Attenuated Salmonella for Oral Immunization

C H A P T E R

22 Attenuated Salmonella for Oral Immunization Kenneth L. Roland1, Qingke Kong2 and Yanlong Jiang3 1

The Biodesign Institute, Arizona State University, Tempe, AZ, United States 2Department of Infectious Diseases & Immunology, College of Veterinary Medicine, University of Florida, Gainesville, FL, United States 3College of Animal Sciences & Technology, Jilin Provincial Engineering Research Center of Animal Probiotics, Jilin Agriculture University, Changchun, China

I. INTRODUCTION Salmonella enterica is a mucosal pathogen that interacts with the host immune system, making it an attractive target for use as an antigen delivery vector. Infection with wild-type Salmonella typically generates a robust immune response that leads to lifelong immunity. Recombinant Salmonella strains expressing heterologous genes can be orally administered to elicit an immune response against the pathogen from which the heterologous gene was derived. While strains of other bacteria, such as Escherichia coli, Listeria, Shigella, and Vibrio, have been and continue to be evaluated as oral vaccines, the invasive nature of Salmonella and its propensity to home to host immune cells make it adept at eliciting B and T cell memory responses with the greatest potential to elicit long-lasting mucosal, humoral, and cellular immunity (Chapter 29: Induction of Local and Systemic Immunity by Salmonella Typhi in Humans, Chapter 30: Oral Shigella Mucosal Vaccines DOI: https://doi.org/10.1016/B978-0-12-811924-2.00022-5

Vaccines and Chapter 31: Cholera Immunity and Development and Use of Oral Cholera Vaccines for Disease Control). Pathogenic S. enterica are introduced into the human body via the gastrointestinal tract by ingestion of contaminated food. The ingested cells must then pass through and survive the low-pH environment of the stomach before reaching the small intestines. The environment in the human gut is characterized by high osmolarity, the presence of antimicrobial peptides such as defensins, short-chain fatty acids, and resident microflora. In the ileum, Salmonella makes its way through the mucus layer that coats the intestinal epithelium, where it adheres and invades enterocytes or the follicle associated epithelium (FAE) that overlays the Peyer’s patches (PPs). The PPs consist of B cell-rich follicles, T cells, macrophages, and dendritic cells (DCs) that constitute a major component of the gut-associated lymphoid tissue (GALT). One hallmark of the FAE is the presence of microfold

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(M) cells. M cells specialize in the transcytosis of intact luminal material such as soluble proteins, bacteria, and viruses (Chapter 28: M CellTargeted Vaccines). After invasion into the PPs, Salmonella rapidly encounters DCs and is phagocytized [1,2]. These Salmonella-containing DCs may interact directly with B cells within the PPs, resulting in IgA switching and production of intestinal IgA [3]. In addition, T cell priming by the Salmonella-containing DCs begins in the PPs and continues in the deeper immunological tissues (e.g., the spleen) [4,5], resulting in activation of B cells and CD41 and CD81 T cells, leading to production of a systemic cell-mediated and humoral immune response [5
7]. Salmonella enterica Typhimurium initially stimulates a strong proinflammatory immune response that assists with effector cell recruitment and DC maturation. During invasion of PPs and intestinal epithelial cells, the host immune system is exposed to numerous pathogen-associated molecular patterns (PAMPs) produced by S. Typhimurium, including flagella, lipopolysaccharide (LPS), and bacterial DNA [8
11]. These PAMPs are recognized by their cognate Toll-like receptors (TLRs), TLR5, TLR4, and TLR9, respectively. The interactions with TLRs result in the secretion of IL-8 and the proinflammatory cytokines IL-1β, IL-6, TNFα, and IFNγ [12
15]. The production of these cytokines recruits and activates neutrophils, monocytes, and DCs [16]. Colonization of PPs by Salmonella is required to facilitate a strong mucosal IgA response [17]. When salmonellae are phagocytosed by a macrophage, their fate is not necessarily death. While most bacterial cells are killed by macrophages, Salmonella is equipped to survive the encounter because of the genes present in Salmonella pathogenicity island 2 (SPI-2), allowing it to grow within the macrophage in a specialized structure called the Salmonellacontaining vacuole. The Salmonella may be subsequently transported by the macrophage to deeper tissues such as spleen or liver, where it interacts further with lymphoid cells. These properties of Salmonella, including its ability to

stimulate mucosal, humoral, and cellular immunity, when adequately attenuated, make it an attractive antigen delivery vaccine vector.

