization of virulence-deficient mutants of Vibrio cholerae. Infect. Immun., 24, 111-116. GOLDBERG,I. & MEKALANOS,J.J. (1986), Effect of a recA mutation on cholera toxin gene amplification and deletion events. J. Bact., 165, 723-731. GOLDBERG,M.B., DIRITA,V.J. & CALDERWOOD,S.B. (1990), Identification of an iron-regulated viruience determinant in Vibris Cholera/using TnphoA mutagenesis. Infect. Immun., 58, 55-60. HERRINGTON,D., HALL,R.H., LOSONSKY,G., MEKALANOS,J.J., TAYLOR,R.K. & LEVlNE,M.M. (1988), Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J. exp. Med., 168, 1487-1492. HOLM~;REN,J., CLEMENS,J., SACK,D.A. & SVENNERHOLM,g. (1989), New Cholera vacines. Vaccine, 7, 94-96.
A TTENUA TED LIVE CHOLERA
VACCINES
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HOLMES,R.K., BAINE,W.B. & VASIL,M.K. (1978), Quantitative measurements of cholera entertoxin in cultures of toxinogenic, wild type, and non-toxinogenic mutant strains of Vibrio cholerae by using a sensitive and specific reversed passive hemagglutinaLion assay for cholera enterotoxin. Infect. Immun., 19, 101-106. KAPER,J.B., LOCKMAN,H., BALDINI,M.M. & L~VINE,M.M. (1984), Recombinant nontoxinoc Vibrio cholerae strains as attenuated cholera vaccine candidates. Nature (Lond.), ,655-658. KETLeY, J.M., KAPER, J.B., HERRINGTON,D.A., LOSONSKY,G. & LEVINe, M.M. (1990), Diminished immunogenicity of recombination deficient derivative of Vibrio cholerae vaccine strain CVDI03. Infect. Immun., 58, 1481-1484. LEvI~, M.M., KAPER,J.B., HERRINOTON,D., LOSONSKY,G., MORRIS,J.G., CLEMem'S,M.L., BLACK,R.E., TALL, B. & HALL, R. (1988a), Volunteer studies of deletion mutants of Vibrio cholerae 01 prepared by recombinant techniques. Infect. Immun., .~6, 161-167. LEVINE,M.M., KAPER,J.B., I'IERRINGTON,D., KETLEY,G., LOSONSKY,G., TACKET,C.O., TALL, B. & CRUZ, S. (1988b), Safety immunogenicity, and effacacy of recombinant live oral cholera vaccines, CVDI03 and CVDI03-HgR. Lancet, I!, 467-470. MEKALANOS,J.J. (1983), Duplication and amplification of toxin genes in Vibrio cholerae. Cell, 35, 253-263. MEKALANOS,J.J°, SWARTZ,D.J., PEARSON,G.D.N., HARFORD,N., GROYNE,F. & DEWILDE,M. (1983), Cholera toxin genes: nucleotide sequence, deletion analysis, and vaccine deveiopment. Nature (Lond.), 306, 551-557. MILLER, V.L. & MEKALANOS,J.J. (1988), A novel suicide vector and its use in construction of insertion mutations: Osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J. Bacterioi., 170, 2575-2583. MILLER, V.L., DIRITA, V.J. & MEKALANOS,J.J. (1989), Identification of toxS, a regulatory gene whose product enhances ToxR-mediated activation of the cholera toxin promoter. J. Bact., 171, 1288-1293. O'BmAN, A.D., CHEN,M.E., HOLMES,R.K. & KAPER,J.B. (1985), Environmental and human isolates of Vibrio cholerae and Vibrio parahaemolyticus produce a S;~igella dysenteriae 1 (Shiga)-like cytotoxin. Lancet, !, 77-78. TAYLOR,R.K., MILLER,V.L., FURLONG,D.B. & MEKALANOS,J.J. (1987), Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc. nat. Acad. Sci. (Wash.), 84, 2833-2837. WOODWARD,W.E., (~ILMAN,R., HORNICK,R., LIBONATI,J. & CASH, R. (1975), Effacacy of a live oral cholera vaccine in human volunteers, in "Proceedings of the 1lth joint conference on cholera, US-Japan cooperative medical science program". National Institutes of Health, Bethesda.
