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Pathogenic and vaccine significance of toxin-coregulated pili of Vibrio cholerae El Tor
Journal of Biotechnology 73 (1999) 109 – 117 www.elsevier.com/locate/jbiotec
Pathogenic and vaccine significance of toxin-coregulated pili of Vibrio cholerae El Tor S.R. Attridge *, E. Voss, P.A. Manning Microbial Pathogenesis Unit, Department of Microbiology and Immunology, The Uni6ersity of Adelaide, Adelaide, South Australia 5005, Australia Received 3 July 1998; accepted 4 January 1999
Abstract Vibrio cholerae O1 strains are classified into one of two biotypes, classical and El Tor, the latter being primarily responsible for cholera cases worldwide since 1961. Recent studies in our laboratory have focused upon the pathogenic and vaccine significance of the toxin-coregulated pili (TCP) produced by strains of El Tor biotype. Mutants in which the tcpA gene (encoding the pilin subunit protein) has been inactivated are dramatically attenuated in the infant mouse cholera model, showing markedly reduced colonisation potential in mixed-infection competition experiments. Significantly, in the vaccine context, antibodies to TCP are sufficient to prevent experimental infection, although our data suggest that this protective effect might be limited to strains of homologous biotype. Since we have shown that tcpA sequences are conserved within a biotype but differ between biotypes, this latter observation suggests that the biotype-restricted pilin epitopes might have greater vaccine significance. Similar studies indicate that TCP also play a critical role in colonisation by strains of the recently-recognised O139 serogroup, which is thought to have evolved from an O1 El Tor strain. In contrast to the effect of introducing mutations in the tcpA gene, strains carrying inactivated mshA genes (encoding the subunit of the m6 annose-s6 ensitive h6 aema6 gglutinin pilus) show unaltered in vivo behaviour. Consistent with this finding is our inability to demonstrate any protective effect associated with antibodies to MSHA. Ongoing approaches to vaccine development are variously aimed at improving the immunogenicity of the current inactivated whole-cell vaccine, or assessing the field efficacy of a promising live attenuated strain. The possible implications of our findings are discussed in relation to both of these options. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Pathogenic; Vaccine; Toxin-coregulated pili; Vibrio cholerae El Tor
1. The discovery of pili in Vibrio cholerae In 1987, Taylor et al. (1987) reported that V. cholerae strains produce pili if cultured appropri* Corresponding author. Fax: +61-8-83034632. E-mail address: [email protected] (S.R. Attridge)
ately in vitro. Individual pilus filaments were aggregated laterally to form typical bundles. Since growth conditions which promoted or repressed pilus production also favoured or inhibited synthesis of the cholera toxin, these structures were called ‘toxin-coregulated pili’ (TCP). The operons responsible for production of both TCP and
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cholera toxin are each part of the so-called toxR regulon, a family of genes whose activation is coordinately controlled by the regulatory protein ToxR (DiRita, 1992). Taylor et al. (1987) went on to show that TCP are essential for efficient gut colonisation by V. cholerae, at least in the infant mouse cholera model (IMCM). This report was quickly followed by human volunteer studies which showed that mutation of either tcpA (encoding the pilin subunit) or toxR dramatically reduced the colonisation potential of a non-toxigenic V. cholerae candidate vaccine strain (Herrington et al., 1988). These reports offered a new approach to those interested in vaccine development, because by analogy with studies performed with other bacterial enteropathogens with similar disease aetiology (such as the K88 pilus-producing enterotoxigenic Escherichia coli (Rutter and Jones, 1973; Rutter et al., 1976)), it was predicted that TCP might elicit antibodies capable of protecting against infection. While this remains to be demonstrated in a clinical setting, further studies in the IMCM soon showed that antibodies to TCP were indeed sufficient to confer passive immunity (Sharma et al., 1989; Sun et al., 1990).
2. The significance of the biotype classification in V. cholerae V. cholerae strains are classified as belonging to either of two biotypes, classical or El Tor, on the basis of biological properties such as bacteriophage- and antibiotic-resistance patterns. The demonstrations of TCP as a critical colonisation factor, and protective antigen, were however restricted to studies using strains of the classical biotype. Southern hybridisation analyses using tcpA-specific probes confirmed that El Tor strains carry a gene homologous to that found in classical V. cholerae (Taylor et al., 1988). However attempts to detect TCP on the surfaces of in vitro-grown El Tor strains were uniformly unsuccessful, despite the testing of a variety of culture conditions (Hall et al., 1988; Sharma et al., 1989; Jonson et al., 1991a). Consistent with this were reports that antibodies to TCP were unable to
mediate significant protection of infant mice against El Tor V. cholerae, even though the same antibodies were effective against classical challenge strains (Sharma et al., 1989; Osek et al., 1994). Since the beginning of the seventh worldwide pandemic in 1961, the vast majority of cholera cases have been attributable to V. cholerae strains of El Tor biotype. It therefore seemed important to elucidate the pathogenic (and hence potential vaccine) significance of TCP for such strains. This became imperative when reports in the early 1990s described a second pilus type in V. cholerae—the mannose-sensitive haemagglutinin (MSHA), so named because of its capacity to agglutinate erythrocytes (Jonson et al., 1991b). These studies suggested that MSHA might be the El Tor analogue of TCP. The latter could only be observed on classical V. cholerae, but during growth in ‘AKI conditions’ (Jonson et al., 1991a) the pilin subunit could be detected by immunoblotting of El Tor strains. In contrast, MSHA could only be visualised on the surfaces of El Tor strains, although classical V. cholerae produced the MshA subunit (Jonson et al., 1991b). We therefore decided to evaluate the significance of TCP as a colonisation factor of El Tor V. cholerae. In particular, we addressed the following issues: 1. do El Tor strains polymerise TcpA into TCP in vitro? 2. would mutation of the tcpA gene affect the colonisation of El Tor strains in the IMCM? 3. are antibodies to TCP sufficient for passive immunity in the same model?
