ADVANCES IN VIRUS RESEARCH. VOL. 41

PROSPECTS FOR ORAL VACCINATION USING RECOMBINANT BACTERIA EXPRESSING VIRAL EPITOPES Florian Schodel Max-Planck-lnstitut fur Biochemie D-8033Martinsried, Germany

I. Introduction A. Immunity to Viral Disease B. Immunity to Bacterial Disease 11. Foreign Antigens Expressed in Salmonella A. Viral Antigens B. Bacterial and Parasitic Antigens 111. Epitope Presentation Systems A. Flagellin B. Hepatitis B Virus Core Particles C. CT-BILT-B D. Miscellaneous Proteins: Comments and Outlook IV. Genetic Stabilization of Foreign Antigen Expression A. Plasmids without Antibiotic Resistance Marker B. Chromosomal Integration V. Bacteria Potentially Useful as Carriers A. Attenuated Salmonella Strains B. Bacille Calmette-Guerin VI. Outlook References

I. INTRODUCTION Live attenuated bacteria are attractive carriers for foreign antigens by the oral route. Salmonella can be attenuated by genetic manipulations so that they are nonpathogenic but retain the ability to invade across the gut epithelium and persist in the gut-associated lymphoid tissue (GALT), in Peyer’s patches, and in the spleen, where they can deliver foreign antigens to the host immune system (for other recent reviews see, e.g., Levine et al., 1989, 1990a,b; Stocker, 1990; Schodel, 1990; Curtiss et al., 1989a,b; Curtiss, 1990; Dougan et al., 1989). The development of genetically defined attenuated strains converges with the power of recombinant gene expression technology, which is most 409 Copyright 0 1992 by Academic Press,Inc. All rights of reproduction in any form reserved.

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FLORIAN SCHODEL

advanced in prokaryotes such as Escherichia coli and Salmonella typhimurium. In theory, every foreign antigen could be transported to defined compartments of the host immune system using attenuated Salmonella or other attenuated bacteria, which are less well studied, as carriers. In addition to systemic immune responses, immune responses can be generated a t mucosal surfaces that are inaccessible t o normal parenteral immunization schedules. The gut- and the bronchiusassociated lymphoid tissues constitute the main part of an immunological system common to all mucosal surfaces: the mucosal-associated lymphoid tissue (MALT) (for reviews see Mestecky, 1987; Brandtzaeg, 1989; Lycke and Svennerholm, 1990). The delivery of antigens by the oral route to the GALT leads to a generalized mucosal immune response, of which the secretory IgA component is the best studied (see, e.g.,Rudzik et al., 1975; McDermott and Bienenstock, 1979; for a recent overview see Lycke and Svennerholm, 1990). Thus oral immunization with a suitable carrier can evoke mucosa! immune responses a t distant sites, e.g., in the lungs. This might be of particular importance, because the vast majority of pathogens invade via mucosal surfaces (for an overview see Mims, 1987). A secretory immune response might be sufficient to prevent reinfection of viruses that are limited in their replication to epithelial cells, whereas viruses that rely on dissemination in the bloodstream to reach their target organs, such as hepatitis A virus, are also susceptible to virus-neutralizing serum antibodies. Dependent on whether systemic immunz responses and/or mucosal immunity are required, bacteria attenuated to varying degrees of invasiveness or bacteria that are restricted to the epithelial layers could be considered as carriers for viral antigens. The type of immunity induced may also depend on whether bacteria capable of intracellular survival or replication are used as carriers. Another important aspect is that live bacteria could potentially be developed into combination vaccines, inducing immune responses against their own wild-type antigens as well as against several coexpressed foreign viral, bacterial, or parasitic antigens. This would be particularly important for vaccination campaigns in the developing world (for a global perspective, see Bloom, 1989). In this review I shall try to describe the current experimental situation using mainly attenuated Salmonella strains to immunize against viral antigens, and some major obstacles and considerations relevant to recombinant live oral antiviral vaccines based on bacteria. It should be noted that protection against a viral disease afforded by immunization with recombinant bacteria expressing viral antigens has thus far not been reported. Although that situation is likely to change soon, for the time being we have to rely on the discussion of immunolog-

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ical phenomenology and wait for the final proof of the validity of our assumptions. The questions I shall try t o consider are as follows: Are there major differences between antibacterial and antiviral immunity that might impede the use of bacteria for immunization against viral disease? How can we stably express viral antigens in bacteria in an immunogenic form? Which bacteria are currently available as prospective carriers for human o r veterinary use and which are under development? Thus the review will fall into three parts: first, a general description of what is assumed to be necessary for antiviral immunity in comparison to immune responses against foreign, viral, bacterial, or parasitic antigens delivered in recombinant bacteria; second, a description of expression systems for foreign epitopes in bacteria and strategies used to genetically stabilize foreign gene expression in bacterial vaccine strains; and third, a description of attenuated bacterial strains currently available or under development, with a bias toward Salmonella strains. A . Immunity to Viral Disease The aim of antiviral immunization is long-lived protection. Many virus infections leave their host with a lifelong protection. This lifelong immunity has been taken as an indication that most virus infections are incompletely cleared and a reservoir of antigen always remains behind, as at least B cell memory can be short-lived in the absence of antigen (Gray and Skarvall, 1988). Viruses rely on intracellular replication in their host, and apart from antibodies and T cell help, readily induce cytotoxic major histocompatability complex (MHC) class Irestricted lymphocytes. Lasting protection after immunizations with subunit antiviral vaccines, such as HBs against hepatitis B virus (HBV) infection, which are assumed t o induce T cell help and antisurface antibodies, proves that these immune responses suffice. I shall limit the discussion to the part of the immune response not linked to the genetics of specific viruses, as, for example, antigenic variation leading to antibody or T cell escape or virus-specific immune modulation (for references see, e.g., Pircher et al., 1990). Specific protection against viral infection can be mediated by virus-neutralizing antibodies blocking infection a t the portal of entry andlor viral dissemination via the bloodstream as well as local reentry into cells of the target tissue. Mucosal slgA can suffice to prevent infection with influenza virus, for example (Renegar and Small, 1991).It should be noted that only preexisting virus-neutralizing antibodies can prevent viral infection; T cells see antigens exclusively in an MHC complex-associated processed form

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FLORIAN SCHODEL

and therefore invariably require infection of target cells before they can recognize viral antigens and directly or indirectly interfere with virus replication. Most viral antigens are T cell dependent and therefore require the induction of CD4+ T cells for antibody production. Sites recognized by CD4+ T helper cells need not be on the antigen that is the target of virus-neutralizing antibodies (Russell and Liew, 1979; Milich et al., 1987). CD4+ T helper cells recognizing, for example, a n amino acid sequence on a n internal viral antigen can provide help for antibody production against viral envelope proteins when these are present in the complete virion as a secondary immunogen, a phenomenon termed intermolecularlintrastructuralT help. CD8' T cells have an important role in virus elimination. CD8+ T cells are restricted by class I MHC antigens and are generally cytolytic T cells. The elimination of virus is, however, not necessarily linked to its cytolytic potential (for a discussion see Lehmann-Grube et al., 1988). The elicitation of virus-specific CD4+ T helper cells may be necesdary for the generation of immunological memory-Tcell memory can, however, be rather short-lived (Zinkernagel, 1990) and its role in the prevention of viral infection by vaccination is not understood.

