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Live cholera vaccines: perspectives on their construction and safety
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Bull. Inst. Pasteur 1995, 93, 255-262
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1995
Live cholera vaccines : perspectives on their construction and safety J.J. Mekalanos ~7 2, (*), M.K. Waldor (l), C.L. Garde1 (‘1, T.S. Coster c3), J. Kenner c3), K.P. Killeen c4), D.T. Beattie c4), A. Trofa @), D.N. Taylor c5) (**) and J.C. Sadoff c5) (***) (I) Department
of Microbiology
and Molecular Genetics, Harvard Medical School, Boston, MA 02115, c2)Shipley Znstitute of Medicine, (3) Clinical Studies Branch, Medical Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-501 I, f4) Virus Research Institute, Cambridge, MA 02139, (‘1 Department of Clinical Trials, Division of Communicable Diseases and Immunology, Walter Reed Army Institute of Research, Washington, DC 20307 (USA)
There have been seven cholera pandemics in recent recorded history. Despite improvements in health care and sanitation, this disease continues to produce significant morbidity and mortality worldwide. Additionally, the aetiological agent of cholera, Vibrio cholerae, has through this century displayed remarkable genetic shifts that testify to its evolutionary resilience. For example, after causing the first six pandemics of cholera, the classical biotype of V. cholerae serogroup 01 was displaced by the 01 El Tor biotype in 1961, while in turn, the El Tor biotype has most recently been replaced in some locales (perhaps only transiently) with a related but different serogroup called 0139 Bengal. Dramatic changes in the epidemiology of cholera have been the norm through the years, with a recent example being the introduction of El Tor cholera into Latin America after a century long cholera-free hiatas. It is our belief that vaccination against cholera can make an impact on the prevalence and incidence of this disease worldwide. The apparent potential for genetic variation by V. cholerae suggests that the most valuable cholera vaccination strategies would be those that are easily adaptable to the appearance of new strains and serogroups of this organism. While killed, whole cell,
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oral vaccines seem to have great potential in their flexibility of manufacture, it remains a concern that such vaccines may not provide sufficient long-term protection to make them effective (Clemens et al., 1990). Our research has focused on live attenuated cholera vaccines in that these offer much promise in terms of ease of administration (single, oral dose), duration of protection (possibly for life-long), and potential for allowing the “vectoring in” of other heterologous (i.e., non-V. cholerae) antigens (Butterton et al., 1995) which could substantially improve the utility (and perhaps cost effectiveness) of these vaccines. Besides the importance of vaccine efficacy, other key concerns associated with the use of live cholera vaccines generally involve safety (Mekalanos, 1994). The safety issues associated with these vaccines fall under two general categories. First, the vaccine should be safe to the vaccinated individual. Second, the vaccine should be safe to the environment as a whole including non-vaccinated “contacts” exposed to the vaccine. Both categories include adverse scenarios such as reversion of the vaccine by back mutation or acquisition of complementing genes from other indigenous microorganisms. Thus, for the vaccine to be considered theoret-
1995.
(*) Corresponding author. (**) Current address: Naval Medical Research Institute Detachment, Lima, Peru, Unit (***) Current address: Merck Research Laboratories, West Point, PA 29486 (USA).
3800,
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2nd FORUM IN THE BULLETIN ically safe for the vaccinee as well as the environment, it would be best if the specific mutations or phenotypic properties associated with the vaccine’s attenuation were well-defined. In this opinion paper, we will comment on what is known about these two critical safety issues and our strategies for satisfying these concerns. Attenuation of V. cholerae and the construction of live vaccines Cholera is an acute secretory diarrhoeal disease caused by V. cholerae, a highly motile Gramorganism. While there is little argument that the majority of the profuse watery diarrhoea characteristic of cholera is caused by a protein enterotoxin (cholera toxin), it is now exceedingly clear that this organism can cause disease by mechanisms that are not directly linked to production of any other known enterotoxin per se. This conclusion is primarily based on the experience of several groups including our own that have sought to develop safe, live, attenuated cholera vaccines through genetic manipulation of V. cholerue. During the last decade of live cholera vaccine research, various strains deleted in the gene encoding the cholera toxin A subunit (c&A) have been constructed and tested in volunteers. With the exception of strains that were grossly defective in intestinal colonization (e.g., strains carrying mutations in toxR, encoding a regulator of ctxAB and tcpA expression, tcpA, encoding the major subunit of a pilus colonization factor and thyA, encoding a essential biosynthetic enzyme) (Herrington et al., 1988; Levine et al., 1988), the majority of these vaccine candidates were found to be “reactogenic” - a term used to denote adverse symptoms in the vaccinees including diarrhoea, loose stools, abdominal cramps, fever, emesis and malaise (Levine and Tacket, 1994). These disappointing results sparked the discovery over the following years of several putative new enterotoxins that were purported to account for the residual virulence properties of reactogenic c&Adeleted strains (Levine and Tacket, 1994). However, to this date, there is no evidence that any single one of these is responsible for the adverse symptoms (particularly diarrhoea) seen in volunteers ingesting El Tor or 0139 vaccine candidates. Thus, for example, CVDllO (Tacket et al., 1993), deleted in ctxA together with hlyA (encoding a haemolysin), zot (encoding zonula occludens toxin), and ace (encoding accessory cholera enterotoxin), constructed by Kaper, Levine et al. at the University of Maryland Center for Vaccine Development, was not significantly more attenuated than El Tor strains deleted in ctx alone (e.g., JBK70) (Levine et al., 1988). Similar results have been obtained by our own group (Taylor et uZ., 1994; Coster et al., 1995) in that strains
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deleted for recA and the entire CTX genetic element including ctxA, zot and ace and the RS elements (for example, strains Peru-3, Bang-3, Bah-3, and Bengal3) showed a similar reactogenicity profile to vaccine candidates such as CVDl 10, JBK70, or CVDl12 (Tacket et al., 1995). A potential breakthrough in understanding the source of the residual symptoms seen in volunteers ingesting ctxA-deleted live cholera vaccines emerged from our work on the role of motility in reactogenicity. Initially, we observed that Peru- 14, a spontaneous filamentous mutant of Peru-3 which exhibits markedly reduced motility in soft agar, produced virtually no adverse side-effects in volunteers ingesting up to lo9 viable cells (Taylor et al., 1994). Peru-14 was not defective in intestinal colonization per se (i.e., the ability to survive and multiply in the intestine) in that volunteers shed Peru- 14 with kinetics that were similar to Peru-3, responded to the vaccine strain immunologically, and were protected from wild-type challenge several months later. Subsequently, we tested in small groups of volunteers, non-motile mutants of previously reactogenic vaccine strains Peru-3 and Bengal-3. The non-motile mutants Peru-15 and Bengal-15 were no more reactogenic than the placebo buffer but still elicited significant and protective immune responses (Coster et al., 1995; Kenner et al., 1995). Both vaccine strains colonized the gastrointestinal tract of the volunteers as evidenced by positive stool cultures and vigorous systemic immune responses. Recently, another nonmotile mutant was tested in a group of volunteers and like its non-motile sister strains, Bah-15 was also greatly reduced in reactogenicity relative to Bah-3 and another derivative (i.e., CVDllO) of the same highly pathogenic parent strain E7946 (Coster et al., unpublished results). We have proposed a hypothesis to explain why non-motile cholera vaccines are reduced in their reactogenicity (Mekalanos and Sadoff, 1994 ; Coster et al., 1995). Here we will term this the “mucus gel penetration = reactogenicity” hypothesis (see fig. 1). In this theory, we postulate that simply close proximity or contact of bacterial cells to the apical surface of the intestinal epithelium causes reactogenicity perhaps through the induction of a local inflammatory response. Ordinarily, the extensive mucus and glycocalyx coat that covers these epithelial cells prevents such intimate contact with bacteria (pathogenic or otherwise) present in the lumen. However, highly motile, chemotactic strains of V. cholerue are known to penetrate the mucus gel very efficiently and swim deeply into the intervillus spaces and crypts. Thus, non-motile strains are predicted to be limited in their interaction in terms of both the types of epithelial cells they could contact and the sheer numbers of epithelial cells they could physically reach. One
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Mucus Gel Penetration = Reactogenicity Hypothesis Mucosal Immune Response
Cvtokine Release? L&al Inflammation? (Reactogenicity)
0EP
Intestinal Lumen Motile
(e.g.,
Peru-3,
Fllementous, (e.g., Peru-14
e
Nonmotile (e.g., Peru-l
Bengal-3) motIlity-deficIent ) 5, Bengal-15)
0EP
No activity within lumen or not made by nonmotile cells
Fig. 1. The mucus gel penetration=reactogenicity hypothesis. The non-motile vaccine strains Peru-15 and Bengal-H, represented by the non-flagellated vibrios are unable to penetrate the mucus gel and contact underlying epithelial cells. The fdamentous strain Peru-14, is motility-deficient, probably due to its long cell length. We speculate that motile strains elicit a cytokine response from a subset of epithelial cells that are usually protected by a mucus gel coat. The cytokine response initiated by close proximity or contact with motile strains is hypothesized to result in a local inflammatory response which, in turn, is the basis for reactogenicity in volunteers ingesting motile vaccine strains. This cyto kine response is induced by an ‘enterotoxic principle (EP)” which may be constitutively produced by both motile and non-motile cells, but which is largely cell-associated and therefore cannot be delivered to mucus protected epithelial cells by non-motile strains. Alternatively, EP might be made only by motile cells and not by non-motile mutants; this block could be regulatory in that the microenvironment present under the mucus coat and close to the apical surface of the epithelial cell triggers the pro duction of EP only by vibrios that reach this privileged site. The immunogenicity of non-motile strains is hypothesized to involve their more intimate interaction with M cells of the lyphoid follicles. Nonmotile vaccines may be taken up by M cells as efftciently as motile strains because M cells lack a significant mucus or glycocalyx apical coating. Transcytosis of non-motile cells across M cells then elicits a mucosal immune response that is of similar magnitude to that elicited by motile strains.
