Typhi Ty21a Research Articles

An innovative method of nonclinical efficacy and toxicological evaluation of recombinant Salmonella typhi Ty21a expressing HPV16 and 18 L1 proteins

Advancement in technology led to development of live attenuated Salmonella typhi Ty21a as enteric vector for expression of foreign proteins. Such vector platform is inevitable for development of vaccine candidate against human papilloma virus (HPV), the etiological agent of cervical cancer with high prevalence in developing nations. The high risk HPVs like type 16 and 18 contributes to 70% of cervical cancer, hence Indian Immunologicals Limited (IIL), Hyderabad, India developed a recombinant HPV vaccine by introducing HPV 16 and 18 L1 protein coding genes into attenuated S. typhi Ty21a vector. Being a genetically engineered enteric vector vaccine, it would be less expensive, with an ease of oral administration, instead of injectable that needs trained personale, is an added advantage for low socioeconomic setup compared to existing HPV vaccines.Establishing the nonclinical efficacy and safety/toxicity as per the national/international regulatory guidelines has become major constrain for such recombinant S. typhi HPV (rSt.HPV) vaccine. Since, the intended clinical mode of rSt.HPV is through oral route, whereas the live attenuated S. typhi Ty21a doesn't colonize in gut of laboratory animals to be used for nonclinical experiments. Hence, an alternate and unconventional method of ‘intranasal drug testing’, was followed for nonclinical efficacy and toxicity evaluations. An array of parameters specified by regulatory agencies were investigated in mice, rat and rabbits administered with rSt.HPV through, intra-peritoneal, intranasal and oral routes, the intended clinical route.•Current unconventional and innovative nonclinical testing procedures helps in exploring the alternate methods by pharmacologist/toxicologist.•Ultimately, such new drugs developed through technology must serve the humankind justifying the guidelines of regulatory agencies.

Alive attenuated Salmonella as a cargo shuttle for smart carrying of gold nanoparticles to tumour hypoxic regions

In the present study, alive attenuated Salmonella typhi Ty21a was introduced as a vehicle for smart delivery of gold nanoparticles to the tumours’ hypoxic regions. At the first step, the uptakes of gold nanoparticles with seven different decorations by S. typhi Ty21a was investigated using flow cytometry and inductively coupled plasma optical emission spectroscopy. The analyses demonstrated that folic acid functionalised gold nanoparticles (FA-GNPs) are the best candidates for producing the Golden Bacteria (GB). Subsequently, the GB and FA-GNPs efficacies for tumour targeting were investigated after intravenous injection to CT-26 tumour-bearing mice. The GB exhibited more GNPs delivery to the tumour in comparison with FA-GNPs. Moreover, GB injection causes more delivery of GNPs to the tumours’ central regions in comparison with tumours’ periphery. This trend is completely in reverse for FA-GNPs injected group. The ratios of peripheral to central regions’ gold concentration of the tumours were 1.95 ± 0.13 and 0.61 ± 0.10 for FA-GNPs and GB groups, respectively. This observation demonstrates higher accumulation of gold nanoparticles in the centre of the tumour due to their active delivery by the S. typhi Ty21a to the deeps of tumours.

Longevity of duodenal and peripheral T-cell and humoral responses to live-attenuated Salmonella Typhi strain Ty21a