Virus-Neutralizing Antibodies Virus-neutralizing antibodies are often directed against highly complex antigenic sites of viral envelope or capsid proteins. The epitopes that these antibodies recognize are conformation dependent, as indeed every epitope is likely to be (I shall come back to that argument later), and sometimes composed of amino acids stemming from more than one protein molecule. Only in a few instances can viral antigens be expressed in prokaryotes in the native conformation found in the complete virion. Until we learn how to model and express a threedimensional antibody-binding site in prokaryotes by genetic engineering, we shall have to rely on the creation of expression systems for peptidic virus-neutralizing epitopes. Ultimately the definition of a viral epitope expressed in bacteria to induce an antibody response recognizing virus surface structures has to be functionally and not structurally defined, even if visualization of three-dimensional antibody/antigen amino acid neighborhoods in their crystalline form has been suggested as the only intellectually satisfying definition of an epitope (Laver et al., 1990). Antibodies cross-reacting with the epitopes of choice have to be virus neutralizing in uiuo. It has been demonstrated for some viruses that virus-neutralizing antibodies can be induced by immunization with synthetic peptides or fusion proteins expressed in bacteria stemming from surface antigens (e.g., for HBV and FMDV; Bittle et al.,

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1982; Gerin et al., 1983; Itoh et al., 1986; Neurath et al., 1986; for a more concise list of peptidic epitopes on viral antigens see van Regenmortel, 1990; Milich, 1989, 1990). The antiviral antibodies recognizing virus surface antigens do not have to correspond to the antibody specificities elicited by natural virus infection. An example of this is a partially protective immune response in chimpanzees against a peptide of the HBV surface antigen that is not recognized by patient sera (Gerin et al., 1983). In general, this approach might be more successful when the surface antigens of viruses are not fixed in a quasi-crystalline capsid, but expose contiguous stretches of amino acids at the surface of a membranous envelope with a relatively high degree of flexibility. These peptidic or continous viral epitopes have to be presented in a n immunogenic form in the carrier bacteria. A number of epitope-presenting systems have been developed for this purpose (see also Table 11)and will be discussed in more detail below. For other viruses and especially RNA viruses such as poliovirus and hepatitis A virus, with the exception of FMDV, approaches using synthetic peptides or bacterial fusion proteins as immunogens to elicit high-titer virus-neutralizing antibodies have been mostly unsuccessful (for review see Minor, 1990). In FMDV, the peptidic epitope giving rise to neutralizing antibodies (Bittle et al., 1982; Pfaff et al., 1982) resides on a n outer loop of VP1 with a apparent high degree of rotational freedom (Acharya et al., 1989).The structure of the exposed amino acids on the capsid surface of most nonenveloped RNA viruses seems to be too rigid to allow high-affinity binding of antibodies directed against contiguous sequences of a single polypeptide. For this class of viruses it can be anticipated that the induction of meaningful antiviral neutralizing antibody titers will only be possible if we either learn to express stably the complete capsids in live bacteria or to model the highly complex epitopes formed by outer loops of several capsid antigens into the outer loops of other viral capsids that can be stably expressed in prokaryotes (like that of hepatitis B virus) (Schodel et al., 1 9 9 0 ~by ) genetic engineering. The first possibility requires that we coexpress several viral capsid proteins in a bacterial cell, mimicking the proteolytic processing found in eukaryotes. The second possibility is highly attractive, but we are far from achieving it with the current lack of adequate prediction of secondary and tertiary structure. It has so far not even been possible to create poliovirus chimeras that would be equally immunogenic for all three serotypes, incorporating capsid outer loops into the other serotype’s respective backgrounds in an immunogenic form, although the primary sequence is relatively similar. A highly speculative option for the induction of antibody against conformation-dependent epitopes that can-

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not be expressed in their native shape in prokaryotes could develop with the successful expression of immunoglobulin Fab or Fv parts in E . coli (Skerra and Pluckthun, 1988; Better et al., 1988; for reviews see Pluckthun and Skerra, 1989; Pluckthun, 1990). If so-called antiidiotypic antibodies mimic the paratope of a first antibody, it should be possible to clone and express antibodies directed against antibodies recognizing a conformational virus epitope in, for example, Salmonella, and induce virus-neutralizing antibodies by this route. This would open the possibility t o raise antiidiotypic slgA antibodies, which to my knowledge has not yet been accomplished.

3.Immunity to Bacterial Disease This section, like the former, is not intended to be a comprehensive review. Instead I will summarize some of the relevant data regarding immunity, mainly against S . typhimurium, as this is one of the better studied bacterial pathogens and a suitable small animal model is available. The focus will be on similarities and differences of antiviral immunity that might be important in the use of these or similar bacteria as carriers. Salmonella typhimurium causes a systemic disease in mice, cattle, horses, and pigs similar to typhoid fever in humans. It is one of the most important human pathogens responsible for food contamination; in humans, S . typhimurium does not cause a systemic typhoidlike disease, but remains restricted to the gut epithelium and induces mainly diarrheal disease. The natural route of Salmonella infection is oral via contaminated food. Salmonella typhimurium is capable of crossing the cells of the intestinal epithelium. This passage through epithelial cells, among them the specialized M cells, in general leaves the epithelium intact, although a cytotoxin gene has been cloned from S . typhimurium (Libby et al., 19901, a n analog of which may be responsible for the occasional bloody diarrhea in typhoid. It has been shown that S . typhimurium can transcytose through polarized cells in vitro (Finlay et al., 1988). After passage of the intestinal epithelium and the lamina propria, S. typhimurium is found in the gut-associated lymphoid tissue and in Peyer’s patches. Salmonella typhimurium enters the bloodstream via the lymphatics, replicates in the spleen and the liver, and a disseminated infection ensues with bacteremia, leading to death. Salmonella typhimurium can invade a number of animal cells and possibly replicates in them, which is a property shared with other gram-negative and gram-positive enteropathogens, for example, some Shigella and Yersinia species and Listeria monocytogenes (for more details on intracellular survival and replication, see Moulder, 1985;

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Finlay and Falkow, 1989). Whereas a t least L. monocytogenes and Shigella flexneri can escape from the lysosome into the cytoplasm by virtue of their hemolysin, which disolves the membrane surrounding them, Salmonella spp. do not escape from the lysosome and are equipped to survive the adverse intralysosomal conditions. One strategy S. typhimurium employ for intracellular survival appears to be the active prevention of secondary lysosome formation (Buchmeier and Heffron, 1991). It is currently unclear how much replication in macrophages contributes to the course of infection (for a controversial view see Hsu, 1989). Resistance or susceptibility to S. typhimurium infection in mice is genetically determined. Three gene loci contributing to resistance (ity, xid, and Ips) have been studied in detail (reviewed in O'Brien, 1986; for discussion see Dougan et al., 1989). In our context it is more interesting to discuss mechanisms of acquired immunity to S. typhirnurium infection. Whereas it has been shown that passive transfer of hyperimmune sera or monoclonal antibodies against lipopolysaccharide (LPS) can increase the LD50 by about 10-fold, immunization with an immunogenic live vaccine strain confers protection to above 1000, or 10,000 X LD5,, (Colwell et al., 1984; Killar and Eisenstein, 1984).Mucosal antibodies against LPS can obviously provide some protection against S. typhimurium infection, because after immunization with Aasd S. typhimurium strains, which are noninvasive but induce some slgA response the wild-type oral LD50is also increased by about 10-fold (Curtiss, 1990). Most bacterial antigens, with the exception of LPS, require the help of CD4+ T cells for induction of antibodies. Resistance to reinfection in the early phase of experimental S. typhimur i u m infection in nude mice or lethally irradiated animals adoptively transferred with selectively depleted preimmune T cells seems not to be controlled by T cells, but clearance of S. typhimurium in the later phase of infection is predominantly mediated by CD4' T cells, with possibly a minor contribution by CD8' cells (Nauciel, 1990). This may reflect the need for T help andlor the activation of professional phagocytes, which are instrumental in bacterial clearance. Unlike in L. monocytogenes infection, wherein sterilizing immunity can be adoptively transferred with CD8' cytotoxic T lymphocytes (CTLs) (references in Kaufmann, 19881, this has not been reported for S. typhimurium. For L. monocytogenes, expression of the hemolysin, mediating escape from the phagocytic vacuole, is necessary for virulence and for MHC class I-restricted presentation of listerial antigens to CD8+ CTLs (Brunt et al., 1990). It has to be noted that hemolysin-deficient L. monocytogenes is also totally avirulent, does not replicate intracellularly, and may therefore never

