Res. Microbiol. 1990, 141, 913-919

(~) INSTITUTPASTEUR/ELsEVIER Paris 1990

H Y B R I D VACCINES U S I N G E S C H E R I C H I A C O L I AS A N A N T I G E N C A R R I E R

T.L. Hale Department of Enteric Infections, Walter Reed Army Institute of Research, Washington, D.C. 20307-5100

Introduction. The distal ileum and large intestine in primates supports a stable flora consisting of several hundred bacterial species with a predominance of strict anaerobes (109 to 10n/g of faeces). In addition to the strictly anaerobic flora, there is usually a small complement of facultative aerobes of the family Enterobacteriaceae (usually 106/g of faeces) and the most commonly isolated component of this flora is Escherichia coil Since the discovery of fertility factors (F+) in conjugative plasmids of E. coli, the transfer of chromosomal DNA from high-frequency recombinant (Hi',-) donors to F- E. coli recipients has become a routine technique of bacterial genetics. Transformation and transduction techniques of gene transfer are also easily accomplished in E. coil recipients. Therefore, the capacity of E. coil to colonize the intestine along with the ability to serve as a recipient for foreign DNA suggest that this speci~.s could be used as a carder of heterologous antigens in oral vaccines.

A non-invasive E. coli hybrid vaccine expressing hetero~ogous antigens. An early attemr~t to construct a hybrid E. coli vaccine expressing heterologous antigens involved the conjugal transfer of the His and Met chromosomal markers from a Shigellaflexneri 2a Hfr into an E. coli 08 recipient (Levine et al., 1977). Since genes encoding the S. flexneri group 3,4 antigen are linked to His and genes encoding the type II serotype are linked to Met, the resulting hybrid (PGAI42-1-15) expressed the 2a somatic antigen of the donor strain. When given to volunteers at a 3 x 10 20 dosage, this vaccine was shed for up to two weeks, and only 2 070 of vaccines experienced nausea or vomiting in conjunction with oral inoculation. Even though the PGA 142-1-15 hybrid transiently colonized the intestine, however, less than 10 070 of vaccinees had a significant rise in serum IgG recognizing the S. flexneri 2a somatic antigen. More significantly, vaccination -vith 2 or 3 doses o f PGAI42-1-15 elicited no protection from a subsequent oral challenge with an ID40 of virulent S. flexneri 2a. The failure of this E. coli..S, flexneri hybrid to evoke a protective immune response against a homologous ,.. flexneri serotype suggests that colonization of the intestinal lumen provides little antigenic stimulus to the gut associated lymphoid tissue (GALT).

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E. eoli hybrid vaccines expressing the Shigella invasive phenotype. Approximately four years after the testing of PGA142-1-15, it was discovered that the genes encoding the enteroinvasive phenotype of Shigella species are located on large mobilizable plasmids. Shortly thereafter, it was found that transfer of the virulence plasmid from S. flexneri serotype 5 into E. coli K12 confers a shigella-like ability to invade HeLa cells in tissue culture (Sansonetti et al., 1983; and table I). This invasive phenotype is closely associated with the ability of shigellae to infect the intestinal epithelium in orally challenged guinea pigs or Rhesus monkeys (LaBrec et al., 1964). Recently it has been shown that virulel~t shigellae preferentially parasitize the membranous follicle-associated epithelial cells (M cells) in ligated rabbit ileal loops (Wassef et al., 1989) and invasive shigellae are also ingested by macrophages more efficiently than are non-invasive mutants (Clerc et al., 1987). These observations sugge:;t that E. coil expressing the shigella invasive phenotype might efficiently deliver antigens to the GALT. An E. coli K12 hybrid vaccine designated EC104 (Formal et al., 1984) was constructed by conjugal mobilization of the S. flexneri 5 virulence plasmid followed by the conjugal transfer of the His and Pro markers from an S. flexneri 2a Hfr. Like PGAI42-1-15, this vaccine expresses the S. flexneri 2a group and type somatic antigen, but it has the additional capacity to invade HeLa cells and to multiply within the cytoplasm of infected host cells. EC 104 evokes an inflammatory response in ligated rabbit ileal loops but, unlike virulent shigellae, this hybrid does not evoke fluid accumulation in ileal loops nor does it evoke keratoconjunctivitis in the corneal epithelium of guinea pigs (Sereny test). EC104 was tested for safety and efficacy in Rhesus monkeys by oral administration of two doses of 1 x 10 n organisms. This vaccine regimen was associated with diarrhoea or dysentery in 15 070 of the monkeys, but the aetiology of this reaction was questionable because, in addition to EC104, S. flexneri 4 was isolated from the symptomatic monkeys. A second group of monkeys was inoculated with three doses of 1 x 10 l0 EC104 and no intestinal symptoms were observed; 30 070 of these animals had significant rises in serum IgG recognizing the S. flexneri 2a somatic antigen, and when the monkeys inoculated with either regimen of EC104 were challenged with virulent S. fiexneri 2a, there was 75 070 protection when compared to unvaccinated controls (Formal et al., 1984). The seroconversion and protective efficacy evoked by EC104 in the monkey model suggests that enteroinvasive E. coli-S, flexneri hybrid vaccines can effectively elicit a mucosal imnmne response in subhuman primates, and these preclinal studies provided support for safety and efficacy trials of such vaccines in humans. In preliminary safety trials, EC104 evoked transient diarrhoea in 25 070 of volunteers, but the doseresponse curve was puzzling in that lower doses (i.e., I x 10T) were reactogenic while higher doses did not evoke intestinal symptoms (S.B. Formal, unpublished data). When a similar vaccine was subsequently tested in volunteers at an oral dosage of 1 x 109, 30 o70 of these individuals experienced transient diarrhoea or dysentery. In contrast, smaller doses (1 x 10~ organisms) colonized the it~testine of volunteers for up to three days in the absence of intestinal sympto~ns, but three doses of this regimen did not evoke protection against a subsequent IDs0 challenge (1 x 103) of S. flexneri 2a (M.M. Levine, unpublished data).