II. APPROACHES FOR ATTENUATION A. Serial passage In the early studies, attenuated Salmonella vaccines were developed by in vitro serial passage or by random mutagenesis with chemical mutagens. The random mutagenesis method was widely used to develop both agricultural vaccines, such as Salmonella enterica Gallinarum 9R for fowl typhoid [18], and human vaccines, including the Salmonella Typhi strain Ty21a for typhoid fever [19]. Both strains are still in use today. While these methods were in use for many decades, the approach is not without drawbacks. One major problem is an incomplete understanding of the basis of attenuation. For example, two easily distinguished phenotypes of Ty21a are its lack of the Vi capsule, present in nearly all wild-type isolates, and its requirement for exogenous galactose in order to produce O-antigen due to a mutation in the galE gene. For many years, these two defects were assumed to constitute the sole basis of attenuation. However, this assumption was proven to be incorrect in a 1988 study in which a derivative of S. typhi Ty2, the parent of Ty21a, carrying a defined deletion in galE and a spontaneous mutation resulting in lack of Vi was found to retain virulence in human volunteers [20]. Subsequent DNA sequence analysis of the Ty21a genome showed that a number of other mutations are present that could affect attenuation [21]. It is likely that the combined impact of mutations generated during random mutagenesis resulted in a general reduction of the strain’s ability to grow and survive in vivo that are responsible for its attenuation. Other drawbacks include low immunogenicity and risk of reversion to virulence. The Ty21a

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vaccine must be given orally in three or four doses, though its genetic structure and attenuation phenotype appear to be quite stable [21]. The S. Gallinarum 9R vaccine must be injected rather than being given orally, and there are ongoing concerns about its potential virulence in some breeds of chickens [22].

B. Deletion mutants With the development of techniques to produce deletions in specific target genes, Salmonella-defined deletion mutants were constructed, providing better control over the genetic composition of vaccine strains. Salmonella mutants deleted for genes in the biosynthetic pathway for aromatic amino acid synthesis were first described as potential vaccines in the 1980s [23]. These mutants affect the shikimate pathway and are unable to synthesize aromatic compounds, including aromatic amino acids and certain vitamins. Salmonella mutants with deletions in aroA, aroC, and/or aroD are immunogenic and have been extensively studied as vaccine candidates [23]. An aroA S. Typhimurium mutant is available commercially for use in poultry [24]. aro mutants are attenuated, largely owing to their inability to replicate in host tissues where aromatic amino acids are limiting. In addition, aroA and aroD mutants exhibit cell wall defects resulting in greater sensitivity to serum and other components of the innate immune system [25]. In a detailed study of a S. Typhimurium aroA mutant, the mutation was found to have pleiotropic effects, including increased sensitivity to complement and phagocytic uptake, reduced motility, and altered expression of several virulence genes, and when delivered intravenously to BALB/c mice, they elicit increased levels of TNF-α [26]. Deletion of certain regulatory genes also attenuates. S. Typhimurium mutants with deletions in cya and/or crp and phoPQ were shown to be attenuated and immunogenic in mice [27,28]. Crp, in conjunction with adenylate cyclase, encoded by cya, regulates expression of

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a number of genes and operons required for transport and catabolism of sugars, as well as a variety of virulence factors, including fimbriae, flagella, and outer membrane proteins. PhoP is a DNA-binding protein that regulates many Salmonella genes in response to magnesium and pH, sensed by the membrane protein PhoQ [29]. PhoPQ constitutes a master regulator of Salmonella virulence, including survival in macrophages [29]. A ΔphoPQ S. typhi mutant was safe in humans [30], while a Δcya Δcrp S. typhi mutant was highly reactogenic [31]. These results highlight the need for caution in translating mouse safety data obtained with S. Typhimurium into S. Typhi strains for humans. Fur (ferric uptake regulator) acts as a repressor of many genes whose products are involved in iron, zinc, and manganese acquisition and uptake [32,33]. In Salmonella, Fur also modulates expression of genes involved in surviving acid shock, adaptation to low pH [34,35], and oxidative stress resistance [36,37]. In addition, Fur plays a role in regulation of the Salmonella pathogenicity island 1 (SPI-1) genes (e.g., hilA and hilD) necessary for invasion [38
40]. An Salmonellaenterica Enteritidis Δfur strain is attenuated, and immunization of mice with this strain results in a decreased bacterial load in systemic organs after challenge with the wildtype strain [41]. An S. Gallinarum Δfur mutant is safe and immunogenic in chickens, eliciting a protective immune response against challenge with virulent S. Gallinarum [42]. A Δfur mutation was employed to improve the safety of a S. Typhimurium ΔssaV mutant (discussed in the next section). Introduction of a Δfur into a ΔssaV strain improved its safety profile in immunocompromised mice without compromising immunogenicity [43].

C. Mutations in Salmonellapathogenicity islands Salmonella carry genomic “islands,” generally with a GC content lower than the rest of