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FLORIAN SCHODEL

reach the same antigenic load per cell regardless of intracellular location. Exogenous antigen normally enters the endosomal pathway and its peptidic fragments are presented in the context of MHC class I1 to CD4' T cells, providing mainly T cell help for antibody production. Peptidic fragments of endogenously synthesized antigens, such as viral antigens, associate with MHc class I antigens in the endoplasmic reticulum and the MHC-peptide complex is then translocated to the surface and presented to CD8' T cells (for details and references see Peters et al., 1991). An ABC peptide transporter-like gene was recently identified and is implicated in the transport of peptides from the cytoplasm to the endoplasmic reticulum (Deverson et al., 1990; Monaco et al., 1990; Spies et al., 1990; Trowsdale et al., 1990).The peptides found in association with class I MHC on the cell surface appear more homogeneous than class II-associated peptides (Falk et al., 1990; Rotschke et al., 1990; van Bleek and Nathenson, 1990), and class I MHC selects less peptide specificities in a given mouse MHC background of any given pathogen. The most efficient inducers of CD8' CTL responses in viuo are viral carriers (for example, vaccinia) (for a list of protective CTL responses induced with recombinant vaccinia viruses, see Moss, 1990) that synthesize the foreign antigens intracellularly. Peptides can be introduced into the cytoplasm of cells via osmotic shock and are presented via the class I pathway (Moore et al., 1988),and MHC class I molecules can be loaded with external peptides (Townsend et al., 1986; Bodmer et al., 1988, Carbone et al., 1988). Immunization with synthetic peptides coupled to immunostimulatory complexes or with lipopeptides and with excess of synthetic peptide in Freund's adjuvants can induce CD8+ CTL responses in mice (Staerz et al., 1987; Carbone and Bevan, 1989; Ishioka et al., 1989; Deres et al., 1989; Takahashi et al., 1990; Schultz et al., 1991). The common denominator of these experiments may be that foreign antigens or fragments thereof are introduced into the cytoplasm of cells by pinocytosis instead of phagocytosis. These exceptions to the original dogma that only endogenously synthesized antigens would be presented via the class I pathway are relatively inefficient however. Even if salmonellae do not regularly leave the phagolysosome antigen fragments might be released into the cytoplasm at some stage of the infectioddegradation and find their way to MHC class I molecules in the endoplasmic reticulum. The important question will, however, remain as to how efficient CD8' CTL induction by recombinant salmonellae will be. A low-level CD8' CTL response might in some instances be detrimental and cause augmented pathology instead of viral clearance (Oehen et al., 1991). Possibly the amount of bacterial antigen presented to MHC class I from Salmonella restricted to the phagolysosomal compartment is the limiting factor for the efficient induction of CTLs. This

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speculation should be experimentally testable by expressing the L. monocytogenes hemolysin in attenuated S. typhimurium strains to let them escape to the cytoplasm of infected cells, provided such bacteria would not become too virulent. It is of interest to note that one of the two groups reporting CD8' CTL responses against plasmodia1 circumsporozoite antigens after oral immunisation with live recombinant S. typhimurium (Aggarwal et al., 1990; Flynn et al., 1990) used an attenuated S . typhimurium strain that is deficient in intracellular survival (Furness and Rowley, 1956; Furness, 1958). 11. FOREIGN ANTIGENSEXPRESSED IN SulmonelZa Only very limited numbers of viral antigens have so far been expressed in live Salmonella and tested for their immunogenicity when administered via the oral route. In addition, a bacterial antigen (Streptococcus pyogenes M protein; Poirier et al., 1988) and a parasitic antigen (Plasmodium berghei circumsporozoite antigen; Sadoff et al., 1988; Aggarwal et al., 1990) that have been expressed in attenuated S. typhimurium and have been shown to confer protective immunity after oral immunization, apparently by different mechanisms, will be described (additional heterologous antigens, which are not discussed, are summarized in Table I, and some proteins potentially useful as carriers of foreign epitopes are discussed in Table 11).

A. Viral Antigens 1. Influenza A Virus Nucleocapsid Antigen

Tite and co-workers expressed the nucleocapsid antigen of influenza A virus in an aroA S. typhimurium strain (Tite et al., 1990). Mice fed these recombinant live S. typhimurium developed serum IgG antibodies recognizing the nucleocapsid antigen and specific CD4' proliferative as well as CD4+ cytolytic splenic T cells. Whereas this antigen readily induces CD8+ CTL responses when administered to the same mouse MHC class I background in a live virus, it failed to do so after immunization with the live recombinant S . typhimurium. The oral immunization with the live recombinant S . typhimurium expressing the influenza virus nucleocapsid alone induced no protective immunity. A reduction in lung tissue virus titer after challenge could be demonstrated when the mice were boosted by intranasal immunization with purified antigen after oral immunization with the live recombinant S. typhimurium.

TABLE I FOREIGN ANTIGENS EXPRESSED IN LIVEATTENUATED SALMONELLAE~ Source Hepatitis B virus nucleocapsid Influenza A virus nucleoprotein Woodchuck hepatitis virus nucleocapstd Dengue 4 virus envelope antigen Bacterial and parasitic Escherichia coli enterotoxin B subunit

Salmonella mutant

S typhimurium AaroA Acya Acrp, S dublin AaroA S typhimurium AaroA S typhimurium Acya Acrp

M

T cell

Ref.

i

p ,o

IgGAM

nt

T help

Schodel et al. (1990~); Schodel et al. (1991a)

i

p , 0, sc

Ig

nt

CD4' help, CTL

Tite et al. (1990)

i

p

Ig

nt

nt

F. Schodel et al. (unpublished observations) Cohen et al. (1990)

S . typhimurium S. dublin

S

RA

nt

nt

0

nt

i.p., o

IgGAM

sIgA

Prolif.

S . typhimurium Acya Acrp

o

IgGAM

sIgA

nt

S. typhi TyZla

i.p.

Ig

nt

nt

AaroA

Clements et al. (1986); Maskell et al. (1987); Schodel and Will Schodel et al. (1990a,b) Schodel et al. (1989a); F. Schodel et al. (unpublished observations) Clements and El-Morshidy (1984)

Shigella sonnei antigen Shigella flexneri 2a antigen Escherichia coli fimbriae P-Galactosidase Streptococcus mutans surface protein antigen (SpaA) Glucosyltransferase (GtfA) Streptococcus pyogenes protein Streptococcus sobrinus SpaA Plasmodium berghei Circum sporozoite antigen (CSA) Plasmodium yoelii CSA Vibrio cholerae LPS Francisella tularensis kDa

S . typhi Ty2la

sIgA

0

S . typhi Ty2la

Ig

0

nt

nt

nt

Formal et al. (1981); Tramont et al. (1984); Black et al. (1987) Baron et al. (1987)

S . typhimurium galE

-

Ig

sIgA

nt

S . typhimurium AaroA S. typhimurium 3aroA S. typhi Ty2la

i v., i.p. nt

k! Ig

sIgA nt

nt DTH

-

-

-

Stevenson and Manning (1985) Dougan et al. (1986) Brown et al. (1987) Curtiss et al. (1986)

S . typhimurium AthyA Aasd, AaroA S . typhimurium AaroA

-

Ig

sIgA

nt

Katz et al. (1987)

o

IgGAM

sIgA

nt

Poirier et al. (1988)

0

Ig

sIgA

nt

cf, Curtiss (1990)

o

nt

sIgA

DTH

cf. Curtiss (1990)

0

-

-

DTH, CD8', CTL

Sadoff et al. (1988); Aggarwal et al.

i.p., o

-

Nt

CD8+,CTL

Flynn et al. (19901

0

IgGAM

sIgA

nt

Ig

nt

nt

La Brooy et al. (1986); Attridge et al. (1990) Sjostedt et al. (1990)

S. typhimurium Aasd, AaroA S. typhimurium Acya Acrp Aasd S . typhimurium

S . typhimurium AaroA S . typhi Ty2la

S. typhimurium Acya Acrp Aasd

TABLE I (continued) Source Clostindium tetani 50-kDa toxin fragment Bordetella pertussis 69-kDa outer membrane protein Treponema pallidum lipoprotein Pseudomonas aeruginosa lipoprotein Neisseria meningitidis 28-kDa outer membrane protein Leishmania major gp63 surface protein Brucella abortus 31-kDa protein

Salmonella mutant

S

RA

M

T cell

Ref.

S . typhimurium AaroA

0,i.v.

IgG

nt

nt

Fairweather et

S. typhimurium AaroA

0,sc

nt

-

-

Fairweather et

S. typhimurium AaroA

nt

nt

-

-

Strugnell et al.

S . dublin AaroA

0

IgGAM

sIgA

nt

S. typhimurium

i.v.

Ig

nt

nt

F. Schodel et al. (unpublished observations); al. (1989b) Tarkka et al. (1989)

S. typhimurium AaroA

o

IgG

nt

CD4' helper

Yang et al. (1990)

S. typhimurium Acya Acrp

o

IgGA

-

-

Stabel et al. (1990)

RA, Route of administration; i.p., intraperitoneal; 0,oral; i.v., intravenous; sc, subcutaneous; S, serum antibody response; globulin; M, mucosal immune response; sIgA, secretory IgA; nt, not tested or indicated; DTH, delayed-type hypersensitivity; proliferative T cell response; CTL, cytotoxic T lymphocytes. For single viral epitopes and epitope presentation systems, refer corresponding text.