3. El Tor V. cholerae produce TCP in vitro Initial immuno-electron microscopic (IEM) studies failed to detect surface TCP on El Tor strains under conditions in which classical V. cholerae displayed typical pilus bundles (Hall et al., 1988; Sharma et al., 1989; Jonson et al., 1991a). However all these studies used antibodies specific for classical TCP, and subsequent findings in our laboratory and elsewhere suggested that
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this might be a significant limitation to the visualisation of El Tor pili. Our early attempts to construct tcpA mutants in El Tor strains by recombining an inactivated tcpA::KmR construct into the chromosome were unsuccessful (Voss, 1990), although this strategy worked well with classical V. cholerae (Manning, 1992). Since the inactivated gene was of classical origin, it seemed feasible that sequence differences between the two biotypic forms of tcpA might have precluded homologous recombination. Southern hybridisation analyses using probes recognising different regions of the classical tcpA gene sequence suggested that the C-terminal region might indeed differ between the biotypes (Voss, 1990; Ogierman et al., 1996). This led to sequencing studies which confirmed that there are indeed two biotypic tcpA sequences, each being conserved within a biotype, but differing significantly in the C-terminal third of the sequence (Iredell and Manning, 1994). In 1991 Jonson et al. (1991a,b) reported that with appropriate in vitro culture conditions, strains of El Tor biotype produce the TcpA subunit, as detected by immunoblotting using antibodies raised against classical TCP. Although the two biotypic pilin proteins evidently shared antigenic epitopes, the use of monoclonal antibodies revealed that there are also epitopes which are biotype-restricted. This was not surprising, given that the predicted primary structures of the proteins showed numerous amino acid differences (Iredell and Manning, 1994). However it strengthened our view that, to maximise the chance of visualising TCP on El Tor vibrios by IEM, we should prepare sera specific for the El Tor TcpA protein. This work is described elsewhere (Voss and Attridge, 1993). Rabbits were immunised with TcpA-positive outer membrane fractions of El Tor bacteria, and the resulting sera extensively absorbed with similar fractions prepared from tcpA mutants of the homologous strain. This approach allowed the first demonstration of TCP on the surfaces of in vitro grown El Tor V. cholerae. Serum prepared against El Tor TcpA detected pili on all El Tor strains which were TcpA-positive by immunoblotting, but reacted only weakly with TCP on classical vibrios, consistent with the fail-
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ure of initial attempts to visualise El Tor TCP with antibodies to classical pili. Having resolved the question of whether El Tor strains can express TCP, it was next of interest to determine whether these pili were involved in colonisation by strains of this biotype.
4. In vivo behaviour of El Tor tcpA mutants Knowledge of the El Tor tcpA sequence (Iredell and Manning, 1994) allowed us to mutate the gene by insertion of a kanamycin-resistance cartridge. The inactivated gene was then introduced into the chromosome using a strategy initially devised for use with classical biotype strains (Manning 1992). Strain construction was confirmed by Southern hybridisation analysis, and immunoblotting and IEM showed that the mutants failed to produce TcpA/TCP (Attridge et al., 1993). Their in vivo behaviour was then assessed in the IMCM, comparing virulence and colonisation potential with that of their wild-type parents. The IMCM was first devised by Ujiiye et al. (1968) and is in widespread use. Infant mice orally inoculated with a suspension of pathogenic V. cholerae experience a potentially fatal diarrhoeal syndrome, with dramatic bacterial growth in the intestine. Simultaneous LD50 titrations can be used to compare the virulence of two strains, while mixed-infection competition experiments (Attridge 1979; Attridge and Rowley, 1983) allow a direct comparison of colonisation potential. Finally, since pre-treatment of the challenge inoculum with antibodies against protective antigens such as lipopolysaccharide results in protection of the mice (compared with controls receiving untreated V. cholerae; Ujiiye and Kobari, 1970), the model can also be used to define new protective antigens of possible vaccine significance. The introduction of an insertionally-inactivated tcpA gene dramatically attenuated each of four El Tor strains, with LD50 doses increasing ca. 300– 10 000-fold (Attridge et al., 1993). Competition experiments showed that three of the four tcpA mutants retained virtually no colonisation potential. Mice were fed mixed suspensions comprising approximately equal numbers of wild-type and
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mutant bacteria, but 24 h later 104 – 105 of the former were recovered for every persisting mutant organism. The mutation had a less dramatic but still pronounced effect in the fourth strain, with output ratios of about 30:1 in favour of wild-type. Because of the potentially polar effect of the tcpA::KmR mutation, this work was repeated using more refined tcpA mutants, in which the wildtype tcpA gene was replaced with one carrying an in-frame deletion. Similar results were obtained (Voss et al., 1996), confirming the pivotal role of TCP in colonisation of infant mice by V. cholerae O1 El Tor. By analogy with the results of earlier studies with strains of classical biotype, it seemed likely that antibodies to El Tor TCP would mediate passive protection of infant mice, presumably by neutralising the colonisation function of these pili.
5. Assessment of protective efficacy of antibodies to El Tor TCP The data in Table 1 show the results of protection tests in which sera specific for either El Tor or classical TCP were assayed for their capacities to confer passive immunity against challenge strains of either biotype. Two main conclusions can be drawn from the experiments involving V. Table 1 Protective efficacy of antibodies to TCPa Challenge strain (biotype)
Antiserum specificity
El Tor TCP
Classical TCP
V. cholerae O1 H1 (El Tor) N16961 (El Tor) 569B (classical)
570 360 B20
B20 B20 275
V. cholerae O139 AI-1838 AI-1854
340 195
NT NT
a Figures show protective titres of antibodies to El Tor or classical TCP when used to pre-treat various challenge strains in the IMCM. NT, not tested. Adapted from Voss et al. (1996).
cholerae O1 challenge strains. First, antibodies to El Tor TCP are clearly sufficient to protect mice from challenge with strains of homologous biotype. Second, the immunity mediated by antibodies to TCP does not extend to challenge strains of heterologous biotype (Table 1). This suggests that the biotype-restricted antigenic epitopes of TcpA are collectively of much greater protective significance than those epitopes common to both biotypic forms of the pilin. It also offers an explanation for numerous earlier failures to observe protection against El Tor V. cholerae when antibodies to classical TCP have been tested in this model (as discussed elsewhere, Voss et al., 1996).
6. The pathogenic significance of MSHA During the course of our studies a second pilus type, MSHA, was described in El Tor strains of V. cholerae (Jonson et al., 1991b). Investigators in Sweden reported that antibodies to these pili showed protective potential in infant mice (Osek et al., 1994) suggesting a role for MSHA in establishing infection and raising the possibility that this might represent another antigen of vaccine significance. However the notion of multiple pilus adhesins was difficult to reconcile with our observations that tcpA mutants of El Tor strains generally retained minimal residual colonisation potential and were virtually avirulent in the IMCM. In a collaborative study with Drs Jonson and Holmgren from Sweden, we decided to examine the in vivo consequences of inactivating the mshA gene encoding the pilin subunit. El Tor strains were constructed in which the wild-type gene was replaced either with an mshA gene carrying an in-frame deletion, or alternatively with a potentially polar mshA::kmR gene. However neither type of mutant was attenuated for infant mice, nor did either show impaired colonisation when competed against wild-type (Attridge et al., 1996). Fig. 1 shows an example of the results obtained in these experiments. Neither selection of a (spontaneous) streptomycin-resistance marker (to enable differentiation in mixed infection experiments),
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ies to MshA were not protective whereas antibodies to TcpA provided passive immunity against each El Tor strain tested (Attridge et al., 1996).