BackgroundWe have previously demonstrated that polyfunctional Ty21a-responsive CD4+ and CD8+ T cells are generated at the duodenal mucosa 18 days following vaccination with live-attenuated S. Typhi (Ty21a). The longevity of cellular responses has been assessed in peripheral blood, but persistence of duodenal responses is unknown. MethodsWe vaccinated eight healthy adults with Ty21a. Peripheral blood and duodenal samples were acquired after a median of 1.5 years (ranging from 1.1 to 3.7 years) following vaccination. Cellular responses were assessed in peripheral blood and at the duodenal mucosa by flow cytometry. Levels of IgG and IgA were also assessed in peripheral blood by enzyme-linked immunosorbent assay. ResultsNo T-cell responses were observed at the duodenal mucosa, but CD4+ T-cell responses to Ty21a and FliC were observed in peripheral blood. Peripheral anti-lipopolysaccharide IgG and IgA responses were also observed. Early immunoglobulin responses were not associated with the persistence of long-term cellular immune responses. ConclusionsEarly T-cell responses which we have previously observed at the duodenal mucosa 18 days following oral vaccination with Ty21a could not be detected at a median of 1.5 years. Peripheral responses were observed at this time. Immunoglobulin responses observed shortly after vaccination were not associated with cellular immune responses at 1.5 years, suggesting that the persistence of cellular immunity is not associated with the strength of the initial humoral response to vaccination.

Stable Chromosomal Expression of Shigella flexneri 2a and 3a O-Antigens in the Live Salmonella Oral Vaccine Vector Ty21a.

We have been exploring the use of the live attenuated Salmonella enterica serovar Typhi Ty21a vaccine strain as a versatile oral vaccine vector for the expression and delivery of multiple foreign antigens, including Shigella O-antigens. In this study, we separately cloned genes necessary for the biosynthesis of the Shigella flexneri serotype 2a and 3a O-antigens, which have been shown to provide broad cross-protection to multiple disease-predominant S. flexneri serotypes. The cloned S. flexneri 2a rfb operon, along with bgt and gtrII, contained on the SfII bacteriophage, was sufficient in Ty21a to express the heterologous S. flexneri 2a O-antigen containing the 3,4 antigenic determinants. Further, this rfb operon, along with gtrA, gtrB, and gtrX contained on the Sfx bacteriophage and oac contained on the Sf6 bacteriophage, was sufficient to express S. flexneri 3a O-antigen containing the 6, 7, and 8 antigenic determinants. Ty21a, with these plasmid-carried or chromosomally inserted genes, demonstrated simultaneous and stable expression of homologous S Typhi O-antigen plus the heterologous S. flexneri O-antigen. Candidate Ty21a vaccine strains expressing heterologous S. flexneri 2a or 3a lipopolysaccharide (LPS) elicited significant serum antibody responses against both homologous S Typhi and heterologous Shigella LPS and protected mice against virulent S. flexneri 2a or 3a challenges. These new S. flexneri 2a and 3a O-antigen-expressing Ty21a vaccine strains, together with our previously constructed Ty21a strains expressing Shigella sonnei or Shigella dysenteriae 1 O-antigens, have the potential to be used together for simultaneous protection against the predominant causes of shigellosis worldwide as well as against typhoid fever.

Protection against inhalation anthrax by immunization with Salmonella enterica serovar Typhi Ty21a stably producing protective antigen of Bacillus anthracis

The national blueprint for biodefense concluded that the United States is underprepared for biological threats. The licensed anthrax vaccine absorbed vaccine, BioThrax, requires administration of at least 3–5 intramuscular doses. The anthrax vaccine absorbed vaccine consists of complex cell-free culture filtrates of a toxigenic Bacillus anthracis strain and causes tenderness at the injection site and significant adverse events. We integrated a codon-optimized, protective antigen gene of B. anthracis (plus extracellular secretion machinery), into the chromosome of the licensed, oral, live-attenuated typhoid fever vaccineTy21a to form Ty21a-PA-01 and demonstrated excellent expression of the gene encoding protective antigen. We produced the vaccine in a 10-L fermenter; foam-dried and vialed it, and characterized the dried product. The vaccine retained ~50% viability for 20 months at ambient temperature. Sera from animals immunized by the intraperitoneal route had high levels of anti-protective antigen antibodies by enzyme-linked immunosorbent assay and anthrax lethal toxin-neutralizing activity. Immunized mice were fully protected against intranasal challenge with ~5 LD50 of B. anthracis Sterne spores, and 70% (7/10) of vaccinated rabbits were protected against aerosol challenge with 200 LD50 of B. anthracis Ames spores. There was a significant correlation between protection and antibody levels determined by enzyme-linked immunosorbent assay and toxin-neutralizing activity. These data provide the foundation for achievement of our ultimate goal, which is to develop an oral anthrax vaccine that is stable at ambient temperatures and induces the rapid onset of durable, high-level protection after a 1-week immunization regimen.