~
Res. z~icrobiol. 1990, 143,893-899
N E W A T T E N U A T E D DERIVATIVES O F VIBRIO C H O L E R A E
G.D.N. Pearson (l), V.J. DiRita (1), M.B. Goldberg (2), S.A. Boyko (2), S.B. C a l d e r w o o d (2) and J . J . Mekalanos (i)
(l) Department of Microbiology and Molecular Ger,e~ics, Harvard Medical School, Boston, MA 02115, and (2) Infectious Disease Unit, Massachusetts General Hospital, Boston, MA 02114 (USA)
Introduction. After more than a century of research on cholera, there is still no effective commercial vaccine. The disease remains a major health problem in the developing countries of Asia and Africa. Despite effective treatments, the rapidity of the disease still results in over 30,000 deaths per year in Bangladesh alone and perhaps over 200,000 worldwide (Holmgren et al., 1989). One approach to the development of an effective cholera vaccine is the construction of attenuated Vibrio cholerae mutants by specific deletion of genes encoding virulence factors. Deletion of the structural gene encoding the toxic A subunit of cholera toxin (ctxA) but not the immunogenic B subunit has yielded a number of derivatives that have been tested in human volunteers (Mekalanos et al., 1983; Kaper et al., 1984; Levine et al., 1988a,b; Herrington et al., 1988). While these strains were strongly immunogenic, they also induced a spectrum of side effects in volunteers that have limited their utility as vaccines. Although there exists one strain that appears less reactogenic than others (Levine et al., 1988b), the molecular definition of relevant properties causing its further attenuation is not straightforward because of the history of the parental strain used in its construction. Deletion mutations in either of two genes (tcpA and toxR) have been defined that are strongly attenuating (Taylor et al., 1987) but which also adversely affect imn,,~nogenicity (Herrington et al., 1988). Thus, additional defined mutations need to be identified which can be incorportated into ctx deleted vaccine strains to give full attenuation but still maintain immunogenicity. Live attenuated strains also suffer from the theoretical drawback of genetic instability in the environment where they may recombine with toxinogenic strains. The cholera toxin genes are located on a genetic element (Mekalanos, 1933; Goldberg and Mekalanos, 1985) that may be picked up by vaccine strains and then complement ctx deletions responsible for attenuation. In this report, we describe new attenuated derivatives of V. cholerae with mutations that cause intermediate reductions in intestinal colonization. Partial reduction in intestinal colonization may be one strategy for further attenuation of cholera live vaccines. In addition, some of these strains have properties that make them safer in terms of their likelihood to revert to toxinogenicity by re-acquisition of functional cholera toxin genes.
894
G.D.N. PEARSON
ET AL.
Materials and methods. Strains and plasmids. - Conditions for the growth and storage of V. cholerae strains have been reported (Kaper et al., 1984; Taylor et al., 1987). Construction of the recombinant suicide plasmids pGP52, pVJ22, and pVJ23 utilized plasmid pJM703.1 (Miller and Mekalanos, 1988) and will be described elsewhere (in preparation). The construction of other strains used in this study are described in the text or in cited references. Biochemical and genetic analysis. - Competition intestinal colonization assays, auto-agglutination selection, Southern blot hybridization and SDS-PAGE were done as described (Taylor et al., 1987).
Results. Characterization o f the ctx genetic element.