would expect that non-motile strains would be restricted to the most superficial layers of the intestinal epithelium and that they might contact
directly only those epithelial cells that had little or no mucus or glycocalyx coating their lumenally exposed surfaces.
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One such class of epithelial cells are the microfold or “M cells” that overlay the mucosa-associated lymphoid follicles. M cells are acknowledged to be the principal antigen sampling cells of the mucosa and are further known to lack an extensive mucus and glycocalyx coat on their apical membrane (Kraehenbuhl and Neutra, 1992). Thus, non-motile vaccine strains might interact with M cells just as well as motile vaccine strains given that the mechanical barrier of mucus and glycocalyx is largely absent. Indeed, if antigen endocytosis by M cells is a prerequisite for a mucosal immune response, then the fact that our non-motile vaccine strains elicit protective immune responses (presumably mucosal in nature) in volunteers is a strong indication that M cell contact was established during colonization by these strains (Taylor et al., 1994; Coster et al., 1995). We envision that motility-deficient vaccine strains multiply predominantly within the intestinal lumen and outer mucus layers, but that these bacterial cells are freely able to bind to M cells and then be endocytosed and processed by the immune cells of the follicle (fig. 1). We currently are exploring various possibilities for why close proximity to or contact of V. cholerae with intestinal epithelial cells might induce adverse reactions in volunteers (see fig. 1). First is the idea that a cytokine cascade might be induced by such epithelial contact alone. Epithelial cells have been found to produce the lymphokine IL6 in response to the presence of adherent E. coli (Hedges et al, 1992). Cultured epithelial cells have also been shown to release the neutrophil chemotactic chemokine IL8 in the presence of Salmonella typhimurium (McCormick et al., 1993). If cytokines are produced by intestinal epithelial cells in response to close proximity or contact by live V. cholerae cells, then these potent signal molecules could initiate a local inflammatory response which could alone account for the adverse symptoms seen in recipients of motile vaccine strains. Non-motile strains might fail to induce this response because they either do not interact as extensively with the mucus coated epithelial cells or alternatively, are not able to deliver the bacterial molecule that initiates this inflammatory response (fig. 1). Mechanistically, the bacterial molecule that initiates the postulated cytokine response and which ultimately causes reactogenicity might be considered formally an enterotoxin. However, if such a molecule is a component of the cell envelope (e.g., lipopolysaccharide, a peptidoglycan precursor/ breakdown fragment, outermembrane protein, etc.) or a bacterial surface structure (e.g., pili, adhesins, flagella, etc.), it is probably best not to consider it an enterotoxin per se. Instead we would like to designate the bacterial component which is responsible for the reactogenicity of motile vaccine strains by the term “enterotoxic principle” for simplicity.
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Motility might then affect the functional activity of the enterotoxic principle (EP) in conceptually different ways. First, in accordance with the “mucus gel penetration=reactogenicity” hypothesis, the delivery of the EP to its target tissue might be affected mechanically by reduced motility. Thus, the EP might always be expressed by bacterial cells (as would be the case if, for example, lipopolysaccharide or peptidoglycan-related material was the enterotoxic principle) but its delivery to the critical responsive host target cells might not occur unless motility brings the EP molecules very close to the effector cell (where local concentration and diffusion of EP would be optimized). Alternatively, the motility phenotype of V. cholerae might directly affect the expression of EP at either the transcriptional or post-transcriptional levels. In support of this possibility, we have observed that the motility phenotype of V. cholerue can dramatically affect the expression of a variety of different molecules implicated in cholera pathogenesis (Garde1 and Mekalanos, submitted). For example, we have isolated both spontaneous hyperswarmer (exhibiting faster swarm rates in soft agar) and spontaneous non-motile V. cholerae mutants and found that hyperswarmer mutants were defective in expression of TCP pili and cholera toxin but significantly, were dramatically upregulated in the expression of proteases. In contrast, all non-motile mutants examined showed significantly increased expression of toxin coregulated pili, cholera toxin, and a cell-as&o ciated haemolysin but decreased levels of fucose-sensitive haemagglutinin and HEp-2 cell adhesins. In general, non-motile mutants displayed little or no defects in intestinal colonization in suckling mice, while hypermotile mutants were highly impaired in colonization. These results strongly suggest that the motility phenotype of V. cholerae is tightly coupled to the expression of multiple ToxR-regulated and non-ToxR-regulated virulence determinants. If one of the phenotypes that are downregulated in non-motile mutants represent the enterotoxic principle, then this could explain the lack of reactogenicity of non-motile vaccine strains. Alternatively, if a molecule that is constitutively expressed or even upregulated in nonmotile mutants (e.g., cell-associated haemolysin) turns out to be the enterotoxic principle, then delivery of EP via the directed movement of vibrios to the proximity of target host cells is likely to be the key step missing in the colonization process by nonmotile strains that accounts for their reduced reactogenicity (see fig. 1). Genetic stability and safety of live cholera vaccines As noted in the beginning of this paper, we believe that the genetic variability of V. cholerue suggests that a cognitive approach to the construc-
CHOLERA tion of attenuated, live cholera vaccines would provide greater flexibility in adjusting the vaccine formulation in response to changes in the bacterium’s biology (i.e., its epidemiology, biotype, serogroup, etc.). The studies described above have moved us substantially closer to the development of a “genetic blueprint” for safe and effective cholera vaccines. Such a genetic blueprint consists in theory of a set of defined mutations which when incorporated into any virulent strain of V. cholerae (regardless of biotype, serogroup, or serotype), can be expected to produce a protective vaccine strain that is sufficiently attenuated for human use and environmental release.
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and Tacket, 1994), along with cep, a gene encoding a putative pilin-like colonization factor (Pearson et al., 1993). This core region is flanked by repetitive sequences called RSl or simply RS sequences (Pearson et al., 1993). The RS sequences encode a site-specific recombination system that mediates recombination between the CTX genetic element and chromosomal copies of attRS1, a 17-18 base pair target sequence present multiple times at various locations within the CTX genetic element (Pearson et al., 1993).