CFA EAF EPEC GALT Hfr icsA

= = = = = =

colonizationfactor antigen. EPEC adherence factor. enteropathogenicEscherichia coil gut-associatedlymphoid tissue. high-frequencyrecombinant(donor). intercellularspread.

kcpA = keratoconjunctivitisprovocation. M cell = membranous follicle-associated epithelial cell. PABA = paraamino benzoic acid. sod = superoxide-dismutase.

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Although human trials suggest that enteroinvasive E. coli-S, flexneri hybrids are attenuated by at least four orders of magnitude when compared to virulent S. flexneri 2a, the residual reactogenicity of these vaccines raises questions concerning the basis of this attenuation. Since the wild-type shigella virulence plasmid has been transferred into the E. coil KI2 background, attenuation in the hybrid vaccine is dependent upon differences between the E. coli and S. flexneri chromosome. One shigella virulence locus that is absent in E. coli KI2 chromosome encodes the aerobactin ironbinding siderophore system (Derbyshire et al., 1989). Although aerobactin mutants of S. flexneri are able to invade and multiply within HeLa cells, insertional inactivation of the aerobactin structural gene (iucA) results in a 10- to 100-fold attenuation in the ligated rabbit loop or Sereny test models (Nassif et al., 1987). Attenuation of E. coli-S, flexneri hybrids is also linked to the kcpA (keratoconjunctivitis provocation) locus that regulates a shigella plasmid gene designated icsA (intercellular spread). Since mutations in either kcpA (Formal et al., 1971) or icsA (Markino et al., 1986) attenuate S. flexneri in the keratoconjunctivitis Sereny test, intercellular spread of invasive organisms is apparently critical for the formation of lesions in the epithelial mucosa. Due to a naturally occurring attenuation of the kcpA locus, the E. coli KI2 chromosome does not support expression of the 120-kDal icsA protein product. As a result, spread of intracellular organisms rarely occurs in tissue culture monolayers infected with E. coil K12 hybrids carrying an S. flexneri invasion plasmid (Pal et al., 1989). An iuc-icsA S. flexneri vaccine designated SC5700 illustrates the attenuation associated with this combination of mutations in a shigella background (Sansonetti and Arondel, 1989). SC5700 elicits limited mucosal pathology in ligated rabbit ileal loops with a small amount of fluid accumulation, and colonoscopic examination of orally vaccinated rhesus monkeys revealed lymphoid hyperplasia suggestive of GALT stimulation (P.J. Sansonetti, submitted for publication). This hyperplasia suggests that the M cells overlying lymphoid follicles are infected by SC5700, but these organisms are unable to spread to the contiguous colonic epithelium. Three doses of 5 × 1010 SC5700 elicits mild mucoid diarrhoea in a minority of rhesus monkeys, and the vaccinated animals are significantly protected against a subsequent challenge with virulent S. flexneri. However, the residual reactogenicity of this iuc-icsA mutant vaccine in animal models suggests insufficient attenuation for human use. The delicate balance between immunogenicity and reactogenicity in vaccines expressing the enteroinvasive phenotype of Shigella species is probably a function of the intracellular lifestyle of these organisms. Both S. flexneri and E. coil KI2 carrying the S. flexneri invasion plasmid rapidly lyse endocytic vacuoles in cultured HeLa cells or macrophages, inhibit host cell protein synthesis, multiply freely within the cytoplasm and rapidly kill infected cells (Clerc et aL, 1987). Therefore, inhibition of intracellular multiplication may be required for adequate attenuation of enteroinvasive vaccines. This approach has been employed in an auxotropic S. flexneri Y vaccine that has a transposon insertion in the aroD chromosomal gene (Lindberg et al., 1988). This mutant requires an exogenous supply of paraamino benzoic acid (PABA), a precursor of folic acid. Since this metabolic intermediate is not available in mammaiian cells, intracellular multiplication of aroD mutants is limited. Likewise, mutations in the S. flexneri superoxide dismutase gene (sodB) limit the survival of shigellae within macrophages or polymorphonuclear leukocytes, and inactivation of sodB greatly decreases the virulence of S. flexneri in the ligated rabbit ileal loop model (Franzon et al., 1990). Potential E. coil hybrid vaccines expressing the Salmonella or Yersinla invasive