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42 1

TABLE I1 PROTEINS POTENTIALLY USEFULAS CARRIERS OF FOREIGN EPITOPES IN LIVEBACTERIA Protein External or secreted Flagellin" Fimbriae Mycobacterium bouis a-antigen IgA protease Outer Membrane LamB OmpA PhoE TraT Periplasm MaIE AP LT-BICT-B"

Cytoplasm &Gal HBc"

Ref. Kuwajims et al. (1988);Newton et al. (1989);Wu et al. (19891 Klemm and Hedegaard (1990) Matsuo et al. (1990) Klauser et al. 11990) Charbit el czl. (1986, 1987, 1988);Leclerc et al. (1989, 1990) Pistor and Hobom (1990) Hogervorst et al. (1990) Harrison et al. (1990) OCallaghan et al. (1990) Freimuth and Steinman (19901 Guzman-Verduzco and Kupersztoch (1987, 1990); Schodel and Will (1989);Schodel et al. (1990a,b, 1991b); Sanchez and Holmgren (1989);Dertzbaugh and Macrina (1989); Dertzbaugh et al. (1990); Clements (1990) Itakura et al. 11977);Young and Davis (1983) Schodel et al. (199Oc, 1991a)

Only flagellin (Wu el al., 19891 and HBc ISchodel et al., 199Oc, 1991a) have been shown to induce antibodies against inserted epitopes when expressed in orally delivered recombinant Salmonella, and LT-B has been shown to induce a proliferative T cell response against a heterologous epitope (Schodel et al., 1990b).

2. Hepatitis B Virus Nucleocapsid Antigen Although full-length core protein of HBV could not be constitutively expressed in attenuated Salmonella strains due to its toxicity, we could express a carboxy-terminal truncated version that retained HBc particulate antigenicity and immunogenicity and the capacity to self-assemble t o nucleocapsid particles (Schodel et al., 1 9 9 0 ~ )The . properties of this expression system are further discussed in Section 111. Several S. typhimurium and Salmonella dublin strains delivered this antigen to the systemic immune system by the oral route in mice. We could demonstrate a high-titered serum anticore response after one or several oral immunizations. The immunoglobulin class and IgG subclass distributions of the serum anti-HBc response where similar to

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FLORIAN SCHODEL

that seen after parenteral immunization, with the notable exception that oral immunization with recombinant salmonellae induced a serum IgA response. The observed IgG subclass distribution is consistent with the induction of functional T cell help. Although it has been reported that immunization with HBc provides a partial protection against HBV infection in chimpanzees (Murray et al., 1984) and, using WHc and HBc, against woodchuck hepatitis B virus (WHV) infection in woodchucks (Roos et al., 1989; Schodel et al., 1992), this protection is not mediated by anti-Hbc antibodies. In addition we have exploited the high immunogenicity of HBc particles expressed in Salmonella to develop an epitope presentation system by genetic fusion or insertion of the foreign epitopes to HBc (Schodel et al., 199Oc; 1991a).As mentioned above, we were also interested in determining whether recombinant SalmoneZla may be able to induce efficiently CD8' cytolytic T cells. Therefore, S . typhimurium expressing a defined CTL epitope of murine cytomegalovirus (CMV) as a C-terminal fusion protein with HBc was used in preliminary experiments to immunize mice of the appropriate MHC class I background. We could not show induction of CD8+ CTLs against the CMV epitope, although serum antibodies against HBc were induced, showing that the antigen was delivered (F.Schodel et al., 1990, unpublished observations). The CMV epitope has been demonstrated to induce CD8' CTLs in the HBc context when administered as a recombinant vaccinia virus construct (Schlicht et al., 19901, so it appears unlikely that there is a problem of processing of the viral CTL epitope in a foreign protein context.

3 . Miscellaneous Viral Antigens Viral proteins or protein fragments stemming from herpesviruses (Bowen et al., 19901, influenza virus (Pistor and Hobom, 19901, rotaviruses (Salas-Vidal et al., 1990; Reeves et al., 1990), dengue viruses (Cohen et al., 19901, and human immunodeficiency virus (HIV) (Stocker, 1990) were also expressed in live attenuated Salmonella strains. Unfortunately, no immunological data were available a t the time of this review. For epitopes expressed in flagellin or HBc, see Section 111.

B. Bacterial and Parasitic Antigens 1. Streptococcus pyogenes M Protein The S. pyogenes M protein forms fibrils on the bacterial surface and protects the bacterium against phagocytosis in the infected host. Poirier et al. (1988),in the group of the late Ed Beachey, have expressed

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the S . pyogenes M protein in aroA S . typhimurium and immunized mice by the oral route with the recombinant salmonellae. The mice developed serum IgGAM antibodies and saliva secretory IgA that opsonized S. pyogenes in uitro. The immunized mice were protected against a lethal S. pyogenes infection by the intranasal and intraperitoneal (i.p.1 route and against a wild-type i.p. S. typhimurium challenge. This was the first report of apparently antibody-mediated protection against a pathogen using recombinant S . typhimurium as orally administered foreign antigen carriers. 2. Plasmodium berghei and Plasmodium yoelii Circ umsporozo i te A ntige ns The Plasmodium berghei circumsporozoite (CS) antigen was expressed by the group of Jerry Sadoff (Sadoff et al., 1988) in an attenuated S. typhimurium strain originally described by Furness (Furness and Rowley, 1956; Furness, 1958) as being defective in survival in macrophages. Plasmodium berghei is a mouse malaria agent. Mice orally immunized with recombinant S . typhimurium expressing CS were partially protected against P. berghei challenge (60%)(Sadoff et al., 1988). Antibodies against CS could not be demonstrated in the immunized mice. The protective effect was later described as mediated by CS-specific CD8' CTLs (Aggarwal et al., 1990). Another group showed the induction of CS-specific CD8' CTLs after orally immunizing mice with aroA S. typhimurium expressing a Plasmodium yoelii circumsporozoite antigen (Flynn et al., 1990). The mice, however, were not protected against P. yoelii challenge.

111. EPITOPEPRESENTATION SYSTEMS As outlined above, peptidic epitopes have been identified for some viruses that are able t o induce virus-neutralizing antibodies or induce protective immunity when administered as immunogens (for reviews see Milich, 1989, 1990; Van Regenmortel, 1989). These peptidic epitopes are usually insufficiently immunogenic when administered as free peptides due to the lack of suitable T cell sites. One approach toward increasing the immunogenicity of peptidic B cell epitopes has therefore focused on including defined T cell sites in synthetic peptides that increase their immunogenicity (see, e.g., Milich et al., 1986, 1987, 1990). This can be achieved by translational fusion or colinear synthesis as well as by chemical coupling methods. The traditional methods of coupling synthetic peptides to carriers such as bovine serum albumin or

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keyhole limpet hemocyanin serve the same purpose in principle. When aiming a t expressing a peptidic epitope in a n immunogenic form in live bacteria, the carrier protein has some additional functions It should (1) stabilize expression of the epitope by protection from proteolysis, (2) ideally be of little toxicity to the host bacterium, ( 3 )transport the epitope t o the desired bacterial compartment, if the location has any role in immunogenicity, and, most importantly (4)present the epitope to the immune system in a fashion that directs a sufficient part of the host immune response toward this particular epitope. The last point is by no means trivial, because a bacterial cell with more than 3000 protein antigens induces strong immune responses only against a minority of its proteins (see, e.g., Brown and Hormaeche, 1988). The regulation of these immune responses is not understood. The regulation of bacterial gene expression during invasion of eukaryotic cells or tissues is probably one factor governing their immunogenicity, but certainly not the only one. The heat-shock proteins are a class of antigen that induces strong host immune responses (Young and Elliott, 1989; Kaufmann, 1990),and this has been linked to their induction following stress, e.g., oxidative stress inside a macrophage (Buchmeier and Heffron, 1990). This argument will also be taken up in Section IV. A list of bacterial or eukaryotic proteins that have been used for the presentation of foreign epitope in bacteria is found in Table 11. Only two of these systems insertion of foreign epitopes into flagellin (Wu et al., 1989) and into the hepatitis B virus nucleocapsid (Schodel et al., 199Oc, 1991a) have been successfully tested in live salmonellae administered by the oral route and will therefore be discussed in more detail. Cytoplasmic, periplasmic, and outer membrane proteins have been found to be immunogenic in live bacteria, thus the location of the immunogen in the bacteria may be of secondary importance. Some of the features of selected epitope presentation systems are described below. For other fusion proteins of potential value but that have only been tested for immunogenicity as purified proteins or in E. coli (which are not useful as live oral carriers), see Table I1 and reviews by Lenstra et al. (19901, Stader and Silhavy (19901, and Uhlen and Moks (1990).