The blueprint we have developed (Mekalanos and Sadoff, 1994; Taylor et al., 1994; Waldor and Mekalanos, 1994a; Coster et al., 1995) involves the introduction of three different classes of mutations. The first step is the deletion of the entire transposable “CTX element” which includes genes encoding cholera toxin (CT), two other potential enterotoxins (Zot and Ace), a potential colonization factor (Cep), and the RS site-specific recombination system (see below). In the second step, the gene encoding the non-toxic B subunit of CT under control of a heatshock promoter is introduced in place of the recA gene, rendering the strains defective in homologous recombination activity. The last step involves introduction of a mutation affecting the motility of the vaccine strain; we previously used spontaneous mutations for this step, but have recently been examining the utility of defined mutations in motility and chemotaxis genes including the V. cholerae homologue of the mofB gene of E. coli (C. Garde1 and I. Mekalanos, unpublished results). We believe that this genetic strategy will allow us to construct additional live attenuated V. cholerae vaccine strains that display similar low levels of reactogenicity, high levels of immunogenicity and maximum levels of environmental safety. Such a cognitive approach should also facilitate the construction of V. cholerae vector vaccines expressing heterologous antigens (Butterton et al., 1995).
The structure and activity of the CTX genetic element suggests that it might be capable of tmnsposing back into vaccine derivatives if transferred by conjugation or transduction from toxigenic strains. Accordingly, we ultimately devised constructions that ensure that the chance of reacquisition of the CTX element by vaccine strains would be remote. The first step in our vaccine constructions has been deletion of the entire CTX element using a pair of Hind111 sites that flank the ends of the element (Pearson et aE., 1993). This “attRS1 deletion” removes the core, all RS and attRS1 sequences. Deletion of all attRS1 sites is presumably required to eliminate CTX element site-specific recombination (Pearson et al., 1993). Thus, we considered spontaneous core deletion mutants which we had previously constructed (Goldberg and Mekalanos, 1986) to be less than satisfactory because they retained copies of attRS1 on each end of the chromosomal RSl sequences that remained in such strains. A recently described vaccine candidate (CVDl 10) suffers from the same theoretical problem since it was constructed in part by spontaneous deletion of the CTX core and thus retains a copy of RSl (and its associated attRS1 sites) on its chromosome (Michalski et al., 1993). Thus, strain CVDllO is still capable in theory of reacquiring the CTX element by site-specific recombination in contrast to our larger attRS1 deletion derivatives (Pearson et al., 1993; Taylor et al., 1994) which are defective in CTX sitespecific recombination.
In regard to genetic stability, live cholera vaccines should be engineered so that they are unable to regain toxigenicity by reversion or recombination. For a variety of early vaccine candidates, recombinant DNA technology allowed the deletion of the gene encoding the toxic A subunit of cholera toxin, thus theoretically eliminating the possibility of reversion (Mekalanos et al., 1983; Levine et al., 1988). However, work in our laboratory established early on that the ctxAB genes of V. cholerae reside on a large genetic element termed the “CTX genetic element” which has the properties of a site-specific transposon and which can amplify its copy number in vivo (Mekalanos, 1983). In addition to ctxAB, two other putative toxin genes, zot and ace have been localized to the central core of the element (Levine
In addition to reacquisition of the CTX element by an RS l-mediated, site-specific recombinational mechanism, vaccine strains could also, in theory, regain cholera toxin genes by homologous recombination after the element was introduced back into the vaccine strain by DNA transfer from a toxigenic donor by transformation, transduction or conjugation. Therefore, after introduction of attRS1 deletions in our constructs, we have performed a second step, the deletion of recA, a gene encoding an enzyme essential for homologous recombination (Taylor et al., 1994; Coster et al., 1995). These deletions were accomplished by a marker exchange reaction that simultaneously deleted recA sequences and replaced them with the ctxB expressed from the powerful heat shock promoter of the htpG gene
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(Waldor and Mekalanos, 1994a). In theory, the combination of the recA and attRS1 deletions provides a level of recombinational safety that is unprecedented in any other live bacterial vaccine currently under evaluation. The suggestion that CTX genes can be mobilized between strains of V. cholerae is far from theoretical. The mapping of the ctx structural genes by conjugation provided an early example of this possibility (Sporecke et al., 1984). More recently, experimental evidence that the ctxA- vaccine strain CVD103-HgR can reacquire c&A from a V. cholerue strain possessing the conjugative sex factor P has been reported (Kaper et al., 1994). There is no easy way to score the frequency of gene transfer and recombination in natural settings, but both undoubtedly occur. For example, it is now quite clear that the new 0139 serogroup of V. cholerae 0139 arose by horizontal gene transfer and recombination between some unknown donor and an El Tor 01 recipient (Waldor and Mekalanos, 1994b; Bik et al., 1995). Furthermore, DNA sequence analysis of the “house-keeping” gene asd in 45 clinical and environmental isolates of V. cholerae suggests that V. cholerae is capable of a higher level of genetic exchange than Salmonella enterica and Escherichia coli species (Karaolis et al., 1995). The most extensively tested, recombinant cholera vaccine strain to date, CVD 103-HgR (Levine and Tacket, 1994), may not fulfill rigorous criteria that should define vaccine strains with full enviromnental safety (Mekalanos, 1994). Although this strain carries a ctxA deletion, the other mutation(s) that renders this strain relatively non-reactogenic in volunteers is undefined and therefore may be subject to reversion. For example, although CVD 103-HgR carries a mercury resistance gene inserted into the hly locus, this mutation has not been shown to be associated with detectable attenuation in other vaccine candidates (Levine and Tacket, 1994). It is clear that CVD 103-HgR can reacquire a wild type ctxA gene by homologous recombination (Kaper et al., 1994) and that it still has multiple copies of the attRS1 sequence (thus providing ample target sites for reacquisition of the CTX element by site-specific recombination). Although a recA mutation has been introduced into a CVD 103-HgR derivative, this mutation adversely affected this strain’s immunogenicity and colonization properties (Ketley et al., 1990). Because several El Tor and 0139 vaccine candidates we have examined have tolerated recA deletions well in volunteer studies (Taylor et al., 1994; Coster et al., 1995), it cannot be concluded that recA is incompatible with the immunogenicity of live cholera vaccines per se. Nonetheless, the recA + strain CVD 103-HgR has been tested in extensive outpatient studies in several countries and is currently the focus of a large field trial in Indone-
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sia (Levine and Tacket, 1994). The risks of widespread use of such a strain are unknown. If the scenario of reacquisition of cholera toxin genes by CVD 103-HgR in fact occurs during field trials or widescale use of this vaccine, then it would result in the appearance of toxigenic strains of the classical biotype. V. cholerae strains of the classical biotype have been entirely replaced by El Tor strains worldwide with the possible exception of Southern Bangladesh. Because CVD 103-HgR is a derivative of strain 569B (Levine and Tacket, 1994), a hypertoxigenic strain of the classical biotype of V. cholerue, it is conceivable that toxigenic recombinants of CVD 103-HgR might exhibit higher levels of toxin production and other characteristics uniquely associated with classical strains. These new characteristics could provide the driving force for expansion of new toxigenic recombinants even in the presence of indigenous resident El Tor strains. For example, it is generally acknowledged that the most important intestinal colonization factor of V. cholerae is the toxin coregulated pilus (TCP). The major subunit of TCP shows only 82% amino acid sequence identity between classical and El Tor strains and this level of sequence divergence is considerably greater than that seen for the cholera toxin subunits between the two biotypes (Rhine and Taylor, 1994). This result strongly suggests that either anti-TcpA immune pressure within cholera endemic populations or selection for alteration in TcpA function (e.g., variation in receptor binding activity) has driven the more rapid evolution of the tcpA gene. Thus, the tcpA gene of CVD 103-HgR might encode an antigen (classical TcpA) which could allow a c&A+ recombinant of this vaccine strain to escape protective immune responses against the El Tor TcpA. We believe that one cannot predict whether a toxigenic classical strain (such as a hypothetical CVD 103HgR ctnA+ recombinant) will be more, or less evolutionarily or epidemiologically “fit”, relative to El Tor strains when introduced for the first time into a human population that has an unknown genetic susceptibility or immune status to cholera. This discussion introduces another aspect to evaluating the environmental safety of live vaccines that has not received much formal consideration (Mekalanos, 1994). That is, to fully assess the environmental safety of live vaccine strains, one should consider the impact of the DNA that a vaccine strain might transfer to indigenous V. cholerae strains; it should be noted that killed vaccines strain also contain DNA, although one presumes that the chances of this DNA being released and then taken up by indigenous strains is presumably low. For example, it might be argued that a risk exists for transfer of a classical copy of the tcpA gene from CVD 103-HgR into resident toxigenic El Tor strains at the Indonesian field trial site. CVD 103-HgR would, of course,