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the chromosome, that have been acquired by horizontal gene transfer. Many of these regions facilitate pathogenesis and are known as pathogenicity islands [44]. Although the number of pathogenicity islands and their specific gene content can vary by serovar, all Salmonella serovars that infect warm-blooded animals carry Salmonella pathogenicity island 1 (SPI-1) and SPI-2 [45]. Both SPI-1 and SPI-2 encode a type 3 secretion system (T3SS), a specialized secretion apparatus that secretes virulence proteins across host cell membranes directly into host cells [46]. The current paradigm is that SPI-1 facilitates invasion of host epithelial cells, and SPI-2 is important for survival in macrophages [47]. The ssaV gene encodes a component of the SPI-2 T3SS and strains lacking ssaV are unable to secrete SPI-2 effector proteins and are unable to survive in macrophages [48]. The ΔssaV ΔaroC S. Typhi strain M01ZH09 is safe and immunogenic when administered orally to human volunteers [49,50]. At the highest dose, all 15 vaccinees were positive for anti-Salmonella IgA antibody-secreting cells (ASCs). By day 28, 75% of the volunteers produced anti-Salmonella serum IgG responses that were at least fourfold above background, suggesting that this strain is a promising typhoid vaccine candidate [50]. Strain M01ZH09 was used to deliver the B subunit of heat-labile toxin (LTB) from E. coli in a clinical trial [51]. The eltB gene, encoding the LTB subunit, was inserted into the chromosome of M01ZH09 and expressed from the in vivoinducible ssaG promoter. At the highest dose, 92% of vaccinees produced anti-Salmonella IgA ASCs, and 63% produced anti-LTB IgA ASCs after one or two doses. When combined with ELISA data measuring serum IgG responses, 67% of the vaccinees demonstrated immune responses against LTB, and 95% of the test subjects produced anti-Salmonella immune responses. To date, these are the most promising results for a Salmonella-vectored vaccine in humans (Chapter 32: Oral Vaccines for Enterotoxigenic Escherichia coli).

D. Vectoring guest antigens As was mentioned in the above example, one method for modifying attenuated Salmonella for antigen delivery is to express a foreign gene from the bacterial chromosome. However, a more common approach is to express foreign genes from multicopy plasmids. While expression from the chromosome ensures that the antigen gene will not be lost from the population, the amount of protein produced from a single copy of the gene is often not adequate to promote a protective immune response. Increasing the gene dosage within the vaccine strains results in higher levels of protein production, thereby increasing the subsequent immune response. To be suitable for antigen delivery by Salmonella, the plasmid vector must carry a promoter to transcribe the guest antigen gene, an origin of plasmid replication, a selectable marker, and a means to maintain the plasmid in the vaccine cell population. The use of plasmids necessitates methods to select for and maintain their presence in the cell. In most nonvaccine applications, a geneencoding antibiotic resistance is used. When the plasmid is first introduced into the bacterium, plasmid-containing cells are selected on media containing the antibiotic whose resistance is encoded by the plasmid. Plasmidbearing cells are then grown in the presence of antibiotics to maintain the plasmid in the population. This approach is not practical for vaccine applications, as antibiotic resistance genes cannot be used. There are a variety of novel methods to select for and maintain plasmids in vaccine strains (for a review, see Ref. [52]). Here, we will discuss only the AsdA-balanced lethal plasmid stabilization system [53], as it is widely used. The asdA gene encodes aspartate semialdehyde dehydrogenase, an enzyme required for the synthesis of arginine, lysine, threonine, and methionine. One intermediate in the lysine

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biosynthetic pathway is diaminopimelic acid (DAP), an essential component of the peptidoglycan layer in the bacterial cell wall. An asdA Salmonella mutant cannot grow, even on rich media, unless the growth medium is supplemented with DAP. In the absence of DAP, asdA mutants undergo lysis [54]. Inclusion of a copy of asdA gene on the plasmid carrying a heterologous gene of interest permits selection of plasmid-bearing ΔasdA strains by simply plating on any rich medium. The plasmid is maintained in the population because any cell that loses the plasmid will lyse. This system has proven to be convenient for the selection and maintenance of plasmids in vaccine strains designed to deliver a wide variety of protein antigens.

E. Antigen delivery —location The final location of the antigen in the Salmonella cell can have a huge impact on immunogenicity. Typically, the protein products of antigen genes are retained in the cytoplasm of the Salmonella vaccine. The importance of antigen location was investigated in a study in which two Δcrp S. Typhimurium mutant strains producing the Streptococcus pneumoniae protein PspA were compared. In one strain, the PspA was retained in the cytoplasm. In the other strain, the PspA was fused to a type 2 secretion signal, resulting in secretion of PspA into the periplasmic space and into the growth medium [55]. The two strains produced equivalent levels of PspA. When used to immunize mice, the strain that secreted PspA elicited significantly greater serum anti-PspA IgG responses than the strain in which PspA was retained in the cytoplasm [56]. This strain also elicited strong anti-PspA mucosal IgA responses in immunized mice [55]. A direct comparison of strains in which the early secretory antigen 6 (ESAT-6) from Mycobacterium tuberculosis was localized either in the cytoplasm, on the bacterial cell surface, or secreted was conducted by using a ΔaroA