A . Flagellin A feature common to the flagellins of gram-negative bacteria is that they have highly conserved regions a t their amino and carboxy termini, whereas the middle regions vary dramatically in size (up to about 30 kDa) and in primary sequence (for review see Joys, 1988). Flagellin has an interesting specialized export pathway; it is believed to be secreted through a central channel in the flagellum and polymerizes

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from the tip of the flagellum (Namba et al., 1990). Even a n N-terminal 183 residue fragment of the 497-residue E . coli flagellin was exported, but failed to assemble (Kuwajima et al., 1989).All the different flagellins polymerize to flagella that differ in their outward appearance but appear to have a structurally similar core, which is a hint that the middle regions might be good recipients for relatively large foreign inserts. This strategy was first used in E. coli flagellin by insertion of ovalbumin epitopes, which could be shown to be immunogenic (Kuwajima et al., 1988). A second approach used two naturally occurring restriction sites in region 4 of the gene for the H1-d flagellin of S. dublin (Newton et al., 1989). A number of foreign epitopes were inserted a t these EcoRv sites and were immunogenic when expressed in attenuated flagellinless aroA S. dublin strains and administered parenterally. Only two of the constructs were reported to be immunogenic after repeated oral adminstration of the recombinant salmonellae (Wu et al., 1989; Stocker, 1990). As natural Salmonella isolates are usually aflagellate and only acquire flagella when cultured in suitable media, and some patients recovering from typhoid have only low titered (if any) antiflagellin serum antibodies, one speculation is that the flagellin expression off the pUC derivative, which carries the lac promoter in a sense orientation with the flagellin promoter, might be down-regulated during invasion. Therefore, flagellin might still be an attractive carrier molecule for foreign epitopes in live bacteria, but its use may require the development of bacteria that express flagella either constitutively or coupled to a transcription unit that is preferentially activated in the host tissue.

B . Hepatitis B Virus Core Particles The hepatitis B virus nucleocapsid antigen (HBc)is a 21.5-kDa monomer of which 180 units assemble to form 27-nm particles (for review see Schodel et al., 1990d). These particles are highly immunogenic and induce T cell-dependent as well as T cell-independent immune responses (Milich and McLachlan, 1986). Expression of the full-length nucleocapsid protein is toxic to all Salmonella strains tested (Schodel and Will, 1989; Schodel et al., 1 9 9 0 ~ )Deletion . of the carboxy-terminal 28 amino acids results in a molecule that forms particles but has lost the RNA-binding properties of the arginine-rich carboxy terminus and can be expressed to high levels in live Salmonella strains, as discussed above (Schodel et al., 1 9 9 0 ~ )It. has previously been found that fusion of foreign epitopes to the amino terminus of HBc exposes these sequences a t the particle surface and renders them highly immunogenic (Clarkeet

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al., 1987). We and others have fused foreign epitopes t o the carboxy terminus of HBc (Borisova et al., 1989; Stahl and Murray, 1989; Schodel et al., 1990e).Although these carboxy-terminal fusion proteins can now be stably expressed in live Salmonella and are highly immunogenic for the HBc moiety, they are not as immunogenic as amino-terminal fusions for the fusion partner (Schodel et al., 1 9 9 0 ~).Such recombinant HBc/pre-S2 particles in aroA S. typhimurium and S . dublin are capable of eliciting high-titered anti-HBc and lower-titered anti-pre-S2 after a single oral immunization dependent upon the carrier strain. Using a Acya Acrp S. typhimurium strain (Curtiss and Kelly, 1987)as a carrier for an HBc/pre-S2 particles expressing plasmid, we could induce high-titered anti-HBc antibodies 2 weeks after a single oral immunization with only 5 x lo6 colony-forming units (CFUs) (F. Schodel and D. R. Milich, unpublished observations, 1991). More recently we found that insertion of foreign epitopes in a predicted outer loop of HBc, between amino acids 75 and 80, makes these inserts more immunogenic than carboxy-terminal fusion and a t least as immunogenic as the amino-terminal fusion (Schodel et al., 1991a; 1992).These core particles have served to deliver epitopes of the pre-S1 region of HBV in a highly immunogenic form to mice as a n oral immunogen in live Acya Acrp S. typhimurium (Schodel et al., 1991a). Several B and T cell sites of other pathogens, such as the V3 loop region of HIV, neutralizing epitopes of Semliki Forest virus, epitopes of coxsackievirus B4, and LCMV, have now been inserted in that region and some of the constructs are currently being tested for immunogenicity (in collaboration with Albrecht von Brunn, Jay Berzofsky, Avigdor Shafferman, Sarah Cohen, Reinhard Kandolf, and Birgit Reimann).

C. CT-BILT-B Subunit B of the heat-labile enterotoxin ofE. coli (LT-B)is a homolog of the cholera toxin subunit B (CT-B).It is an 11.5-kDa protein that is secreted into the periplasm in gram-negative bacteria, assembles as pentamers, and associates with the adenylate cyclase-activating subunit A in a ratio of 5: 1 as holotoxin complexes. LT-BICT-B are nontoxic and mediate receptor (ganglioside GM11 binding. Secretory antibodies against CT-B/LT-B can neutralize holotoxin activity. CT-B/LT-B are among the few proteins that are highly immunogenic when administered by the oral route as purified polypeptides (Aizpurna and RussellJones, 1988). Diarrhea mediated by enterotoxicogenic E. coli and Vibrio cholerae is in part due to holotoxin action, which can be neutralized by (cross-reacting) secretory antibodies against LT-B/CT-B. LT-B was

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first expressed in attenuated Salmonella typhi and S . typhimurium strains in a n effort to create hybrid oral vaccines protecting from typhoid, cholera, and enterotoxicogenic E . coli (ETEC) (Clements and El-Morshidy, 1984; Clements et al., 1986).Although these vaccines may not be very efficient against cholera or ETEC-associated disease, which are caused by additional toxic effects of the microorganisms, it has been demonstrated that LT-B can be stably expressed in attenuated Salmonella strains and is immunogenic when delivered in recombinant salmonellae by the oral route (Clements and El-Morshidy, 1984; Clements et al., 1986; Maskell et al., 1987; Schodel and Will, 1989; Schodel et al., 1990a,b).It has since become one of the foreign antigens used to demonstrate the ability of other attenuated bacteria, for example, Yersinia enterocolitica (Sory and Cornelis, 1990), to serve as foreign antigen carriers that elicit systemic and mucosal immune responses by the oral route. LT-B/CT-B are very strong immunogens when administered by the oral route, in the absence of a bacterial carrier (see, e.g., Lycke and Holmgren 19861, and therefore information about the usefulness of carrier strains to deliver antigens to the systemic immune systems as derived from CT-B expression experiments is limited CT-B secreted by V . cholerae elicits systemic antibodies; in Vibrio cholerae CT-B is secreted not only to the periplasm but to the extracellular milieu by a specialized secretion apparatus. CT-B also elicits systemic antibodies when coadministered with killed vibriones (see, e.g., Czerkinsky et al., 1991). The fact that CT-B and LT-B are highly immunogenic in live bacteria and as purified immunogens makes them interesting carrier molecules, both for the expression of foreign epitopes in attenuated carrier bacteria and for the delivery of foreign antigens across mucosal surfaces in the form of translational fusions t o LT-B/CT-B. We have fused a number of hepatitis B virus and woodchuck hepatitis B virus B cell and T cell epitopes to the carboxy teminus of LT-B; the fusion genes were expressed in attenuated S. dublin strains (Schodel and Will, 1989; Schodel et al., 1990a,b). Unfortunately, those fusion proteins, tested by oral administration of recombinant salmonellae to mice, retained the high T and B cell immunogenicity of LT-B but elicited few, if any, antibodies to the carboxy-terminal fused viral sequences. A T cell epitope of the HBV nucleocapsid protein fused to LT-B was recognized by mouse splenic cells in a proliferative assay (Schodel et al., 1990b). We have recently shown that the fused sequences are immunogenic when the purified fusion protein is used for oral or parenteral immunization, but the antibody response t o LT-B/CT-B is about 20 to 30 times higher than against the fused sequence (Schodel et al., 1991b). A similar experience was made with other amino-terminal fused epitopes (Sanchez et al., 1990; Dertzbaugh et al., 1990). CT-B and LT-B could still be inter-

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esting carrier molecules for foreign epitopes if it becomes possible to transfer the high immunogenicity of the native molecules to the inserted sequences.