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need to acquire a DNA mobilizing system (e.g., a conjugative sex factor or transducing phage) but once this occurred, tcpA or any other gene associated uniquely with the classical biotype might then be mobilized into El Tor strains. If these classical biotype genes increased the fitness of resident toxigenie El Tor strains within an El Tor immune population, then a new epidemic or pandemic clone might emerge. Similarly, a possibility exists for the transfer of the genes encoding the 0139 lipopolysaccharide 0 antigen from an 0139 vaccine strain to resident, toxigenic El Tor 01 strains in a locale that might initially lack indigenous 0139 strains. In this regard, we have recently observed that a recA mutation eliminates P factor and non-P-factor-mediated conjugative donor activity in live oral 0139 vaccine Bengal-15 (Haider et al., submitted). Loss of recA resulted in a 2-3 log reduction in the ability of Bengal-15 to transfer an antibiotic resistance gene to El Tor recipient strains and this defect could be complemented by a cloned copy of the wild-type recA gene. Furthermore, because recA mutations render bacterial organisms highly sensitive to ultraviolet light, our vaccine constructs Bengal- 15 and Peru-15 are much more sensitive to killing by sunlight and thus are, in theory, crippled in terms of their ability to survive in environmental reservoirs outside of the host’s gastrointestinal tract. Low survival of the vaccine strain outside of the host should also dramatically decrease the chances of recombination between strains. Such results reaffirm our assertion that recA mutations greatly improve the theoretical environmental safety of live cholera vaccines and thus is one of the more valued features of our recently described El Tor 01 and 0139 vaccine candidates (Taylor et al., 1994; Coster et al., 1995). Clearly the use of any vaccine (live or dead) ultimately involves an evaluation of the relative risks. We should prudently try to minimize the chance that a rare genetic event involving the vaccine strain could produce recombinant strains with pathogenic potential. However, such a goal needs to be balanced with the extreme need for effective cholera vaccines in epidemic and endemic settings where the environmental “load” of fully pathogenic strains is already quite high. For example, the threat of cholera in refugee camps is so high and its impact potentially so devastating that theoretical arguments about the environmental safety and stability of live cholera vaccine strains should not play a major role in deciding whether or not to implement their use in such a setting. Similarly, the use of safe, attenuated live cholera vaccines by travelers in developed countries where environmental contamination with toxigenic V. cholerue is already minimal or nonexistent should be considered very low risk. In developed countries, the absence of endemic cholera
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and the relatively higher levels of sanitation and hygiene would together make it highly unlikely that live V. cholerue vaccine strains would contact resident toxigenic recipient or donor strains in the gastrointestinal tract of vaccinees or in the environment. High levels of public sanitation would also not be conducive to the spread of rare recombinant strains to vaccinee contacts even if they did manage to somehow emerge.
Conclusions As a field, we still know little about the ecological and molecular aspects of the emergence of new V. cholerue strains, persistence of these strains in host populations and the environment, and finally the displacement of resident strains by newly emerged variants. Clearly a cognitive approach to the construction of live cholera vaccines is highly desirable in terms of safety considerations, the adaptablity of such an approach to changes in the epidemiology and biology of V. cholerue and lastly, to further development of this organism as a vector for delivery of heterologous antigens to the mucosal immune system. Live attenuated cholera vaccines should contain ideally a defined set of mutations responsible for the observed attenuation. It also seems highly prudent to consider broad use of only live attenuated cholera vaccines that have been engineered to minimize their chance of reacquiring through recombination the critical virulence determinants that were deleted in their construction (e.g., cholera toxin). Such strains should also be designed to reduce the likelihood of transfer of DNA from the vaccine strains to indigenous environmental or clinical isolates of toxigenic V. cholerue residing in cholera endemic and epidemic locales.
References Bik, E.M., Bunschoten, A.E., Gouw, R.D. & Mooi, F. (1995), Genesis of the novel epidemic Vibrio cholerue 0139 strain: evidence for horizontal transfer of genes involved in polysaccharide synthesis. EMBO J., 14, 209-216. Butterton, J.R., Beattie, D.T., Gardel, C.L., Carroll, P.A., Hyman, T., Killeen, K.P., Mekalanos, J.J. & Calderwood, S.B. (1995), Heterologous antigen expression in Vibrio cholerae vector strains. Infect. Immun., 63, 2689-2696. Clemens, J.D., Sack, D.A., Harris, J.R., Loon, F.V., Chakraborty, J., Ahmed, F., Rao, M.R., Khan, M.R., Yunus, M. & Huda, N. (1990), Field trial of oral cholera vaccines in Bangladesh: results from threeyear follow-up. Lance& 335, 270-273. Coster, T.S., Killeen, K.P., Waldor, M.K., Beattie, D., Spriggs, D., Kenner, J.R., Trofa, A., Sadoff, J., Mekalanos, J.J. & Taylor, D.N. (1995), Safety, immunog-
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enicity and efficacy of a live attenuated Vibrio cholerae 0139 vaccine protype, Bengal-15 Lancer, 345, 949-952. Goldberg, I. & Mekalanos, J.J. (1986), Effect of a recA mutation on cholera toxin gene amplification and deletion events. J. Bacterial., 165, 723-731. Hedges, S., Svensson, M. & Svanborg, C. (1992), Interleukin-6 response of epithelial cell lines to bacterial stimulation in vitro. Infect, Immun., 60, 1295-1301. Herrington, D.A., Hall, R.H., Losonsky, G., Mekalanos, J.J., Taylor, R.K. & Levine, M.M. (1988), Toxin, toxin-coregulated pili, and the roxR regulon are essential for Vibrio cholerae pathogenesis in humans. J. Exp. Med., 168, 1487-1492. Kaper, J.B., Michalski, J., Ketley, J.M. & Levine, M.M. (1994), Potential for reacquisition of cholera enterotoxin genes by attenuated Vibrio cholerae vaccine strain CVD 103-HgR. Infect. Zmmun., 62, 1480-1483. Karaolis, D.K., Lan, R. & Reeves, P.R. (1995), The sixth and seventh cholera pandemics are due to independent clones separately derived from environmental, nontoxigenic, non-01 Vibrio cholerae. J. Bacterial., 177, 3191-3198. Kenner, J., Coster, T., Trofa, A., Taylor, D., Barrera-Oro, M., Hyman, T., Adams, J., Beattie, D., Killeen, K., Mekalanos, J.J. & Sadoff, J.C. (1995), Peru-15, a live, attenuated oral vaccine candidate for Vibrio cholerae 01 El Tor. J. Infect. Dis. (in press). Ketley, J.M., Kaper, J.B., Herrington, D.A., Losonsky, G. & Levine, M.M. (1990). Diminished immunogenicity of a recombination-deficient derivative of Vibrio cholerae vaccine strain CVD103. Infect. Immun., 58, 1481-1484. Kraehenbuhl, J.P. & Neutra, M.R. (1992), Molecular and cellular basis of immune protection of mucosal surfaces. Physiol. Rev., 72, 853-879. Levine, M.M., Kaper, J.B., Herrington, D., Losonsky, G., Morris, J.G., Clements, M., Black, R.E., Tall, B. & Hall, R. (1988), Volunteer studies of deletion mutants of Vibrio cholerae 01 prepared by recombinant techniques. Infect. Immun., 56, 161-167. Levine, M.M. & Tacket, C.O. (1994), Recombinant live cholerae vaccines, in “Vibrio cholerae and cholera” (Wachsmuth, I.K., Blake, P.A. and Olsvik, O., ed.) (pp. 395-413). ASM Press. Washington, D.C. McCormick, B.A., Colgan, S.P., Archer, CD., Miller, S.I. & Madara, J.L. (1993), Salmonella typhimurium attachment to human intestinal epithelial monolayers : transcellular signalling to subepithelial neutrophil. .I. Cell Biol., 123, 895907.