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S. Typhimurium strain [57]. As in the previous study, all strains produced similar levels of the antigen, varying only in where the antigen was localized. Compared to the cytoplasmic construct, surface-expressed and secreted constructs elicited significantly greater numbers of splenic ESAT-61 T helper 1 (Th1) cells. No IFNγ1 splenic T cells could be detected by using an ESAT-6 MHC II tetramer from mice immunized with the cytoplasmic construct; by contrast, the levels in mice immunized with the other two constructs achieved 3%
30% of total splenic ESAT-61 CD41 T cells. The impact on serum IgG responses was not evaluated for the cytoplasmic construct, but the strains that secreted ESAT-6 to the cell surface or into the extracellular environment elicited high titers of anti-ESAT-6 IgG. In this regard, the two strains were similar, although on day 21, the IgG titers in mice immunized with the surface-expressed strain were significantly lower than mice receiving the secreted construct. However, on days 35 and 42, there was no significant difference in the titers between groups. These results provide further evidence that antigen location is an important consideration in designing Salmonella-vectored vaccines. Technologies for enhancing the immunogenicity of Salmonella vaccines. Traditionally attenuated Salmonella vaccines rely on mutations designed to weaken the strain, making it less virulent. One caveat of this approach is that the resulting strains exhibit a reduced capacity to survive host defenses and interact with host immune cells, leading to reduced immunogenicity. To overcome these issues and enhance the ability of Salmonella to survive and replicate in target immune tissues, Roy Curtiss and colleagues have described a variety of novel approaches for vaccine development, including regulated delayed attenuation, regulated delayed antigen synthesis, and regulated delayed lysis (for a detailed review of these approaches, see Ref. [58]). In this scenario, the vaccine strains are not burdened by debilitating mutations or

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excessive heterologous antigen synthesis at the time of administration and during the initial stages of infection. Once the strain reaches target immune tissues, the attenuation phenotype is expressed, and heterologous antigen synthesis begins.

F. Regulated delayed attenuation Vaccine strains with regulated delayed attenuation display wild-type characteristics when grown in media containing appropriate sugars, providing them with a full complement of virulence factors required to survive transit through the gastrointestinal tract and to carry out the initial stages of infection. Once inside host tissues, expression of specific virulence genes or attributes shuts off, resulting in a fully attenuated strain. Several methods have been used to construct strains with the regulated delayed attenuation phenotype. For example, deletion of the pmi, encoding 6-phosphomannose isomerase, or galE, encoding uridine diphosphategalactose-4-epimerase, results in strains that are dependent on exogenous mannose or galactose, respectively, for the synthesis of O-antigen. O-antigen is a cell surface carbohydrate polymer that serves to protect Salmonella from the action of complement [59], and is important for penetration of the mucus that overlays the intestinal epithelium [60]. Strains are grown with the appropriate sugar prior to administration, resulting in full-length O-antigen. Free mannose and free galactose are not present in host tissues, and the cells gradually lose their O-antigen as they divide. However, pmi deletion mutants of S. Typhimurium are only partially attenuated in mice [61], and Salmonella Typhi galE mutants remain virulent for humans [20]. Thus, pmi and galE mutations cannot be used as the sole basis of attenuation but may serve as secondary mutations to make the cell more susceptible to host defenses. The regulation of virulence genes can be modified by replacing the native promoter

with a sugar-inducible promoter. The arabinose-regulated PBAD promoter, along with the gene encoding the arabinosesensitive transcriptional activator AraC, has been frequently used for this purpose. In one study, the araC PBAD promoter was used to drive expression of individual Salmonella virulence genes, including crp, phoP, rpoS, and/or fur [62]. When the vaccine is grown prior to administration, arabinose is added to the culture medium, and the arabinose-regulated virulence gene(s) is expressed. Thus upon immunization, the strain is producing its full complement of virulence factors. After immunization, when the vaccine strain reaches host tissues where free arabinose is not present, virulence gene expression ceases, and their protein products are lost by dilution as the bacteria divide. When administered orally, strains carrying arabinose-regulated crp, phoP, or rpoS genes were highly attenuated, while arabinose-regulated fur mutants were partially attenuated [62]. The safety and immunogenicity of vaccine strains designed by using this approach have been demonstrated for S. Typhimurium in mice and for S. Gallinarum in chickens [63,64]. However, since key virulence genes are expressed in response to sugars, arabinose in particular, it is possible that host diet could affect the virulence of these strains. While this question has not been specifically addressed for S. Typhimurium in mouse studies, it was investigated in chickens using a S. Gallinarum vaccine. Rhode Island red chicks were immunized with a highly virulent S. Gallinarum strain attenuated by replacement of the crp promoter with araC PBAD [64], a construct essentially identical to the one used in the S. Typhimurium studies. Note that the parent S. Gallinarum strain has an oral LD50 of less than 1 3 106 CFU [42]. Two groups of chicks were orally inoculated at 4 and 18 days of age with the vaccine strain. After the first inoculation, one group of birds was supplied with water

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containing 0.2% arabinose, and the other group was given water with no arabinose. All chickens survived, indicating that dietary arabinose intake does not affect the virulence of strains attenuated in this manner. The immunized birds in both groups were equally protected against challenge with the parent S. Gallinarum strain at 4 weeks of age.

