Vaccines against Infections Caused by <i>Salmonella</i>, <i>Shigella</i>, and Pathogenic <i>Escherichia coli</i>
CARLOS A. GUZMAN,1* STEFAN BORSUTZKY,1 DIDIER FAVRE,2 AND GUIDO DIETRICH2
[SECTION EDITORS: FERRIC FANG AND MARTIN KAGNOFF]
Posted December 29, 2004
Vaccine Research Group, Division of Microbiology, GBF—German Research Centre for Biotechnology, Mascheroder Weg 1, Braunschweig, Germany,1 and Berna Biotech Ltd., Rehhagstr. 79, CH-3018 Berne, Switzerland2
*Corresponding author. Phone: (49) 531-6181558, Fax: (49) 531-6181411, E-mail:
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Different mechanisms contribute to protection against infections caused by enteropathogenic bacteria. The general clearance mechanisms of the mucosal barrier (e.g., mucus, ciliary activity, lytic enzymes, and pH) and the innate immune system constitute the first nonspecific line of defense against infections. The adaptive immune response represents a more refined and pathogen-specific second line of defense, which also leads to memory responses able to protect against subsequent challenges. The cellular components of the innate immune system (e.g., dendritic cells) provide a critical bridge between the innate and the adaptive immune system. Pathogen-specific responses can be stimulated by previous infections or through vaccination, which constitutes the most cost-efficient prevention strategy. However, there are diseases caused by enteropathogenic bacteria for which vaccines are not available or the available vaccines are not completely satisfactory in terms of safety, efficacy, and costs. Thus, it is essential to develop and implement better, safer, and cheaper vaccines able to promote long-lasting protection. In this context, vaccines that are able to stimulate not only systemic but also mucosal immune responses have several advantages. The stimulation of local responses at the portal of entry may protect against both disease and infection (i.e., colonization), thereby reducing the risk of pathogen transmission to other susceptible hosts.
Different approaches have been successfully pursued to develop vaccines against Salmonella, Shigella, and E. coli. These include inactivated whole-cell vaccines, live attenuated vaccines, and subunit vaccines (see Table 1 for the specific properties, strengths, and limitations of the different strategies). The first approach is the use of whole-cell vaccines, which are obtained by inactivating virulent organisms. These vaccines can be administered to immune-deficient hosts. For many years, heat inactivation was the preferred strategy to produce whole-cell vaccines. All virulence factors expressed by the pathogen are preserved by this method. However, the destruction of heat-labile antigens during preparation can compromise efficacy. This problem has in part been solved by the use of chemicals for inactivation (e.g., formaldehyde, glutaraldehyde, and phenol). However, chemical agents may also disrupt antigenic structures. In addition, the quality control (e.g., potency tests, killing) of inactivated vaccines may be very difficult.
Table 1Properties of inactivated, attenuated, and subunit vaccines |
The second approach is based on the use of attenuated bacteria that are unable to cause clinical disease but trigger a self-limiting infection, leading to the stimulation of protective immunity (36). Attenuated vaccines are also able to elicit cellular immune responses, which play an important role in the clearance of intracellular pathogens. However, key virulence factors may be missing in an attenuated vaccine strain depending on the specific attenuating mutation, whereas most virulence factors can be expressed by strains used in the preparation of an inactivated vaccine. On the other hand, antigens that are only expressed in vivo will not be present in an inactivated vaccine preparation. The expression of virulence factors is responsible for the main difficulty associated with the design of live vaccines: achieving the optimal balance between safety and immunogenicity. In addition, the use of attenuated microorganisms is associated with the potential risk of reversion to a virulent state. This risk has been largely overcome by the use of multiple well-defined attenuating deletion mutations (36). Nevertheless, live bacterial vaccines require a careful and exhaustive evaluation of the potential impact of environmental release. It is essential to assess the duration and rate of shedding and the risk of horizontal gene transfer. This potential disadvantage is balanced by the simplicity of vaccine administration via the mucosal route, which is also associated with a higher acceptance by vaccinees and more simple and inexpensive administration logistics (36). The mucosal route also provides better safety, since parenteral vaccination is an important cause of spreading of blood-borne diseases (e.g., HIV and hepatitis) in the developing world. The ability of most attenuated vaccines to replicate in the host results in the elicitation of strong and long-lasting immune responses, which mimic those stimulated by natural infections (129, 170). However, the presence of preexisting or cross-reactive immune responses against the vaccine strain may affect overall efficacy.
A third approach is the use of subunit vaccines that are generated by using purified subcellular components from pathogenic bacteria. These components are obtained from wild-type organisms (native antigens) or by recombinant DNA technology. Subunit vaccines generally have an improved safety profile, since all the components present in the formulation are well defined. However, this may depend on the route of administration; for example, oral attenuated vaccines against typhoid fever are less reactogenic than parenteral inactivated ones (see below). Moreover, the immunogenicity of subunit vaccines is usually poorer than that of cellular vaccines, which contain immune stimulatory components (e.g., cell wall components, DNA), requiring the use of adjuvants (Table 1). Subunit vaccines often have higher production costs than cellular vaccines but are less reactogenic. An important aspect that needs to be considered is that a subunit vaccine may lose efficacy as a result of changes in the antigenic profile of circulating strains (139, 169). This risk is minimized when multiple antigens or whole organisms are used in the formulation. However, the presence of multiple antigens may lead to a reduction in vaccine efficacy, as a result of the suppression of immune responses against subdominant antigens by immune-dominant ones.
Typhoid fever is an acute, life-threatening, febrile illness resulting from infections caused by the Salmonella enterica serovar Typhi and, to a lesser extent, by Salmonella enterica serovar Paratyphi types A and B (48). Global estimates indicate that over 17 million new cases of typhoid fever occur each year, resulting in more than 600,000 deaths (83, 147). Different Salmonella serovars can also cause localized enteric syndromes for which no vaccines are available. Although antibiotics have been used successfully to cure typhoid fever in the past, the emergence of multi-drug-resistant strains has rendered the clinical management of infected patients increasingly difficult (137). This situation underscores the importance of preventive strategies, such as improved hygiene, and highlights the role of vaccination for the control of typhoid fever. The most common sources of transmission are contaminated food and water. Doses of as few as 105 organisms have resulted in typhoid fever in up to 55% of volunteers in studies performed in endemic areas (106). In the initial stages of infection, bacteria survive in the acidic content of the stomach, reach the small intestine and penetrate the mucosal barrier (Fig. 1 and 2), eventually reaching the mesenteric lymph nodes via lymphatics. The main host defenses at this initial stage are the local and systemic immune responses, particularly secretory antibodies. An effective immune response may prevent bacteria from reaching the lamina propria, thereby blocking subsequent stages of the infection process. Following penetration of the mucosal epithelium in the nonimmune host, microorganisms may translocate via the blood stream to the reticuloendothelial cells of the liver and spleen, in which they replicate. Immune responses against circulating extracellular bacteria encompass antilipopolysaccharide (O), anticapsular (Vi), and antiflagella (H) antibodies (Fig. 3). However, these immune responses stimulated by typhoid infection do not result in protection from relapse and reinfections, thereby confirming the important role of a safe and efficacious vaccine.
Inactivated Parenteral Vaccines against Typhoid Fever.
The original concept of a vaccine to protect against typhoid fever was first introduced in England in 1896 and resulted in the production of a parenteral whole-cell vaccine. During the 1960s and 1970s, trials conducted by the World Health Organization (WHO) determined that the efficacy of a vaccine consisting of S. enterica serovar Typhi inactivated by heat and phenol, or, alternatively, by acetone, ranged between 51% and 88% in children and young adults, respectively, and lasted for up to 12 years (8, 71, 180). Although these early parenteral vaccines were clearly able to confer protection against typhoid fever, their global use as public health tools for routine vaccination was undermined by a high incidence of adverse reactions, such as fever (6 to 30%), headache (10%), and severe local pain (up to 35%) (7, 45). Likewise, a whole-cell killed vaccine consisting of serovar Typhi, together with S. enterica serovar Paratyphi types A and B (TAB) was found to induce strong local side reactions and systemic symptomatology in over 65% of vaccinated individuals (32). The first human trials using attenuated strains against typhoid fever were made in the early 1970s with a streptomycin-dependent mutant of serovar Typhi (107). Although this vaccine conferred no significant protection, more effective alternative vaccines have replaced these initial prototypes, namely the parenteral Vi-based subunit vaccines and the live attenuated oral vaccine Ty21a.
Vi-based Parenteral Vaccines against Typhoid Fever.
The Vi (virulence)-capsular polysaccharide (Fig. 3A) consists of (α1-4),2-deoxy-2-N-acetylgalacturonic acid, which is partially O-acetylated at carbon 3 and forms a capsule that protects bacteria against complement-mediated lysis and phagocytosis (121, 157). For vaccine production, the Vi antigen is isolated from the virulent wild-type serovar Typhi Ty2 strain. The vaccine is administered as a single intramuscular or subcutaneous dose containing 25 μg of nondenatured Vi antigen; revaccination is recommended after 3 years (73).
Immunization with Vi antigen results in the induction of anti-Vi antibody titers in vaccinees in endemic and nonendemic areas (a fourfold rise in anti-Vi antibodies is defined as seroconversion). Previous exposure to Salmonella does not seem to influence the immune response (1, 94, 174). The protective efficacy of the Vi polysaccharide was assessed in field trials in Nepal and South Africa in the 1980s. In the South African trial performed in children, the vaccine exhibited a protective efficacy of 64% during the first 21 months after vaccination and an efficacy of 55% over 3 years (94, 95). In Nepal, the vaccine resulted in 75% protective efficacy over 20 months in children and adults (1, 153). Vi capsular polysaccharide is well tolerated and safe. The most important reported side effects are fever, pain, redness, and swelling at the injection site. In very rare cases, allergic reactions and rashes have been observed (73).