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Mekalanos, J.J. (1983), Duplication and amplification of toxin genes in Vibrio cholerae. Cell, 35, 253-263. Mekalanos, J.J. (1994), Live bacterial vaccines: environmental aspects. Curr. Opin. Biotechnol., 5, 3 12-3 19. Mekalanos, J.J. & Sadoff, J.C. (1994), Cholera vaccines: fighting an ancient scourge. Science, 265, 1387-1389. Mekalanos, J.J., Swartz, D.J., Pearson, G.D., Harford, N., Groyne, F. & de Wilde, M. (1983), Cholera toxin genes : nucleotide sequence, deletion analysis and vaccine development. Nature (Lond.), 306, 551-557. Michalski, J., Galen, J.E., Fasano, A. & Kaper, J.B. (1993), CVDllO, an attenuated Vibrio cholerae 01 El Tor live oral vaccine strain. Infect. Immun., 61,44624468. Pearson, G.D.N., Woods, A., Chiang, S.L. & Mekalanos, J.J. (1993), CTX genetic element encodes a site-specific recombination system and an intestinal colonization factor. Proc. Natl. Acad. Sci. USA, 90, 37503754. Rhine, J.A. & Taylor, R.K. (1994), TcpA pilin sequences and colonization requirements for 01 and 0139 Vibrio cholerae. Mol. Microbial., 13, 1013-1020. Sporecke, I., Castro, D. & Mekalanos, J.J. (1984). Genetic mapping of Vibrio cholerae enterotoxin structural genes. J. Bacterial., 157, 253-261. Tacket, C., Losonsky, G., Nataro, J., Comstock, L., Michalski, J., Edelman, R., Kaper, J. & Levine, M. (1995), Initial clinical studies of CVD 112 Vibrio cholerae 0139 live oral vaccine: safety and efficacy against experimental challenge. J. Znfect. Dis., 172, 883-886. Tacket, CO., Losonsky, G., Nataro, J.P., Cryz, S.J., Edelman, R., Fasano, A., Michalski, J., Kaper, J.B. & Levine, M.M. (1993), Safety and immunogenicity of live oral cholera vaccine candidate CVD 110, a AcrxA Azot Aace derivative of El Tor Ogawa Vibrio cholerae. J. Infect. Dis., 168, 1536-1540. Taylor, D.N., Killeen, K.P., Hack, D.C., Kenner, J.R., Coster, T.S., Beattie, D.T., Ezzell, J., Hyman, T., Trofa, A., Sjogren, M.H., Friedlander, A., Mekalanos, J.J. & Sadoff, J.C. (1994), Development of a live, oral, attenuated vaccine against El Tor cholera. J. Infect. LG., 170, 1518-1523. Waldor, M.K. & Mekalanos, J.J. (1994a), Emergence of a new cholera pandemic: molecular analysis of virulence determinants in Vibrio cholerae 0139 and development of a live vaccine prototype. J. Infect. Dis., 170, 278-283. Waldor, M.K. & Mekahmos, JJ. (1994b), Vibrio cholerae 0139 specific gene sequences. Lancet, 343, 1366.
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Live cholera vaccines : perspectives on their construction and safety J.J. Mekalanos ~7 2, (*), M.K. Waldor (l), C.L. Garde1 (‘1, T.S. Coster c3), J. Kenner c3), K.P. Killeen c4), D.T. Beattie c4), A. Trofa @), D.N. Taylor c5) (**) and J.C. Sadoff c5) (***) (I) Department
of Microbiology
and Molecular Genetics, Harvard Medical School, Boston, MA 02115, c2)Shipley Znstitute of Medicine, (3) Clinical Studies Branch, Medical Division, U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick, Frederick, MD 21702-501 I, f4) Virus Research Institute, Cambridge, MA 02139, (‘1 Department of Clinical Trials, Division of Communicable Diseases and Immunology, Walter Reed Army Institute of Research, Washington, DC 20307 (USA)
There have been seven cholera pandemics in recent recorded history. Despite improvements in health care and sanitation, this disease continues to produce significant morbidity and mortality worldwide. Additionally, the aetiological agent of cholera, Vibrio cholerae, has through this century displayed remarkable genetic shifts that testify to its evolutionary resilience. For example, after causing the first six pandemics of cholera, the classical biotype of V. cholerae serogroup 01 was displaced by the 01 El Tor biotype in 1961, while in turn, the El Tor biotype has most recently been replaced in some locales (perhaps only transiently) with a related but different serogroup called 0139 Bengal. Dramatic changes in the epidemiology of cholera have been the norm through the years, with a recent example being the introduction of El Tor cholera into Latin America after a century long cholera-free hiatas. It is our belief that vaccination against cholera can make an impact on the prevalence and incidence of this disease worldwide. The apparent potential for genetic variation by V. cholerae suggests that the most valuable cholera vaccination strategies would be those that are easily adaptable to the appearance of new strains and serogroups of this organism. While killed, whole cell,
Received September
11,
oral vaccines seem to have great potential in their flexibility of manufacture, it remains a concern that such vaccines may not provide sufficient long-term protection to make them effective (Clemens et al., 1990). Our research has focused on live attenuated cholera vaccines in that these offer much promise in terms of ease of administration (single, oral dose), duration of protection (possibly for life-long), and potential for allowing the “vectoring in” of other heterologous (i.e., non-V. cholerae) antigens (Butterton et al., 1995) which could substantially improve the utility (and perhaps cost effectiveness) of these vaccines. Besides the importance of vaccine efficacy, other key concerns associated with the use of live cholera vaccines generally involve safety (Mekalanos, 1994). The safety issues associated with these vaccines fall under two general categories. First, the vaccine should be safe to the vaccinated individual. Second, the vaccine should be safe to the environment as a whole including non-vaccinated “contacts” exposed to the vaccine. Both categories include adverse scenarios such as reversion of the vaccine by back mutation or acquisition of complementing genes from other indigenous microorganisms. Thus, for the vaccine to be considered theoret-
1995.
(*) Corresponding author. (**) Current address: Naval Medical Research Institute Detachment, Lima, Peru, Unit (***) Current address: Merck Research Laboratories, West Point, PA 29486 (USA).
3800,
APO
AA
34031.