Characterization of the cloned ctx genes from several strains of V. cholerae (Kaper et al., 1984; Mekalanos, 1983; Goldberg and Mekalanos, 1986) has led to the proposal that the ctx genes reside on a 7-kb genetic element that has a structure similar to complex transposons (i.e., it is flanked by repeated sequences called RS sequences which are analogous to insertion sequences). We have characterized the RS sequence: associated with the ctx element in an assay designed to detect their transposition or site-specific recombination properties. The assay tests the ability of RS sequences to mediate integration of a conditionally replication-deficient plasmid into the chromosome of a strain of V. cholerae (2740-80) that lacks the entire ctx element. The plasmid pGP52 carries cloned RS sequences and can be mobilized by conjugation into strain 2740-80. As shown in table I, pGP52 could integrate into the 2740-80 chromosome at very high frequency. This integration event occurred at a specific site on the chromosome and at a frequency at least a million-fold higher than that of the control plasmid pVM55 (Miller and Mekalanos, 1988) bearing several hundred bases pairs of homology. The DNA on the 2740-80 chromosome where integration of pGP52 occurs was sequenced and compared to the sequences at the ends of several RS elements. The chromosome shares only a 17 nucleotide sequence (CCTAGTGCGCATTATGT) with the ends of RS1 which we have named ER2. Southern blot hybridization confirmed that the ER2 sequence is present at the ends of the ctx genetic element in several strains of V. cholerae. These data suggest that the ctx genetic element is a site-specific transposon that can integrate at high frequency into ER2 sequences on the V. cholerae chromosome.
TABLE I. - - Integration frequency of suicide plasmids pGP52 and pVMSS into various strains of V. eholerae. Recipient strain
Plasmid transferred
Transfer frequency
2740-80 2740-80 recA8 2740-80 2740-80 recA8
pGP52 pGP52 pVM55 pVM55
1.5 x 105 1.0 x 104 < 0.1 < 0.1
Integration was selectedafter conjugativetransfer of plasmids from E. coil as described (Miller and Melakanos, 1988). Transfer frequency is expressedas ampicillin-resistantrecipients per donor cell after 3 h of mating.
A TTENUA TED LIVE CHOLERA
VACCINES
895
We also tested the ability of pGP52 to integrate into strains of V. cholerae carrying the recA8 mutation (this represents a deletion in the recA gene constructed by recombinant methods). Integration was decreased 16-fold in the 2740-80 recA8 background (table I) and about 20-fold in the 0395-N1 recA8 background. Thus, the recA8 mutation may be capable of significantly reducing the probability of a non-toxinogenic vaccine strain aquiring the cholera toxin genes by either homologous recombination or site-specific recombination with environmental strains. Recently, Ketley et al. (1990) have reported that a recA mutant of V. cholerae vaccine strain CVDI03 was deficient in colonization of human volunteers. Because CVD103 is a derivative of strain 569B, a strain that has defects in a number of colonization properties (Baselski et al., 1978; 1979; see below), we examined the colonization properties of strain 0395-N1R, a recA8 derivative of strain 0395-N1. 0395-N1 is known to intestinally colonize mice and humans efficiently (Herrington et al., 1988; Taylor et al., 1987). In mixed infections in suckling mice, 0395-N1R was only outcompeted by about 5-10-fold by its parental RecA ÷ strain 0395-N1. This level of colonization defect is far less than was observed for mutants defective in tcpA or toxR, which are outcompeted about 500-1000-fold in suckling mice (Taylor et al., 1987). We conclude that recA alleles may have utility as stabilizing and attenuating mutations in some vaccine candidates like 0395-N1 without adversely affecting immunogenicity. Construction o f toxS deletion derivatives o f strain 0395-N1. The reduced colonization properties of strain 569B relative to other strains of V. cholerae might be explained by the fact that this strain has a deletion in a regulatory gene called toxS (Miller et al., 1989). The toxS gene stimulates the expression of toxRcontrolled genes of V. cholerae such as the gene for the major outer membrane protein OmpU. Because strain 569B is reduced in OmpU production, we sought to isolate toxS-defective mutants of strain 0395-N1 and then test them for OmpU production and colonization properties. We constructed a deletion mutation in the toxS gene of strain 0395-N1 by enriching for