A novel method to generate Salmonella Typhi Ty21a ghosts exploiting the λ phage holin-endolysin system.

Human typhoid fever caused by Salmonella Typhi still poses a severe global disease burden in developing countries despite the availability of commercial vaccines. In this study, we constructed a non-living S. Typhi Ty21a vaccine candidate by employing a lambda (λ) phage-derived holin-endolysin system to efficiently construct bacterial ghosts. The lysis plasmid pJHL464 harbors an R lysis cassette that is stringently regulated by dual promoters containing cI857/λPR and ParaBAD/araC components. The plasmid was introduced into an asd gene-deleted S. Typhi Ty21a strain designated JOL1675. The in vitro expression of endolysin (~17.76 kDa) in the subsequent JOL1675 vaccine construct when grown under lysis inducible conditions was validated by immunoblotting. In scanning electron microscopy analysis, surface transmembrane tunnels and a collapsed body were visualized in the ghosts. Following 48 h of lysis, no viable JOL1675 cells remained, indicating that lysis of all cells was achieved. Subcutaneous immunizations of mice with the JOL1675 ghosts produced significantly increasing titers of serum IgG and vaginal wash secretory IgA antibodies against JOL1675 outer membrane proteins during the observational period. Further, serum collected at 6 weeks post-immunization of rabbits exhibited effective bactericidal activity against wild type S. Typhi in the presence of complement. These data showed that JOL1675 ghosts are highly immunogenic and elicit humoral and mucosal responses expected to correlate with protective immunity against S. typhi. Collectively, our findings support the conclusion that incorporating a λ phage holin-endolysin-mediated lysis construct into S. Typhi is an efficient strategy for developing a novel and safe non-living typhoid vaccine candidate.

A novel approach for construction of an inactivated typhoid vaccine candidate that effectively augments both humoral and cellular immune responses

Salmonella enterica serovar Typhi ghost was constructed as a vaccine candidate against typhoid fever. An asd+ plasmid pJHL187 harboring a ghost cassette comprised of PhiX 174 E lysis gene stringently controlled under the convergent promotor components and was transformed into the asd gene-deleted mutant S. Typhi Ty21a strain (STG). Twenty female BALB/c mice randomly assigned into two groups were subcutaneously vaccinated at 5weeks of age to assess immunogenic characteristics of the constructed STG. The level of serum IgG in the immunized mice was significantly increased during the observational period (P<0.001) as the mice showed the significant elevation of secretory IgA at week 6 compared to those in the non-immunized mice (P<0.05). The CD3+CD4+ T cell subpopulation in the primed splenocytes showed approximately twofold increase in the immunized group. Further, the gene expression of various immunomodulatory cytokines associated with Th-1, Th-2 and Th-17 immunity was observed in in vitro restimulated splenocytes isolated from the immunized mice. Serum Bactericidal activity of antibodies produced in the rabbits immunized with STG was proved by the elimination of almost all of wild-type S. Typhi in the presence of exogenous complement over an hour at week 6 after the first immunization. The immuno-stimulatory traits of STG demonstrated that the construct effectively enhanced the immunological responses, providing a potential of STG as the vaccine candidate against typhoid fever.