2nd FORUM IN THE BULLETIN ically safe for the vaccinee as well as the environment, it would be best if the specific mutations or phenotypic properties associated with the vaccine’s attenuation were well-defined. In this opinion paper, we will comment on what is known about these two critical safety issues and our strategies for satisfying these concerns. Attenuation of V. cholerae and the construction of live vaccines Cholera is an acute secretory diarrhoeal disease caused by V. cholerae, a highly motile Gramorganism. While there is little argument that the majority of the profuse watery diarrhoea characteristic of cholera is caused by a protein enterotoxin (cholera toxin), it is now exceedingly clear that this organism can cause disease by mechanisms that are not directly linked to production of any other known enterotoxin per se. This conclusion is primarily based on the experience of several groups including our own that have sought to develop safe, live, attenuated cholera vaccines through genetic manipulation of V. cholerue. During the last decade of live cholera vaccine research, various strains deleted in the gene encoding the cholera toxin A subunit (c&A) have been constructed and tested in volunteers. With the exception of strains that were grossly defective in intestinal colonization (e.g., strains carrying mutations in toxR, encoding a regulator of ctxAB and tcpA expression, tcpA, encoding the major subunit of a pilus colonization factor and thyA, encoding a essential biosynthetic enzyme) (Herrington et al., 1988; Levine et al., 1988), the majority of these vaccine candidates were found to be “reactogenic” - a term used to denote adverse symptoms in the vaccinees including diarrhoea, loose stools, abdominal cramps, fever, emesis and malaise (Levine and Tacket, 1994). These disappointing results sparked the discovery over the following years of several putative new enterotoxins that were purported to account for the residual virulence properties of reactogenic c&Adeleted strains (Levine and Tacket, 1994). However, to this date, there is no evidence that any single one of these is responsible for the adverse symptoms (particularly diarrhoea) seen in volunteers ingesting El Tor or 0139 vaccine candidates. Thus, for example, CVDllO (Tacket et al., 1993), deleted in ctxA together with hlyA (encoding a haemolysin), zot (encoding zonula occludens toxin), and ace (encoding accessory cholera enterotoxin), constructed by Kaper, Levine et al. at the University of Maryland Center for Vaccine Development, was not significantly more attenuated than El Tor strains deleted in ctx alone (e.g., JBK70) (Levine et al., 1988). Similar results have been obtained by our own group (Taylor et uZ., 1994; Coster et al., 1995) in that strains
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deleted for recA and the entire CTX genetic element including ctxA, zot and ace and the RS elements (for example, strains Peru-3, Bang-3, Bah-3, and Bengal3) showed a similar reactogenicity profile to vaccine candidates such as CVDl 10, JBK70, or CVDl12 (Tacket et al., 1995). A potential breakthrough in understanding the source of the residual symptoms seen in volunteers ingesting ctxA-deleted live cholera vaccines emerged from our work on the role of motility in reactogenicity. Initially, we observed that Peru- 14, a spontaneous filamentous mutant of Peru-3 which exhibits markedly reduced motility in soft agar, produced virtually no adverse side-effects in volunteers ingesting up to lo9 viable cells (Taylor et al., 1994). Peru-14 was not defective in intestinal colonization per se (i.e., the ability to survive and multiply in the intestine) in that volunteers shed Peru- 14 with kinetics that were similar to Peru-3, responded to the vaccine strain immunologically, and were protected from wild-type challenge several months later. Subsequently, we tested in small groups of volunteers, non-motile mutants of previously reactogenic vaccine strains Peru-3 and Bengal-3. The non-motile mutants Peru-15 and Bengal-15 were no more reactogenic than the placebo buffer but still elicited significant and protective immune responses (Coster et al., 1995; Kenner et al., 1995). Both vaccine strains colonized the gastrointestinal tract of the volunteers as evidenced by positive stool cultures and vigorous systemic immune responses. Recently, another nonmotile mutant was tested in a group of volunteers and like its non-motile sister strains, Bah-15 was also greatly reduced in reactogenicity relative to Bah-3 and another derivative (i.e., CVDllO) of the same highly pathogenic parent strain E7946 (Coster et al., unpublished results). We have proposed a hypothesis to explain why non-motile cholera vaccines are reduced in their reactogenicity (Mekalanos and Sadoff, 1994 ; Coster et al., 1995). Here we will term this the “mucus gel penetration = reactogenicity” hypothesis (see fig. 1). In this theory, we postulate that simply close proximity or contact of bacterial cells to the apical surface of the intestinal epithelium causes reactogenicity perhaps through the induction of a local inflammatory response. Ordinarily, the extensive mucus and glycocalyx coat that covers these epithelial cells prevents such intimate contact with bacteria (pathogenic or otherwise) present in the lumen. However, highly motile, chemotactic strains of V. cholerue are known to penetrate the mucus gel very efficiently and swim deeply into the intervillus spaces and crypts. Thus, non-motile strains are predicted to be limited in their interaction in terms of both the types of epithelial cells they could contact and the sheer numbers of epithelial cells they could physically reach. One
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Mucus Gel Penetration = Reactogenicity Hypothesis Mucosal Immune Response
Cvtokine Release? L&al Inflammation? (Reactogenicity)
0EP
Intestinal Lumen Motile
(e.g.,
Peru-3,
Fllementous, (e.g., Peru-14
e
Nonmotile (e.g., Peru-l
Bengal-3) motIlity-deficIent ) 5, Bengal-15)
0EP
No activity within lumen or not made by nonmotile cells
Fig. 1. The mucus gel penetration=reactogenicity hypothesis. The non-motile vaccine strains Peru-15 and Bengal-H, represented by the non-flagellated vibrios are unable to penetrate the mucus gel and contact underlying epithelial cells. The fdamentous strain Peru-14, is motility-deficient, probably due to its long cell length. We speculate that motile strains elicit a cytokine response from a subset of epithelial cells that are usually protected by a mucus gel coat. The cytokine response initiated by close proximity or contact with motile strains is hypothesized to result in a local inflammatory response which, in turn, is the basis for reactogenicity in volunteers ingesting motile vaccine strains. This cyto kine response is induced by an ‘enterotoxic principle (EP)” which may be constitutively produced by both motile and non-motile cells, but which is largely cell-associated and therefore cannot be delivered to mucus protected epithelial cells by non-motile strains. Alternatively, EP might be made only by motile cells and not by non-motile mutants; this block could be regulatory in that the microenvironment present under the mucus coat and close to the apical surface of the epithelial cell triggers the pro duction of EP only by vibrios that reach this privileged site. The immunogenicity of non-motile strains is hypothesized to involve their more intimate interaction with M cells of the lyphoid follicles. Nonmotile vaccines may be taken up by M cells as efftciently as motile strains because M cells lack a significant mucus or glycocalyx apical coating. Transcytosis of non-motile cells across M cells then elicits a mucosal immune response that is of similar magnitude to that elicited by motile strains.
would expect that non-motile strains would be restricted to the most superficial layers of the intestinal epithelium and that they might contact
directly only those epithelial cells that had little or no mucus or glycocalyx coating their lumenally exposed surfaces.
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One such class of epithelial cells are the microfold or “M cells” that overlay the mucosa-associated lymphoid follicles. M cells are acknowledged to be the principal antigen sampling cells of the mucosa and are further known to lack an extensive mucus and glycocalyx coat on their apical membrane (Kraehenbuhl and Neutra, 1992). Thus, non-motile vaccine strains might interact with M cells just as well as motile vaccine strains given that the mechanical barrier of mucus and glycocalyx is largely absent. Indeed, if antigen endocytosis by M cells is a prerequisite for a mucosal immune response, then the fact that our non-motile vaccine strains elicit protective immune responses (presumably mucosal in nature) in volunteers is a strong indication that M cell contact was established during colonization by these strains (Taylor et al., 1994; Coster et al., 1995). We envision that motility-deficient vaccine strains multiply predominantly within the intestinal lumen and outer mucus layers, but that these bacterial cells are freely able to bind to M cells and then be endocytosed and processed by the immune cells of the follicle (fig. 1). We currently are exploring various possibilities for why close proximity to or contact of V. cholerae with intestinal epithelial cells might induce adverse reactions in volunteers (see fig. 1). First is the idea that a cytokine cascade might be induced by such epithelial contact alone. Epithelial cells have been found to produce the lymphokine IL6 in response to the presence of adherent E. coli (Hedges et al, 1992). Cultured epithelial cells have also been shown to release the neutrophil chemotactic chemokine IL8 in the presence of Salmonella typhimurium (McCormick et al., 1993). If cytokines are produced by intestinal epithelial cells in response to close proximity or contact by live V. cholerae cells, then these potent signal molecules could initiate a local inflammatory response which could alone account for the adverse symptoms seen in recipients of motile vaccine strains. Non-motile strains might fail to induce this response because they either do not interact as extensively with the mucus coated epithelial cells or alternatively, are not able to deliver the bacterial molecule that initiates this inflammatory response (fig. 1). Mechanistically, the bacterial molecule that initiates the postulated cytokine response and which ultimately causes reactogenicity might be considered formally an enterotoxin. However, if such a molecule is a component of the cell envelope (e.g., lipopolysaccharide, a peptidoglycan precursor/ breakdown fragment, outermembrane protein, etc.) or a bacterial surface structure (e.g., pili, adhesins, flagella, etc.), it is probably best not to consider it an enterotoxin per se. Instead we would like to designate the bacterial component which is responsible for the reactogenicity of motile vaccine strains by the term “enterotoxic principle” for simplicity.