Since immune responses against polysaccharides do not involve T cells, immunological memory is not established, and booster effects against polysaccharide antigens are absent. Furthermore, these vaccines are not very effective in young children. The linkage of T-independent polysaccharide antigens to a T-dependent protein carrier molecule can overcome these limitations. Covalent binding of Vi polysaccharide to a nontoxic recombinant Pseudomonas aeruginosa exotoxin A (rEPA) resulted in the induction of higher and more sustained IgG antibody responses than pure Vi polysaccharide. Enhanced immunogenicity was not only observed in adults, but also in children 5 to 14 years of age. This vaccine also stimulated a booster response in 2- to 4-year-old children. A two-dose immunization schedule resulted in 92% protection of children 2 to 5 years of age in a recent field trial conducted in Vietnam (115).
Live Attenuated Oral Vaccines against Typhoid Fever.
The development of a typhoid fever vaccine suitable for oral administration received a major boost in 1975 with the generation of attenuated serovar Typhi strain Ty21a. This mutant was developed by chemical mutagenesis and has a GalE- and Vi-negative phenotype (60). The mutation of the galE gene results in a complete deficiency of the enzyme uridine diphosphate (UDP)-galactose-4-epimerase, which is responsible for the conversion of UDP-glucose to UDP-galactose and vice versa. Because of this enzyme deficiency, UDP-galactose cannot be metabolized and accumulates in the cytoplasm to cause cell lysis and attenuation, when excess galactose is present in the growth medium. However, galE deficiency alone was found to result in premature lysis when administered to mice, thereby preventing the elicitation of an adequate immune response. Therefore, strain Ty21a resulted from a further selection for reduced levels of enzymes involved in the synthesis of UDP-galactose from exogenous galactose, namely galactose permease, galactokinase, and galactose-1-phosphate-uridyltransferase (30). Since galactose is incorporated into the lipopolysaccharide (LPS) core moiety via UDP-galactose, the absence of galE leads to the formation of rough LPS, that is, LPS devoid of part of the core and the O-antigen. Since the O-antigen is the main antigenic determinant on the cell surface, Ty21a is supplied with a source of external galactose during production of the vaccine. This enables bacteria to generate UDP-galactose by an alternative route, thereby expressing complete immunogenic LPS. Thus, although the immunogenic properties of wild-type serovar Typhi are maintained when Ty21a is grown under appropriate conditions, the galE phenotype contributes to strain attenuation in vivo.
As a result of the mutagenesis process used during the generation of the vaccine strain, further spontaneous mutations were generated, including mutations in the via and ilvD genes, leading to the loss of the Vi capsular polysaccharide and an auxotrophic phenotype for isoleucine and valine, respectively. An additional mutation in the rpoS gene, which also contributes to the avirulence of the Ty21a strain (29), was inherited from wild-type parental strain Ty2. It is likely that the poor capacity of Ty21a to survive starvation conditions and resist various environmental stresses results, at least in part, from the rpoS mutation (156).
The Ty21a strain is the active constituent of Vivotif (Berna Biotech Ltd., Berne, Switzerland), currently the only licensed live oral vaccine against typhoid fever (109). It is available in two commercial formulations, either a lyophilized vaccine in capsules that are resistant to the stomach’s acids or a liquid formulation consisting of two sachets, one containing the lyophilized vaccine and the other containing bicarbonate buffer to temporarily neutralize stomach acidity. The contents of the two sachets are dissolved in 100 ml of water, which is ingested by the vaccinee. The liquid formulation is particularly suited for vaccination of children. Both formulations are given orally, one hour before meals, and in three doses (four doses in the United States and Canada) of 2 × 109 to 10 × 109 live bacteria within a time frame of one week.
As stressed before, protection against Salmonella is mediated by mucosal and serum antibodies (mucosal IgA and serum IgG) and cell-mediated immunity (CMI) (162) (Fig. 4). The oral administration of Ty21a mimics the process of natural infection, inducing not only systemic immunity-like parenteral vaccines, but also local antibody responses, thereby protecting against infection rather than only against disease (35). Increased serum IgG antibodies and gut-derived IgA antibody-secreting cells (ASC) against the O antigen are the best surrogate markers of protection. Vaccination with three doses of Ty21a induces strong serum IgG and IgA responses, which can be boosted by a fourth dose 14 months after primary immunization (191). An increase in O-specific fecal IgA was observed 1 to 8 months after immunization (144). Because of its invasive properties, Ty21a also triggers CMI, which is crucial for protection against intracellular bacterial pathogens (138). Ty21a induced strong systemic CD4+ T-helper type 1 responses in vaccinees, which were characterized by the production of gamma interferon (IFN-γ) in the absence of interleukin-4 (IL-4) (191). Vaccination with Ty21a also elicited strong CD8+ cytotoxic T cells (CTL), which persisted for at least 2 years after immunization. A strong correlation was found between the CTL activity and the frequency of IFN-γ-secreting CD8+ T cells (141).
The efficacy of Ty21a was analyzed in a large number of clinical trials (Table 2), with over 600,000 vaccinated adults and children. Excellent tolerability and an overall protective efficacy of 50 to 96% (applying the current administration schedule) were demonstrated for up to 7 years. During these studies, the liquid formulation was found to be slightly more immunogenic than the enteric-coated capsules. The field studies conducted in Santiago (Chile) confirmed the efficacy and tolerability of Ty21a and also provided evidence of herd immunity (109). The incidence of typhoid fever fell in the unvaccinated population during the years in which vaccination was performed and started to rise again as soon as vaccination with Ty21a was stopped in and around Santiago (17, 52). Two possible mechanisms have been suggested for the herd immunity effect of Ty21a. First, individuals vaccinated with Ty21a have significantly reduced excretion of virulent salmonellae than the nonvaccinated population, thereby resulting in reduced contamination of water supplies. Second, fewer temporary carriers (i.e., children with subclinical or incubating acute infections) may reduce the number of cases of direct fecal-oral transmission of the disease. Although the booster schedule for Ty21a has not been determined for travelers to endemic regions, Ty21a has been shown to confer protection against typhoid fever in 70 to 80% of vaccinees (109, 110). It is recommended that a reimmunization dose replicating the initial vaccination be administered every 5 years under conditions of repeated or continued exposure to typhoid fever (2).
Table 2Clinical trials performed with Ty21a |
The excellent safety and tolerability profile of Ty21a was further confirmed in more than 200 million vaccinees during its more than 20 years of use worldwide. Recent postmarketing surveillance has identified only mild and infrequent adverse events associated with Ty21a (65). From 1990 to 2000, more than 38 million people were vaccinated with Ty21a with only 743 spontaneous reports of adverse events, an incidence of 0.002%. The most common adverse events reported with Ty21a were mild and transient gastrointestinal disturbances, followed by general symptoms such as pyrexia. The multiple mutations of Ty21a collectively render it genetically stable. Reversion to virulence has not been observed in vitro or in vivo (61). This excellent safety profile is astonishing, particularly in the light of clinical data generated with a similar mutant strain (79). The galE via mutant strain Ty2H1, which was developed by site-directed mutagenesis of the parent strain Ty2, did not exhibit the same safety profile as Ty21a. Despite the attenuated phenotype showed in mice, Ty2H1 promoted typhoid-like disease with fever and bacteremia in two of four volunteers in a phase 1 clinical trial (79). This can be explained, at least in part, by the additional mutations present in Ty21a. Clinical trials have also shown either a limited and transient level of shedding or a complete lack of shedding in the stools of volunteers, depending on the administered dose of Ty21a (111). With a 1010 CFU dosage (higher than commercial formulations of Vivotif), a low rate of excretion, mainly on day 1 after vaccination, was observed (61). Further studies showed a lack of fecal excretion of Ty21a upon administration of the commercial formulation, and an inability to culture Ty21a from the small intestine suggests that the strain has a limited ability to proliferate in vivo (61). Neither person-to-person transmission (192) nor invasion of the bloodstream (16) has been observed in vaccinees. The very low excretion rate of Ty21a combined with its genetic attenuation significantly reduces its ability to survive in humans and the environment.
One of the drawbacks associated with the use of Ty21a is the need to administer multiple doses to elicit a protective response. Thus, as an alternative to the Ty21a strain, several attenuated serovar Typhi strains have been engineered from Ty2 with the intention to develop a vaccine strain with optimal efficacy after just a single oral dose. Among the most extensively evaluated vaccine candidates are strains CVD908 (Ty2 aroC aroD) and CVD908-htrA (Ty2 aroC aroD htrA) (175, 178, 179). Deletions in the aro genes render these bacteria auxotrophic for aromatic amino acids and for p-aminobenzoic acid and 2,3-dihydroxybenzoate. The mutant bacteria become attenuated under in vivo conditions because they are unable to scavenge these compounds that are unavailable in the human gut. The htrA gene encodes a periplasmic serine protease that degrades aberrant proteins during extracytoplasmic stress conditions. The htrA deletion attenuates Salmonella strains by impairing their response to stress and ability to survive inside macrophages (122). Upon a single oral immunization in humans, both strains have been found to be strongly immunogenic (175, 178, 179). However, although CVD908 was well tolerated, it was found to enter the bloodstream of individuals vaccinated with high doses, so its development was discontinued. Similarly, an aroC aroD derivative of the serovar Typhi isolate ISP1820 (strain CVD906) and aroA aroD (strain PBCC211) or aroA aroD htrA (strain PBCC222) derivatives of the CDC10-80 strain were found to cause fever and other adverse reactions, including vaccinemia (37). In contrast CVD908htrA was never found in blood cultures (175, 178, 179) and is therefore considered to be safe.