7. The emergence of V. cholerae O139
Fig. 1. In vivo consequences of mutating tcpA or mshA genes in V. cholerae O1 strain 174. The figure shows results of three competition experiments performed in the same batch of mice, in which the colonisation capacity of wild-type strain 174 was compared with that of a spontaneous streptomycin-resistant (SR) variant of 174 (Expt. 1), the SR variant carrying a mutated mshA gene (Expt. 2), or the SR variant carrying a mutated tcpA gene (Expt. 3). Mice were fed mixed bacterial suspensions and 24 h later their intestines were excised, homogenised, and plated in the presence or absence of streptomycin to allow the separate enumeration of wild-type and mutant bacteria. Each dot represents the (log10) ratio of wild-type: mutant bacteria recovered from a single mouse, with data from Expts. 1 and 2 plotted according to the left axis and from Expt. 3 according to the right axis. Input ratios (IRs) of wild-type: mutant bacteria and median output ratios (MORs) were as follows (seven mice per group): Expt. 1—IR 0.97, MOR 1.3; Expt. 2—IR 0.74, MOR 1.4; Expt. 3—IR 0.72, MOR 36 500.
nor the additional introduction of a deleted mshA gene, significantly affected the colonisation potential of El Tor strain 174. In contrast, inactivating tcpA had the expected dramatic impact on the level of in vivo persistence. Very recently, workers at the Centre for Vaccine Development in Baltimore have conducted volunteer trials (Tacket et al., 1998) which suggest that the relative significance of TCP and MSHA is the same for human infection as we and others (Taylor et al., 1987; Thelin and Taylor, 1996) have described in the infant mouse model. Subsequently we tested antibodies to MSHA and TCP for protective activity in infant mice, using challenge bacteria grown under culture conditions conducive for production of the relevant pilus type. Consistent with the results of the virulence and colonisation experiments which failed to demonstrate an in vivo role for MSHA, antibod-
Until recently only V. cholerae strains of O1 serogroup had shown the potential to establish cholera epidemics, but in 1992 outbreaks of disease occurred in India and Bangladesh which were eventually attributed to isolates of a novel serogroup, O139 (Cholera Working Group, 1993; Nair et al., 1994). For a short period O139 strains displaced V. cholerae O1 as the primary cause of cholera in some regions. The incidence of O139 disease then fell dramatically and remained low for several years, before returning to predominate in Calcutta towards the end of 1996 and through into the middle of 1997 (Ghosh et al., 1997). Although the factors governing the relative incidence of O1 and O139 cholera remain unknown, the emergence of the latter poses a new challenge in terms of vaccine development. V. cholerae O139 evidently arose from a pre-existing O1 strain of El Tor biotype. A region of the genome involved in O-antigen biosynthesis was replaced by horizontal gene transfer, resulting in a novel lipopolysaccharide structure as well as an additional capacity for capsule formation (Bik et al., 1995; Stroeher et al., 1995). The epidemiology of O139 disease differs from that described for O1 cholera in that among inhabitants of endemic regions, the incidence of disease is similar in all age-groups (Cholera Working Group, 1993; Nair et al., 1994). This indicates that naturally acquired pre-existing immunity to V. cholerae O1 provides no immunity against O139 strains, suggesting that vaccines designed to combat O1 disease might be similarly ineffective against V. cholerae O139. This inference is supported by experimental studies which have failed to detect any degree of cross-protection between strains of the two serogroups (Albert et al., 1994). V. cholerae O139 strains carry the El Tor tcpA gene and produce TCP when cultured appropriately in vitro. Studies in the IMCM and in human volunteers indicate that these pili play the same
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pivotal role in O139 pathogenesis as earlier described for V. cholerae O1. O139 tcpA mutant strains fail to colonise infant mice (Attridge et al., 1996; Thelin and Taylor, 1996) or human volunteers (Tacket et al., 1998). Conversely, antibodies to TCP are sufficient for protection of infant mice against O139 challenge strains (Table 1; Voss et al., 1996). As with V. cholerae O1, mutation of the mshA gene does not reduce the colonisation potential of O139 strains (Attridge et al., 1996; Thelin and Taylor, 1996; Tacket et al., 1998).
8. Implications of these studies for vaccine development The numerous field trials conducted to evaluate the efficacy of various parenteral vaccine formulations were collectively disappointing and led to a general acceptance that to be effective a cholera vaccine would need to be orally administered. V. cholerae are non-invasive, so a sufficiently immunogenic vaccine would prime the mucosal immune system of the gut, eliciting IgA antibodies whose location and isotype make them the most appropriate for enteric defence. Two main approaches are being pursued in the quest for an effective cholera vaccine.
8.1. Inacti6ated whole-cell 6accine (WCV) Holmgren and collaborators in Sweden have produced an inactivated vaccine comprising heatand formalin-killed V. cholerae of the four main biotype/serotype combinations. Two parallel formulations have been prepared, one of which is supplemented with the isolated B subunit of the cholera toxin (Holmgren et al., 1994). The rationale for this is that cholera is a toxin-mediated disease, so antibodies which neutralise this factor might be beneficial. Studies in a rabbit model had indicated that antibacterial and antitoxic immune responses can act synergistically in mediating protection (Holmgren et al., 1977). The twin WCV9B preparations were evaluated in an extensive field-trial in Bangladesh. Both were reasonably effective, with cumulative protective efficacies (PEs) of 50% ( +B subunit) and
52% during the first 3 years after vaccination (Clemens et al., 1990); neither conferred any benefit thereafter (van Loon et al., 1996). These vaccines were significantly more effective in recipients who were older than five at the time of immunisation (protection rates of 63% (+ B) and 68%, compared with 26% (+ B) and 23% in recipients aged 2–5 years). They also conferred a higher level of protection against cholera caused by classical V. cholerae (PE 58% (+ B) and 60%) than disease caused by El Tor Strains (PE 39% (+ B) and 40%). Although this vaccine is reasonably effective in the field, various modifications are being considered to improve immunogenicity (particularly in younger recipients at greatest risk of disease) and thereby protective efficacy. One means to achieve this would be to include novel protective antigens. Because the vaccine was developed before the discovery of TCP and MSHA, the vaccine organisms were not cultured appropriately for pilus expression. Initially the addition of MSHA appeared an attractive option (Holmgren et al., 1994), but since then studies in the IMCM (Attridge et al., 1996; Thelin and Taylor, 1996) and recently in human volunteers (Tacket et al., 1998) have failed to support any role for these pili in pathogenesis. On this basis it would seem unlikely that immunity to MSHA would be of any benefit, and indeed our mouse studies found that antibodies to MSHA were not protective (Attridge et al., 1996). The incorporation of TCP into the WCV formulations would seem more appropriate, given the pivotal role of these pili in colonisation by both biotypes of V. cholerae, and data from the infant mouse model showing that antibodies to TCP are sufficient to mediate protection. Since our studies (Voss et al., 1996) suggest that the biotype-restricted pilin epitopes are more relevant for the induction of protective antibodies, the current epidemiology of cholera would indicate that inclusion of El Tor TCP would be more important. However if both biotypes of vaccine organisms were separately cultured for expression of these pili, it might be possible to improve the vaccine’s efficacy against all V. cholerae strains.