Evaluation of Regulated Delayed Attenuation Strategies for Salmonella enterica Serovar Typhi Vaccine Vectors in Neonatal and Infant Mice

We developed regulated delayed attenuation strategies for Salmonella vaccine vectors. In this study, we evaluated the combination of these strategies in recombinant attenuated Salmonella enterica serovar Typhi and Salmonella enterica serovar Typhimurium vaccine vectors with similar genetic backgrounds in vitro and in vivo. Our goal is to develop a vaccine to prevent Streptococcus pneumoniae infection in newborns; thus, all strains delivered a pneumococcal antigen PspA and the impact of maternal antibodies was evaluated. The results showed that all strains with the regulated delayed attenuated phenotype (RDAP) displayed an invasive ability stronger than that of the S. Typhi vaccine strain, Ty21a, but weaker than that of their corresponding wild-type parental strains. The survival curves of different RDAP vaccine vectors in vitro and in vivo exhibited diverse regulated delayed attenuation kinetics, which was different from S. Typhi Ty21a and the wild-type parental strains. Under the influence of maternal antibody, the persistence of the S. Typhimurium RDAP strain displayed a regulated delayed attenuation trend in nasal lymphoid tissue (NALT), lung, and Peyer's patches, while the persistence of S. Typhi RDAP strains followed the curve only in NALT. The bacterial loads of S. Typhi RDAP strains were lower in NALT, lung, and Peyer's patches in mice born to immune mothers than in those born to naive mothers. In accordance with these results, RDAP vaccine strains induced high titers of IgG antibodies against PspA and against Salmonella lipopolysaccharides. Immunization of mothers with S. Typhi RDAP strains enhanced the level of vaginal mucosal IgA, gamma interferon (IFN-γ), and interleukin 4 (IL-4) and resulted in a higher level of protection against S. pneumoniae challenge.

Asymmetric pore occupancy in crystal structure of OmpF porin from Salmonella typhi

OmpF is a major general diffusion porin of Salmonella typhi, a Gram-negative bacterium, which is an obligatory human pathogen causing typhoid. The structure of S. typhi Ty21a OmpF (PDB Id: 3NSG) determined at 2.8 Å resolution by X-ray crystallography shows a 16-stranded β-barrel with three β-barrel monomers associated to form a trimer. The packing observed in S. typhi Ty21a rfOmpF crystals has not been observed earlier in other porin structures. The variations seen in the loop regions provide a starting point for using the S. typhi OmpF for structure-based multi-valent vaccine design. Along one side of the S. typhi Ty21a OmpF pore there exists a staircase arrangement of basic residues (20R, 60R, 62K, 65R, 77R, 130R and 16K), which also contribute, to the electrostatic potential in the pore. This structure suggests the presence of asymmetric electrostatics in the porin oligomer. Moreover, antibiotic translocation, permeability and reduced uptake in the case of mutants can be understood based on the structure paving the way for designing new antibiotics.

A method to generate recombinant Salmonella typhi Ty21a strains expressing multiple heterologous genes using an improved recombineering strategy

Live attenuated Salmonella enterica serovar Typhi Ty21a (Ty21a) is an important vaccine strain used in clinical studies for typhoid fever and as a vaccine vector for the expression of heterologous antigens. To facilitate the use of Ty21a in such studies, it is desirable to develop improved strategies that enable the stable chromosomal integration and expression of multiple heterologous antigens. The phage λ Red homologous recombination system has previously been used in various gram-negative bacteria species to mediate the accurate replacement of regions of chromosomal DNA with PCR-generated 'targeting cassettes' that contain flanking regions of shared homologous DNA sequence. However, the efficiency of λ Red-mediated recombineering in Ty21a is far lower than in Escherichia coli and other Salmonella typhimurium strains. Here, we describe an improved strategy for recombineering-based methods in Ty21a. Our reliable and efficient method involves the use of linear DNA-targeting cassettes that contain relatively long flanking 'arms' of sequence (ca. 1,000bp) homologous to the chromosomal target. This enables multiple gene-targeting procedures to be performed on a single Ty21a chromosome in a straightforward, sequential manner. Using this strategy, we inserted three different influenza antigen expression cassettes as well as a green fluorescent protein gene reporter into four different loci on the Ty21a chromosome, with high efficiency and accuracy. Fluorescent microscopy and Western blotting analysis confirmed that strong inducible expression of all four heterologous genes could be achieved. In summary, we have developed an efficient, robust, and versatile method that may be used to construct recombinant Ty21a antigen-expressing strains.