Based on a different attenuating concept, the Ty2-derivative Ty800 was generated with deletions in the phoP-phoQ regulon, which controls bacterial survival in phagosomes. This strain was very well tolerated and immunogenic in a phase I clinical trial, eliciting anti-O-antigen IgG and IgA in young adults (76). Curtiss and Kelly have developed an attenuated Ty2 derivative based on the deletion of the cya and crp genes. The cya gene encodes adenylate cyclase, whereas crp codes for the cyclic AMP receptor protein. These two factors constitute a global regulatory system that controls a large number of genes found to be important for the virulence of serovar Typhimurium (31). Additional mutants were generated, such as the strain X4073, which carries mutations in both the cya crp regulator genes and the virulence gene cdt involved in bacterial dissemination from the gut to deeper tissues. This strain was shown to be safe in phase I clinical trials and promoted the elicitation of IgG and IgA antibodies against the O-antigen (176). Finally, the recently developed strain ZH9 lacks the aroC and ssvA genes, the latter of which encodes a protein involved in protein secretion via a type III secretion system. This strain was also shown to be safe and immunogenic in a phase I clinical study (74).
Cross-protection against S. enterica serovar Paratyphi.
Although serovar Typhi is by far the most significant causative agent of typhoid fever, S. enterica serovar Paratyphi causes over 30% of enteric fever cases. Like serovar Typhi, serovar Paratyphi is considered to be a pathogen increasingly associated with multidrug resistance. From an immunological perspective, serovar Paratyphi types A and B lack the Vi antigen, rendering Vi polysaccharide-based serovar Typhi vaccines ineffective. However, these strains share the somatic O12-antigen with serovar Typhi, thus providing a basis for cross-protection against serovar Paratyphi from a serovar Typhi-specific vaccine that induces protection against the O12-antigen (Fig. 5). Accordingly, immunological studies have shown that volunteers vaccinated with Ty21a displayed a significant increase in cross-reactive antibacterial activity against serovar Paratyphi types A and B (144). These findings have been supported by the clinical trials discussed previously, involving more than 200,000 subjects (17, 108).
Salmonellae can cause a wide range of acute or chronic diseases (e.g., enteritis, septicemia, and abortion) in many animal species (e.g., cattle, swine, and poultry), leading to important economic losses. Their incidence rates have increased worldwide because of the intensification of livestock production. Neither hygienic measures nor chemotherapeutic agents have been able to substantially reduce the occurrence of salmonellosis. Furthermore, the risk of emergence and spread of antibiotic resistance and new regulatory standards for food products have led to a general ban on the use of antimicrobial drugs in food production. Infections caused by salmonellae do not only represent a threat to the health of farm animals but can also affect susceptible human hosts upon contact with contaminated products. Thus, infected animals are a major reservoir of virulent salmonellae, with serovar Typhimurium the second most important cause of salmonellosis in humans.
Therefore, the efficient prevention of Salmonella infections in animals is a critical issue from both public health and financial standpoints. Vaccination is the best alternative to tackle this problem since the use of vaccines can reduce (i) the clinical symptoms and consequent economic impact of veterinary diseases, (ii) the risk of carrier development, and (iii) bacterial shedding and horizontal transfer to susceptible hosts. Two major vaccination approaches have been successfully exploited to prevent Salmonella infections in farm animals: immunization with inactivated whole-cell lysates (i.e., bacterins) and vaccination with live attenuated bacterial strains. The number of inactivated or attenuated classical bacterial vaccines is consistently larger in the veterinary than in the human field because of the need to develop vaccines customized for specific animal species. New experimental approaches such as the use of DNA vaccines will not be addressed in this chapter, since they have not yet been implemented in the field.
Inactivated vaccines have been the most widely used. This can be explained, at least in part, by the broader margins in terms of acceptable side effects and the critical need to keep costs low. Classical vaccines such as Talovac 109SE (Lohmann Animal Health, Germany) consist of killed Salmonella coadministered with an oil adjuvant. To increase the in vitro expression of disease-relevant antigens, a serovar Enteritidis vaccine is produced under the conditions of iron restriction (Salenvac, Intervet, The Netherlands). Field trials in poultry have demonstrated that this vaccine reduces the shedding of Salmonella and enhances productivity (50). For cattle there are vaccines such as Murivac, a bacterin derived from serovar Typhimurium (Impfstoffwerk Dessau Tornau, Germany), which is able to protect calves against oral infection with virulent bacteria (173).
The use of live attenuated Salmonella as vaccines has become more attractive in recent years, since these vaccines are more effective than killed or subunit vaccines (126). Their capacity for the stimulation of cellular responses seems to be important for the eradication of this facultative intracellular pathogen. Serovars Gallinarum and Pullorum cause fowl typhoid and pullorum disease, respectively, in birds. The clinical manifestations include anorexia, diarrhea, hepatitis, splenitis, myocarditis, pneumonia, ophthalmitis, and high mortality due to septicemia, which can lead to decreased egg production and fertility. Rough (R) derivatives of serovars Gallinarum, Pullorum, Choleraesuis, and Dublin have been developed by passage in a medium with low nutritional content (171). Although these vaccines were generated by using empiric approaches and the underlying mechanisms of attenuation were not well understood, the 9R strain of serovar Gallinarum is still commercially available (Nobilis SG 9R, Intervet). The 9R strain confers strong protection against fowl typhoid in layer flocks after intramuscular injection, does not spread to egg content, and does not interfere with serologic monitoring (51). In an attempt to obtain a better vaccine, an aroA mutant was developed, but this strain was found to be less effective (64). Additional studies have been performed with nuoG (NADH dehydrogenase I) mutants. A single oral immunization with a serovar Gallinarum nuoG derivative reduced the mortality in 2-week-old chickens from 75% to less than 8% following challenge with virulent serovar Gallinarum (202). Temperature-sensitive (ts) mutants that have lost their capacity to proliferate at host body temperatures were also used. The fact that they do not carry any deletion in either metabolic genes or virulence factors results in a conserved pool of antigens. ts-serovar Enteritidis mutants efficiently protected chickens against lethal bacterial challenge and reduced Salmonella shedding (20). However, the main drawback of this ts attenuation was the risk of reversion, since it depended on a single point mutation.
Another strategy to obtain attenuated vaccines was based on the use of streptomycin-dependent (Smd) mutants (133, 134, 135), in which the ribosomes have been modified to make the presence of streptomycin essential for protein translation and bacterial growth, due to a mutation in the rpsL gene. Upon administration of such strains to animals, bacterial protein synthesis and growth are arrested. Oral vaccination of calves with a Smd-derivative of serovar Dublin resulted in the decline of clinical cases of salmonellosis, the reduction of bacterial excretion in feces, and a drop in calf losses (133, 134, 135). However, an intensive immunization regime was required (134). Serovar Typhimurium carrying a mutation in the galE gene (59) were also able to confer protection against oral challenge with virulent strains in calves (197). Salmonella excretion in the feces and the number of animals becoming persistent carriers were also significantly reduced in poultry receiving this type of vaccine by oral or parenteral route (198, 201). However, other studies suggested that galE mutants can retain some virulence in mice, chicken, and pigs, leading to concerns in terms of their implementation in farm animals (145, 197, 198, 201).
Auxotrophic bacteria defective in the metabolic pathways leading to the synthesis of aromatic compounds (aro) or purines (pur) depend on specific supplementation (77, 119). Vaccination with a live attenuated aro mutant of serovar Typhimurium conferred significant protection against challenge with virulent bacteria in calves (159). Different attenuations have been combined in the development of commercial vaccines to avoid the risk of reversion to the wild-type phenotype, thereby increasing the safety of the vaccine strain. Mutants carrying both aro and pur mutations exhibit limited survival in vaccinated animals while providing protection even after a single immunization (119). In eastern Germany (former GDR) such vaccines were first implemented for the control of Salmonella infections in farm animals (135). There are commercially available vaccines against (i) serovar Typhimurium for poultry and cattle (Zoosaloral H and Zoosaloral R, both pur and his), (ii) serovar Dublin for cattle (Bovisoral, pur and thiA), (iii) serovar Choleraesuis for swine (Suisaloral, R/pur) and (iv) serovar Enteritidis (Salmovac SE, ade and his) (172).
Some antibiotics target structures that are essential for cellular metabolism (e.g., nalidixic acid, rifampin, streptomycin, or nourseothricin), thereby leading to decreased proliferation and virulence. Thus, antibiotic targets are attractive structures for attenuation. However, the resulting mutants are also resistant to the antibiotics for which the molecular target has been mutated. To isolate such attenuated mutants, bacteria are grown under antibiotic selection pressure. Depending on the specific mutations, the phenotypes may exhibit a broad range of attenuation. Thus, this strategy allows the generation of mutants with nearly any desired level of attenuation (120). In the case of the Salmonella Vac T TAD (Lohmann Animal Health, Germany) vaccine strain, the so-called "metabolic drift" mutations were obtained by growing serovar Typhimurium on medium containing nalidixic acid and rifampin. This led to reduced growth, most likely due to mutations in the genes encoding gyrase and RNA polymerase. These mutations were then combined with other attenuating mutations, which control bacterial survival in the environment. The Salmonella Vac T TAD vaccine against serovar Typhimurium and serovar Enteritidis was launched in 2003 as a live oral vaccine for poultry. Three doses administered into drinking water give protection throughout the period of laying.
Salmonella transits across several anatomic niches during natural infection. Thus, the expression of virulence genes is tightly controlled. This allows a fine-tuning of gene expression according to the specific need, avoids the metabolic burden associated with the expression of unnecessary products, and prevents the display of nonessential bacterial ligands, which may constitute a target for host clearance mechanisms. Therefore, genes involved in signaling cascades have been targeted to attenuate Salmonella strains. In this context, strains carrying deletions in the genes encoding adenylate cyclase (cya) and the cAMP receptor (crp) have been also used as a basis for veterinary vaccines. cAMP and its receptor are required for the transcription of genes and operons involved in the importation of catabolites and in the biosynthesis of fimbriae and flagella. Although the cya crp mutants are able to attach and to invade lymphatic organs, they are unable to colonize mesenteric lymph nodes and spleen, thereby leading to an improved safety profile (31). Serovars Typhimurium and Choleraesuis lacking crp and cya have been found to be effective as live vaccines in chicken and pigs, respectively (70, 88). Live serovar Typhimurium vaccines based on these mutations have been commercially available in the United States since 1998 (MeganVac1; Megan Health, Inc., St. Louis, Mo.).