phenotype.

The cloning of chromosomal gene libraries from Salmonella typhi (Elsinghorst et al., 1989) or Yersinia pseudotuberculosis (Isberg et al., 1987) has allowed the ex-

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pression of the invasive phenot~l~es of these species in an E. coli background (table ]3. Although there is no published application of this technology to hybrid vaccine construction, it is possible E. coli hybrids expressing these invasion determinants cot dd be used as oral vaccines targeting heterologous antigens to the M cells and Peyer's patches of the ileum and jejunum. For example, the ability of S. typhimurium to invade Henle 407 cells correlates with the ability of these orgnisms to colonize the Peyer's patches in orally challer~ged mice (Galan and Curtiss, 1989). By analogy, the invasive phenotype cloned from S. typhi into E. coli could allow the coloniza1:ion of Pcyer's patches in humans. T~te association of Y. pseudotuberculosis with mesenteric lymphadenitis and terminal ileitis in humans suggests that E. coil expressing the invasive phenotype of this species could also preferentially colonize the ileal lymphoid tissue in humans. The invasion determinants of S. typhi Ty2 include at least four loci (invABCD) distributed within a 33-kilobase chromosomal region, and invasion of Henle 407 ceds by E. coli HB101 carrying this cloned region occurs at only 10 °7o of the leve'l seen with the Ty2 parent (Elsinghorst et al., 1989). Maintenance of such a large segment of foreign DNA as an exrrachromosomal element in vivo will be difficult and further genetic manipulation of the invasion region will probably be required ~Loimprove both the expression and the stability of the invasive phenotype in vaccine constructs. In contrast, the invasive phenotype of Y. pseudotuberculosis can be transferred to E. coli by a single chromosomal locus (inv) encoding a 103-kDa outer membrane protein designated invasin (Isberg et al., 1987). Invasin promotes bacterial endocytic uptake by binding to glycoprotein "integins" in the mammalian !t31asma membrane including the ~4[~1(VLA-4) variety that is found on the surface of T lymphocytes in Peyer's patches (Isberg and Leong, 1990).

']'ABLE I. - - Invasion and adherence determinants that can be expressed ia E. coU. Genetic element Sh!geilaflexneri invasion determinant

Source 140-mDa plasmid

Phenotype in E. coli

Possible vaccine function

Transconjugants in KI2 Invasionof the colonic invade HeLa cells, epitheliumby hybrids lyse endocyticvacuoles, expressingheterologous and multiply intracellularly antigens Salmonella typhi Chromosome Clones in HB101 Invasion of the ileal invasion determinant invade Henle 407 c e l l s epithelium by but do not multiply hybrids expressing intracellularly heterologous antigens Yersini~ Chromosome Clones in HB101 Same pseudotuberculosis invade HEp-2 cells inv locus but do not multiply intracellularly EAF 60.MDa Transconjugants in K12 Colomzationof the plasmid exhibit localized adherence small intestine by to HEp-2 cells and brush hybridsexpressing border adherence to isolated heterologcus antigens human enterocytes ETEC colonization 60-MDa CFA plasmid-mediateddiffuse Same factor (CFA/I) plasmid adherence to Caco-2 cells, brush border adherence to isolatedhuman enterocytes,and colonization of rabbit intestine