English

We have developed a recombinant Salmonella typhi Ty21a vaccine candidate (rSTvacIII) displaying a multi-epitopes mycobacterial antigen called the VacIII (containing ubiquitin and 4 immunogenic mycobacterial epitopes of ESAT-6, PhoS1, Hsp 16.3 and MTB 8.4 genes) on the surface of S. typhi Ty21a (Ty21a) using PgsA protein as a carrier. The expression of VacIII protein on the surface of Ty21a was verified by western blot. Immunization in BALB/c mice with rSTvacIII induced strong Th1-type responses based on high level of in vitro IFN-γ but low IL-4 secretion. Since the protective immunity against tuberculosis is mediated by Th1-type response, the rSTvacIII have potential as effective vaccine candidate against tuberculosis. Key words: VacIII antigen, PgsA anchoring motif, tuberculosis, Salmonella typhi Ty21a.

Mucosal priming of newborn mice with S. Typhi Ty21a expressing anthrax protective antigen (PA) followed by parenteral PA-boost induces B and T cell-mediated immunity that protects against infection bypassing maternal antibodies

The currently licensed anthrax vaccine has several limitations and its efficacy has been proven only in adults. Effective immunization of newborns and infants requires adequate stimulation of their immune system, which is competent but not fully activated. We explored the use of the licensed live attenuated S. Typhi vaccine strain Ty21a expressing Bacillus anthracis protective antigen [Ty21a(PA)] followed PA-alum as a strategy for immunizing the pediatric population. Newborn mice primed with a single dose of Ty21a(PA) exhibited high frequencies of mucosal IgA-secreting B cells and IFN-γ-secreting T cells during the neonatal period, none of which was detected in newborns immunized with a single dose of PA-alum. Priming with Ty21a(PA) followed by PA-boost resulted in high levels of PA-specific IgG, toxin neutralizing and opsonophagocytic antibodies and increased frequency of bone marrow IgG plasma cells and memory B cells compared with repeated immunization with PA-alum alone. Robust B and T cell responses developed even in the presence of maternal antibodies. The prime-boost protected against systemic and respiratory infection. Mucosal priming with a safe and effective S. Typhi-based anthrax vaccine followed by PA-boost could serve as a practical and effective prophylactic approach to prevent anthrax early in life.

Parallel analysis of mucosally derived B- and T-cell responses to an oral typhoid vaccine using simplified methods

There is a great need for simple methods for analysis of immune responses to mucosal vaccines that can be used on small blood volumes in field trials in both children and adults. We have investigated if mucosally derived B- and T-cell responses can be monitored in parallel by analysis of antibodies and T-cell effector molecules in culture supernatants from circulating blood lymphocytes obtained from orally vaccinated Swedes. Immunization with a live oral model vaccine, i.e. Salmonella enterica serovar Typhi Ty21a, gave rise to secretion of typhoid specific IgA and IgM antibodies from peripheral blood mononuclear cells (PBMCs) and this response could be equally well detected by ELISA and ELISPOT 7 days after vaccination. The ELISA based assay could be further simplified by using buffy coat cells, but not by using whole blood or frozen PBMCs. Vaccine induced T-cell responses appeared later than the B-cell responses, but could be detected by ELISA assessment of IFN-γ and granzymes in supernatants from antigen stimulated PBMCs 21 days after vaccination. Thus, both B- and T-cell responses could be detected using simple ELISA based assays that would be practical to use in large-scale vaccine trials.