G. Regulated delayed antigen synthesis The strongest case for developing Salmonella vaccines is its utility as an antigen delivery vector. Typically, genes encoding one or more antigens derived from a pathogen of interest are introduced into attenuated Salmonella on a plasmid. The choice of promoters to drive antigen gene(s) expression is an important parameter influencing the efficacy of the vaccine. The strong, constitutive Ptrc promoter can drive high levels of antigen synthesis in cells grown in vitro. In mouse studies, this promoter was shown to be a good, but not ideal, choice for eliciting optimal immune responses [65]. The suboptimal results using the Ptrc promoter are likely to be related to the fact that expression is unregulated in Salmonella. Unregulated heterologous antigen synthesis consumes cellular resources, reducing the ability of the vaccine strain to grow and to cope with host defenses. To overcome this problem, the heterologous antigen gene is placed under transcriptional control of a promoter that is active only in vivo. For example, the PssaG or PpagC promoters are turned on in macrophages [66,67], while the PnirB promoter is expressed under anaerobic conditions [68]. As an alternative approach, a method to regulate heterologous gene expression from the Ptrc promoter in Salmonella was developed. The Ptrc promoter carries a binding site for the transcriptional repressor LacI. The lacI gene is not native to Salmonella, so an arabinose-regulated lacI gene was introduced into the S. Typhimurium chromosome. This results in

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expression of lacI when the strain is grown in the presence of arabinose. When the LacI repressor is synthesized, it binds to Ptrc, thereby reducing or eliminating transcription of the heterologous passenger gene [69]. When arabinose is absent (as in host tissues), lacI is no longer transcribed. The LacI concentration in the cell decreases by dilution as the cell divides, allowing transcription from Ptrc to proceed. Synthesis of heterologous antigen increases, reaching maximum levels after approximately nine cell divisions due to dilution of LacI [69]. One potential drawback of this technology is that overproduction of LacI can reduce Salmonella virulence, which may reduce the immunogenicity of vaccine strains [70]. Despite this, the presence of an arabinose-inducible lacI gene was found to enhance the immunogenicity of a vaccine strain carrying a Ptrc-driven heterologous antigen gene, but reduces immunogenicity in strains carrying other, in vivoinducible promoters, such as PssaG, that do not bind LacI [71]. Thus, it is likely that the presence of the Ptrc promoter on a multicopy plasmid titrates the LacI so that its negative effect on virulence and immunogenicity is minimized. Despite its potential drawbacks, this system may be more flexible than in vivoregulated promoters, since the regulated Ptrc promoter system is compatible with a wider variety of attenuation strategies [71]. For example, promoters that rely on PhoP for activation, such as PpagC and PssaG, will not function properly in a ΔphoP background. Live attenuated Salmonella vaccine strains for antigen delivery have been constructed that utilize both regulated delayed attenuation and regulated delayed antigen synthesis. S. Typhimurium χ9558 includes arabinoseregulated crp and fur genes in addition to a Δpmi mutation [72]. This strain also carries an arabinose-regulated lacI gene to control heterologous antigen gene expression and a ΔasdA mutation to allow use of the AsdA-based

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FIGURE 22.1

Overview of the regulated delayed lysis system. (A) Arabinose regulation of wild-type araBAD. When arabinose is bound to AraC, it binds to the araI1 and araI2 sites. AraC and the cAMP receptor protein (CRP) activate transcription of from the PBAD promoter, driving expression of araBAD and araC. (B) In the absence of arabinose, AraC binds to the araO2 and araI1 regions, forming a DNA loop, repressing the transcription of all four genes. (C) The RASV lysis system is composed of plasmid and chromosomal components. Synthesis of MurA, C2, and LacI from chromosome and of MurA and AsdA from the plasmid occurs when arabinose is present. In the absence of arabinose, LacI is not made, and antigen genes are transcribed from Ptrc. murA and asdA are no longer expressed, leading to elimination of MurA and AsdA by dilution as the cell divides, eventually resulting in cell lysis.

balanced-lethal plasmid maintenance system. Immunization of mice with strain χ9558 carrying an antigen from S. pneumoniae resulted in a greater level of protection against lethal S. pneumoniae challenge than a traditionally attenuated S. Typhimurium strain producing the same antigen [72]. Strain χ9558 derivatives carrying Yersinia antigens have also been shown to protect mice against lethal challenge with Yersinia pestis [73]. Several S. Typhi vaccine candidates with genotypes similar to χ9558 were used to deliver PspA in a clinical trial. The strains were safe but only weakly immunogenic [74]. However, it should be noted that each of these S. Typhi strains carried 10 or more mutations, which may have resulted in over attenuation.