D. Miscellaneous Proteins: Comments and Outlook Apart from the three epitope presentation systems described above, any protein that can be stably expressed in prokaryotes without impairing their physiology too much and that has strong T helper cell sites could probably be used as a carrier for heterologous epitopes. Two protein carriers of HBV and poliovirus epitopes have been extensively studied in E . coli by Maurice Hofnung and co-workers; these carrier proteins are LamB and MalE (for overview see O’Callaghan et al., 1990; see also references in Table 11).The selection of proteins that are potentially useful as carriers of foreign epitopes given in Table I1 is by no means exhaustive. For additional gene fusion systems extensively used in E . coli to stabilize expression or to help purify recombinant products, see Lenstra et al., (1990), Stader and Silhavy (1990), and Uhlen and Moks (1990). Intracellular localization of the recombinant protein in the carrier bacteria seems not to drastically influence immunogenicity, because there are immunogenic proteins in all compartments (see references in this section and Section I1 and Tables I and 11).A systematic comparison of the differential immunogenicity of the same epitope or antigen is hampered by the fact that expressing the same epitope within the same carrier molecule in different bacterial compartments also implies changing its amino acid sequence surroundings, with a potential impact on the availabilty of T cell help even if both locations are equally surface accessible in the purified molecule. In addition, every chimera or antigen will fold differently if, for example, a leader sequence for periplasmic secretion is provided, therefore making comparisons difficult. Predictably, a good bacterial carrier antigen would be one that induces high-titered antibody responses during natural infection (see, e.g., Brown and Hormaeche, 1988) or one that has a high degree of inherent immunogenicity, such as the particulate hepatitis B virus nucleocapsid (Schodel et al., 1 9 9 0 ~ ) . OF FOREIGN ANTIGEN EXPRESSION IV. GENETICSTABILIZATION

Most experiments described in this review were performed using high-copy-number plasmids carrying antibiotic resistance markers and a consititutively active promoter element. An inherent problem of this

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approach is plasmid segregation i n vivo and therefore loss of expression of the foreign antigens. Also, the presence of antibiotic resistance markers may not be desirable for safety reasons in strains that are released into the environment. Two strategies are currently pursued to overcome these problems. (1)Plasmids were designed that complement in trans a lethal mutant phenotype of the bacterial host. (2) Expression cassettes were integrated into the chromosome by homologous recombination, or in the case of a BCG expression system, by taking advantage of a phage integration system (Stover et al., 1990).

A . Plasrnids without Antibiotic Resistance Marker Roy Curtiss and colleagues showed that mutants of S . typhirnurium that are unable to synthesize the enzyme aspartate-P-semialdehyde dehydrogenase (Asd), important in cell wall synthesis, and lyse in the absence of diaminopimelic acid, are highly attenuated in mice (Curtiss, 1985). They cloned first the Streptococcus mutans and later the S. typhimurium asd genes and used them to construct expression plasmids that complement Aasd Salmonella strains in trans (Nakayama et al., 1988; Galan et al., 1990). Trans-complementation restores full virulence to wild-type Aasd strains. This does not prevent plasmid segregation, but cells that lose the plamids lyse. Using these plasmids, several foreign genes could be stably expressed in attenuated Salmonella strains. A transposon has been constructed that allows insertion of the asd gene in any expression vector, retaining or deleting the antibiotic resistance genes as required (Galan et al., 1990).This general strategy could be extended to other balanced lethal mutations. It provides a high level of containment. On top of a negative selection against vaccine strains that have lost their plasmid it would be desirable to prevent plasmid segregation i n uiuo. Most Salmonella species harbor a virulence plasmid (see also next section). This plasmid is unusually stable and it may never be lost i n uiuo (Gulig, 1990).The partitioning region of this plasmid has been cloned and it has been shown that it can be used to stabilize some high-copy-number plasmids (Tinge and Curtiss, 1990). Where tocicity of the recombinant product for the carrier strain is a problem, it may be possible to use promoter constructs that are only active in a part of the bacterial population, so that normal replication and invasiveness are conserved in the off-state and the vaccine strain continously segregates cells that produce the recombinant antigen in high quantities. An expression cassette, based on the A leftward promoter placed between invertible G segment sequences of bacteriophage

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Mu, which is inverted by the gin invertase under thermosensitive cl repressor control, has recently been described (Yan et al., 1990). The only cells that express the gene of interest are those in which the orientation of the P L promoter is directed toward the promoterless gene.

B. Chromosomal Integration Two groups have so far published systems for the chromosomal integration of expression cassettes via homologous recombination. Hone et al. used the cys gene as a target gene for the integration of the K88 fimbrial gene (Hone et al., 1988a), the group of Gordon Dougan used aroC as a target gene (Strugnell et al., 1990). Flynn et al., (1990) have reported a defective transposon carrying an expression cassette that integrates into random sites on the chromosome. They have described expression of a P. yoelii circumsporozoite antigen by one of these chromosomally integrated constructs (Flynn et al., 1990). An interesting alternative has been successfully used in BCG. The cloned and sequenced inu gene of a mycobacterial phage that undergoes site-specific integration into the chromosome was put on a plasmid directing synthesis of LacZ and functioned to integrate the LacZ expression cassette (Stove et al., 1990). Chromosomal integration might be the most attractive way to create stable antibiotic-sensitive vaccine strains. Copy number might, however, often be a problem a t the levels of expression achieved: so far, levels of expression by chromosomally integrated expression cassettes have not been reported; in the published data, the foreign antigens were a t best visualized on Western blots. Our experience is that even a highly immunogenic molecule such as LT-B induces little immune response when administered orally in live recombinant Salmonella if the level of expression is below approximately 100 ng/ml (overnight culture) (F. Schodel et al., unpublished observations). The use of stronger promoters, optimized translation signals, and integration closer to the chromosomal origin of replication might solve the problem of low-level expression. It is not clear whether such chromosomally integrated constructs under strong promoter control will be sufficiently stable. Alternatively, it might be possible to use promoters that are most active a t certain stages of the infection, for example, thegroE promoter or other promoters that are up-regulated in macrophages (Buchmeier and Heffron, 1990). It remains speculative whether preferential intracellular synthesis of foreign antigens would contribute to their immunogenicity.

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43 1

V. BACTERIA POTENTIALLY USEFULAS CARRIERS Probably many bacteria are good candidates as carriers for heterologous antigens to the immune system. A carrier organism ultimately useful for human use should not only be safe and immunogenic, but ideally also should provide protection against disease caused by the wild-type strain it is derived from. Our aim should not be to develop more vaccines, but rather vaccines better suited for the purpose of the vaccination, and, where possible, multivalent vaccines. While some proposed bacterial carriers, such as lactobacilli (Gerritse et al., 19901, may be intrinsically safe, but they certainly do not protect against any wildtype disease. Among the potential bacterial carriers, attenuated Salmonella strains have been tested most extensively in animal models and S . typhi strains that might be useful as typhoid vaccines and carriers of heterologous antigens are being developed. Therefore, Salmonella will be most extensively discussed; S. typhimurium and S . typhi strains will be discussed in parallel because the information currently available about the latter is limited and S . typhimurium causes a disease resembling typhoid in mice. A second potentially important carrier bacterium is BCG, not least because it is the bacterial vaccine with the widest distribution worldwide. Even though the current route of administration of BCG is intradermal and thus extrapolation of its usefulness as an oral carrier remains somewhat speculative, it has historically been used by this route and deserves further discussion. Additionally, it is possible to endow intrinsically noninvasive bacteria such as E. coli K12, by genetic means, with the invasion aparatus of, e.g., Shigella enterocoLitica and transform them into potential carriers. This possibility has been reviewed recently by Hale (1990, Hale and Formal, 1989) and will not be considered here.