The efficacy of live vaccine candidates against serovar Choleraesuis (e.g., Argus SC), which causes infections in pigs, has also been evaluated. Vaccinated pigs were able to maintain normal weight after challenge and field studies. These results demonstrated that this type of vaccine is able to reduce the prevalence of Salmonella in swineherds (104). The phoP-phoQ two component regulatory system, which is required for both Salmonella persistence in macrophages and resistance to defensins, constitutes an additional target for attenuations (57, 132, 136). Other mutant strains have been evaluated less extensively, such as mutants lacking the DNA adenine methylase (Dam). This enzyme is involved in the initiation of DNA replication and the repair of mismatches and in the regulation of gene expression (58). The resulting mutants are highly attenuated for virulence and confer protection against oral challenge with homologous and heterologous Salmonella serovars in mice and chicken broilers (41). The existence of experimental vaccines obtained by recombinant DNA technology using well-defined molecular targets for attenuation suggests that a new generation of more efficient and safer vaccines will arrive soon in the market.
There are other important issues related to the use of vaccines in the control of salmonellosis in farm animals that need to be considered. One is whether immunization with one serovar can induce cross-protection against other serovars. In this context, farm- specific vaccines have been proposed to eradicate salmonellosis (193). Since vaccination is compulsory for poultry meat producers in several countries, and local regulations also require monitoring of broiler-breeder flocks with slaughtering of infected flocks (e.g., in the European Community), efforts have been made to develop efficacious multivalent vaccines for poultry (201). One of those vaccines consists of iron-restricted inactivated serovars Typhimurium and Enteritidis. After intramuscular vaccination, chickens were protected against an oral challenge, but colonization still occurred (22).
An additional problem is that it might be extremely difficult to differentiate between immune antibody responses elicited in vaccinated and naturally infected animals. This becomes critical if regulations require the slaughter of infected animals. Therefore, significant efforts need to be made to develop either marked vaccines and/or adequate diagnostic systems (9). Antigens that are not required for either bacterial viability or the elicitation of a protective response can be deleted from the vaccine strains. This would allow naturally infected animals to develop antibodies against these antigens, whereas vaccinated animals would not, permitting discrimination between natural infections and vaccinated animals by simple assessment of humoral immune responses.
Shigella species are the causative agents of shigellosis, a diarrheal disease that is transmitted via contaminated food and water or through person-to-person contact. It is caused by four main species, S. flexneri, S. dysenteriae, S. sonnei, and S. boydii. As few as 10 organisms can cause disease. The spectrum of illness ranges from moderate diarrhea to a dysentery characterized by fever, severe abdominal cramps, and mucoid bloody diarrhea. Infections caused by S. dysenteriae type 1 can also lead to severe complications such as hemolytic-uremic syndrome. Shigellosis is endemic worldwide and an important cause of morbidity and mortality, primarily among children in developing countries (68, 97, 103). Between 1966 and 1997 the annual number of episodes worldwide was estimated to be 164.7 million, with approximately 500,000 deaths occurring each year (103).
Shigella infections start in the terminal ileum and the colon where incoming bacteria gain access to the subepithelium through the M cells present in the epithelial dome associated with lymphoid follicles. There the bacteria rapidly invade macrophages, which undergo apoptosis with a concomitant release of IL-1β and induction of inflammation. Polymorphonuclear leukocytes migrate from the circulation into the gut lumen via the submucosa, an event disrupting the integrity of the epithelial barrier. As a consequence, massive numbers of Shigella enter through this breach and invade the colonic epithelium. The proteins encoded by the ipa locus govern this process, which is under control of the two-component OmpR-EnvZ system. The bacteria multiply intracellularly and spread from cell to cell through the recruitment and polymerization of cytoskeletal proteins mediated by products of the icsA and ipaB/C genes. LPS delivered in the cytoplasm of infected cells triggers the production of proinflammatory cytokines and chemokines such as IL-8. This leads to an inflammatory cascade that eventually destroys the epithelial barrier with sustained bacterial invasion and subsequent hemorrhage (164). Shigella can also multiply in the extracellular environment, a process facilitated by the iucABCD-iut genes that code for the iron-acquiring aerobactin receptor system (151). Intracellular survival of Shigella depends on intact thymine, purine, and aromatic metabolic pathways.
Evidence indicates that recovery from either naturally acquired or experimentally induced infection can confer a high degree of protection, correlating with a rise in serotype-specific antibodies directed against the O-antigen (26, 42, 43, 72). Furthermore, conversion of S. flexneri serotype Y to X by the glucosyltransferase gene of phage SfX resulted in a 75% protection in guinea pigs against the X serotype and loss of protection against the Y serotype, thereby confirming the critical importance of O-antigen-specific immunity in protection against shigellosis (80). Other surface antigens, such as the so-called invasion plasmid antigens (Ipa), stimulate immune responses during natural infections, but their significance in protection is unclear (67, 118, 184). Thus, most of the vaccination approaches against Shigella rely on presenting the O-antigen in an immunogenic form. The Shigella species relevant for inclusion in a vaccine formulation are one of fourteen serotypes of group A (S. dysenteriae type 1, the most virulent type present in both endemic and epidemic forms), two types of the group B (S. flexneri types 2a and 1a; type 2a being the most common in developing countries), and the unique type of group D (S. sonnei, which is ubiquitously present and most prevalent in developed countries) (158).
Since Shigella spp. are mucosal pathogens, it would be expected that mucosal immunization is the best approach to protect against colonization through the stimulation of a sIgA response. However, this notion has been challenged by the protection obtained using various parenteral vaccines, which proved the contribution of serum antibodies. In addition, the relatively high frequency of dysentery among HIV patients and observed T-cell activation during shigellosis (81, 82) and in a challenge model for S. dysenteriae 1 (163) point to a significant contribution of CMI in shigellosis (12, 18). Vaccine development has been complicated by the fact that humans and some non-human primates are the only known hosts for Shigella. In addition, the infective dose in monkeys (i.e., 109 to 1010) is much higher than in humans (i.e., 102 to 103) (44). Therefore, a vaccine reverting to virulence at low frequency could prove safe in monkeys yet lead to disease in humans.
Soon after the isolation of S. dysenteriae in 1898, heat-killed cultures were tested to assess their potential as vaccines. However, there is still no vaccine available. In the 1940s and 1950s parenteral Shigella vaccines were developed consisting of heat- or acetone-inactivated whole cells. Although high anti-LPS titers were observed, these failed to provide protection. Furthermore, the early vaccines were quite reactogenic, possibly because of the presence of endotoxins. These early failures coupled with the observation that shigellosis rates are highest in children in endemic areas suggested that orally administered live vaccines that would mimic the course of natural infection might provide protection (118, 151). A further key observation was the association of Shigella virulence with invasion of the colonic epithelium. Thus, it was concluded that the mucosal presentation of relevant Shigella antigens in the absence of invasion could lead to protection without the signs of disease.
Clinical trials in the 1960s were conducted with orally administered, noninvasive live S. sonnei and S. flexneri strains capable of growth only in the presence of streptomycin (Sm-dependent), thereby preventing propagation and invasion. Unfortunately, these early vaccines proved to be genetically unstable, poorly immunogenic, and therefore unfit for use in humans (131). Nevertheless, these pioneering studies demonstrated the essential soundness of this approach and confirmed that a vaccine against Shigella was an attainable goal. In subsequent years, various strategies were devised for the development of live oral Shigella vaccines, including the generation of (i) noninvasive avirulent mutants, (ii) attenuated strains with deletions in virulence genes, and (iii) vaccine carriers expressing relevant antigens, principally the O-antigen.
Noninvasive Vaccine Strains.
An avirulent and noninvasive strain called 2457O was derived from the wild-type S. flexneri 2a strain 2457T isolated in 1965. Despite the good safety profile shown in monkeys, this strain produced dysentery in some vaccinees, and virulent revertants were isolated in the feces of these subjects (42, 43, 53). Another avirulent vaccine was derived by serial passage of an S. flexneri 2a strain (130). The resulting strain, called Istrati T32, was shown to be noninvasive due to an extensive deletion in the invasion plasmid (188). The strain was well tolerated at doses of 2 × 1011 CFU and conferred 85% protection when five doses 3 days apart and two boosters per year were given. Despite the high number of doses, this vaccine has been used on a routine basis in Romania (Vadizen). This vaccine promoted serotype-independent protection in monkeys and human volunteers. This might be explained by the stimulation of immune responses against antigens shared by all serotypes. However, Ipa antigens are not expressed in this strain because of the large plasmid deletion. Thus, at the present time, there is no clear explanation for this finding (118, 151).
Invasive Vaccine Strains.
Numerous studies carried out in the 1980s and 1990s on the genetic basis of Shigella pathogenicity and the encouraging results obtained with Salmonella spp. fostered the rational design of live attenuated Shigella vaccines. However, it was soon clear that it would be difficult to develop an ideal vaccine because of the relatively narrow window between safety and efficacy. Various vaccine strains were constructed carrying single and combined deletions in (i) aro genes, which are part of the aromatic metabolic pathway leading to the formation of folic acid, para-aminobenzoic acid, and 2,3-dihydrofolic acid; (ii) the icsA/virG gene that is involved in cell-to-cell spread, alone or together with the siderophore aerobactin encoded in the iucA gene; and (iii) the guaBA operon that regulates de novo synthesis of purines, alone or together with the set and sen genes encoding two putative enterotoxins.