H. Regulated delayed vaccine lysis Another innovative technology is the design of vaccine strains that undergo regulated delayed vaccine lysis [75]. This system was developed to address a number of goals, including biocontainment, release of nonsecreted protein antigens, and DNA vaccine delivery. The basic components of this system are outlined in Fig. 22.1. These vaccine strains feature a ΔasdA deletion mutation and arabinose-controlled expression murA. The MurA gene product, like AsdA, is required for synthesis of the peptidoglycan component of the bacterial cell wall. The murA gene is essential, and its absence cannot be corrected by adding a nutrient to the growth medium, so a

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conditional lethal mutation was constructed by placing the gene under transcriptional control of araC PBAD. The plasmid component of this system carries arabinose-controlled asdA and murA genes, along with antigen genes of interest. The Ptrc promoter drives antigen gene expression for delivery of protein antigens, and is regulated by an arabinose-regulated lacI gene present in the chromosome. S. Typhimurium lysis strains designed by using this technology have been used to deliver proteins from Gram-positive pathogens and from influenza A virus. In the first chapter describing this system, mice were orally immunized with lysis strains producing the S. pneumoniae protein PspA. Immunized mice produced both humoral and mucosal responses against PspA and Salmonella proteins [75]. Humoral responses against Salmonella antigens were strongly Th1, while a mixed Th1/Th2 response was elicited against PspA. In another study, a lysis strain carrying a plasmid encoding a woodchuck hepatitis virus-like particle (VLP) modified to produce the influenza A M2e protein was used to orally or intranasally immunize mice twice at a 3-week interval [76]. The strain elicited both humoral IgG and mucosal IgA responses against M2e. Intranasal immunization resulted in a more rapid antibody response, but by 6 weeks, the two groups showed similar levels of IgG in the serum. These responses were greater than those in a control group that received the VLP-M2e delivered by a nonlysis Δcya Δcrp S. Typhimurium strain. Five days after being challenged with influenza virus, immunized mice had lower titers of virus in their lungs compared to nonimmunized controls, suggesting mucosal protection, although the difference was not statistically significant. Immunized mice also showed a significant increase in weight gain and increased survival compare to Salmonellaonly controls. A similar strain, further modified with a ΔsifA deletion to allow escape from the endosome prior to lysis [77], was used to orally

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deliver the influenza A NP protein in three or four doses, eliciting a strong Th1-type response [78]. The vaccine elicited protection against weight loss and led to increased survival after influenza challenge. A lysis strain was used to deliver Clostridium perfringens antigens to chickens. C. perfringens causes necrotic enteritis in chickens, a disease characterized in part by intestinal lesions. Chickens were orally immunized with two doses of a Salmonella lysis strain producing two relevant antigens, alpha toxoid and NetB toxoid from a plasmid [79]. Overall, serum responses were low, with maximum IgY, the chicken equivalent of IgG, titers about four-fold greater than those in controls. However, the vaccine elicited strong mucosal responses, with maximum intestinal IgA, IgM, and IgY titers against both antigens that were 30- to 60-fold greater than those in controls. After challenge, immunized birds exhibited fewer and milder lesions than Salmonella-only controls. Protection was not as great when a nonlysis strain delivering the same antigens was used as the immunogen (Jiang, Roland, and Curtiss, unpublished). A Salmonella lysis strain is also useful for delivering DNA vaccines. In this context, the plasmid component of the lysis system is modified to include a eukaryotic promoter and nuclear targeting sequences. A number of additional mutations were included in the vaccine strain to facilitate efficient DNA delivery, including ΔsifA [80]. The system was used to deliver a gene encoding the influenza hemagglutinin antigen (HA). Results from the study showed that the lysis DNA delivery system was effective, eliciting high serum IgG titers against HA and protection against challenge with influenza virus. Mucosal responses were not reported. Taken together, results from the studies described in this section suggest that lysis strains are superior to nonlysis strains for eliciting protective mucosal responses, at

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least against the pathogens tested. Thus far, this approach has not been tested in a clinical trial, so how useful and applicable it is for human vaccines remains an open question. One concern about this system relates to its potential to trigger sepsis due to release of lipid A during lysis. While symptoms of sepsis have not been reported in animal systems, this remains a potential obstacle that must be addressed before this system can be approved for human trials. In the next section, I will discuss strategies for lowering the toxicity of lipid A by altering its structure in vivo.

I. Strategies for reducing lipid A toxicity Lipid A, also known as endotoxin, is a component of the complex molecule called LPS which forms the outer monolayer of the outer membrane of Salmonella. LPS is composed of lipid A, core sugars, and the highly immunogenic, multimeric O-antigen polysaccharide. Salmonella lipid A is itself a complex molecule consisting of a β(1
6)-linked glucosamine disaccharide, with phosphate groups at the 1 and 40 positions (Fig. 22.2). The disaccharide is typically hexa-acylated with 12- to 16-carbon acyl groups, although the exact number and length of the acyl groups can vary depending on the environment. Lipid A is responsible for the toxic effects of LPS (endotoxin), which include fever and sepsis [81]. Lipid A containing two phosphate groups, located at the 1 and 40 positions in the molecule, and six acyl chains that are 12
14 carbons in length activates proinflammatory responses through the TLR4MD2-CD14 pathway, while lipid A variants with fewer acyl chains, such as tetra- or pentaacylated lipid A species, have significantly diminished immunostimulatory activity [82,83]. A monophosphoryl lipid A derivative of E. coli lipid A (MPL), in which the 1-phosphate and several acyl groups are