A. Attenuated Salmonella Strains An attenuated Salmonella strain should be safe and immunogenic. This balance may be difficult to obtain depending on the nature of the immune response that is necessary, as systemic immunogenicity will partly be dependent on the invasiveness of the strain. Yet several mutants of various Salmonella species that have been described retain invasiveness but are of low virulence. Although our knowledge about genes important for virulence increases, all the attenuated Salmonella strains available have been derived empirically, not by deliberate manipulation of “virulence” genes, even if some authors consider their

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methods of attenuation more rational as others. It can be envisaged that a more detailed knowledge of genes important in virulence regulation, such as genes required for attachment, cell invasion, or intracellular survival, will ultimately be exploited to create attenuated strains with well-characterized traits. Safety on the geneic level implies that the mutation should have a very low probability of reversion; this criterion is met by deletions in defined gene loci, whereas biochemically defined mutants might be single-point mutations that have too high a chance to revert. Probably the genetically safest strains are those carrying deletions in independent gene loci that could not revert after a single transduction event. The attenuation of auxotrophs is dependent on the lack of certain precursor substances in the host tissue. Thus gene defects causing auxotrophy might in addition have a t least the theoretical possibility of phenotypic reversion, if the compound required is present as a dietary component in high amounts, for example, and thus becomes available in host tissues that normally do not supply the compound and thus limit bacterial growth. Mutations in about 5%)of the genes of S . typhimurium reduce virulence when bacteria are introduced orally; mutations in about 2% of the genes reduce virulence when bacteria are introduced by the intraperitoneal (i.p.1 route (F. Heffron, personal communication, 1991). This would amount to about 60-200 mutants (only considering one per gene) that are still to be tested. At this point it has to be remembered that only one mutant S. typhi strain is currently licensed as an oral vaccine and that this strain is by no means satisfactory for the reasons outlined below. Although testing of a large number of mutant genes for S. typhimurium in a mouse model is feasible and will be done, it will take a long time until even a small number of these mutants is tested in humans. Despite the persisting need for a better typhoid vaccine and the emerging potential of S. typhi as carriers, there is currently one center in the world (the Center for Vaccine Development in Maryland) where new S. typhi strains are clinically tested in a systematic manner. 1 . galE

Salmonella typhimurium strains with defects in the gene for the UDPgalactose epimerase gene are nonvirulent (Germanier and Fiirer, 1971; Hone et a.Z., 1987).Galactose epimerase (GalE)is necessary for the synthesis of complete LPS; galE strains are rough when grown in the absence of galactose. When low amounts of galactose are added to the medium, smooth strains can be grown but have a tendency to acquire a galactose-resistant phenotype, which is rough, noninvasive, and nonimmunogenic. When Germanier and Fiirer (1975) isolated a nonvi-

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rulent S. typhi mutant after random nitrosoguanidine mutagenesis, it turned out to be a gulE mutant and it was assumed that the galE mutation is the basis of attenuation. In the meantime, it has become clear that galE cannot be the main attenuating mutation in Ty2la. GalE+ revertants of Ty2la remain avirulent (Silva-Salinas et al., 1985) and a genetically defined galE mutation introduced into S. typhi by David Hone caused typhoid in volunteers (Hone et al., 1988b). A S. typhimurium strain harboring the samegalE mutation is avirulent and immunogenic (Hone et al., 1987). Ty2la has a number of additional mutant phenotypes that may contribute to its avirulence (discussed in Hone et al., 1988a,b).Ty2la is a particularly labile strain and success or lack of success in immunization studies has depended on the mode of application and the manufacturing process (Levine et al., 1990a,b). Although it is the only currently licensed and an empirically safe live typhoid vaccine strain, it is probably not very useful in the currently marketed preparations. In general, Ty2la has been disappointing as a typhoid vacine strain outside of few carefully controlled trials and is therefore unlikely to be a good candidate carrier strain.

2 . Plasmid -Cured Strains Many species and serotypes of SalmonelLa, with the notable exception of S. typhi, carry large plasmids (50-100kbp) associated with virulence (for review see Gulig, 1990). These virulence plasmids are stably transmitted. Plasmid-cured derivatives of S. typhimuriilm are attenuated by oral administration (Hackett et al., 1986; Pardon et al., 1986; Gulig and Curtiss, 1987) but retain significant virulence when administered i.p. The absence of the virulence plasmid of S . typhimurium affects the tissue distribution after oral infection. Plasmid-cured derivatives of S . typhimurium are impaired in their ability t o reach the mesenteric lymphnodes and spleen (Gulig and Curtiss, 1987). Curing of the virulence plasmids alone is insufficient to generate safe, attenuated vaccine strains. 3 . A uxotrop hs

It was noted by Bacon and colleagues in the 1950s that S. typhimurium strains that are auxotrophs for aromatic compound precursors or purines are nonvirulent in mice (Bacon et al., 1950a,b, 1951). This notion was first systematically exploited by Stocker and colleagues to create genetically defined auxotrophic mutants by transposon mutagenesis (Hoiseth and Stocker, 1981; Robertson et al., 1983; Smith et al., 1983; McFarland and Stocker, 1987; Edwards and Stocker, 1988). Mutants in several genes of the aromatic compound biosynthesis pathway

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FLORIAN SCHODEL TABLE 111 MUTATIONS THAT ATTENUATE Salmonella STRAINS

Gene Auxotrophs Pub pabB asp" his" cys" aroA aroC aroD Pur asd

daP purA purEb purHD nadA pncB gua.4 guaB LPS synthesis galE pmi rfc

Ref. Bacon et al. (1950b) Stocker (1990) Bacon et al. (1950a,b, 1951); Curtiss (1990) Bacon et al. (1950a,b, 1951); Fields et al. (1986) Bacon et al. (1950a,b, 1951) Hoiseth and Stocker (1981); Dougan et al. (1987); Mukkur et al. (1987); Lascelles et al. (1988) Dougan et al. (1986) Miller et al. (1989b) Bacon et al. (1950a,b, 1951);McFarland and Stocker (1987); Fields et al. (1986) Curtiss (1985) Clarke and Gyles (1987) Brown and Stocker (1987) OCallaghan et al. (1988) Edwards and Stocker (1988) Wilson and Stocker (1988) Wilson and Stocker (1988) Fields et al. (1986) Fields et al. (1986) Germanier and Fiirer (1971, 1975); Nnalue and Stocker (1987); Hone et al. (1987) Collins et al. (1991) Collins et al. (1991) (continued)

of S. typhimurium and S. dublin are highly attenuated yet invasive enough to provide good protection against wild-type challenge after oral immunization in small rodents and cattle (see above, see also Eisenstein et al., 1984; Killar and Eisenstein, 1984, 1985; Mukkur et al., 1987; O'Callaghan et al., 1988; Carsiotis et al., 1989). The attenuation is probably due to a requirement forp-aminobenzoic acid, which is not present in mammalian tissues (Stocker, 1990). Purine-dependent auxotrophs of S. typhimurium were also found to be highly attenuated in mice. Double insertion/deletion mutants in aroA purA were therefore created in S. typhi (Edwards and Stocker, 1988) and were tested for safety and immunogenicity in human volunteers (Levine et al., 1987). Unfortunately, the strains were safe but elicited only marginal im-

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TABLE 111 (continued) Gene Regulatory CrP phoP PhoQ ompR recA recBC fruR Others CYa Pa& htrA mviS ompFh T”d Strde MY

Ref. Curtiss and Kelly (1987) Fields et al. (1989); Galan and Curtiss (1989);Miller et al. (1989a,b) Miller et al. (1989a,b) Dorman et al. (1989) Lipps and Heffron (1989) Lipps and Heffron (1989) Saier and Chin (1990) Curtiss and Kelly (1987) Miller and Mekalanos (1990) Johnson et al. (1990) Carsiotis et al. 11989) Dorman et al. (1989) Fahey and Cooper (1970); Hormaeche etal. (1981); Ohta et al. (19871 Reitman (1967); DuPont et al. (1970);Cvjetanovic et al. (1970) Furness and Rowley (1956);Furness (1958); Fields et al. (1986)