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One note of caution in the present context is a retrospective study of immune responses of human volunteers and cholera patients which questioned the immunogenicity of TCP antigens (Hall et al., 1991). If confirmed, this finding would imply that induction of significant anti-TCP responses might not be trivial. However, poor immunogenicity could be a consequence of transient TCP expression in vivo, in which case the administration of several doses of (vast numbers of) inactivated, piliated V. cholerae might circumvent this potential difficulty. In either event, other observations support the view that vaccine organisms should be piliated. TCP expression by live attenuated vaccine strains dramatically improves the vibriocidal responses elicited in human volunteers (Herrington et al., 1988), presumably by promoting colonisation of the mucosal surface. The presence of TCP might similarly facilitate interactions between inactivated vibrios and the intestinal epithelium, and lead to improved immunogenicity. This vaccine approach has already been applied to the development of a formulation which also contains inactivated bacteria of O139 serogroup, in an effort to simultaneously immunise against both O1 and O139 cholera (Jertborn et al., 1996). Since TCP play a similar role in the pathogenesis of both serogroups, the above comments would also apply to the O139 component of this combined vaccine.
8.2. Li6e attenuated V. cholerae 6accines Levine’s group in Baltimore have been attempting to develop a safe yet effective live oral cholera vaccine. The rationale for this approach has been detailed elsewhere (Levine and Tacket, 1994). Essentially, if a pathogenic strain of V. cholerae could be attenuated by deletion of genes encoding the cholera toxin, then the mutated strain might be safe to administer yet retain the propensity to colonise the gut. It would therefore provide a more enduring stimulus to the mucosal immune system, such that a single vaccination might elicit sustained protective immunity. An additional attraction of this approach is that viable organisms would retain the capacity to synthesise those com-
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ponents relevant to pathogenesis in response to in vivo stimuli; among such factors might be as yet unrecognised protective antigens. Progress in this area was for many years hampered by the finding that a succession of vaccine candidates, although indeed capable of eliciting strong immunity after a single immunisation, proved unacceptably reactogenic in human volunteers (Levine and Tacket, 1994). However, a strain called CVD 103-HgR, derived from classical strain 569B, looks most promising. This construct does not cause any higher level of side-effects than placebo vaccines yet is highly immunogenic in a range of recipient groups, including inhabitants of regions where cholera is endemic. It evokes enduring immunity after a single dose and is currently being assessed for efficacy in the field (Levine and Tacket, 1994). The only limitation of CVD 103-HgR apparent from the volunteer studies conducted to date is that it is less protective against challenge strains of El Tor biotype (Levine and Tacket, 1994). The field evaluation of CVD 103-HgR currently underway in Indonesia will indicate whether this vaccine is sufficiently effective in conferring immunity against the much lower doses of El Tor vibrios likely to be encountered during environmental exposure. Several attempts have already been made to construct an El Tor analogue of CVD 103-HgR, with variable success in terms of residual vaccine reactogenicity (Tacket et al., 1993; Kenner et al., 1995; Tacket et al., 1997). Alternatively it might be possible to exploit the demonstrated safety of CVD 103-HgR and slightly modify this strain in response to any demonstrated limitation in cross-biotypic protective efficacy. The only biotype-associated protective antigens described to date are the biotype-restricted epitopes of TCP. It would be quite straightforward to derive from CVD 103HgR a strain in which the classical tcpA gene is replaced with its El Tor homologue. This construct should be non-reactogenic but might be more protective against the El Tor V. cholerae primarily responsible for cholera today. A vaccine comprising a mixed suspension of CVD 103-HgR strains carrying alternative biotypic forms of tcpA might offer adequate protection against disease caused by isolates of either biotype.
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Attempts are also underway to develop an attenuated oral vaccine to combat O139 disease. Two candidate vaccines have conferred encouraging levels of protection in small groups of volunteers (Coster et al., 1995; Tacket et al., 1995) but further testing will be required to establish the safety of these strains.
Acknowledgements We acknowledge the contribution of our collaborators, Gunhild Jonson and Jan Holmgren, and thank the Australian National Health and Medical Research council for financial support.
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S.R. Attridge et al. / Journal of Biotechnology 73 (1999) 109–117 Nair, G.B., Ramamurthy, T., Bhattacharya, S.K., Mukhopadhyay, A.K., Garg, S., Bhattacharya, M.K., Takeda, T., Shimada, T., Takeda, Y., Deb, B.C., 1994. Spread of Vibrio cholerae O139 Bengal in India. J. Infect. Dis. 169, 1029 – 1034. Ogierman, M.A., Voss, E., Meaney, C., Faast, R., Attridge, S.R., Manning, P.A., 1996. Comparison of the promoter proximal regions of the toxin-coregulated tcp gene cluster in classical and El Tor strains of Vibrio cholerae O1. Gene 170, 9 – 16. Osek, J., Jonson, G., Svennerholm, A-M., Holmgren, J., 1994. Role of antibodies against biotype-specific Vibrio cholerae pili in protection against experimental classical and El Tor cholera. Infect. Immun. 62, 2901–2907. Rutter, J.M., Jones, G.W., 1973. Protection against enteric disease caused by Escherichia coli —a model for vaccination with a virulence determinant? Nature 242, 531–532. Rutter, J.M., Jones, G.W., Brown, G.T.H., Burrows, M.R., Luther, P.D., 1976. Antibacterial activity in colostrum and milk associated with protection of piglets against enteric disease caused by K88-positive Escherichia coli. Infect. Immun. 13, 667 – 676. Sharma, D.P., Thomas, C., Hall, R.H., Levine, M.M., Attridge, S.R., 1989. Significance of the toxin-coregulated pili as protective antigens of Vibrio cholerae in the infant mouse model. Vaccine 7, 451–456. Stroeher, U.H., Jedani, K.E., Dredge, B.K., Morona, R., Brown, M.H., Karageorgos, L.E., Albert, M.J., Manning, P.A., 1995. Genetic rearrangements in the rfb regions of Vibrio cholerae O1 and O139. Proc. Natl. Acad. Sci. USA 92, 10374 – 10378. Sun, D., Mekalanos, J.J., Taylor, R.K., 1990. Antibodies directed against the toxin-coregulated pilus isolated from Vibrio cholerae provide protection in the infant mouse experimental cholera model. J. Infect. Dis. 161, 1231– 1236. Tacket, C.O., Losonsky, G., Nataro, J.P., Cryz, S.J., Edelman, R., Fasano, A., Michalski, J., Kaper, J.B., Levine, M.M., 1993. Safety, immunogenicity, and transmissibility of live oral cholera vaccine candidate CVD110, a Dctx Dzot Dace derivative of El Tor Ogawa Vibrio cholerae. J. Infect. Dis. 168, 1536 – 1540. Tacket, C.O., Losonsky, G., Nataro, J.P., Comstock, L., Michalski, J., Edelman, R., Kaper, J.B., Levine, M.M., 1995. Initial clinical studies of CVD112 Vibrio cholerae
