Salmonella enterica serovar typhimurium surA mutants are attenuated and effective live oral vaccines

A previously described attenuated TnphoA mutant (BRD441) of Salmonella enterica serovar Typhimurium C5 (I. Miller, D. Maskell, C. Hormaeche, K. Johnson, D. Pickard, and G. Dougan, Infect. Immun. 57:2758-2763, 1989) was characterized, and the transposon was shown to be inserted in surA, a gene which encodes a peptidylprolyl-cis, trans-isomerase. A defined surA deletion mutation was introduced into S. enterica serovar Typhimurium C5 and the mutant strain, named S. enterica serovar Typhimurium BRD1115, was extensively characterized both in vitro and in vivo. S. enterica serovar Typhimurium BRD1115 was found to be defective in the ability to adhere to and invade eukaryotic cells. Furthermore, S. enterica serovar Typhimurium BRD1115 was attenuated by at least 3 log units when administered orally or intravenously to BALB/c mice. Complementation of the mutation with a plasmid carrying the intact surA gene almost completely restored the virulence of BRD1115. In addition, S. enterica serovar Typhimurium BRD1115 demonstrated potential as a vaccine candidate, since mice immunized with BRD1115 were protected against subsequent challenge with S. enterica serovar Typhimurium C5. S. enterica serovar Typhimurium BRD1115 also showed potential as a vehicle for the effective delivery of heterologous antigens, such as the nontoxic, protective fragment C domain of tetanus toxin, to the murine immune system.     

Phase 2 clinical trial of attenuated Salmonella enterica serovar typhi oral live vector vaccine CVD 908-htrA in U.S. volunteers

Salmonella enterica serovar Typhi strain CVD 908-htrA is a live attenuated strain which may be useful as an improved oral typhoid vaccine and as a vector for cloned genes of other pathogens. We conducted a phase 2 trial in which 80 healthy adults received one of two dosage levels of CVD 908-htrA in a double-blind, placebo-controlled, crossover study. There were no differences in the rates of side effects among volunteers who received high-dose vaccine (4.5 x 10(8) CFU), lower-dose vaccine (5 x 10(7) CFU), or placebo in the 21 days after vaccination, although recipients of high-dose vaccine (8%) had more frequent diarrhea than placebo recipients (0%) in the first 7 days. Seventy-seven percent and 46% of recipients of high- and lower-dose vaccines, respectively, briefly excreted vaccine organisms in their stools. All blood cultures were negative. Antibody-secreting cells producing antilipopolysaccharide (LPS) immunoglobulin A (IgA) were detected in 100 and 92% of recipients of high- and lower-dose vaccines, respectively. Almost half the volunteers developed serum anti-LPS IgG. Lymphocyte proliferation and gamma interferon production against serovar Typhi antigens occurred in a significant proportion of vaccinees. This phase 2 study supports the further development of CVD 908-htrA as a single-dose vaccine against typhoid fever and as a possible live vector for oral delivery of other vaccine antigens.     

Immune responses to recombinant pneumococcal PspA antigen delivered by live attenuated Salmonella enterica serovar typhimurium vaccine

Attenuated Salmonella enterica serovar Typhimurium expressing recombinant antigens from other pathogens elicits primarily a Th1-type dominant immune response to both recombinant and Salmonella antigens. The immunogenicity and appropriate subcellular location of the recombinant antigen in the Salmonella vaccine strain may contribute to augmenting immune responses by facilitating adequate exposure of recombinant antigen to antigen-presenting cells for processing. To allow for secretion from gram-negative bacteria and overexpression of antigen, a DNA fragment encoding a highly antigenic alpha-helical region of PspA (pneumococcal surface protein A) was subcloned downstream from the beta-lactamase signal sequence in the multicopy Asd(+) pYA3493 vector to create pYA3494. pYA3493 was derived from a class of Asd(+) vectors with reduced expression of Asd to minimize selective disadvantage and enhance immunization of expressed recombinant antigens. The S. enterica serovar Typhimurium vaccine strain was constructed by the introduction of deletion mutations Delta crp-28 and Delta asdA16. Approximately 50% of the recombinant PspA (rPspA) expressed in a Salmonella strain harboring pYA3494 was detected in the combined supernatant and periplasmic fractions of broth-grown recombinant SALMONELLA: After a single oral immunization in BALB/c mice with 10(9) CFU of the recombinant Salmonella vaccine strain carrying pYA3494, immunoglobulin G (IgG) antibody responses were stimulated to both the heterologous antigen rPspA and Salmonella lipopolysaccharide (LPS) and outer membrane proteins (OMPs). About half, and even more at later times after immunization, of the antibodies induced to rPspA were IgG1 (indicating a Th2-type response), whereas 60 to 70% of the antibodies to LPS and 80 to 90% of those to OMPs were IgG2a (indicating a Th1-type response). A sublethal infection with Streptococcus pneumoniae WU2 boosted PspA antibody levels and maintained IgG2a/IgG1 ratios similar to those seen before the challenge. Oral immunization with Salmonella-PspA vaccine protected 60% of immunized mice from death after intraperitoneal challenge with 50 times the 50% lethal dose of virulent S. pneumoniae WU2.     