Not all mutants are attenuated and not all have been tested for immunogenicity. These mutants are only slightly attenuated. galE is not attenuating in S . typhi (Hone et al., 1988b) and not always in S. choleraesum (Nnalue and Stocker, 19861. Temperature sensitive, restricted growth a t 37°C. Streptomycin dependent. Mutants defective in survival in macrophages. a

mune responses. Apparently the purA mutation had caused an overattenuation of the strains, a finding that was confirmed for S. typhimurium purA mutants in mice (Sigwart et al., 1989).In the meantime, several double mutants in the aro genes of S . typhi have been constructed and are currently being tested (Dougan et al., 1988; Levine et al., 1990a,b). Analogous aro double mutants of S. typhimurium have been shown to be highly attenuated while preserving immunogenicity (Dougan et al., 1988). Other auxotrophs that are attenuated but have not been extensively used as carriers, some because they are overattenuated or not sufficiently attenuated, are listed in Table 111. 4 . Regulatory Genes and Others

The group of Roy Curtiss has demonstrated that deletion mutations in the adenylate cyclase and the cyclic AMP receptor protein genes (cya and crp) render various Salmonella species avirulent while retaining their protective effects (Curtiss and Kelly, 1987).The two mutations in

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different gene loci can be combined and result in strains that are as attenuated and immunogenic as the single mutants but are highly unlikely to revert to wild type. The reason for attenuation is unknown; the strains have a reduced generation time. Cyclic AMP binds to the CAMPreceptor protein to exert a positive regulatory effect a t the transcriptional level on the expression of many genes, for example, those required for carbohydrate and amino acid transport and expression of surface proteins (for more detailed discussion see Curtiss, 1990). More recently, inactivation of another regulatory gene, phoP, has also been demonstrated to attenuate S. typhimurium strains (Fields et al., 1989; Galan and Curtiss, 1989; Miller et al., 1989). The phoP gene regulates the expression of various other genes, among them those encoding gene products that confer resistance to defensins, a class of intracellular microbicidal molecules present in mammalian hosts (Fields et a1., 1989), and pug a gene of unknown function homologous t o a Yersinia virulence gene ail (Pulkinnen and Miller, 1991). Both phoP mutants, which are unable to express pug, and pugc mutants, in which the expression of pug has been uncoupled from transcriptional control and rendered constitutive, have a reduced virulence and are immunogenic (Miller and Mekalanos, 1990). pugc Mutants have a n only slightly increased LDS0 compared to wild-type. The phoN deletion does not appear to attenuate S. typhimurium (Groisman et al., 1989; Fields et al., 1989). Mutations in ompR, important in the control of expression of outer membrane proteins, also reduce the virulence of S. typhimurium, as does a mutant in one of the porins encoded by ompF, if only to a lesser degree (Dorman et al., 1989). A number of attenuated mutants were derived by phage Tn p h o A insertion mutagenesis in virulent S. typhimurium and testing for avirulence in an oral infection model (Miller et al., 1989). Of the avirulent smooth S. typhimurium mutants, two were found to be insertions in htrA a homolog of an E . coli heat-shock protein gene ( Johnson et al., 1990). Mutants in recA and recBC were also found t o be attenuated and a combination of each with aroA led strains with a virulence similar to the original aroA mutant (Lipps and Heffron, 1989). These could be interesting carriers, as foreign genes might be more stably maintained in them. It is probably not interference with homologous recombination in these mutants but with the SOS salvage pathway that makes recA and recBC S . typhimurium less virulent. They might be more susceptible t o oxidative stress inside phagocytic cells (Lipps et al., 1991).These mutants have so far only been tested by the i.p. route; it is not clear whether they would retain sufficient invasiveness for immunogenicity

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by the oral route. A V. cholerae recA mutant was reported to be of reduced immunogenicity when administered by the oral route (Kettley et al., 1990).

B . Bacille Calmette-Guerin Bacille Calmette-Guerin (BCG)is an attenuated derivative of Mycobacterium bovis attenuated by growth on ox bile by Calmette and Guerin (1908) and was used as an oral vaccine in a classical experiment 1921 on a child infected with Mycobacterium tuberculosis. Although of doubtful value in the prevention of mycobacterial disease (see discussion in Barletta et al. 19901, it is probably the most widely distributed vaccine worldwide. BCG is administered intradermally shortly after birth and by that route is an extraordinarily safe vaccine; the major complication is local tuberculous (granulomatous)lymphadenitis due t o overdosage. This happens occasionally when differences in virulence between different strains are not taken into account in the dosage regimen. The reasons for the lack of protection against M . tuberculosis hominum following immunization with BCG seen in some studies are not clear; among them might be a lack of cross-reaction of critical antigens, which could eventually be overcome by developing attenuated derivatives of M. tuberculosis hominum. Historically, BCG was used until 1978 as an oral vaccine (Calmette, 1928; Zeyland and Piasecka-Zeyland, 1929; Symposium, 1958). Problems associated with oral use were occasional lymphadenitis colli, which can be a serious complication, and the lack of effectiveness by the oral route due to neutralization of BCG by stomach acidity. The group of Barry Bloom has developed transformation protocols for mycobacteria and shuttle plasmids from E . coli ( Jacobs et al., 1987; Snapper et al., 1988).Expression of foreign antigens in mycobacteria has been achieved both from extrachromosomal replicons and from chromosomally integrated expression cassettes (Snapper et al., 1988; Husson et al., 1990; Stover et al., 1990). The transcription and translation signals of mycobacteria are being studied (Barletta et al., 1990). Mycobacteria as facultative intracellular microorganisms elicit CD8 ' CTL responses (for review see Kaufmann, 1988) and might be interesting carriers for the presentation of antigens to the class I pathway. To exploit the currently available network for immunization with BCG, however, would require continued intradermal application of recombinant BCG. To exploit its potential as an orally administered carrier, additional research has to be done and the current immunization network will not present as much of a n advantage.

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VI. OUTLOOK What then, is the prospect for oral vaccination using recombinant bacteria expressing viral epitopes? Live recombinant bacteria stably expressing viral antigens would be cheap to produce, and if they can be given orally are easy to administer. Most of the relevant information so far stems from the use of attenuated S . typhimurium strains. Salmonella typhimurium can be attenuated t o nonvirulence by defined genetic manipulations while retaining invasiveness, and can be used to elicit mucosal and systemic immune responses against carried foreign antigens in several animal models. Both antibody and T helper responses against recombinant antigens can be generated using such strains for oral immunization. Epitope expression systems are being developed that allow induction of high-titered persistent antibodies against contiguous foreign epitopes. Using these systems, live recombinant antiviral vaccines can be constructed for viruses wherein peptidic epitopes that induce virus-neutralizing antibodies have been defined. An important future effort will be to develop epitope presentation systems that allow the modeling of three-dimensional antibody-binding sites. Unfortunately we are far from achieving this goal. Future efforts will also have to include the expression of multiple viralibacteriali parasitic epitopes in a single carrier strain to create multivalent vaccines. Although it may also be possible to generate CD8+ CTL responses, this may not be necessary or beneficial for most vaccination purposes. Genetic means to express stably sufficient quantities of foreign antigens without antibiotic resistance markers are being developed. An important bottleneck is the absence of sufficiently tested attenuated S . typhi strains for human use that combine nonvirulence with immunogenicity. As immunogenicity will predictably require invasiveness, this balance might be difficult to achieve. Both Acya Acrp and AaroC AaroD S. typhi are currently undergoing their first clinical trials (Levine et al., 1990a,b; R. Curtiss and M. Levine, personal communication) and may be promising candidate carrier strains. Suitable oral immunization techniques and protocols will have to be developed for BCG. The impact of previous immunizations with a carrier strain or exposure t o wild-type infections on immune responses to a carried foreign antigen will have to be determined. Finally, a current irrational hostility toward recombinant DNA technology in some developed countries might become an important impediment for further exploration of live bacteria as carriers of recombinant antigens.

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ACKNOWLEDGMENTS We thank Fred Heffron and David Milich for critically reading the manuscript. Work in the laboratory was supported by grants from the Wilhelm-Sander Stiftung and the Walter-Schulz Stiftung.

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Prospects for oral vaccination using recombinant bacteria expressing viral epitopes.

ADVANCES IN VIRUS RESEARCH. VOL. 41 PROSPECTS FOR ORAL VACCINATION USING RECOMBINANT BACTERIA EXPRESSING VIRAL EPITOPES Florian Schodel Max-Planck-ln...
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