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O139 live oral vaccine: safety and efficacy against experimental challenge. J. Infect. Dis. 172, 883 – 886. Tacket, C.O., Kotloff, K.L., Losonsky, G., Nataro, J.P., Michalski, J., Kaper, J.B., Edelman, R., Levine, M.M., 1997. Volunteer studies investigating the safety and efficacy of live oral El Tor Vibrio cholerae O1 vaccine strain CVD111. Am. J. Trop. Med. Hyg. 56, 533 – 537. Tacket, C.O., Taylor, R.K., Losonsky, G., Lim, Y., Nataro, J.P., Kaper, J.B., Levine, M.M., 1998. Investigation of the roles of toxin-coregulated pili and mannose-sensitive haemagglutinin pili in the pathogenesis of Vibrio cholerae O139 infection. Infect. Immun. 66, 692 – 695. Taylor, R.K., Miller, V.L., Furlong, D.B., Mekalanos, J.J., 1987. Use of phoA gene fusions to identify a pilus colonisation factor coordinately regulated with cholera toxin. Proc. Natl. Acad. Sci. USA 84, 2833 – 2837. Taylor, R., Shaw, C., Peterson, K., Mekalanos, J., 1988. Safe, live Vibrio cholerae vaccines? Vaccine 6, 151 – 154. Thelin, K.H., Taylor, R.K., 1996. Toxin-coregulated pilus, but not mannose-sensitive haemagglutinin, is required for colonization by Vibrio cholerae O1 El Tor biotype and O139 strains. Infect. Immun. 64, 2853 – 2856. Ujiiye, A., Kobari, K., 1970. Protective effect on infections with Vibrio cholerae in suckling mice caused by the passive immunization with milk of immune mothers. J. Infect. Dis. 121, S50 – S55. Ujiiye, A., Nakatomi, M., Utsunomiya, A., Mitsui, K., Sogame, S., Iwanaga, M., Kobari, K., 1968. Experimental cholera in mice. I. First report on the oral infection. Trop. Med. 10, 65 – 71. van Loon, F.P.L., Clemens, J.D., Chakraborty, J., Rao, M.R., Kay, B.A., Sack, D.A., Yunus, M., Ali, M., Svennerholm, A-M, Holmgren, J., 1996. Field trial of inactivated oral cholera vaccines in Bangladesh: results from 5 years of follow-up. Vaccine 14, 162 – 166. Voss, E., 1990. The significance of the toxin co-regulated pilus in the pathogenesis of Vibrio cholerae El Tor. Honours thesis, The University of Adelaide. Voss, E, Attridge, S.R., 1993. In vitro production of toxincoregulated pili by Vibrio cholerae El Tor. Microb. Pathog. 15, 255 – 268. Voss, E., Manning, P.A., Attridge, S.R., 1996. The toxincoregulated pilus is a colonization factor and protective antigen of Vibrio cholerae El Tor. Microb. Pathog. 20, 141 – 153.
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Pathogenic and vaccine significance of toxin-coregulated pili of Vibrio cholerae El Tor S.R. Attridge *, E. Voss, P.A. Manning Microbial Pathogenesis Unit, Department of Microbiology and Immunology, The Uni6ersity of Adelaide, Adelaide, South Australia 5005, Australia Received 3 July 1998; accepted 4 January 1999
Abstract Vibrio cholerae O1 strains are classified into one of two biotypes, classical and El Tor, the latter being primarily responsible for cholera cases worldwide since 1961. Recent studies in our laboratory have focused upon the pathogenic and vaccine significance of the toxin-coregulated pili (TCP) produced by strains of El Tor biotype. Mutants in which the tcpA gene (encoding the pilin subunit protein) has been inactivated are dramatically attenuated in the infant mouse cholera model, showing markedly reduced colonisation potential in mixed-infection competition experiments. Significantly, in the vaccine context, antibodies to TCP are sufficient to prevent experimental infection, although our data suggest that this protective effect might be limited to strains of homologous biotype. Since we have shown that tcpA sequences are conserved within a biotype but differ between biotypes, this latter observation suggests that the biotype-restricted pilin epitopes might have greater vaccine significance. Similar studies indicate that TCP also play a critical role in colonisation by strains of the recently-recognised O139 serogroup, which is thought to have evolved from an O1 El Tor strain. In contrast to the effect of introducing mutations in the tcpA gene, strains carrying inactivated mshA genes (encoding the subunit of the m6 annose-s6 ensitive h6 aema6 gglutinin pilus) show unaltered in vivo behaviour. Consistent with this finding is our inability to demonstrate any protective effect associated with antibodies to MSHA. Ongoing approaches to vaccine development are variously aimed at improving the immunogenicity of the current inactivated whole-cell vaccine, or assessing the field efficacy of a promising live attenuated strain. The possible implications of our findings are discussed in relation to both of these options. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Pathogenic; Vaccine; Toxin-coregulated pili; Vibrio cholerae El Tor
1. The discovery of pili in Vibrio cholerae In 1987, Taylor et al. (1987) reported that V. cholerae strains produce pili if cultured appropri* Corresponding author. Fax: +61-8-83034632. E-mail address: [email protected] (S.R. Attridge)
ately in vitro. Individual pilus filaments were aggregated laterally to form typical bundles. Since growth conditions which promoted or repressed pilus production also favoured or inhibited synthesis of the cholera toxin, these structures were called ‘toxin-coregulated pili’ (TCP). The operons responsible for production of both TCP and
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cholera toxin are each part of the so-called toxR regulon, a family of genes whose activation is coordinately controlled by the regulatory protein ToxR (DiRita, 1992). Taylor et al. (1987) went on to show that TCP are essential for efficient gut colonisation by V. cholerae, at least in the infant mouse cholera model (IMCM). This report was quickly followed by human volunteer studies which showed that mutation of either tcpA (encoding the pilin subunit) or toxR dramatically reduced the colonisation potential of a non-toxigenic V. cholerae candidate vaccine strain (Herrington et al., 1988). These reports offered a new approach to those interested in vaccine development, because by analogy with studies performed with other bacterial enteropathogens with similar disease aetiology (such as the K88 pilus-producing enterotoxigenic Escherichia coli (Rutter and Jones, 1973; Rutter et al., 1976)), it was predicted that TCP might elicit antibodies capable of protecting against infection. While this remains to be demonstrated in a clinical setting, further studies in the IMCM soon showed that antibodies to TCP were indeed sufficient to confer passive immunity (Sharma et al., 1989; Sun et al., 1990).