FIGURE 22.2 Diagram of Salmonella lipid A. The figure illustrates the basic structure of lipid A, including glucosamines, acyl groups, and phosphate groups. Carbons comprising the glucosamine moieties are numbered. Depending on conditions, the phosphate groups may be modified with 4-amino-4 deoxy-L-arabinose or phosphoethanolamine moieties, and additional acyl groups or modified acyl groups may be present.

chemically removed, is much less toxic while retaining its ability to bind to TLR4 and is a potent mucosal adjuvant [84]. Kong and associates have taken a unique approach to enhancing Salmonella vaccine safety by modifying the lipid A in living cells to the less toxic, 40 -monophosphoryl form while retaining its adjuvant properties [85]. To achieve this, the Francisella tularensis lpxE gene, encoding an inner membrane phosphatase, was introduced into the Salmonella chromosome to facilitate removal of the 1-phosphate group from lipid A. Constitutive expression of lpxE from the chromosome, along with deletion of several Salmonella genes involved in lipid A modification, resulted in a strain that produced lipid A that was essentially 100% dephosphorylated at the 1 position. The resulting 40 -monophosphorylated lipid A

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was purified and used to stimulate the human monocytic cell line MM6, inducing significantly lower levels of IL-6 than lipid A isolated from wild-type S. Typhimurium [85]. In a rabbit ligated loop assay, the S. Typhimurium strain producing 40 -monophosphorylated lipid A did not elicit a detectable inflammatory response, while its wild-type parent was highly inflammatory, as characterized by flattening of epithelial villi, substantial PMN infiltration, and significant tissue destruction. An attenuated S. Typhimurium strain modified to produce 40 -monophosphorylated lipid A retained immunogenicity and was able to deliver the pneumococcal protein PspA, eliciting antiPspA serum IgG and mucosal IgA, resulting in significant protection against challenge with S. pneumoniae. This seems to be a promising approach for overcoming potential issues with the delayed lysis technology. However, as yet, there are no reports of lysis strains engineered to produce modified lipid A. Vaccine strains with lipid A with the 1 and 40 phosphates removed have been explored. However, this modification resulted in lower immunogenicity [86]. Lipid A toxicity can also be reduced by modification of the acyl groups attached to the glucosamine component of lipid A [87]. Deletion of the waaN (msbB) gene results in pentaacylated lipid A missing a myristoyl group [88]. This modification results in a loss of virulence and decreased endotoxicity linked to a reduced ability of the penta-acylated lipid A to induce TNF-α in human monocytes [89]. A waaN mutant of S. Typhimurium strains with modified acyl groups has been investigated for use as an antigen delivery vector [90]. Introduction of a ΔwaaN mutation into an attenuated ΔpabA ΔpabB mutant enhanced mucosal IgA responses to a vectored antigen, PspA, although anti-PspA serum IgG responses were delayed. This is a promising alternative approach to 1-dephosphorylation for detoxifying lipid A in lysis strains.

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J. Sugar-inducible acid resistance Attenuated S. Typhi strains are the preferred Salmonella vector for human vaccines. However, S. Typhi is more sensitive to the low pH environment of the human stomach than other enteric pathogens such as S. Typhimurium, E. coli or Shigella [91]. Many attenuated S. Typhi strains exhibit a further increase in sensitivity to low pH. To survive the low gastric pH in humans, oral Salmonella vaccines are typically given with an agent designed to increase the gastric pH, such as bicarbonate, or they are incorporated into an enteric-coated capsule. While this approach is helpful, it precludes the Salmonella vaccine from sensing an important environmental signal—low pH—signifying its entry into a host and, in the case of encapsulation, reduces overall efficacy. Increasing the acid resistance of S. Typhi vaccines would preclude the need to bypass stomach acidity, enhancing its ability to reach the host mucosa and initiate invasion of local tissues in the gut, leading to enhanced immunogenicity. One caveat of this approach is that fewer attenuated S. Typhi cells may be required to elicit a protective immune response, thus lowering the effective vaccine dose and perhaps also the frequency of vaccination. Enteric bacteria have evolved a number of different strategies to survive low-pH environments, including amino acid decarboxylase systems. Several groups have taken advantage of these existing genes and have engineered S. Typhi vaccine strains to express them from sugar-inducible promoters such as araC PBAD. In my laboratory, we modified the native S. Typhi acid resistance genes adiA and adiC, encoding arginine decarboxylase and the arginine
agmatine antiporter, such that their expression is driven by a sugarinducible promoter [92]. Addition of the appropriate sugar results in expression of adiA and adiC and a concomitant increase in acid resistance. Our results demonstrated that