spontaneously occurring mutants deficient in TCP pili production by selecting for the non-autoagglutinating phenotype (Agg-) (Taylor et al., 1987). The strain isolated (Agg-2) appears to carry the same deletion in toxS that is carried by strain 569B and which apparently also occurred spontaneously in this strain's history (data not shown). When analysed by SDS-PAGE (fig. 1), outer membranes of Agg-2 appear similar in OmpU/OmpT ratio to those of strain 569B or to 0395 pVJ22 (the latter strain carries a null insertion mutation of pVJ22 into toxS). The defect in OmpU expression seen in strain Agg-2 and 569B was complemented by pVJ23 which carries an active copy of the toxS gene. Finally, strain M 13, a hypotoxinogenic mutant strain (Holmes et al., 1978), appears identical in OmpT/OmpU ratio and in its response to complementation by pVJ23 to that seen with its parent strain 569B. This result suggests that MI 3 is probably not completely defective in ToxR function or this strain would not make any OmpU even in the presence of pVJ23 (Miller and Mekalanos, 1988). It also suggests that the low reactogenicity of this strain (Woodward et ai., 1975) may in fact be due in part to its unusual outer membrane profile as well as its reduced toxin production phenotype (Holmes et al., 1978). Under optimal culture conditions, Agg-2 still expresses significant levels of TCP pili and cholera toxin B subunit, but never produces wild type levels of the OmpU protein. We reasoned that if the intestinal environment presents optimal signals for induction of toxR-regulated genes, then toxS mutants might be only mildly defective in colonization unless OmpU plays an essential role in this process. We analysed the colonization properties of Agg-2 by competition assay with 0395. We found that Agg-2 was about 16 times more efficient at intestinal colonization than strain JJM43 (carrying the toxR43 allele (Herrington et al., 1988; Taylor et al., 1987). Thus, Agg-2
896
G.D.N. PEARSON
. . . . .
ET AL.
~
2tJ~ I
i
O395
I
~-2
i
~gB
i
M13
FiG. I. - - SDS-PAGE analysis of total proteins produced by various V. cholerae strains. Strain background~ are indicated below. Lanes marked with plasmids represent derivatives carrying integrated versions of pVJ22 (causing a toxS null phenotype) or pVJ23 (complementing toxS mutations). The positions of the outer membrane proteins OmpT and OmpU are indicated togther with molecular weight standards.
is intermediate in its colonization properties between 0395-N1 (its highly immunogenic parent) and JJM43 (a poorly immunogenic toxR mutant derivative). These data suggest that Agg-2 is not highly defective in production of TCP pili and other colonization properties in vivo, but rather has a mild colonization defect perhaps due to reduced expression of OmpU. Construction o f irgA mutants o f strain 0395-N1. The irgA locus of V. cholerae encodes a 77-kDa outer membrane protein that is expressed best only when the organism is grown under iron limited conditions (Goldberg et al., 1990). Recently we have cloned the irgA gene and determined a portion of its DNA sequence corresponding to its 5' end. The deduced amino acid sequence encoded by irgA was found to be highly homologous to the FepA protein of Escherichia coli. A portion of this homology is shown in figure 2. Because FepA functions as the ferrienterochelin receptor in E. coli, it seems plausible that irgA encodes an outer membrane receptor for a V. cholerae siderophore and therefore may play an important role in iron optake in the intestine. Consistent with this conclusion, an irgA::TnphoA mutant (MBG40) of strain 0395 is about 10-fold reduced in intestinal colonization of suckling mice by a competition
A TTENUA TED LIVE CHOLERA
VACCINES
IrgA
132
Q A I E R I E V I R G P M S
FepA
139
E M I E R I E V I R G P A R
897
FIG. 2. - Region of homology between the peptide encoded by irgA and that encoded by FepA. The number of residues from the amino terminal end of each protein to the start of the alignment is indicated on the left.
assay (Goldberg et al., 1990). However, this irgA mutant is about 100-fold less virulent as assessed by LDs0, suggesting that irgA may play multiple roles in the pathogenesis of cholera. Thus, a mutation in irgA may be an excellent candidate for a second attenuating mutation in cholera live vaccine strains. In this regard, we have constructed a strain (SBC20) which carries a irgA mutation in the 0395-N1 background and are currently examining this mutant's colonization properties. Discussion.