The prototype aro vaccine was the S. flexneri Y strain SFL114, which contains a Tn10 insertion in the aroD gene (117). However, for safety reasons a deletion mutation was created in the aroD gene, thereby generating the SFL124 Δ aroD strain, which was initially tested for safety and immunogenicity in Swedish volunteers. The strain was well tolerated and triggered the elicitation of O-antigen-specific antibodies in serum and feces and LPS-specific ASC, which were more numerous after three doses than after one dose (116). Two further clinical trials in Vietnamese adults and in children 4 to 9 years old confirmed the good tolerability and immunogenicity of the vaccine in individuals from endemic areas (113, 114). The nature of the immune response in this setting suggested secondary responses, indicating that the vaccine might be used to boost immunity due to natural infection. A similar vaccine was produced using the S. flexneri 2a strain 2457T (SFL 1070 Δ aroD) as a backbone (86). This vaccine displayed dose-dependent reactogenicity and immunogenicity, with the 108 CFU dose providing the best balance. These results demonstrated the importance of the wild-type parent strain selected for construction of an attenuated vaccine. SFL1070 and SFL124 carry the same deletion, but the former was markedly more virulent than the latter, probably reflecting differences in the virulence of the wild-type progenitors. Nevertheless, these results suggested that aro mutations alone are not sufficiently attenuating and that it will be essential to combine them with additional mutations.
P. Sansonetti’s group at the Pasteur Institute has constructed several vaccine candidate strains mostly based on the deletion of the icsA/virG gene (13). One of the most promising S. flexneri 2a vaccine candidates, labeled SC602, was constructed from the fully virulent strain 454 by removing the icsA/virG gene and the chromosomal iucA-iut siderophore aerobactin locus (11). The latter mutation limits the capacity of the strain to colonize tissues by disabling its ability to scavenge transferrin-bound iron. Challenge studies with SC602 indicated that the vaccine, although conferring protection against severe shigellosis, was too reactogenic at the 104 dose for routine human use. This, along with regulatory authorities’ concern that the vaccine strain might spread to nonvaccinated contacts (28, 182, 183), may discourage further development. A recent trial performed with SC602 on 34 healthy adults demonstrated that the vaccine can protect against the more severe symptoms of shigellosis. However, the relatively significant reactogenicity of this vaccine strain was also confirmed (87). A S. sonnei vaccine candidate designated WRSS1, similarly attenuated by a virG deletion, was also tested in a phase I study in human volunteers. Although highly immunogenic, mild fever occurred in 22% of the volunteers. This vaccine will be further tested for safety, immunogenicity, and potential secondary spread to household contacts in phase II studies in Israel (102, 183).
Another vaccine against S. dysenteriae type 1, WRSd1, containing deletions of the icsA/virG gene and of a 20-kb chromosomal region encompassing the Shiga toxin coding genes (stxAB), was evaluated for safety, immunogenicity, and protective efficacy using the guinea pig keratoconjunctivitis model (189). WRSd1 was Sereny test negative, and two applications of this strain to the cornea triggered a protective response against three different S. dysenteriae type 1 virulent strains. WRSd1 given intragastrically to rhesus monkeys was also proven to be safe and immunogenic. Furthermore, WRSd1 was tested in guinea pigs in combination with SC602 (S. flexneri 2a) and WRSS1 (S. sonnei), since a vaccine protecting against multiple Shigella species is desirable for most areas where Shigella is endemic. Guinea pigs vaccinated with a mixture of equal amounts of the three vaccine strains were protected against challenge with each of the homologous virulent strains. However, unlike WRSS1 and SC602, the level of protection afforded by WRSd1 in the combination vaccine was lower than the protection elicited by a pure culture.
M. Levine and colleagues at the Center for Vaccine Development in Baltimore have constructed several virG S. flexneri 2a vaccines. Building on progressive experience, the group genetically fine-tuned its vaccine strains with the hope of achieving the correct balance between safety and efficacy. All strains were derived from the wild-type S. flexneri 2a 2457T known to cause disease. Therefore, one of the goals in constructing these strains was to be able to compare various attenuating mutations in the same genetic background (Table 3). CVD1203, a Δ aroA Δ virG mutant, produced strong immune responses in human volunteers and was adequately attenuated up to doses of 106 CFU. However, unacceptable dose-dependent side reactions were noted at doses above 108 CFU (100). These symptoms might have been caused by activation of the innate immune system due to the large number of organisms rather than to specific illness, as judged by the early onset of disease in comparison with the wild-type strain. However, the level of attenuation of this mutant can still be appreciated when one considers that the wild-type parental strain 2457T produces full-blown disease with an infective dose of about 103 CFU. In any case, the need for further attenuation was evident, and the effect of guaBA deletions was examined. The rationale for this lies in early observations (128), demonstrating that mutations affecting the adenine nucleotide pathway (purA and purB) were excessively attenuating (i.e., poor immunogenicity in humans), whereas mutations in either the common purine pathway (purF, purG, purC, purJHD) or the guanidine nucleotide pathway (guaB or guaA) lead to mildly reduced virulence. Therefore, guaBA deletions were expected to be able to further attenuate Δ aro Δ virG mutant strains without affecting their immunogenicity. Surprisingly, it turned out that a single Δ guaBA deletion mutant termed CVD 1204 showed 30-fold lower infectivity than the parental strain 2457T or the Δ aroA strain CVD 1201 (146). Nevertheless, CVD 1204 caused fever and diarrhea at a dose of 107 CFU (183). Thus, derivatives containing additional mutations, such as strains CVD 1205 (Δ guaBA Δ virG), CVD 1207 (Δ guaBA Δ virG Δ sen Δ set), and CVD 1208 (Δ guaBA Δ sen Δ set) were developed. Clinical trials suggested that CVD1207 is safe but poorly immunogenic. The number of anti-LPS IgA ASC was modest (5.3 × 106 to 6.1 × 106 peripheral blood mononuclear cells [PBMCs]), even in comparison with SC602 (18 × 106 PBMC), whereas the wild-type strain 2457T elicited 239 × 106 PBMC (101). Therefore, the protective capacity of CVD 1207 may be weak. In contrast, CVD 1208, like CVD 1204, was more immunogenic and caused only short-term fever without diarrhea at a dosage of up to 109 CFU (183).
Table 3Live attenuated vaccines against shigellosis |
Carrier-Based Vaccines.
The difficulty of obtaining the correct balance between safety and efficacy in attenuated Shigella strains has prompted investigators to use known avirulent strains for expression of the Shigella O-antigen. In initial studies, the region coding for group and type O-antigen of the S. flexneri 2a was conjugationally transferred into an E. coli O8 strain, which was unable to invade the colonic epithelium. Unfortunately, the resulting strain PGAI41-1-15 did not elicit any significant anti-LPS antibody titers and was unable to confer protection against wild-type S. flexneri 2a challenge in volunteers (112).
The genes coding for the S. sonnei serotype D O-antigen were also inserted in the chromosome of the Vibrio cholerae vaccine strain CVD 103-HgR, a licensed live attenuated vaccine against cholera. The resulting strain, CH3, synthesized the O-antigen as a loose capsule. Past experience using a serovar Typhi-based carrier (53) suggested that presentation of unbound O-antigen might not provide adequate immune stimulation. Addition of the R1 LPS core, which is compatible with the Shigella core (strain CH9), was not sufficient to ensure linkage of the O-antigen to the core. Covalent linkage of the S. sonnei O-antigen to the underlying V. cholerae LPS core was finally obtained in a CVD103-HgR derivative possessing a deletion spanning the rfbA and rfbB genes, which prevented the expression of the homologous O-antigen (strain CH22) (49). A phase I clinical trial showed that, while CH22 was safe, only modest anti-S. sonnei O-antigen responses were elicited. The development of this strain was therefore discontinued (unpublished results).
The disappointing results obtained using noninvasive carrier strains indicated the need to use an invasive strain for enhanced immunogenicity. Thus, enteroinvasive E. coli-Shigella hybrids were prepared by sequential conjugational transfer of the S. flexneri 5 and 2a rfb determinants and group factors 3 and 4 in E. coli K12 395-1. However, the resulting strain, EcSf2a-1, was reactogenic at the dose of 1 × 109 CFU, and vaccine efficacy was only modest (20%) (98). A second strain was constructed by introducing an aroD deletion in an icsA-positive variant of EcSf2a-1. Three doses of this vaccine, named EcSf2a-2, protected Rhesus monkeys against homologous challenge and prompted a series of human trials. However, a dose of 2 × 109 CFU was found to be too reactogenic, and at 1 × 109 CFU the vaccine elicited no significant protection (98, 99). These studies had the merit of showing that a minimum of fourfold increase in mean geometric average of circulating IgA anti-2a ASC may be necessary for protection against Shigella. Despite these disappointing results, EcSf2a-2 was tested under military field conditions in Israel, assuming that even a modest immune response may be sufficient in endemic conditions. Gastrointestinal reactions were reported by 6% of the vaccinees versus 3% of those who received placebo. However, protective efficacy of the vaccine could not be evaluated because of the virtual disappearance of S. flexneri 2a from Israel at the time of the trials (25, 181).
A further vaccine strain was generated by transferring the mega plasmid of S. sonnei into the serovar Typhi Ty21a vaccine strain. The resulting strain, 5076-1-C, expressed the S. sonnei O-antigen in a capsule-like form but none of the Shigella invasion genes. Protection against intraperitoneal challenge was observed in mice. Serum and mucosal immune responses were observed in volunteers, as well as 64% protection against challenge with S. flexneri. Unfortunately, further plans for field trials with 5076-1C were cancelled since subsequent vaccine lots were not protective for unexplained reasons (67).