Towards a human oral vaccine for anthrax: The utility of a Salmonella Typhi Ty21a-based prime-boost immunization strategy

We previously demonstrated the ability of an orally administered attenuated Salmonella enterica serovar Typhimurium strain expressing the protective antigen (PA) of Bacillus anthracis to confer protection against lethal anthrax aerosol spore challenge [Stokes MG, Titball RW, Neeson BN, et al. Oral administration of a Salmonella enterica-based vaccine expressing Bacillus anthracis protective antigen confers protection against aerosolized B. anthracis. Infect Immun 2007;75(April (4)):1827–34]. To extend the utility of this approach to humans we constructed variants of S. enterica serovar Typhi Ty21a, an attenuated typhoid vaccine strain licensed for human use, which expressed and exported PA via two distinct plasmid-based transport systems: the Escherichia coli HlyA haemolysin and the S. Typhi ClyA export apparatus. Murine immunogenicity studies confirmed the ability of these constructs, especially Ty21a expressing the ClyA-PA fusion protein, to stimulate strong PA-specific immune responses following intranasal immunization. These responses were further enhanced by a subsequent boost with either parenterally delivered recombinant PA or the licensed US human alum-adsorbed anthrax vaccine (AVA). Anthrax toxin neutralizing antibody responses using this prime-boost regimen were rapid, vigorous and broad in nature. The results of this study demonstrate the feasibility of employing a mucosal prime with a licensed Salmonella Typhi vaccine strain followed by a parenteral protein boost to stimulate rapid protective immunity against anthrax.

Correlation of T cell response and bacterial clearance in human volunteers challenged with Helicobacter pylori revealed by randomised controlled vaccination with Ty21a-based Salmonella vaccines

Background:Helicobacter pylori remains a global health hazard, and vaccination would be ideal for its control. Natural infection appears not to induce protective immunity. Thus, the feasibility of a vaccine for...

Salmonella enterica Serovar Typhi Ty21a Expressing Human Papillomavirus Type 16 L1 as a Potential Live Vaccine against Cervical Cancer and Typhoid Fever

Human papillomavirus (HPV) vaccines based on L1 virus-like particles (VLPs) can prevent HPV-induced genital neoplasias, the precursors of cervical cancer. However, most cervical cancers occur in developing countries, where the implementation of expensive vaccines requiring multiple injections will be difficult. A live Salmonella-based vaccine could be a lower-cost alternative. We previously demonstrated that high HPV type 16 (HPV16)-neutralizing titers are induced after a single oral immunization of mice with attenuated Salmonella enterica serovar Typhimurium strains expressing a codon-optimized version of HPV16 L1 (L1S). To allow the testing of this type of vaccine in women, we constructed a new L1-expressing plasmid, kanL1S, and tested kanL1S recombinants of three Salmonella enterica serovar Typhi vaccine strains shown to be safe in humans, i.e., Ty21a, the actual licensed typhoid vaccine, and two highly immunogenic typhoid vaccine candidates, Ty800 and CVD908-htrA. In an intranasal mouse model of Salmonella serovar Typhi infection, Ty21a kanL1S was unique in inducing HPV16-neutralizing antibodies in serum and genital secretions, while anti-Salmonella responses were similar to those against the parental Ty21a vaccine. Electron microscopy examination of Ty21a kanL1S lysates showed that L1 assembled in capsomers and capsomer aggregates but not well-ordered VLPs. Comparison to the neutralizing antibody response induced by purified HPV16 L1 VLP immunizations in mice suggests that Ty21a kanL1S may be an effective prophylactic HPV vaccine. Ty21a has been widely used against typhoid fever in humans with a remarkable safety record. These finds encourage clinical testing of Ty21a kanL1S as a combined typhoid fever/cervical cancer vaccine with the potential for worldwide application.