Mucosal and cellular immune responses elicited by recombinant Lactococcus lactis strains expressing tetanus toxin fragment C

The mucosal and cellular responses of mice were studied, following mucosal-route administration of recombinant Lactococcus lactis expressing tetanus toxin fragment C (TTFC), which is a known immunogen protective against tetanus. A TTFC-specific T-cell response with a mixed profile of T-helper (Th) subset-associated cytokines was elicited in the intestine, with a Th2 bias characteristic of a mucosal response. These results correlated with the humoral response, where equivalent titers of anti-TTFC immunoglobulin G1 (IgG1) and IgG2a in serum were accompanied by an elevated IgA-specific response at more than one mucosal site. The route of vaccination had an important role in determining the immune response phenotype, as evidenced by the fact that an IgG1-biased subclass profile was obtained when lactococci were administered parenterally. Stimulation of splenic or mesenteric lymph node cells with lactococci resulted in their proliferation and the secretion of gamma interferon via antigen-specific and innate immune mechanisms. The data therefore provide further evidence of the potential of recombinant lactococcal vaccines for inducing systemic and mucosal immune responses.     

Clinical and field trials with attenuated Salmonella typhi as live oral vaccines and as "carrier" vaccines

In recent years there has been a resurgence of research to develop new and improved attenuated strains of Salmonella typhi to function as live oral vaccines against typhoid fever and to serve as "carrier" vaccines to express foreign antigens of other pathogens and deliver them to the immune system. Strain Ty21a has served as a prototype in clinical and field trials to identify the optimal formulations and dosage schedules for live vaccines and to quantitate the duration of protection that can be achieved. Clinical trials with three new attenuated S. typhi candidate vaccines, a Vi+ variant of Ty21a, an aroC,aroD double mutant recombinant strain and a cya,crp double mutant, are underway or will be initiated shortly.     

Candidate live, attenuated Salmonella enterica serotype Typhimurium vaccines with reduced fecal shedding are immunogenic and effective oral vaccines

Environmental shedding of genetically manipulated microorganisms is an issue impeding the development of new live vaccines. We have investigated the immunogenicity of a number of novel Salmonella enterica serotype Typhimurium oral vaccine candidates that express the fragment C (TetC) component of tetanus toxin and harbor combinations of additional mutations in genes shdA, misL, and ratB that contribute to the persistence of serotype Typhimurium's colonization of the intestine. Serotype Typhimurium aroA (TetC) derivatives harboring additional mutations in either shdA or misL or combinations of these mutations exhibited a marked decrease in shedding of the vaccine strain in the feces of orally vaccinated mice. However, equivalent levels of anti-TetC and anti-Salmonella lipopolysaccharide immunoglobulin G (IgG), IgG1, IgG2a, and IgA were detected in sera of the vaccinated but not of the control mice. Cellular immune responses to TetC were detected in all vaccinated mice, regardless of the presence of the additional mutations in shdA or misL. Further, immunization with serotype Typhimurium aroA candidate vaccines harboring shdA and misL afforded complete protection against challenge with a virulent strain of serotype Typhimurium.     