The attenuated S. flexneri aroD strain SFL124 has also been used to express antigens from S. dysenteriae 1. The rfp and rfb gene clusters, which code for S. dysenteriae serotype 1 O-antigen biosynthesis, were inserted into the attenuated S. flexneri aroD serotype Y strain SFL124, as well as an R derivative (89, 90, 91). The resulting clones efficiently expressed homologous S. flexneri Y and heterologous S. dysenteriae 1, or only heterologous O-antigens, respectively. Mucosal vaccination with the resulting candidates stimulated the production of O-antigen-specific antibodies. The recombinant clones were tagged with a nonantibiotic resistance marker, making them easily distinguishable from endemic shigellae. One of the clones led to 47% full protection and 53% partial protection against challenge with wild-type S. dysenteriae 1 in an experimental animal model system. Introduction of the rol (wzz) genes of S. dysenteriae 1 and E. coli K-12 into the carrier strains expressing the heterologous S. dysenteriae type 1 O-antigen allowed optimization of the length of the O-antigen chains, thereby leading to an improved immunogenicity (91). The gene coding for the B subunit of the Shiga toxin (StxB) was also introduced into the recombinant strain expressing the O-antigens of S. flexneri Y and S. dysenteriae 1, by generating a fusion with the C terminus of E. coli hemolysin A. The resulting chimeric gene was combined into an expression cassette with the determinants of the hemolysin accessory translocator proteins HlyB and HlyD (187). Polyclonal antibodies raised against the StxB'-'HlyA fusion exhibited Shiga-toxin-neutralizing activity in cytotoxicity assays. In a related approach, a chimeric E. coli LamB protein encompassing an epitope of the B subunit has been surface displayed in an S. flexneri vaccine strain. Vaccinated animals showed a peptide-specific humoral response able to neutralize toxin activity for HeLa cells (161).
In parallel with the development of live vaccines, other approaches involving nonliving formulations have been attempted. In view of the current concept of live vaccines mimicking natural infection to obtain optimal protection against enteric pathogens, these nonliving preparations proved to be surprisingly efficient at eliciting strong immune responses and conferring significant protection against Shigella species. Among them are the vaccine prototypes based on the so-called proteosomes (123). Proteosomes are specific vaccine formulations consisting in preparations of outer membrane vesicles from Neisseria meningitidis, which were originally shown to improve the immunogenicity of parenterally administered peptides and other antigens noncovalently complexed to them (123). Preparations consisting of S. flexneri 2a or S. sonnei O-antigen complexed to proteosomes were found to be immunogenic and protective in a murine model following intragastric or intranasal immunization (125). These results prompted a phase I dose escalating clinical trial using a S. flexneri 2a proteosome-LPS vaccine in healthy adults. The two-dose vaccine was well tolerated, with mild dose-related rhinorrhea and nasal stuffiness as the most common side reactions. At doses of 1.0 and 1.5 mg of vaccine, 90% of the 35 volunteers exhibited an anti-LPS IgA-ASC response, whereas all responded with an ASC response of at least one isotype. Two- to fivefold rises in serum IgA and IgG antibodies, and two- to threefold increases in IgM serum antibodies were also noted (56). Altogether, the magnitude of the immune response was similar to that observed using live vaccine candidates associated with protective efficacy in human challenge models. The S. flexneri 2a proteosome-LPS vaccine was further evaluated in a placebo-controlled double-blind study in which 27 volunteers received two intranasal doses of the vaccine and were challenged 4 to 9 weeks after vaccination. Protective efficacy for dysentery, diarrhea, and fever ranged from 24 to 32%. For volunteers challenged 4 to 6 weeks after vaccination who had high levels of anti-LPS IgA, protection was 56 to 78% against severe disease/fever or diarrhea/any disease, respectively. Although showing only a moderate level of protection, this trial demonstrated the potential of intranasal immunization for protection against enteric diseases and lends further credence to the concept of a common mucosal immune system (183).
The notion that stimulation of mucosal immunity by a live vaccine will be required for efficient protection against shigellosis has been further challenged by the construction and successful testing of at least two parenterally administered candidate vaccine formulations. The observation that serum IgG is present in the gut in concentrations similar to secreted IgA (152), coupled with the successes obtained using Haemophilus influenzae type B polysaccharide conjugate vaccines, prompted the National Institutes of Health investigator John Robbins and colleagues in the early 1990s to develop parenteral Shigella O-polysaccharide conjugate vaccines. S. dysenteriae O-antigen was coupled to the tetanus toxoid, whereas both S. flexneri 2a and S. sonnei O-antigens were coupled to rEPA. The resulting vaccine candidates were safe, causing only mild reactions in less than half of the human volunteers. Four weeks after vaccination, the serum anti-O-antigen antibody levels were as high as in patients infected with the corresponding pathogen (158). These results warranted field-efficacy trials, which were performed in Israel. Double-blind, randomized, vaccine-controlled trials (n = 1,446) with the S. sonnei-rEPA were carried out from 1993 to 1995 in Israeli soldiers using the EcSf2a-2 strain (see above) and a tetravalent meningococcal vaccine as controls. Cases of shigellosis caused by S. sonnei occurred in four companies under surveillance. The attack rate in controls was 6.2 to 20.7% and 0 to 11.8% in vaccinated recruits, depending on the company. The efficacy of the vaccine in three of the groups where shigellosis occurred 70 to 155 days after vaccination was 74%. However, in the fourth group, in which shigellosis cases were detected 1 to 17 days after vaccination, the efficacy of the vaccine was about 43%. These results suggested that such a vaccine could be useful in outbreak situations. However, the epidemiology of Shigella infections and the wide confidence intervals from this study underscore the need for additional work (23, 24).
In a study of children 4 to 7 years of age, both the S. sonnei-rEPA- and S. flexneri-rEPA-conjugated vaccines were found to be safe, with only mild local reactions. Significant IgG, IgM, and IgA serum responses were elicited and persisted for up to 6 months after vaccination. A booster response was stimulated by a second dose administered 6 weeks after the first vaccination (149). S. sonnei and S. flexneri 2a O-antigens were also conjugated to native or succinylated rEPA or to the nontoxic derivative CRM9 of the diphtheria toxin and further tested in human volunteers. Succinylation was expected to enhance the immune response of the conjugates (148). The S. sonnei formulations stimulated high levels of O-antigen-specific antibodies that persisted for up to 26 weeks after vaccination. The S. flexneri 2a-rEPAsucc was more immunogenic than its CRM9succ counterpart and was therefore evaluated in children. In 1- to 4-year-old children, the most susceptible age group for shigellosis, the vaccines were safe and immunogenic. Vaccination with the S. sonnei-CRM9 and S. flexneri 2a-rEPAsucc prototypes elicited a more than fourfold increase in anti-O-antigen antibodies in 92 and 85% of the children, respectively, for up to 2 years after vaccination. A booster dose of the S. sonnei vaccine, but not the S. flexneri 2a vaccine, induced a significant increment in IgG anti-LPS. In contrast, booster doses of both vaccines elicited a rise in anti-LPS IgG in both adults and older children. Taken together, these results suggest that conjugate vaccines are promising candidates for prevention of shigellosis from early childhood to adulthood. They also demonstrate that exuded serum IgG in the intestinal lumen can efficiently inactivate infecting pathogens. The drawback may be related to cost, since polysaccharide-protein conjugates are expensive to manufacture and, to our knowledge, no commercial partner has yet adopted the product.
Finally, ribosomal preparations from Shigella strains, first made in the USSR in 1978, were found to induce local IgA and prime mucosal immune memory after subcutaneous injection. The vaccines were highly protective in guinea pigs and monkeys. A S. sonnei preparation gave 89% protection in monkeys against homologous challenge with only mild local reactions. The O-antigen was found to contaminate the preparations, and an adjuvant effect of the ribosomes was postulated. However, the precise mechanism underlying protection against shigellosis was never elucidated (105, 118).
In summary, despite intensive efforts, no Shigella vaccine has as yet been registered for human use. However, as Shigella pathogenicity and relevant immune mechanisms are increasingly understood, several very promising vaccine candidates have been developed with the concomitant hope that safe and efficient Shigella vaccines will be available in the near future.
Although E. coli strains are important members of the normal gut flora of human and animals, there are pathogenic E. coli strains that can cause enterotoxic or enterotoxemic diseases, especially in young individuals and in animals. E. coli strains can be distinguished by their surface antigens, which are classified as O-antigens, K-antigens (capsular polysaccharides), H-antigens (flagellar antigens), and heat-labile F-antigens (fimbriae or pili). Toxicity depends on the production of specific toxins, which are responsible for the main symptoms of the infection. Enterotoxins, such as the heat-labile enterotoxin (LT) and heat-stable enterotoxin (ST), are produced by enterotoxigenic E. coli (ETEC), act in the intestine, and cause diarrhea. Other E. coli strains can also produce Shiga toxins (Stx1 and Stx2), which were previously called Shiga-like toxins (SLT-1 and SLT-2) or verotoxins (VT1 and VT2). The E. coli strains producing Stx are referred to as Shiga-toxin-producing E. coli (STEC) or verotoxigenic E. coli (VTEC). In contrast to enterotoxins, the Shiga toxins exhibit cytolytic activity and are able to penetrate the epithelial barrier, reaching the blood circulation. In swine, the Shiga toxin variant Stx2e acts as a vasotoxin on vascular endothelial cells, leading to edema and neurological dysfunction (paddling movements, recumbence, and ataxia). STEC strains able to cause bloody diarrhea and hemolytic uremic syndromes in humans are referred to as enterohemorrhagic E. coli (EHEC), which have a significant impact as emerging food-borne human pathogens. Enteropathogenic E. coli (EPEC) are a predominant cause of diarrhea in infants in developing countries and substantially contribute to the high morbidity rates in this age group. In the developed world EPEC is less prevalent, but sporadic outbreaks in daycare centers and nurseries have been reported (143). Both EPEC and STEC are able to induce attaching and effacing lesions on infected enterocytes. This process is characterized by local effacement of microvilli, tight attachment of bacteria to the eukaryotic cell surface, and subsequent reorganization of filamentous actin to form pedestal-like structures (Fig. 1B) that cup individual bacteria (39). The "locus of enterocyte effacement" is a chromosomal pathogenicity island that encodes the critical virulence factors for this process (127). Another type of pathogenic E. coli are the enteroinvasive E. coli (EIEC), which trigger a disease mimicking shigellosis characterized by fever and bloody diarrhea that is caused by bacterial invasion of colonic epithelial cells. More recently, enteroaggregative E. coli (EAEC) have been implicated as etiologic agents of diarrhea lasting for at least 14 days both in children from developing and developed countries. EAEC are characterized by their unique stacked brick-like adherence pattern. Finally, uropathogenic E. coli (UPEC) are the most common cause of urinary tract infections, and some E. coli strains can cause neonatal meningitis, but these agents will not be discussed further in this chapter.