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Motility might then affect the functional activity of the enterotoxic principle (EP) in conceptually different ways. First, in accordance with the “mucus gel penetration=reactogenicity” hypothesis, the delivery of the EP to its target tissue might be affected mechanically by reduced motility. Thus, the EP might always be expressed by bacterial cells (as would be the case if, for example, lipopolysaccharide or peptidoglycan-related material was the enterotoxic principle) but its delivery to the critical responsive host target cells might not occur unless motility brings the EP molecules very close to the effector cell (where local concentration and diffusion of EP would be optimized). Alternatively, the motility phenotype of V. cholerae might directly affect the expression of EP at either the transcriptional or post-transcriptional levels. In support of this possibility, we have observed that the motility phenotype of V. cholerue can dramatically affect the expression of a variety of different molecules implicated in cholera pathogenesis (Garde1 and Mekalanos, submitted). For example, we have isolated both spontaneous hyperswarmer (exhibiting faster swarm rates in soft agar) and spontaneous non-motile V. cholerae mutants and found that hyperswarmer mutants were defective in expression of TCP pili and cholera toxin but significantly, were dramatically upregulated in the expression of proteases. In contrast, all non-motile mutants examined showed significantly increased expression of toxin coregulated pili, cholera toxin, and a cell-as&o ciated haemolysin but decreased levels of fucose-sensitive haemagglutinin and HEp-2 cell adhesins. In general, non-motile mutants displayed little or no defects in intestinal colonization in suckling mice, while hypermotile mutants were highly impaired in colonization. These results strongly suggest that the motility phenotype of V. cholerae is tightly coupled to the expression of multiple ToxR-regulated and non-ToxR-regulated virulence determinants. If one of the phenotypes that are downregulated in non-motile mutants represent the enterotoxic principle, then this could explain the lack of reactogenicity of non-motile vaccine strains. Alternatively, if a molecule that is constitutively expressed or even upregulated in nonmotile mutants (e.g., cell-associated haemolysin) turns out to be the enterotoxic principle, then delivery of EP via the directed movement of vibrios to the proximity of target host cells is likely to be the key step missing in the colonization process by nonmotile strains that accounts for their reduced reactogenicity (see fig. 1). Genetic stability and safety of live cholera vaccines As noted in the beginning of this paper, we believe that the genetic variability of V. cholerue suggests that a cognitive approach to the construc-
CHOLERA tion of attenuated, live cholera vaccines would provide greater flexibility in adjusting the vaccine formulation in response to changes in the bacterium’s biology (i.e., its epidemiology, biotype, serogroup, etc.). The studies described above have moved us substantially closer to the development of a “genetic blueprint” for safe and effective cholera vaccines. Such a genetic blueprint consists in theory of a set of defined mutations which when incorporated into any virulent strain of V. cholerae (regardless of biotype, serogroup, or serotype), can be expected to produce a protective vaccine strain that is sufficiently attenuated for human use and environmental release.
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and Tacket, 1994), along with cep, a gene encoding a putative pilin-like colonization factor (Pearson et al., 1993). This core region is flanked by repetitive sequences called RSl or simply RS sequences (Pearson et al., 1993). The RS sequences encode a site-specific recombination system that mediates recombination between the CTX genetic element and chromosomal copies of attRS1, a 17-18 base pair target sequence present multiple times at various locations within the CTX genetic element (Pearson et al., 1993).
The blueprint we have developed (Mekalanos and Sadoff, 1994; Taylor et al., 1994; Waldor and Mekalanos, 1994a; Coster et al., 1995) involves the introduction of three different classes of mutations. The first step is the deletion of the entire transposable “CTX element” which includes genes encoding cholera toxin (CT), two other potential enterotoxins (Zot and Ace), a potential colonization factor (Cep), and the RS site-specific recombination system (see below). In the second step, the gene encoding the non-toxic B subunit of CT under control of a heatshock promoter is introduced in place of the recA gene, rendering the strains defective in homologous recombination activity. The last step involves introduction of a mutation affecting the motility of the vaccine strain; we previously used spontaneous mutations for this step, but have recently been examining the utility of defined mutations in motility and chemotaxis genes including the V. cholerae homologue of the mofB gene of E. coli (C. Garde1 and I. Mekalanos, unpublished results). We believe that this genetic strategy will allow us to construct additional live attenuated V. cholerae vaccine strains that display similar low levels of reactogenicity, high levels of immunogenicity and maximum levels of environmental safety. Such a cognitive approach should also facilitate the construction of V. cholerae vector vaccines expressing heterologous antigens (Butterton et al., 1995).
The structure and activity of the CTX genetic element suggests that it might be capable of tmnsposing back into vaccine derivatives if transferred by conjugation or transduction from toxigenic strains. Accordingly, we ultimately devised constructions that ensure that the chance of reacquisition of the CTX element by vaccine strains would be remote. The first step in our vaccine constructions has been deletion of the entire CTX element using a pair of Hind111 sites that flank the ends of the element (Pearson et aE., 1993). This “attRS1 deletion” removes the core, all RS and attRS1 sequences. Deletion of all attRS1 sites is presumably required to eliminate CTX element site-specific recombination (Pearson et al., 1993). Thus, we considered spontaneous core deletion mutants which we had previously constructed (Goldberg and Mekalanos, 1986) to be less than satisfactory because they retained copies of attRS1 on each end of the chromosomal RSl sequences that remained in such strains. A recently described vaccine candidate (CVDl 10) suffers from the same theoretical problem since it was constructed in part by spontaneous deletion of the CTX core and thus retains a copy of RSl (and its associated attRS1 sites) on its chromosome (Michalski et al., 1993). Thus, strain CVDllO is still capable in theory of reacquiring the CTX element by site-specific recombination in contrast to our larger attRS1 deletion derivatives (Pearson et al., 1993; Taylor et al., 1994) which are defective in CTX sitespecific recombination.