Effect of attenuated Salmonella enterica serovar Typhimurium expressing a Streptococcus mutans antigen on secondary responses to the cloned protein

Attenuated Salmonella enterica serovar Typhimurium has been used for targeted delivery of recombinant antigens to gut- and nose-associated lymphoid tissues. Contradictory reports have described the effect of preexisting immunity to the antigen delivery vehicle. We decided to examine this discrepancy by studying the effect of immunizing mice by the intranasal (i.n.) route with Salmonella expressing an insoluble protein and to study the ability to augment recall responses by boosting with either Salmonella-expressed protein or purified soluble protein alone. The glucan-binding domain (GLU) of the enzyme glucosyltransferase (GTF), which is an important virulence factor of Streptococcus mutans, was recombinantly expressed in the insoluble phase in S. enterica serovar Typhimurium, and the immunogenicity of this construct was studied in mice. We examined the induction of primary immune responses by insoluble GLU polypeptide delivered in Salmonella at week 1 (groups 1 and 2) and recall responses after a week 15 boost with either Salmonella expressing GLU (group 1) or purified GLU polypeptide (groups 2 and 3). Group 4 served as the control and received phosphate-buffered saline alone by the i.n. route. Significant anti-GLU serum immunoglobulin G (IgG) levels were seen in groups 1, 2, and 3 at week 18 (P < 0.001), i.e., 3 weeks after the booster immunization. Mice in group 2, who received Salmonella followed by GLU, had the highest GLU-specific IgG levels among all groups. The serum IgG levels persisted in all responding groups for at least 7 weeks after the boost (week 22). The IgG2a/IgG1 subclass ratio of serum anti-GLU antibodies in group 1 significantly increased after the boost. These results support the induction of a type 1-like immune response to GLU after primary and booster immunizations with Salmonella expressing GLU. On the other hand, group 2 mice, which received Salmonella expressing GLU as the primary dose and soluble protein as the booster dose, exhibited a shift from a type 1-like to a more type 2-like immune response to GLU following the boost. These results indicate that S. enterica serovar Typhimurium is an excellent delivery vehicle for the insoluble and recombinantly expressed GLU of GTF and that this construct was especially effective in priming the host for a secondary response to soluble GLU polypeptide.     

Tetanus toxin fragment C expressed in live Salmonella vaccines enhances antibody responses to its fusion partner Schistosoma haematobium glutathione S-transferase

Tetanus toxoid has been used widely as an adjuvant. The atoxic fragment C from tetanus toxin (TetC) is potently immunogenic when expressed in Salmonella vaccine strains and has been used as a fusion partner for antigens (Ag). However, there has been no formal comparison of the immunomodulatory impact of TetC on its fusion partners. In this study, we have addressed this important issue. The protective 28-kDa glutathione S-transferase (GST) from Schistosoma haematobium (Sh28GST) was expressed either as a fusion to TetC or as the full-length Sh28GST alone in a nonvirulent aroA-attenuated strain of Salmonella enterica serovar Typhimurium. The Sh28GST proteins were soluble and stably expressed in Salmonella, as evaluated by Western blotting with TetC and/or Sh28GST antisera. Mice were immunized orally with a single dose of the live recombinant Salmonella. The constructs were stable in mice but, dramatically, only the strain expressing the TetC-Sh28GST fusion elicited significant antibody (Ab) responses directed against Sh28GST as determined by enzyme-linked immunosorbent assay. An analysis of the isotype profiles showed that these mice also produced anti-Sh28GST immunoglobulin A and GST-neutralizing assays revealed high levels of neutralizing Abs in sera. These are important correlates of protection in schistosomiasis. In addition, stimulation of spleen cells from immunized mice with Sh28GST Ag showed that both strains, expressing Sh28GST alone or the TetC-Sh28GST fusion, were able to stimulate the secretion of Th1-related cytokines (gamma interferon and interleukin 2) to comparable levels. Thus, TetC has modulated the immune responses generated against its fusion partner, Sh28GST, by markedly enhancing the Ab responses elicited. These results have important implications in the rational development of live vaccines.     