2. The significance of the biotype classification in V. cholerae V. cholerae strains are classified as belonging to either of two biotypes, classical or El Tor, on the basis of biological properties such as bacteriophage- and antibiotic-resistance patterns. The demonstrations of TCP as a critical colonisation factor, and protective antigen, were however restricted to studies using strains of the classical biotype. Southern hybridisation analyses using tcpA-specific probes confirmed that El Tor strains carry a gene homologous to that found in classical V. cholerae (Taylor et al., 1988). However attempts to detect TCP on the surfaces of in vitro-grown El Tor strains were uniformly unsuccessful, despite the testing of a variety of culture conditions (Hall et al., 1988; Sharma et al., 1989; Jonson et al., 1991a). Consistent with this were reports that antibodies to TCP were unable to
mediate significant protection of infant mice against El Tor V. cholerae, even though the same antibodies were effective against classical challenge strains (Sharma et al., 1989; Osek et al., 1994). Since the beginning of the seventh worldwide pandemic in 1961, the vast majority of cholera cases have been attributable to V. cholerae strains of El Tor biotype. It therefore seemed important to elucidate the pathogenic (and hence potential vaccine) significance of TCP for such strains. This became imperative when reports in the early 1990s described a second pilus type in V. cholerae—the mannose-sensitive haemagglutinin (MSHA), so named because of its capacity to agglutinate erythrocytes (Jonson et al., 1991b). These studies suggested that MSHA might be the El Tor analogue of TCP. The latter could only be observed on classical V. cholerae, but during growth in ‘AKI conditions’ (Jonson et al., 1991a) the pilin subunit could be detected by immunoblotting of El Tor strains. In contrast, MSHA could only be visualised on the surfaces of El Tor strains, although classical V. cholerae produced the MshA subunit (Jonson et al., 1991b). We therefore decided to evaluate the significance of TCP as a colonisation factor of El Tor V. cholerae. In particular, we addressed the following issues: 1. do El Tor strains polymerise TcpA into TCP in vitro? 2. would mutation of the tcpA gene affect the colonisation of El Tor strains in the IMCM? 3. are antibodies to TCP sufficient for passive immunity in the same model?
3. El Tor V. cholerae produce TCP in vitro Initial immuno-electron microscopic (IEM) studies failed to detect surface TCP on El Tor strains under conditions in which classical V. cholerae displayed typical pilus bundles (Hall et al., 1988; Sharma et al., 1989; Jonson et al., 1991a). However all these studies used antibodies specific for classical TCP, and subsequent findings in our laboratory and elsewhere suggested that
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this might be a significant limitation to the visualisation of El Tor pili. Our early attempts to construct tcpA mutants in El Tor strains by recombining an inactivated tcpA::KmR construct into the chromosome were unsuccessful (Voss, 1990), although this strategy worked well with classical V. cholerae (Manning, 1992). Since the inactivated gene was of classical origin, it seemed feasible that sequence differences between the two biotypic forms of tcpA might have precluded homologous recombination. Southern hybridisation analyses using probes recognising different regions of the classical tcpA gene sequence suggested that the C-terminal region might indeed differ between the biotypes (Voss, 1990; Ogierman et al., 1996). This led to sequencing studies which confirmed that there are indeed two biotypic tcpA sequences, each being conserved within a biotype, but differing significantly in the C-terminal third of the sequence (Iredell and Manning, 1994). In 1991 Jonson et al. (1991a,b) reported that with appropriate in vitro culture conditions, strains of El Tor biotype produce the TcpA subunit, as detected by immunoblotting using antibodies raised against classical TCP. Although the two biotypic pilin proteins evidently shared antigenic epitopes, the use of monoclonal antibodies revealed that there are also epitopes which are biotype-restricted. This was not surprising, given that the predicted primary structures of the proteins showed numerous amino acid differences (Iredell and Manning, 1994). However it strengthened our view that, to maximise the chance of visualising TCP on El Tor vibrios by IEM, we should prepare sera specific for the El Tor TcpA protein. This work is described elsewhere (Voss and Attridge, 1993). Rabbits were immunised with TcpA-positive outer membrane fractions of El Tor bacteria, and the resulting sera extensively absorbed with similar fractions prepared from tcpA mutants of the homologous strain. This approach allowed the first demonstration of TCP on the surfaces of in vitro grown El Tor V. cholerae. Serum prepared against El Tor TcpA detected pili on all El Tor strains which were TcpA-positive by immunoblotting, but reacted only weakly with TCP on classical vibrios, consistent with the fail-
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ure of initial attempts to visualise El Tor TCP with antibodies to classical pili. Having resolved the question of whether El Tor strains can express TCP, it was next of interest to determine whether these pili were involved in colonisation by strains of this biotype.
4. In vivo behaviour of El Tor tcpA mutants Knowledge of the El Tor tcpA sequence (Iredell and Manning, 1994) allowed us to mutate the gene by insertion of a kanamycin-resistance cartridge. The inactivated gene was then introduced into the chromosome using a strategy initially devised for use with classical biotype strains (Manning 1992). Strain construction was confirmed by Southern hybridisation analysis, and immunoblotting and IEM showed that the mutants failed to produce TcpA/TCP (Attridge et al., 1993). Their in vivo behaviour was then assessed in the IMCM, comparing virulence and colonisation potential with that of their wild-type parents. The IMCM was first devised by Ujiiye et al. (1968) and is in widespread use. Infant mice orally inoculated with a suspension of pathogenic V. cholerae experience a potentially fatal diarrhoeal syndrome, with dramatic bacterial growth in the intestine. Simultaneous LD50 titrations can be used to compare the virulence of two strains, while mixed-infection competition experiments (Attridge 1979; Attridge and Rowley, 1983) allow a direct comparison of colonisation potential. Finally, since pre-treatment of the challenge inoculum with antibodies against protective antigens such as lipopolysaccharide results in protection of the mice (compared with controls receiving untreated V. cholerae; Ujiiye and Kobari, 1970), the model can also be used to define new protective antigens of possible vaccine significance. The introduction of an insertionally-inactivated tcpA gene dramatically attenuated each of four El Tor strains, with LD50 doses increasing ca. 300– 10 000-fold (Attridge et al., 1993). Competition experiments showed that three of the four tcpA mutants retained virtually no colonisation potential. Mice were fed mixed suspensions comprising approximately equal numbers of wild-type and
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mutant bacteria, but 24 h later 104 – 105 of the former were recovered for every persisting mutant organism. The mutation had a less dramatic but still pronounced effect in the fourth strain, with output ratios of about 30:1 in favour of wild-type. Because of the potentially polar effect of the tcpA::KmR mutation, this work was repeated using more refined tcpA mutants, in which the wildtype tcpA gene was replaced with one carrying an in-frame deletion. Similar results were obtained (Voss et al., 1996), confirming the pivotal role of TCP in colonisation of infant mice by V. cholerae O1 El Tor. By analogy with the results of earlier studies with strains of classical biotype, it seemed likely that antibodies to El Tor TCP would mediate passive protection of infant mice, presumably by neutralising the colonisation function of these pili.