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this system significantly increases in survival for ΔaroD and ΔphoPQ S. Typhi strains at both pH 3.0 and pH 2.5 [92] and in a low gastric pH mouse model [93]. We also examined the use of the glutamate decarboxylase (GAD) system from E. coli. This system allows E. coli to survive at a lower pH than S. Typhi [94]. The GAD system is composed of two homologous decarboxylases (GadA and GadB) and a glutamate/ γ
aminobutyric acid antiporter (GadC) [95]. GadA and GadB are biochemically indistinguishable, and only GadB is strictly required for survival at pH 2.5 in E. coli [96]. However, both are required for survival at pH 2 [96,97]. In E. coli, this system maintains an internal pH between 4 and 5 [98]. We introduced an arabinose-inducible glutamate decarboxylase system (gadBC) into S. Typhi guaBA, phoPQ, and fur mutants. The presence of gadBC enhanced survival to acid shock in vitro and survival during passage through the gastrointestinal tract in a low gastric pH mouse model (Brenneman and Roland, unpublished). Dennis Kopecko’s group introduced the gadA gadBC system from Shigella flexneri into the licensed typhoid vaccine strain Ty21a under transcriptional control of araC PBAD [99]. The inclusion of this system resulted in significant increases in survival at pH 3.0 and pH 2.5 compared to Ty21a without this system. The impact of this approach on immunogenicity has not yet been tested in a clinical trial.

K. Modification of fimbriae Bacterial fimbriae are extracellular structures whose primary function is to present adhesins that enable the bacterium to bind to surfaces. In addition to adherence to surfaces, fimbriae are involved in other functions, including conjugation, biofilm formation, and evasion of host phagocytes. Although fimbriae have been used

as vaccine antigens, the role of fimbriae in vaccine design to influence host immune responses has been an understudied area. However, there have been a few reports suggesting that host immune responses can be influenced by the fimbrial composition of the vaccine strain. When the E. coli CFA/I fimbriae were produced in an S. Typhimurium vaccine, there was a shift in the subsequent early immune response from Th1- to Th2-type, compared with a strain that does not produce CFA/I [100]. This was due to altered interaction with host macrophages, resulting in a reduction in the early proinflammatory cytokine responses [101]. In another study, four S. Typhimurium fimbrial operons—agf, saf, sti, and stc—were identified as being expressed in the mouse spleen. In the context of an attenuated S. Typhimurium vaccine strain delivering PspA, constitutive expression of either sti, saf, or stc significantly enhanced protection against challenge with S. pneumoniae [42], suggesting that this approach has promise for vectored vaccines. We have been investigating the influence of modifying the S. Typhi fimbrial profile as a means to improve immunogenicity. The S. Typhi stg fimbrial operon facilitates adherence to enterocytes [102], while the S. Typhimurium lpf fimbrial operon facilitates adherence to M cells, leading to efficient colonization of PPs with subsequent stimulation of innate immune responses [103]. We have found that deleting the stg fimbrial operon and introducing lpf in wildtype S. Typhi resulted in increased adherence and invasion of M cells, with subsequent increases in release of the proinflammatory cytokine interleukin-8 [104]. More important, these modifications had a profound effect on the adherence and invasion of M cells by the licensed vaccine strain Ty21a (Tafoya, Roland unpublished), suggesting that this approach may significantly enhance immune responses to this vaccine.

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III. VACCINES AGAINST NONTYPHOIDAL SALMONELLA Bacteremia due to invasive nontyphoidal Salmonella (iNTS) is an emerging disease in parts of sub-Saharan Africa. The primary serovars associated with this disease are S. Typhimurium and S. Enteritidis, although other serovars may also be involved. This situation has driven efforts to develop live attenuated vaccines to prevent infections by iNTS. Prior to these efforts, only two attenuated S. Typhimurium vaccines were tested in humans: the ΔaroC ΔssaV mutant WT05 [49] and a ΔphoPQ mutant expressing the urease gene from Helicobacter pylori [105]. Both strains were safe and immunogenic, although the WT05 strain was shed in feces for up to 22 days. However, these early results indicate that NTS strains may be suitable as human vaccines. iNTS isolates were used to construct S. Typhimurium and S. Enteritidis strains carrying ΔguaBA ΔclpP or ΔguaBA ΔclpX mutations as human vaccines. Preclinical studies evaluating these strains are underway [106] (Chapter 29: Mucosal Vaccines for Salmonella typhi Infection).

IV. CONCLUDING REMARKS Salmonella has unique characteristics that make it an ideal antigen delivery vector. As our understanding of Salmonella pathogenesis has improved, so has our ability to harness Salmonella’s usefulness. From putting “Salmonella on a string” to induce attenuation and/or lysis after infection to advancing safety by modifying its lipid A and enhancing immunogenicity by inactivating immunosuppressive genes, we are finally poised to make human Salmonella vaccines a reality. Thus, the technology for constructing Salmonella vaccines is reaching its maturity. The lack of informative animal models for S. Typhi leaves human trials as the best option

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for testing S. Typhi vaccines. As of 2013, there were only 13 clinical trials testing Salmonella vectoring antigens. Advanced tissue culture models are being developed, but it is unclear how well these will mimic the complicated interplay between Salmonella and host. What is needed now are more clinical studies to test the innovative ideas outlined in this chapter and lead the way to achieving the great potential of Salmonella vaccines.

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IV. CURRENT AND NEW APPROACHES FOR MUCOSAL VACCINE DELIVERY