Recombinant attenuated Salmonella enterica serovar typhimurium expressing the carboxy-terminal domain of alpha toxin from Clostridium perfringens induces protective responses against necrotic enteritis in chickens

Clostridium perfringens-induced necrotic enteritis (NE) is a widespread disease in chickens that causes high mortality and reduced growth performance. Traditionally, NE was controlled by the routine application of antimicrobials in the feed, a practice that currently is unpopular. Consequently, there has been an increase in the occurrence of NE, and it has become a threat to the current objective of antimicrobial-free farming. The pathogenesis of NE is associated with the proliferation of C. perfringens in the small intestine and the secretion of large amounts of alpha toxin, the major virulence factor. Since there is no vaccine for NE, we have developed a candidate live oral recombinant attenuated Salmonella enterica serovar Typhimurium vaccine (RASV) that delivers a nontoxic fragment of alpha toxin. The 3' end of the plc gene, encoding the C-terminal domain of alpha toxin (PlcC), was cloned into plasmids that enable the expression and secretion of PlcC fused to a signal peptide. Plasmids were inserted into Salmonella enterica serovar Typhimurium host strain chi8914, which has attenuating pabA and pabB deletion mutations. Three-day-old broiler chicks were orally immunized with 10(9) CFU of the vaccine strain and developed alpha toxin-neutralizing serum antibodies. When serum from these chickens was added into C. perfringens broth cultures, bacterial growth was suppressed. In addition, immunofluorescent microscopy showed that serum antibodies bind to the bacterial surface. The immunoglobulin G (IgG) and IgA titers in RASV-immunized chickens were low; however, when the chickens were given a parenteral boost injection with a purified recombinant PlcC protein (rPlcC), the RASV-immunized chickens mounted rapid high serum IgG and bile IgA titers exceeding those primed by rPlcC injection. RASV-immunized chickens had reduced intestinal mucosal pathology after challenge with virulent C. perfringens. These results indicate that oral RASV expressing an alpha toxin C-terminal peptide induces protective immunity against NE.     

Comparison of the human immune response to live oral, killed oral or killed parenteral Salmonella typhi TY21A vaccines

The live oral typhoid vaccine Ty21a has proved to confer protection against the disease at least as effectively as killed parenteral vaccines, whereas killed oral vaccines have not been protective in field trials. This prompted us to compare the immune response of subjects vaccinated either with live oral, killed oral or killed parenteral Salmonella typhi Ty21a vaccine. The immune responses were studied by analysis of peripheral blood antibody-secreting cells (ASC), believed to reflect the mucosal immune response. Live and killed bacteria administered by the oral route elicited immune responses of similar specificity and Ig class profile (IgA dominating), but the response to the live vaccine was significantly stronger and lasted longer. The administration route, on the other hand, influenced the antigenic specificity of the ASC response suggesting different processing of the antigen by the systemic and local immune systems. Thus, the response after oral vaccination was almost exclusively directed to the surface O-antigen, whereas after parenteral vaccination an equally strong response was seen to the O-antigen, to lipopolysaccharide core and to flagella.     

Novel attenuated Salmonella enterica serovar Choleraesuis strains as live vaccine candidates generated by signature-tagged mutagenesis

Salmonella enterica serovar Choleraesuis is a host-adapted pathogen that causes swine paratyphoid. Signature-tagged mutagenesis (STM) was used to understand the pathogenicity of S. enterica serovar Choleraesuis in its natural host and also to develop novel attenuated live vaccine candidates against this disease. A library of 960 signature-tagged mutants of S. enterica serovar Choleraesuis was constructed and screened for attenuation in pigs. Thirty-three mutants were identified by the STM screening, and these mutants were further screened for attenuation by in vivo and in vitro competitive growth. Of these, 20 mutants targeting the outer membrane, type III secretion, transporter, lipopolysaccharide biosynthesis, and other unknown proteins were confirmed for attenuation. Five highly attenuated mutants (SC2D2 [ssaV], SC4A9 [gifsy-1], SC6F9 [dgoT], SC12B12 [ssaJ], and SC10B1[spiA]) were selected and evaluated for safety and protective efficacy in pigs by comparison with a commercially available vaccine strain. STM-attenuated live vaccine strains SC4A9 (gifsy-1) and SC2D2 (ssaV) were superior to commercially available live vaccine because they provided both safety and a protective immune response against challenge in pigs.     