5. Assessment of protective efficacy of antibodies to El Tor TCP The data in Table 1 show the results of protection tests in which sera specific for either El Tor or classical TCP were assayed for their capacities to confer passive immunity against challenge strains of either biotype. Two main conclusions can be drawn from the experiments involving V. Table 1 Protective efficacy of antibodies to TCPa Challenge strain (biotype)
Antiserum specificity
El Tor TCP
Classical TCP
V. cholerae O1 H1 (El Tor) N16961 (El Tor) 569B (classical)
570 360 B20
B20 B20 275
V. cholerae O139 AI-1838 AI-1854
340 195
NT NT
a Figures show protective titres of antibodies to El Tor or classical TCP when used to pre-treat various challenge strains in the IMCM. NT, not tested. Adapted from Voss et al. (1996).
cholerae O1 challenge strains. First, antibodies to El Tor TCP are clearly sufficient to protect mice from challenge with strains of homologous biotype. Second, the immunity mediated by antibodies to TCP does not extend to challenge strains of heterologous biotype (Table 1). This suggests that the biotype-restricted antigenic epitopes of TcpA are collectively of much greater protective significance than those epitopes common to both biotypic forms of the pilin. It also offers an explanation for numerous earlier failures to observe protection against El Tor V. cholerae when antibodies to classical TCP have been tested in this model (as discussed elsewhere, Voss et al., 1996).
6. The pathogenic significance of MSHA During the course of our studies a second pilus type, MSHA, was described in El Tor strains of V. cholerae (Jonson et al., 1991b). Investigators in Sweden reported that antibodies to these pili showed protective potential in infant mice (Osek et al., 1994) suggesting a role for MSHA in establishing infection and raising the possibility that this might represent another antigen of vaccine significance. However the notion of multiple pilus adhesins was difficult to reconcile with our observations that tcpA mutants of El Tor strains generally retained minimal residual colonisation potential and were virtually avirulent in the IMCM. In a collaborative study with Drs Jonson and Holmgren from Sweden, we decided to examine the in vivo consequences of inactivating the mshA gene encoding the pilin subunit. El Tor strains were constructed in which the wild-type gene was replaced either with an mshA gene carrying an in-frame deletion, or alternatively with a potentially polar mshA::kmR gene. However neither type of mutant was attenuated for infant mice, nor did either show impaired colonisation when competed against wild-type (Attridge et al., 1996). Fig. 1 shows an example of the results obtained in these experiments. Neither selection of a (spontaneous) streptomycin-resistance marker (to enable differentiation in mixed infection experiments),
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ies to MshA were not protective whereas antibodies to TcpA provided passive immunity against each El Tor strain tested (Attridge et al., 1996).
7. The emergence of V. cholerae O139
Fig. 1. In vivo consequences of mutating tcpA or mshA genes in V. cholerae O1 strain 174. The figure shows results of three competition experiments performed in the same batch of mice, in which the colonisation capacity of wild-type strain 174 was compared with that of a spontaneous streptomycin-resistant (SR) variant of 174 (Expt. 1), the SR variant carrying a mutated mshA gene (Expt. 2), or the SR variant carrying a mutated tcpA gene (Expt. 3). Mice were fed mixed bacterial suspensions and 24 h later their intestines were excised, homogenised, and plated in the presence or absence of streptomycin to allow the separate enumeration of wild-type and mutant bacteria. Each dot represents the (log10) ratio of wild-type: mutant bacteria recovered from a single mouse, with data from Expts. 1 and 2 plotted according to the left axis and from Expt. 3 according to the right axis. Input ratios (IRs) of wild-type: mutant bacteria and median output ratios (MORs) were as follows (seven mice per group): Expt. 1—IR 0.97, MOR 1.3; Expt. 2—IR 0.74, MOR 1.4; Expt. 3—IR 0.72, MOR 36 500.
nor the additional introduction of a deleted mshA gene, significantly affected the colonisation potential of El Tor strain 174. In contrast, inactivating tcpA had the expected dramatic impact on the level of in vivo persistence. Very recently, workers at the Centre for Vaccine Development in Baltimore have conducted volunteer trials (Tacket et al., 1998) which suggest that the relative significance of TCP and MSHA is the same for human infection as we and others (Taylor et al., 1987; Thelin and Taylor, 1996) have described in the infant mouse model. Subsequently we tested antibodies to MSHA and TCP for protective activity in infant mice, using challenge bacteria grown under culture conditions conducive for production of the relevant pilus type. Consistent with the results of the virulence and colonisation experiments which failed to demonstrate an in vivo role for MSHA, antibod-
Until recently only V. cholerae strains of O1 serogroup had shown the potential to establish cholera epidemics, but in 1992 outbreaks of disease occurred in India and Bangladesh which were eventually attributed to isolates of a novel serogroup, O139 (Cholera Working Group, 1993; Nair et al., 1994). For a short period O139 strains displaced V. cholerae O1 as the primary cause of cholera in some regions. The incidence of O139 disease then fell dramatically and remained low for several years, before returning to predominate in Calcutta towards the end of 1996 and through into the middle of 1997 (Ghosh et al., 1997). Although the factors governing the relative incidence of O1 and O139 cholera remain unknown, the emergence of the latter poses a new challenge in terms of vaccine development. V. cholerae O139 evidently arose from a pre-existing O1 strain of El Tor biotype. A region of the genome involved in O-antigen biosynthesis was replaced by horizontal gene transfer, resulting in a novel lipopolysaccharide structure as well as an additional capacity for capsule formation (Bik et al., 1995; Stroeher et al., 1995). The epidemiology of O139 disease differs from that described for O1 cholera in that among inhabitants of endemic regions, the incidence of disease is similar in all age-groups (Cholera Working Group, 1993; Nair et al., 1994). This indicates that naturally acquired pre-existing immunity to V. cholerae O1 provides no immunity against O139 strains, suggesting that vaccines designed to combat O1 disease might be similarly ineffective against V. cholerae O139. This inference is supported by experimental studies which have failed to detect any degree of cross-protection between strains of the two serogroups (Albert et al., 1994). V. cholerae O139 strains carry the El Tor tcpA gene and produce TCP when cultured appropriately in vitro. Studies in the IMCM and in human volunteers indicate that these pili play the same
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pivotal role in O139 pathogenesis as earlier described for V. cholerae O1. O139 tcpA mutant strains fail to colonise infant mice (Attridge et al., 1996; Thelin and Taylor, 1996) or human volunteers (Tacket et al., 1998). Conversely, antibodies to TCP are sufficient for protection of infant mice against O139 challenge strains (Table 1; Voss et al., 1996). As with V. cholerae O1, mutation of the mshA gene does not reduce the colonisation potential of O139 strains (Attridge et al., 1996; Thelin and Taylor, 1996; Tacket et al., 1998).
8. Implications of these studies for vaccine development The numerous field trials conducted to evaluate the efficacy of various parenteral vaccine formulations were collectively disappointing and led to a general acceptance that to be effective a cholera vaccine would need to be orally administered. V. cholerae are non-invasive, so a sufficiently immunogenic vaccine would prime the mucosal immune system of the gut, eliciting IgA antibodies whose location and isotype make them the most appropriate for enteric defence. Two main approaches are being pursued in the quest for an effective cholera vaccine.