With about 1.5 billion episodes and 3 million deaths a year, diarrheal diseases are the second leading cause of morbidity and mortality due to infectious diseases worldwide, mainly in developing countries (142, 194). With an estimated 400 million cases a year, and one million deaths, mainly among children, ETEC are the most common cause of diarrhea in developing countries, so that the WHO has long recognized this pathogen as one of the priorities for vaccine development. E. coli is also the leading cause of traveler’s diarrhea, accounting for at least 8 million cases among travelers from just the United States alone every year. No vaccine against ETEC diarrhea is yet commercially available. A vaccine conferring high-level protection against this pathogen would be an important contribution to international health and will be the main focus of this subsection.
ETEC isolates that cause diarrhea express several virulence factors that play important roles in the disease process. They include two enterotoxins, LT and ST, and bacterial surface adhesins called pili, which allow the organisms to colonize the intestinal tract. Some clinical ETEC isolates have been shown to produce either LT or ST, whereas other isolates express both toxins. Strains that possess LT tend to be associated with more severe cases of traveler’s diarrhea, whereas ETEC strains that produce only ST cause milder diarrhea.
As for the other mucosal pathogens discussed above, the most practical approach for the prevention of the widespread morbidity and mortality caused by diarrheal disease due to intestinal contamination with ETEC strains would be by means of vaccination. Three different types of E. coli antigens have been shown to be effective as immunogens, providing protection against challenge with ETEC strains in experimental models. First, immunization with either the E. coli LT holotoxin or its B subunit (LT-B) arouses an antitoxin response, which provides protection against active challenge with either the toxin itself or viable bacteria producing LT alone or together with ST (92, 93). Second, vaccination with somatic antigens, usually in the form of whole killed bacteria, prevents diarrhea by means of reducing bacterial growth within the small intestine. However, this extends only to homologous somatic serotypes and not to heterologous serotypes, of which 164 antigenically dissimilar somatic serotypes of E. coli are recognized (196). Third, immunization with the specific fimbrial antigen responsible for bacteria adherence and colonization also provides protection, but this does not extend to ETEC strains possessing antigenically different fimbrial antigens (141). In humans, more than 20 different pili types have been described, some of them being coexpressed in the same organism. Thus, CS3, which is detected in 23% of ETEC isolates, occurs either alone or in combination with CS1 or CS2. CS6, which is detected in 21% of ETEC isolates, occurs either alone or in combination with CS4 and CS5. Colonization factor CFA/I occurs alone in about one third of all ETEC isolates. These data are consistent with the idea that a vaccine composed of CFA/I, CS3, and CS6 may cover about 75% of all ETEC cases. This proportion could be increased to 85% by inclusion of an inactivated LT, LT-B, or the homologous cholera toxin B subunit (CT-B), as immunologically cross-reactive component in the vaccine (196).
Live Attenuated Vaccines against ETEC.
As stated above, live attenuated vaccines represent an attractive strategy for vaccination against mucosal pathogens, including ETEC. One obvious approach is to use attenuated ETEC strains as live vaccines, which could be administered orally. A nontoxigenic ETEC strain (E1392/75-A2) that expresses CFA/II was tested as an oral vaccine in a human volunteer challenge study in the 1980s and found to elicit 75% protective efficacy. However, the vaccine caused mild diarrhea in about 15% of the vaccinees (177). Therefore, the strain was further attenuated by deletion of the aroC and ompR genes (strain PTL-002) or the aroC, ompC, and ompF genes (strain PTL-003), respectively. Both of these strains were shown to be safe in a phase I clinical trial and able to trigger the elicitation of anti-CFA/II IgA-ASC (185).
As an alternative to the attenuation of ETEC strains, already-attenuated live bacterial strains have been exploited for the expression of heterologous ETEC antigens. Recombinant expression of antigens, such as the LT-B subunit and the aforementioned pili, may enhance protection. Thus, a vaccine formulation might contain LT-B as a separate adjunct or secreted from a carrier strain, together with a mixture of ETEC pili either as purified structures or produced from a live bacterial carrier. Pili of ETEC have been expressed in attenuated serovar Typhimurium and S. flexneri 2a vaccine strains. The structural genes for the biogenesis of CFA/I fimbriae were transformed into an attenuated S. enterica strain, which was shown to express the corresponding pili. Oral immunization of mice resulted in the development of CFA/I-specific serum IgG and IgA (199). Likewise, Salmonella was used to express CS3 pili, as well as a fusion protein consisting of LT-B and ST. This strain was found to elicit humoral immune responses against all three antigens after oral immunization of mice (200). In a similar approach, the attenuated S. flexneri 2a strain CVD1204 (a Δ guaBA strain) was assessed as a carrier for a variety of ETEC antigens. CFA/I, CS2, CS3, and CS4 pili were successfully expressed in CVD1204, and the strain was also shown to be suitable for expression of a detoxified version of LT (LThK63). These strains were assessed for their immunogenicity following intranasal immunization of guinea pigs. When used as monovalent vaccine, all strains were found to elicit serum IgG and mucosal IgA immune responses against the respective antigens. Mice immunized with a mixed inoculum of all five strains were found to elicit immune responses against all five antigens, demonstrating that this approach may be suitable for the development of a multivalent ETEC vaccine (5, 6, 10, 96). However, the candidate vaccines based on attenuated Salmonella and Shigella carrier strains still await assessment in clinical studies.
Subunit Vaccines against ETEC.
As an alternative to the oral administration of subunit vaccines against ETEC, transcutaneous immunization (TCI) was recently assessed. In TCI, the antigens are applied to the skin together with an adjuvant via a patch or an alternative topical formulation. Diffusion of the antigens into the skin facilitated by adequate adjuvants leads to their uptake by Langerhans cells, highly potent dendritic cells localized in the skin, thereby promoting strong immune responses. The most efficient adjuvants tested so far for TCI are CT and LT. Patches with LT were found to trigger the elicitation of serum IgG and mucosal IgA in a phase I study (62). It was an obvious next step to assess the TCI technology for vaccine development against ETEC. Patches were developed which contained either CS6 alone or in combination with LT. In a phase I study, the patches containing CS6 plus LT were found to induce anti-CS6 serum IgG and IgA in 68 and 53% of vaccinees, respectively (66). In addition, all vaccinees exhibited anti-LT IgG and IgA responses. In contrast, no specific immune responses were observed in vaccinees receiving the CS6 patches without adjuvant. Both formulations were found to be safe. However, 74% of the vaccinees receiving the CS6-LT patch developed a delayed-type hypersensitivity reaction at the site of vaccination, whereas this was not observed in vaccinees receiving CS6 alone.
Inactivated Vaccines against ETEC.
Based on the strategy of an orally administered killed cholera vaccine (78) and the fact that the CT-B component of this vaccine afforded short-term partial cross-protection against ETEC (21, 150), two ETEC-inactivated vaccines have been similarly designed and tested in humans. The first such vaccine was prepared from the wild-type O78 ETEC strain H10407, which produced CFA/I pili and both LT and ST enterotoxins. The bacteria were killed by treatment with colicin E2, a potent endonuclease that penetrates target cells without disrupting their integrity. Twenty-two young adult volunteers received two doses of 3 × 1010 vaccine cells one month apart. Seventeen of 22 vaccinees demonstrated an incremental increase in anti-CFA/I and anti-LT IgA antibodies, and 90.9% had an immune response to at least one of these two antigens. About 75% efficacy was obtained after challenge with either a CFA/I-positive O63:H- strain or a CFA/II-positive O6:H16 strain (46, 47).
The second vaccine, dubbed ETEC/rCTB, consists of formalin-inactivated ETEC cells globally expressing the CFA/I and the CFA/II pili family composed of the CS1, CS2, and CS3 fimbriae. This mixture of cells is supplemented with purified recombinant CT-B subunit. The latter component was added to provide cross-immunity against the closely related B subunit of ETEC enterotoxin mentioned above. This initial vaccine was orally administered in three doses at 2-week intervals to 20 Swedish adult volunteers and provided significant antifimbria and antitoxin IgA titers in intestinal lavages of most volunteers (CFA/I, 82%; CFA/II, 82%; and CT-B, 91%). The titers were similar to those observed in patients convalescing from ETEC infection. Serum antifimbria and antitoxin IgA were also present (4). These encouraging results prompted the addition of strains expressing the CFA/IV fimbriae (CS3, CS4, and CS6) and the further testing of the new vaccine mixture. Two vaccine doses from two different lots were safe and stimulated strong mucosal immune responses against the CT-B component and various fimbriae in the majority of immunized Swedish volunteers. In contrast, the vaccine elicited little antifimbrial serum antibody response (84). Further human trials confirmed the high safety level and promising immunogenicity of the vaccine (3, 85). On the basis of these promising results, various phase II clinical trials were initiated.