Influence of preimmunization with tetanus toxoid on immune responses to tetanus toxin fragment C-guest antigen fusions in a Salmonella vaccine carrier.

We have previously described a new system for the delivery of recombinant antigens in live Salmonella vaccines as genetic fusions to the C terminus of fragment C of tetanus toxin (TetC) driven by the anaerobically inducible nirB promoter. It has been reported that preimmunization with tetanus toxoid (TT) can suppress the antibody response to peptides chemically coupled to TT (epitope-specific suppression) in both animals and humans, which could interfere with efficacy of the Salmonella-TetC delivery system. We report that preimmunization of BALB/c mice with TT in alum did not suppress the response to either of two protective antigens of Schistosoma mansoni, the full-length S. mansoni P28 glutathione S-transferase (P28) and a construct consisting of eight tandem copies of the protective peptide comprising amino acids 115 to 131 of P28. The guest antigens were expressed in the aroA Salmonella typhimurium SL3261 vaccine strain as fusions to TetC. Preimmunization with TT 10 weeks before administration of the recombinant salmonellae did not alter the antibody response to the full-length P28, whereas the response to the peptide comprising amino acids 115 to 131 was increased by preimmunization with TT, with the increase seen mainly in the immunoglobulin G1 isotype. The antitetanus response was increased by preimmunization with TT in all groups receiving salmonellae expressing TetC. The results could be important when one is considering the use of the Salmonella-TetC delivery system in populations preimmunized with TT.     

Oral and nasal DNA vaccines delivered by attenuated Salmonella enterica serovar Typhimurium induce a protective immune response against infectious bronchitis in chickens

Several studies have reported that intramuscular injection of DNA vaccines against infectious bronchitis virus (IBV) induces protective immune responses. In the present study, we developed oral and nasal DNA vaccines that carried the S1 gene and N gene of IBV delivered by attenuated Salmonella enterica serovar Typhimurium strains SL/pV-S1 and SL/pV-N, respectively. The safety and stability of recombinant Salmonella vaccine were evaluated. Following oral and nasal administration to chickens, the serum and mucosal samples were collected and antibodies against IBV were measured. Chickens were then challenged with IBV strain M41 by the nasal-ocular route 3 weeks after boosting. The results showed that oral and nasal immunization with coadministered SL/pV-S1 and SL/pV-N elicited significant IBV-specific humoral and mucosal immune responses and conferred protective efficacy against IBV challenge higher than that in chickens immunized only with SL/pV-S1. The current study shows that novel DNA vaccines delivered by attenuated S. Typhimurium may be promising candidates for the prevention of infectious bronchitis (IB).These vaccines are efficacious, easily produced economically, and able to be delivered orally and nasally rather than injected. Coadministration of SL/pV-S1 and SL/pV-N may represent an effective mucosal vaccination regimen.     

In vivo characterization of the murine intranasal model for assessing the immunogenicity of attenuated Salmonella enterica serovar Typhi strains as live mucosal vaccines and as live vectors

Attenuated Salmonella enterica serovar Typhi live vector vaccine strains are highly immunogenic in mice following intranasal but not orogastric inoculation. To elucidate the relationship between organs within which vaccine organisms are found and the induction of specific serum immunoglobulin G (IgG) antibodies, we examined the in vivo distribution of serovar Typhi vaccine strain CVD 908-htrA following intranasal administration. Vaccine organisms were cultured from the nasal lymphoid tissue (NALT), lungs, and Peyer's patches 2 min after intranasal inoculation. Vaccine organisms persisted longer in NALT than in other organs. By decreasing the volume of intranasal inoculum containing 10(9) CFU (from a single 30- or 10-microl dose to four 2.5-microl doses given over the course of 1 h), we were able to significantly reduce the number of vaccine organisms isolated from the lungs (P < 0.05) without reducing the number of vaccine organisms in NALT. Reducing the number of vaccine organisms in the lungs resulted in a significant decrease in the serum tetanus antitoxin response elicited by CVD 908-htrA expressing tetanus toxin fragment C under the control of the redox-responsive nir15 promoter. In contrast, a similar construct expressing tetanus toxin fragment C under control of the constitutive lpp promoter stimulated a strong serum IgG tetanus antitoxin response with both inoculation regimens. The data suggest that following intranasal inoculation, NALT is a sufficient inductive site for elicitation of an immune response against both the live vector and heterologous antigen and, as occurs following oral inoculation of humans, attenuated serovar Typhi vaccine organisms elicit serum IgG responses.     