Three new live, attenuated cholera vaccine derivatives have been proposed based on the preliminary work presented here (table II). The three new attenuated strains noted here are all derivatives of 0395-N1. Of interest is the fact that 0395-N1, as a live attenuated cholera vaccine is relatively low in reactogenicity and high in immunogenicity in human volunteers. For example, at a dose of 108 0395-N1 organisms, only 1 of 13 (8 07o)volunteers experienced scorable diarrhoea compared to 6/46 (13 070)for recepients of CVDI03 at the same dose (Herrington et aL, 1988; Levine et al., 1988b). These same volunteers averaged geometric mean vibriocidal titres of 8273 and 1260 for 0395-N1 versus CVDI03, respectively (Herrington et ai., 1988; Levine et al., 1988b). While 0395-N1 appeared slightly more reactogenic than CVDI03 in categories other than diarrhoea (e.g. fevers above 37.8°C), it is clearly a highly immunogenic vaccine with relatively low reactogenicity that is suitable for further investigation, particularly if additional attenuating mutations can be found.
TABLE II.
--
New
proposed cholera live vaccines.
Strain
Properties
0395-NIR
Derivative of strain 0395-N1 (Mekalanos et al., 1983); deleted for the recA gene; reduced in intestinal colonization about 5-10 fold; reduced in homologous and RS mediated site-specific recombination. Derivative of strain 0395-N1 (Makalanos et al., 1983); deleted for the toxS gene; reduced in intestinal colonization about 10-fold; altered in the expression of OmpU. Derivative of strain 0395-N1 (Mekalanos et al., 1983); carries an insertional inactivated irgA gene; reduced in intestinal colonization about 10-fold; presumably altered in iron uptake.
Agg-2 SBC20
898
G.D.N. PEARSON
ET AL.
All three new mutants proposed here show about 10-fold reduced colonization when compared with their parental strain 0395-N1. We believe that stepwise reduction in colonization capacity is the best way to accurately examine the relationship between reaetogenicity and immunogenicity in volunteers. The three new mutations examined (recA, toxS and irgA) each reduce colonization by presumably different mechanisms (growth rate reduction, regulatory alteration, iron uptake defect, respectively). We anticipate that a series of strains can be produced by sequentially adding these mutations to the 0395-N 1 background, thus providing a continuum of vaccines for study in different human age groups or geographical populations. In this way, an immunogenic but non-reactogenic vaccine can be identified in volunteer studies in North America and then be tested in volunteers from a cholera endemic population. If such a vaccine fails to colonize the endemic volunteers, it would be a relatively simple matter to move one mutation back in the series to a better colonizing strain that presumably would overcome the immune, genetic or physiological factors responsible for poor colonization of the vaccine in endemic volunteers. Such a cognitive approach is not possible for strains like CVD103 and its derivatives (Levine et aL, 1988b) because its parent strain 569B starts out as a poor colonizing strain with many undefined mutant phenotypes (Miller and Melakanos, 1988; Ketley et al., 1990; Baselski et aL, 1978, 1979; Miller et aL, 1989). The difference in reactogenicity of 569B versus 0395 derivatives has been proposed to be related to the production of a shiga-like toxin (O'Brien et al., 1985). This now seems less likely for two reasons. First, the production of shiga-like toxin by 0395 has not been detected reproducibly in our hands. Second, we have cloned DNA fragments from 0395 showing detectable homology to E. coil sit-1 gene probes, and when they were sequenced we found no significant homology to the sit-1 coding sequence. It might finally be emphasized that 0395-NIR, the derivative that carries a deletion in the recA, is not only reduced in colonization properties but is also about 20-fold reduced in its ability to integrate pGP52 into its chromosome. The relative resistance to RS-mediated site-specific recombination and homologous recombination that 0395-NIR shows should make it less likely to revert in a cholera endemic area by picking up wild type cholera toxin genes through conjugation, transduction or transformation. KEY-WORDS: Vibrio cholerae, Cholera, Live vaccine; Attenuated mutants, Intestinal colonization.
References.