In regard to genetic stability, live cholera vaccines should be engineered so that they are unable to regain toxigenicity by reversion or recombination. For a variety of early vaccine candidates, recombinant DNA technology allowed the deletion of the gene encoding the toxic A subunit of cholera toxin, thus theoretically eliminating the possibility of reversion (Mekalanos et al., 1983; Levine et al., 1988). However, work in our laboratory established early on that the ctxAB genes of V. cholerae reside on a large genetic element termed the “CTX genetic element” which has the properties of a site-specific transposon and which can amplify its copy number in vivo (Mekalanos, 1983). In addition to ctxAB, two other putative toxin genes, zot and ace have been localized to the central core of the element (Levine
In addition to reacquisition of the CTX element by an RS l-mediated, site-specific recombinational mechanism, vaccine strains could also, in theory, regain cholera toxin genes by homologous recombination after the element was introduced back into the vaccine strain by DNA transfer from a toxigenic donor by transformation, transduction or conjugation. Therefore, after introduction of attRS1 deletions in our constructs, we have performed a second step, the deletion of recA, a gene encoding an enzyme essential for homologous recombination (Taylor et al., 1994; Coster et al., 1995). These deletions were accomplished by a marker exchange reaction that simultaneously deleted recA sequences and replaced them with the ctxB expressed from the powerful heat shock promoter of the htpG gene
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(Waldor and Mekalanos, 1994a). In theory, the combination of the recA and attRS1 deletions provides a level of recombinational safety that is unprecedented in any other live bacterial vaccine currently under evaluation. The suggestion that CTX genes can be mobilized between strains of V. cholerae is far from theoretical. The mapping of the ctx structural genes by conjugation provided an early example of this possibility (Sporecke et al., 1984). More recently, experimental evidence that the ctxA- vaccine strain CVD103-HgR can reacquire c&A from a V. cholerue strain possessing the conjugative sex factor P has been reported (Kaper et al., 1994). There is no easy way to score the frequency of gene transfer and recombination in natural settings, but both undoubtedly occur. For example, it is now quite clear that the new 0139 serogroup of V. cholerae 0139 arose by horizontal gene transfer and recombination between some unknown donor and an El Tor 01 recipient (Waldor and Mekalanos, 1994b; Bik et al., 1995). Furthermore, DNA sequence analysis of the “house-keeping” gene asd in 45 clinical and environmental isolates of V. cholerae suggests that V. cholerae is capable of a higher level of genetic exchange than Salmonella enterica and Escherichia coli species (Karaolis et al., 1995). The most extensively tested, recombinant cholera vaccine strain to date, CVD 103-HgR (Levine and Tacket, 1994), may not fulfill rigorous criteria that should define vaccine strains with full enviromnental safety (Mekalanos, 1994). Although this strain carries a ctxA deletion, the other mutation(s) that renders this strain relatively non-reactogenic in volunteers is undefined and therefore may be subject to reversion. For example, although CVD 103-HgR carries a mercury resistance gene inserted into the hly locus, this mutation has not been shown to be associated with detectable attenuation in other vaccine candidates (Levine and Tacket, 1994). It is clear that CVD 103-HgR can reacquire a wild type ctxA gene by homologous recombination (Kaper et al., 1994) and that it still has multiple copies of the attRS1 sequence (thus providing ample target sites for reacquisition of the CTX element by site-specific recombination). Although a recA mutation has been introduced into a CVD 103-HgR derivative, this mutation adversely affected this strain’s immunogenicity and colonization properties (Ketley et al., 1990). Because several El Tor and 0139 vaccine candidates we have examined have tolerated recA deletions well in volunteer studies (Taylor et al., 1994; Coster et al., 1995), it cannot be concluded that recA is incompatible with the immunogenicity of live cholera vaccines per se. Nonetheless, the recA + strain CVD 103-HgR has been tested in extensive outpatient studies in several countries and is currently the focus of a large field trial in Indone-
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sia (Levine and Tacket, 1994). The risks of widespread use of such a strain are unknown. If the scenario of reacquisition of cholera toxin genes by CVD 103-HgR in fact occurs during field trials or widescale use of this vaccine, then it would result in the appearance of toxigenic strains of the classical biotype. V. cholerae strains of the classical biotype have been entirely replaced by El Tor strains worldwide with the possible exception of Southern Bangladesh. Because CVD 103-HgR is a derivative of strain 569B (Levine and Tacket, 1994), a hypertoxigenic strain of the classical biotype of V. cholerue, it is conceivable that toxigenic recombinants of CVD 103-HgR might exhibit higher levels of toxin production and other characteristics uniquely associated with classical strains. These new characteristics could provide the driving force for expansion of new toxigenic recombinants even in the presence of indigenous resident El Tor strains. For example, it is generally acknowledged that the most important intestinal colonization factor of V. cholerae is the toxin coregulated pilus (TCP). The major subunit of TCP shows only 82% amino acid sequence identity between classical and El Tor strains and this level of sequence divergence is considerably greater than that seen for the cholera toxin subunits between the two biotypes (Rhine and Taylor, 1994). This result strongly suggests that either anti-TcpA immune pressure within cholera endemic populations or selection for alteration in TcpA function (e.g., variation in receptor binding activity) has driven the more rapid evolution of the tcpA gene. Thus, the tcpA gene of CVD 103-HgR might encode an antigen (classical TcpA) which could allow a c&A+ recombinant of this vaccine strain to escape protective immune responses against the El Tor TcpA. We believe that one cannot predict whether a toxigenic classical strain (such as a hypothetical CVD 103HgR ctnA+ recombinant) will be more, or less evolutionarily or epidemiologically “fit”, relative to El Tor strains when introduced for the first time into a human population that has an unknown genetic susceptibility or immune status to cholera. This discussion introduces another aspect to evaluating the environmental safety of live vaccines that has not received much formal consideration (Mekalanos, 1994). That is, to fully assess the environmental safety of live vaccine strains, one should consider the impact of the DNA that a vaccine strain might transfer to indigenous V. cholerae strains; it should be noted that killed vaccines strain also contain DNA, although one presumes that the chances of this DNA being released and then taken up by indigenous strains is presumably low. For example, it might be argued that a risk exists for transfer of a classical copy of the tcpA gene from CVD 103-HgR into resident toxigenic El Tor strains at the Indonesian field trial site. CVD 103-HgR would, of course,
CHOLERA
need to acquire a DNA mobilizing system (e.g., a conjugative sex factor or transducing phage) but once this occurred, tcpA or any other gene associated uniquely with the classical biotype might then be mobilized into El Tor strains. If these classical biotype genes increased the fitness of resident toxigenie El Tor strains within an El Tor immune population, then a new epidemic or pandemic clone might emerge. Similarly, a possibility exists for the transfer of the genes encoding the 0139 lipopolysaccharide 0 antigen from an 0139 vaccine strain to resident, toxigenic El Tor 01 strains in a locale that might initially lack indigenous 0139 strains. In this regard, we have recently observed that a recA mutation eliminates P factor and non-P-factor-mediated conjugative donor activity in live oral 0139 vaccine Bengal-15 (Haider et al., submitted). Loss of recA resulted in a 2-3 log reduction in the ability of Bengal-15 to transfer an antibiotic resistance gene to El Tor recipient strains and this defect could be complemented by a cloned copy of the wild-type recA gene. Furthermore, because recA mutations render bacterial organisms highly sensitive to ultraviolet light, our vaccine constructs Bengal- 15 and Peru-15 are much more sensitive to killing by sunlight and thus are, in theory, crippled in terms of their ability to survive in environmental reservoirs outside of the host’s gastrointestinal tract. Low survival of the vaccine strain outside of the host should also dramatically decrease the chances of recombination between strains. Such results reaffirm our assertion that recA mutations greatly improve the theoretical environmental safety of live cholera vaccines and thus is one of the more valued features of our recently described El Tor 01 and 0139 vaccine candidates (Taylor et al., 1994; Coster et al., 1995). Clearly the use of any vaccine (live or dead) ultimately involves an evaluation of the relative risks. We should prudently try to minimize the chance that a rare genetic event involving the vaccine strain could produce recombinant strains with pathogenic potential. However, such a goal needs to be balanced with the extreme need for effective cholera vaccines in epidemic and endemic settings where the environmental “load” of fully pathogenic strains is already quite high. For example, the threat of cholera in refugee camps is so high and its impact potentially so devastating that theoretical arguments about the environmental safety and stability of live cholera vaccine strains should not play a major role in deciding whether or not to implement their use in such a setting. Similarly, the use of safe, attenuated live cholera vaccines by travelers in developed countries where environmental contamination with toxigenic V. cholerue is already minimal or nonexistent should be considered very low risk. In developed countries, the absence of endemic cholera
VACCINES
and the relatively higher levels of sanitation and hygiene would together make it highly unlikely that live V. cholerue vaccine strains would contact resident toxigenic recipient or donor strains in the gastrointestinal tract of vaccinees or in the environment. High levels of public sanitation would also not be conducive to the spread of rare recombinant strains to vaccinee contacts even if they did manage to somehow emerge.
Conclusions As a field, we still know little about the ecological and molecular aspects of the emergence of new V. cholerue strains, persistence of these strains in host populations and the environment, and finally the displacement of resident strains by newly emerged variants. Clearly a cognitive approach to the construction of live cholera vaccines is highly desirable in terms of safety considerations, the adaptablity of such an approach to changes in the epidemiology and biology of V. cholerue and lastly, to further development of this organism as a vector for delivery of heterologous antigens to the mucosal immune system. Live attenuated cholera vaccines should contain ideally a defined set of mutations responsible for the observed attenuation. It also seems highly prudent to consider broad use of only live attenuated cholera vaccines that have been engineered to minimize their chance of reacquiring through recombination the critical virulence determinants that were deleted in their construction (e.g., cholera toxin). Such strains should also be designed to reduce the likelihood of transfer of DNA from the vaccine strains to indigenous environmental or clinical isolates of toxigenic V. cholerue residing in cholera endemic and epidemic locales.
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