Effect of preexisting immunity to Salmonella on the immune response to recombinant Salmonella enterica serovar typhimurium expressing a Porphyromonas gingivalis hemagglutinin

Recombinant Salmonella strains expressing foreign heterologous genes have been extensively studied as live oral vaccine delivery vectors. We have investigated the mucosal and systemic immune responses following oral immunization with a recombinant Salmonella enterica serovar Typhimurium expressing the hemagglutinin HagB from Porphyromonas gingivalis, a suspected etiological agent of adult periodontal disease. We have previously shown a primary mucosal and systemic response following oral immunization with chi4072/pDMD1 and recall responses following boosting at 14 weeks after primary immunization. In this study, we examined the effects of earlier boosting as well as the effects of deliberately induced immunity to the Salmonella carrier strain on subsequent immune responses. Mice boosted at week 7 following immunization, a point which corresponded to the peak of the primary response, generally showed lower responses than those boosted at week 14. When mice were preimmunized with the Salmonella carrier alone and then immunized with the recombinant strain 7 or 14 weeks later, significant reductions were seen for serum immunoglobulin G (IgG) antibodies at week 14 and for salivary IgA at week 7. No reductions were seen in serum IgA or vaginal wash IgA antibodies. Mice appear to be refractory to boosting with orally administered salmonellae at 7 weeks. Deliberate immunization with the carrier strain did not appreciably affect recall responses at 14 weeks, with the exception of the serum IgG responses, nor did it affect colonization of the Peyer's patches.     

Enhancement of immune responses by an attenuated Salmonella enterica serovar Typhimurium strain secreting an Escherichia coli heat-labile enterotoxin B subunit protein as an adjuvant for a live Salmonella vaccine candidate

A plasmid harboring eltB, the gene encoding heat-labile enterotoxin (LTB), was constructed by insertion of eltB into an Asd(+) β-lactamase signal plasmid (pMMP65). This was introduced into the Δlon ΔcpxR Δasd Salmonella enterica serovar Typhimurium strain and designated the LTB adjuvant strain. LTB protein production and secretion from the strain were demonstrated with an immunoblot assay and enzyme-linked immunosorbent assay. The LTB strain was evaluated for enhancement of immunity and protection efficacy induced by a previously constructed live Salmonella vaccine candidate. In addition, immunization strategies using the LTB strain were optimized for effective salmonellosis protection. Seventy female BALB/c mice were divided into seven groups (A to G; n = 10 mice per group). Mice were primed at 6 weeks of age and boosted at 9 weeks of age. All mice were orally challenged with a virulent wild-type strain at week 3 postbooster. Serum IgG and IgA titers from mice immunized with the LTB strain alone or with a mixture of the LTB strain and the vaccine candidate were significantly increased. The secretory IgA titers from mice immunized with the LTB strain alone or with the mixture were at least 2.2 times greater than those of control mice. In addition, all group E mice (primed with the vaccine-LTB mixture and boosted with the vaccine candidate) were free of clinical signs of salmonellosis and survived a virulent challenge. In contrast, death due to the challenge was 100% in control mice, 80% in group A mice (single immunization with the vaccine candidate), 60% in group B mice (primed and boosted with the vaccine candidate), 40% in group C mice (single immunization with the LTB strain), 30% in group D mice (primed and boosted with the LTB strain), and 30% in group F mice (primed and boosted with the vaccine-LTB mixture). These results suggest that vaccination with the LTB strain, especially when added at the prime stage only, effectively enhances immune responses and protection against salmonellosis.     