BASELSKI,V.S., MEDINA,R.A. & PARKER,C.D. (1978), Isolation and phen0typic characterization of virulence deficient mutants of Vibrio cholerae. Infect. lmmun., 22, 181188. BASELSKI,V.S., MEDINA,R.A. & PARKER,C.D. (1979), In vivo and in vitro characterization of virulence-deficient mutants of Vibrio cholerae. Infect. Immun., 24, 111-116. GOLDBERG,I. & MEKALANOS,J.J. (1986), Effect of a recA mutation on cholera toxin gene amplification and deletion events. J. Bact., 165, 723-731. GOLDBERG,M.B., DIRITA,V.J. & CALDERWOOD,S.B. (1990), Identification of an iron-regulated viruience determinant in Vibris Cholera/using TnphoA mutagenesis. Infect. Immun., 58, 55-60. HERRINGTON,D., HALL,R.H., LOSONSKY,G., MEKALANOS,J.J., TAYLOR,R.K. & LEVlNE,M.M. (1988), Toxin, toxin-coregulated pili, and the toxR regulon are essential for Vibrio cholerae pathogenesis in humans. J. exp. Med., 168, 1487-1492. HOLM~;REN,J., CLEMENS,J., SACK,D.A. & SVENNERHOLM,g. (1989), New Cholera vacines. Vaccine, 7, 94-96.
A TTENUA TED LIVE CHOLERA
VACCINES
899
HOLMES,R.K., BAINE,W.B. & VASIL,M.K. (1978), Quantitative measurements of cholera entertoxin in cultures of toxinogenic, wild type, and non-toxinogenic mutant strains of Vibrio cholerae by using a sensitive and specific reversed passive hemagglutinaLion assay for cholera enterotoxin. Infect. Immun., 19, 101-106. KAPER,J.B., LOCKMAN,H., BALDINI,M.M. & L~VINE,M.M. (1984), Recombinant nontoxinoc Vibrio cholerae strains as attenuated cholera vaccine candidates. Nature (Lond.), ,655-658. KETLeY, J.M., KAPER, J.B., HERRINGTON,D.A., LOSONSKY,G. & LEVINe, M.M. (1990), Diminished immunogenicity of recombination deficient derivative of Vibrio cholerae vaccine strain CVDI03. Infect. Immun., 58, 1481-1484. LEvI~, M.M., KAPER,J.B., HERRINOTON,D., LOSONSKY,G., MORRIS,J.G., CLEMem'S,M.L., BLACK,R.E., TALL, B. & HALL, R. (1988a), Volunteer studies of deletion mutants of Vibrio cholerae 01 prepared by recombinant techniques. Infect. Immun., .~6, 161-167. LEVINE,M.M., KAPER,J.B., I'IERRINGTON,D., KETLEY,G., LOSONSKY,G., TACKET,C.O., TALL, B. & CRUZ, S. (1988b), Safety immunogenicity, and effacacy of recombinant live oral cholera vaccines, CVDI03 and CVDI03-HgR. Lancet, I!, 467-470. MEKALANOS,J.J. (1983), Duplication and amplification of toxin genes in Vibrio cholerae. Cell, 35, 253-263. MEKALANOS,J.J°, SWARTZ,D.J., PEARSON,G.D.N., HARFORD,N., GROYNE,F. & DEWILDE,M. (1983), Cholera toxin genes: nucleotide sequence, deletion analysis, and vaccine deveiopment. Nature (Lond.), 306, 551-557. MILLER, V.L. & MEKALANOS,J.J. (1988), A novel suicide vector and its use in construction of insertion mutations: Osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J. Bacterioi., 170, 2575-2583. MILLER, V.L., DIRITA, V.J. & MEKALANOS,J.J. (1989), Identification of toxS, a regulatory gene whose product enhances ToxR-mediated activation of the cholera toxin promoter. J. Bact., 171, 1288-1293. O'BmAN, A.D., CHEN,M.E., HOLMES,R.K. & KAPER,J.B. (1985), Environmental and human isolates of Vibrio cholerae and Vibrio parahaemolyticus produce a S;~igella dysenteriae 1 (Shiga)-like cytotoxin. Lancet, !, 77-78. TAYLOR,R.K., MILLER,V.L., FURLONG,D.B. & MEKALANOS,J.J. (1987), Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. Proc. nat. Acad. Sci. (Wash.), 84, 2833-2837. WOODWARD,W.E., (~ILMAN,R., HORNICK,R., LIBONATI,J. & CASH, R. (1975), Effacacy of a live oral cholera vaccine in human volunteers, in "Proceedings of the 1lth joint conference on cholera, US-Japan cooperative medical science program". National Institutes of Health, Bethesda.
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