8.1. Inacti6ated whole-cell 6accine (WCV) Holmgren and collaborators in Sweden have produced an inactivated vaccine comprising heatand formalin-killed V. cholerae of the four main biotype/serotype combinations. Two parallel formulations have been prepared, one of which is supplemented with the isolated B subunit of the cholera toxin (Holmgren et al., 1994). The rationale for this is that cholera is a toxin-mediated disease, so antibodies which neutralise this factor might be beneficial. Studies in a rabbit model had indicated that antibacterial and antitoxic immune responses can act synergistically in mediating protection (Holmgren et al., 1977). The twin WCV9B preparations were evaluated in an extensive field-trial in Bangladesh. Both were reasonably effective, with cumulative protective efficacies (PEs) of 50% ( +B subunit) and
52% during the first 3 years after vaccination (Clemens et al., 1990); neither conferred any benefit thereafter (van Loon et al., 1996). These vaccines were significantly more effective in recipients who were older than five at the time of immunisation (protection rates of 63% (+ B) and 68%, compared with 26% (+ B) and 23% in recipients aged 2–5 years). They also conferred a higher level of protection against cholera caused by classical V. cholerae (PE 58% (+ B) and 60%) than disease caused by El Tor Strains (PE 39% (+ B) and 40%). Although this vaccine is reasonably effective in the field, various modifications are being considered to improve immunogenicity (particularly in younger recipients at greatest risk of disease) and thereby protective efficacy. One means to achieve this would be to include novel protective antigens. Because the vaccine was developed before the discovery of TCP and MSHA, the vaccine organisms were not cultured appropriately for pilus expression. Initially the addition of MSHA appeared an attractive option (Holmgren et al., 1994), but since then studies in the IMCM (Attridge et al., 1996; Thelin and Taylor, 1996) and recently in human volunteers (Tacket et al., 1998) have failed to support any role for these pili in pathogenesis. On this basis it would seem unlikely that immunity to MSHA would be of any benefit, and indeed our mouse studies found that antibodies to MSHA were not protective (Attridge et al., 1996). The incorporation of TCP into the WCV formulations would seem more appropriate, given the pivotal role of these pili in colonisation by both biotypes of V. cholerae, and data from the infant mouse model showing that antibodies to TCP are sufficient to mediate protection. Since our studies (Voss et al., 1996) suggest that the biotype-restricted pilin epitopes are more relevant for the induction of protective antibodies, the current epidemiology of cholera would indicate that inclusion of El Tor TCP would be more important. However if both biotypes of vaccine organisms were separately cultured for expression of these pili, it might be possible to improve the vaccine’s efficacy against all V. cholerae strains.
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One note of caution in the present context is a retrospective study of immune responses of human volunteers and cholera patients which questioned the immunogenicity of TCP antigens (Hall et al., 1991). If confirmed, this finding would imply that induction of significant anti-TCP responses might not be trivial. However, poor immunogenicity could be a consequence of transient TCP expression in vivo, in which case the administration of several doses of (vast numbers of) inactivated, piliated V. cholerae might circumvent this potential difficulty. In either event, other observations support the view that vaccine organisms should be piliated. TCP expression by live attenuated vaccine strains dramatically improves the vibriocidal responses elicited in human volunteers (Herrington et al., 1988), presumably by promoting colonisation of the mucosal surface. The presence of TCP might similarly facilitate interactions between inactivated vibrios and the intestinal epithelium, and lead to improved immunogenicity. This vaccine approach has already been applied to the development of a formulation which also contains inactivated bacteria of O139 serogroup, in an effort to simultaneously immunise against both O1 and O139 cholera (Jertborn et al., 1996). Since TCP play a similar role in the pathogenesis of both serogroups, the above comments would also apply to the O139 component of this combined vaccine.
8.2. Li6e attenuated V. cholerae 6accines Levine’s group in Baltimore have been attempting to develop a safe yet effective live oral cholera vaccine. The rationale for this approach has been detailed elsewhere (Levine and Tacket, 1994). Essentially, if a pathogenic strain of V. cholerae could be attenuated by deletion of genes encoding the cholera toxin, then the mutated strain might be safe to administer yet retain the propensity to colonise the gut. It would therefore provide a more enduring stimulus to the mucosal immune system, such that a single vaccination might elicit sustained protective immunity. An additional attraction of this approach is that viable organisms would retain the capacity to synthesise those com-
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ponents relevant to pathogenesis in response to in vivo stimuli; among such factors might be as yet unrecognised protective antigens. Progress in this area was for many years hampered by the finding that a succession of vaccine candidates, although indeed capable of eliciting strong immunity after a single immunisation, proved unacceptably reactogenic in human volunteers (Levine and Tacket, 1994). However, a strain called CVD 103-HgR, derived from classical strain 569B, looks most promising. This construct does not cause any higher level of side-effects than placebo vaccines yet is highly immunogenic in a range of recipient groups, including inhabitants of regions where cholera is endemic. It evokes enduring immunity after a single dose and is currently being assessed for efficacy in the field (Levine and Tacket, 1994). The only limitation of CVD 103-HgR apparent from the volunteer studies conducted to date is that it is less protective against challenge strains of El Tor biotype (Levine and Tacket, 1994). The field evaluation of CVD 103-HgR currently underway in Indonesia will indicate whether this vaccine is sufficiently effective in conferring immunity against the much lower doses of El Tor vibrios likely to be encountered during environmental exposure. Several attempts have already been made to construct an El Tor analogue of CVD 103-HgR, with variable success in terms of residual vaccine reactogenicity (Tacket et al., 1993; Kenner et al., 1995; Tacket et al., 1997). Alternatively it might be possible to exploit the demonstrated safety of CVD 103-HgR and slightly modify this strain in response to any demonstrated limitation in cross-biotypic protective efficacy. The only biotype-associated protective antigens described to date are the biotype-restricted epitopes of TCP. It would be quite straightforward to derive from CVD 103HgR a strain in which the classical tcpA gene is replaced with its El Tor homologue. This construct should be non-reactogenic but might be more protective against the El Tor V. cholerae primarily responsible for cholera today. A vaccine comprising a mixed suspension of CVD 103-HgR strains carrying alternative biotypic forms of tcpA might offer adequate protection against disease caused by isolates of either biotype.
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Attempts are also underway to develop an attenuated oral vaccine to combat O139 disease. Two candidate vaccines have conferred encouraging levels of protection in small groups of volunteers (Coster et al., 1995; Tacket et al., 1995) but further testing will be required to establish the safety of these strains.
Acknowledgements We acknowledge the contribution of our collaborators, Gunhild Jonson and Jan Holmgren, and thank the Australian National Health and Medical Research council for financial support.
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