Mucosal and systemic immune responses to chimeric fimbriae expressed by Salmonella enterica serovar typhimurium vaccine strains

Recombinant live oral vaccines expressing pathogen-derived antigens offer a unique set of attractive properties. Among these are the simplicity of administration, the capacity to induce mucosal and systemic immunity, and the advantage of permitting genetic manipulation for optimal antigen presentation. In this study, the benefit of having a heterologous antigen expressed on the surface of a live vector rather than intracellularly was evaluated. Accordingly, the immune response of mice immunized with a Salmonella enterica serovar Typhimurium vaccine strain expressing the Escherichia coli 987P fimbrial antigen on its surface (Fas(+)) was compared with the expression in the periplasmic compartment (Fas(-)). Orally immunized BALB/c mice showed that 987P fimbriated Salmonella serovar Typhimurium CS3263 (aroA asd) with pCS151 (fas(+) asd(+)) elicited a significantly higher level of 987P-specific systemic immunoglobulin G (IgG) and mucosal IgA than serovar Typhimurium CS3263 with pCS152 (fasD mutant, asd(+)) expressing 987P periplasmic antigen. Further studies were aimed at determining whether the 987P fimbriae expressed by serovar Typhimurium chi4550 (cya crp asd) could be used as carriers of foreign epitopes. For this, the vaccine strain was genetically engineered to express chimeric fimbriae carrying the transmissible gastroenteritis virus (TGEV) C (379-388) and A (521-531) epitopes of the spike protein inserted into the 987P major fimbrial subunit FasA. BALB/c mice administered orally serovar Typhimurium chi4550 expressing the chimeric fimbriae from the tet promoter in pCS154 (fas(+) asd(+)) produced systemic antibodies against both fimbria and the TGEV C epitope but not against the TGEV A epitope. To improve the immunogenicity of the chimeric fimbriae, the in vivo inducible nirB promoter was inserted into pCS154, upstream of the fas genes, to create pCS155. In comparison with the previously used vaccine, BALB/c mice immunized orally with serovar Typhimurium chi4550/pCS155 demonstrated significantly higher levels of serum IgG and mucosal IgA against 987P fimbria. Moreover, mucosal IgA against the TGEV C epitope was only detected with serovar Typhimurium chi4550/pCS155. The induced antibodies also recognized the epitopes in the context of the full-length TGEV spike protein. Hence, immune responses to heterologous chimeric fimbriae on Salmonella vaccine vectors can be optimized by using promoters known to be activated in vivo.     

Optimization of plasmid maintenance in the attenuated live vector vaccine strain Salmonella typhi CVD 908-htrA

The broad objective of the research presented here is to develop a noncatalytic plasmid maintenance system for the stabilization of multicopy expression plasmids encoding foreign antigens in a Salmonella typhi live-vector vaccine strain such as CVD 908-htrA. We have enhanced the maintenance of expression plasmids at two independent levels. First, we removed dependence upon balanced-lethal maintenance systems that involve catalytic enzymes expressed from multicopy plasmids; we accomplished this through incorporation into expression plasmids of a postsegregational killing system based on the noncatalytic hok-sok plasmid addiction system from the antibiotic resistance factor pR1. We also included at least one naturally occurring plasmid partition function in our expression plasmids, which eliminates random segregation of these plasmids, thereby enhancing their inheritance and stability; to accomplish this, we incorporated either the par locus from pSC101, the parA locus from pR1, or both. We monitored the stability of optimized expression plasmids within CVD 908-htrA by quantitating expression of a variant of green fluorescent protein (GFPuv) by using flow cytometry. In this report, we demonstrate the utility of this novel plasmid maintenance system in enhancing the stability of our expression plasmids and go on to show that as the copy number of stabilized plasmids increases, the toxicity of GFPuv synthesis also increases. The implications of these observations for the rational design of immunogenic and protective bacterial live vector vaccines are discussed.     

Bacteremia associated with live attenuated chi8110 Salmonella enterica serovar Typhi ISP1820 in healthy adult volunteers

Live attenuated chi8110 Salmonella enterica serovar Typhi ISP1820 vaccine was given in a dose-escalation trial to healthy, adult volunteers. Positive stool and blood cultures were noted, but limited, as were immune responses measured by ELISA and ELISPOT. Only volunteers with bacteremia developed immune responses; however, no symptoms were associated with bacteremia. The vaccine was insufficiently immunogenic for use as a vaccine. It is possible that reduced survival in the gut and reduced immunogenicity may have been due to the thawing of frozen inocula immediately prior to use.