In Egypt, the vaccine has been tested in several phase II trials in adults, schoolchildren, toddlers, and infants. The vaccine delivered in two doses at 2-week intervals was found to be safe, and credible ASC and serum responses were elicited (69, 166, 167). Significantly, there was a good correlation between IgG and IgA serum responses and ASC, so that serum responses may be taken as a surrogate of ASC responses in future studies (69). This is important since insufficient blood samples can be obtained from infants for ASC evaluation. In children less than 2 years old a third dose led to an increase in antitoxin titers, although antifimbria responses were not increased (168). This finding was instrumental in the decision to use three doses instead of two doses in subsequent trials in children. In Bangladesh the vaccine was tested in adults, children (mean age = 4.5 years), and toddlers (18 to 36 months of age). The vaccine was well tolerated in all groups. Antifimbrial ASC responses increased from 29- to 46-fold in adults and 13- to 24-fold in children and were also more elevated in vaccinated toddlers than in the placebo group. Serum antifimbrial IgA and anti-CT-B IgG and IgA were also increased in vaccinees. In these studies, fecal IgA was present in most responders, making this type of assay possibly useful for assessing vaccine efficacy in infants (154, 155, 194). In Israel ETEC infections are one of the leading causes of diarrhea among soldiers, eliciting a great interest in a corresponding vaccine. Two doses of two lots of the vaccine were administered to 155 healthy adult volunteers and found to be generally safe. However, one of the two vaccine lots stimulated vomiting after the second dose in 17% of the volunteers, whereas placebo recipients did not have this adverse event. The rate of ASC was higher for CT-B, CFA/I, and CS1 than for CS2, CS4, and CS5. In addition, as seen in other trials, the immune response to CT-B, but not to fimbriae, was increased after the second dose. Rates of serum antibody responses were less frequent than ASC responses for both CT-B and fimbriae. Based on these results an efficacy trial has been initiated among the Israeli Armed Forces (27). Phase III trials of the vaccine are currently being completed in Egyptian children and American travelers to Central America. In the latter study, which involves 1,350 volunteers, there was no statistical difference in diarrheal episodes between the vaccine and control groups. However, ETEC-associated cases were less severe in the vaccinated group. A complete evaluation of the results will allow assessment of true vaccine efficacy in this setting (183). A further development of the ETEC/rCTB vaccine may involve the addition of CS17 and PCFO166 antigens to increase protective coverage (78). In conclusion, an intense effort toward an ETEC vaccine is currently being undertaken. It is likely that one or more safe and efficacious vaccines against this deadly disease will be available in the coming years.
Our increasing knowledge of the basic virulence mechanisms of pathogenic E. coli have led to the rational development of new vaccines against infections in farm animals, such as cattle and pigs. As for vaccines for humans, two different groups of vaccine targets have been exploited for vaccine development: adhesion molecules and toxins. The rationale is that antibodies directed against the adhesion molecules will prevent colonization, whereas those specific for toxins can neutralize their activity.
Vaccines against Animal Infections Caused by ETEC.
The most successful approach to protect neonates against ETEC infections is to immunize pregnant dams, thereby eliciting neutralizing antibodies that are transferred into colostrum and milk. By suckling, neonates establish and maintain sufficient levels of antibodies in the intestine to prevent infection. However, this passive immunity is limited to the suckling period. Although highly efficient, immunization of pregnant dams with attenuated strains is considered a risk because of the potential infection of neonates via the dams’ feces after birth. Therefore, most established immunization approaches against ETEC are based on the use of subunit vaccines.
Since the F-antigens are critical for bacterial attachment to host tissues, they have been exploited as target antigens for vaccine development. F-antigens to a great extent determine host specificity: the expression of F5 (or K99) correlates with virulence in cattle, whereas F4 (or K88), F5, F6 (or 978P), and F18 mediate adhesion in swine. Therefore, vaccine formulations for cattle differ from those for swine. In 1973 it was demonstrated that vaccination of dams with the F4 antigen conferred protection to suckling piglets against ETEC infections (160). Subsequent studies carried out in other species with the corresponding F-antigens confirmed the value of this vaccination concept. Since F-antigens do not induce cross-protection against other fimbriae, most vaccines currently used include F5 for cattle and F4, F5, and F6 for swine. Other fimbrial antigens (F1 and F41) have been identified, which are prevalently expressed by ETEC strains infecting farm animals. However, their efficacy as vaccine antigens in cattle and pigs is less well characterized. Since most ETEC strains producing F1 or F41 also express F4, F5, and F6, vaccination with the latter antigens is sufficient for protection. In fact, immunization with F5 protects calves and pigs against ETEC strains producing F5 and F41. In contrast, vaccination with F1 or F41 was not able to protect against challenge with an ETEC strain expressing F1, F5, and F41, but vaccination with F41 promoted protection against F5-F41 producing strains (140).
Since the pathogenesis of ETEC infections requires not only adhesion to intestinal tissues but also the actions of enterotoxins LT and ST, vaccine strategies have additionally focused on the induction of toxin-neutralizing antibodies. In this context, LT-based vaccination of dams was demonstrated to protect suckling piglets against LT-producing ETEC (38). Since most ETEC strains infecting swine and calves produce ST, it would be desirable to induce ST-neutralizing immunity. However, the development of such vaccines faces the problem that ST is poorly immunogenic. Even coupling of ST to a carrier moiety was insufficient to protect piglets against challenge (55, 140).
ETEC also causes a high incidence of infection in postweaning pigs, in association with social and/or physical stresses. Most postweaning E. coli infections are caused by strains expressing F4 and/or F18 fimbriae (195), and fimbriae-specific antibodies correlate with protection of convalescent pigs (165). Thus, these antigens have been exploited for active vaccination. The fact that ETEC colonizes epithelial tissues suggested that efficient immunity against infection will require a strong local mucosal response. This can usually be achieved by administering antigens by the mucosal route. However, the administration of pili-based subunit vaccines by the oral route may be problematic since most antigens from pili become degraded in the intestine or might induce tolerance (15). Thus, killed F4-expressing strains have been used for peritoneal and oral vaccination in the United States. Only a few reports have demonstrated the efficacy of these vaccines. An alternative approach to stimulate intestinal responses against ETEC is the use of live vaccines (54) that temporarily colonize the small intestine. Oral vaccination with a live avirulent E. coli strain expressing F4 and the LT-B subunit conferred protection to postweaning pigs, whereas F18-expressing strains promoted the elicitation of F18-specific neutralizing IgA immunity in nonweaned pigs (14).
Vaccines against Animal Infections Caused by STEC.
Primarily subunit vaccines have been proposed for protection of animals against infections caused by STEC, using bacterial adhesins and detoxified Shiga toxins as antigens. Like ETEC, STEC strains infecting postweaning swine express the fimbrial antigen F18 (195). Live E. coli vaccines expressing F18 were demonstrated to confer protection against ETEC (14), and it has been proposed that F18-specific immunity can also protect against F18 expressing STEC (190). Another colonization factor of STEC strains is intimin, which is required for bacterial attachment in neonatal calves and pigs (33). It has recently been demonstrated that parenteral immunization with adjuvanted intimin results in the stimulation of specific IgA, which is transferred by suckling and protects weaning pigs against challenge (34). In an attempt to neutralize the Shiga toxins produced by STEC, Stx2 has been used as an antigen. Pigs were protected against naturally occurring and experimentally induced edema disease by vaccination with chemically inactivated Stx2 (124). It was also demonstrated that protection against lethal doses of Stx2 is mediated by antibodies (63). However, vaccinated pigs had a reduced rate of weight gain compared with controls (63). In contrast, vaccination with genetically modified Stx2 (E167Q) did not affect weight, and immunized pigs were protected against Stx2-expressing E. coli in comparison with nonvaccinated littermates (19, 63). A serovar Typhimurium aroA vaccine carrier strain has been engineered to efficiently express and export a chimeric protein, fusing the B subunit of Shiga toxin lle and the C terminus of E. coli hemolysin A (186). However, the resulting candidate vaccine against swine edema disease has not yet been tested in the field.
Pathogenic E. coli can cause potentially lethal mastitis in cattle. To increase the resistance of cows against mastitis, immunoprophylactic approaches have been developed. A formalin-killed vaccine based on the E. coli strain J5 (O111:B4) is commercially available. Clinical signs were reduced in vaccinated animals following intramammary challenge with a heterologous E. coli strain. However, this vaccine was not capable of preventing infection (75). The vaccine increases leukocyte infiltration into the mammary glands and modulates the immune response to subsequent infection (40).
Many different vaccine candidates have been developed to prevent infections caused by Salmonella, Shigella, and pathogenic E. coli in both humans and domestic animals. Some vaccines have exhibited adequate safety and efficacy profiles, allowing successful implementation in the field. This has led to a significant reduction in human suffering and the costs associated with disease. However, the safety and efficacy of available vaccines can be further improved, and there are some diseases for which vaccines are still not available (e.g., shigellosis and gastrointestinal disorders caused by E. coli). Current research efforts are focused on the development of a new generation of more efficient and safer vaccines. This has been rendered possible by our expanded knowledge of microbial pathogenesis, immunology, and vaccinology. New molecular targets for attenuation have been identified, allowing the construction of safer attenuated vaccine strains carrying multiple mutations. Novel candidate vaccine antigens have also been identified that are suitable for incorporation in subunit vaccines. Finally, a better understanding of the mechanisms responsible for bacterial clearance and the availability of technology platforms for the optimal delivery of selected antigens will facilitate the rational design of new vaccines.
We thank M. Rohde, A. Fürer, S. Spreng, and Unikum, Berne, for providing us with TEM and FESEM micrographs as well as graphics, and H.-J. Selbitz, M. Iburg, M. Griot-Wenk, K. Goodwin Burry, I. Metcalfe, P. D. Becker, and J. Pearman for helpful discussions and critical reading of the manuscript.
This work was supported in part by grants from DFG (GU 482/2-1 and GU482/2-2) and BMBF (PathoGenoMik 601III4-1/1VIIZV-21) to C.A.G.
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