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The benign intracellular life-style of E. coli hybrids expressing the invasive phenotype of S. typhi or Y. pseudotuberculosis is also a possible advantage for vaccine development. In contrast to hybrids expressing the enteroinvasive phenotype of Shigella species, these organisms are always found within endocytic vacuoles that limit intracellular bacterial multiplication. Metabolic function is maintained within infected cells, and this may account for the absence of gastroenteritis during the acute intestinal invasion phase of typhoid or Y. pseudotuberculosis infections. Therefore, it is possible that E. coli hybrids expressing the invasive phenotype of either S. typhi or Y. pseudotuberculosis would be more attenuated than enteroinvasive E. coli-S, flexneri vaccines. On the other hand, the immunogenicity of these hybrids could be diminished by the absence intracellular bacterial multiplication. Adherence factors that could aid colonization of the ileal mucosa by E. coli hybrid vaccines.

Although cloning of S. typhi or Y. pseudotuberculosis invasion genes into laboratory or commensal strains of E. coli could confer a proclivity for uptake of these hybrids by ileal Peyer's patches, this potential might not be realized in the absence of bacterial colonization factors that promote adherence to the ileal epithelium. Enteropathogenic or enterotoxigenic strains of E. coil express naturally occurring adherence factors that may be of use in vaccine strains targeted to the small intestine. For example, a conjugative colicinogenic plasmid of human enteropathogenic E. coli (EPEC) encodes a poorly characterized EPEC adherence factor (EAF) that mediates the attachment of EPEC stains to the human intestinal mucosa (table I). Transconjugants of E. coil K12 adhere to human foetal small intestine after acquisition of the 60-mDa EAF plasmid (Williams et al., 1978), but these adherent hybrids do not elicit the EPEC "attaching and effacing" lesions because they do not harbour the chromosomal eae locus of EPEC strains (Donnenberg and Yu, 1990). A possibie alternative to EAF would be the well characterized fimbial colonization factor antigens (CFA) of ETEC (table I). CFA/I mediates adherence of ETEC to human intestinal epithelial cells (Cheney and Boedeker, 1983), and this plasmid-encoded colonization factor has been cloned and expressed in E. coli KI2 (Willshaw et al., 1985). Conclusions.

Achieving an optimal balance of immunogenicity and reactogenicity in vaccines expressing the shigella enteroinvasive phenotype remains an elusive goal, but five years of development and testing of E. coli-S, flexneri hybrids in primates and humarls have clarified the problems and confirmed the potential of these vaccines. Possible alternative approaches for construction of E. coli hybrid vaccines may utilize cloned invasion genes from the chromosome of Salmonella typhi or Yersina pseudotuberculosis in combination with plasmid genes from ETEC or EPEC that mediate bacterial attachment to the lymphocyte-rich ileal and jejunal mucosa. I~Y-woP,vS: Escherichia coli, Enterotoxigenicity, Shigella flexneri; Hybrid vaccine. Addendum.

Recently, an aroD E. coli K12/S. flexneri hybrid vaccine has been tested for efficacy in rhesus monkeys (Newland et al., manuscript in preparation) and for safety and efficacy in humans at the University of Maryland Center for Vaccine Develop-

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ment (Kotloff et al., unpublished observation). This PABA-requiring auxotrophic hybrid carries the S. flexneri 5 invasion plasmid and the His, Pro, and Arg chromosomal markers of S. flexneri 2a. Oral immunization of monkeys with three vaccine doses elicited significant serum immune responses against the S. flexneri 2a somatic antigen expressed by the hybrid, and the vaccinated animals were significantly protected against an oral challenge with S. flexneri 2a. Oral immunization of humans ",vith three vaccine doses elicited no diarrhoea or dysentery, but a few volunteers experience intestinal cramping, Subsequent challenge with S. flexneri 2a indicated significant protection against bloody diarrhoea or dysentery in vaccinees. Protection against fever was also obseryed, but the differential between vaccinees and controls did not reach statistical significance in this preliminary study. Nonetheless, the results of preclinical and clinical studies suggest that enteroinvasive aroD E. coli KI2/S. flexneri hybrids may be useful for safe delivery of antigens to the GALT in humans, and further studies are planned to assess the safety and efficacy of these vaccine constructs. The vie~,:sof the author do not purport to reflect the position of the Department of the Army or the Department of Defense (para. 4-3, AR 360-5).

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