Core-linked LPS expression of Shigella dysenteriae serotype 1 O-antigen in live Salmonella Typhi vaccine vector Ty21a: Preclinical evidence of immunogenicity and protection

Shigella dysenteriae serotype 1 (S. dysenteriae 1) causes severe shigellosis that is typically associated with high mortality. Antibodies against Shigella serotype-specific O-polysaccharide (O-Ps) have been shown to be host protective. In this study, the rfb locus and the rfp gene with their cognate promoter regions were PCR-amplified from S. dysenteriae 1, cloned, and sequenced. Deletion analysis showed that eight rfb ORFs plus rfp are necessary for biosynthesis of this O-Ps. A tandemly-linked rfb-rfp gene cassette was cloned into low copy plasmid pGB2 to create pSd1. Avirulent Salmonella enterica serovar Typhi (S. Typhi) Ty21a harboring pSd1 synthesized S. Typhi 9, 12 LPS as well as typical core-linked S. dysenteriae 1 LPS. Animal immunization studies showed that Ty21a (pSd1) induces protective immunity against high stringency challenge with virulent S. dysenteriae 1 strain 1617. These data further demonstrate the utility of S. Typhi Ty21a as a live, bacterial vaccine delivery system for heterologous O-antigens, supporting the promise of a bifunctional oral vaccine for prevention of shigellosis and typhoid fever.

Use of the α-hemolysin secretion system of Escherichia coli for antigen delivery in the Salmonella typhi Ty21a vaccine strain

This study examined the suitability of the hemolysin secretion system of Escherichia coli for expression and delivery of α-hemolysin (HlyA) by the S. typhi Ty21a strain, the only live oral Salmonella vaccine strain licensed for human use, under in vitro and in vivo conditions. For this purpose, two plasmid vectors encoding either the whole α-hemolysin of E. coli (pANN202-812/pMOhly2) or the hemolysin secretion signal (pMOhly1) were transferred into S. typhi Ty21a. S. typhi Ty21a carrying pANN202-812/pMOhly2 revealed efficient secretion of hemolysin in vitro. After formulation according to a process suitable for commercial production of Salmonella-based live bacterial vaccines, plasmids were shown to be stable in Ty21a and hemolysin secretion was demonstrated even after storage of the strains under real-time and stress conditions. After intranasal immunization of mice with S. typhi Ty21a/pANN202-812 plasmids are stable in vivo, and immunization induced a profound immune response against the heterologous HlyA antigen. Therefore, the combination of the hemolysin secretion system and S. typhi Ty21a could form the basis for a new generation of live bacterial vaccines.

Enteric pathogens as vaccine vectors for foreign antigen delivery.

Live attenuated bacteria are attractive vaccine vectors that can elicit host immune responses to foreign antigens. Bacterial vectors may mimic natural infection and therefore interact with the mucosal, humoral, and cellular compartments of the immune system. A plethora of foreign (heterologous) antigens derived from bacterial, fungal, viral, or parasitic organisms have been expressed within bacterial vectors. Delivery of contraceptive and tumor antigens, cytokines, and adjuvants has also been explored. The result is a type of “vaccine factory” (29) in which the bacterial vector produces a foreign protein. Since bacterial vectors replicate within the host, it is hoped that they provide sustained exposure to the antigen, potentially augmenting the type and level of immune response. In addition, bacteria naturally possess immunostimulatory molecules such as lipopolysaccharide (LPS) and flagella that can stimulate immune responses (56). Internationally, there are three live bacterial vaccines commercially available and in clinical use: Salmonella enterica serovar Typhi Ty21a, Vibrio cholerae CVD 103-HgR, and Mycobacterium bovis BCG. These strains plus many other attenuated strains have been used as vectors to express heterologous antigens. Live bacterial vector vaccines have been evaluated in numerous animal and human studies, but there are currently no licensed live bacterial vector vaccines being used clinically. The most compelling rationales for the development of live bacterial vectors are the possibility of oral delivery and protection at mucosal surfaces. This review will cover attenuated bacterial enteric pathogens used as vectors to express foreign (heterologous) antigens, with emphasis on those that have reached clinical study, including Salmonella, Shigella, V. cholerae, and Listeria. This review will not cover bacterial vectors used to deliver DNA vaccines or subunit and conjugate vaccines.

Impact of vector-priming on the immunogenicity of a live recombinant Salmonella enterica serovar typhi Ty21a vaccine expressing urease A and B from Helicobacter pylori in human volunteers

Orally administered recombinant Salmonella vaccines represent an attractive option for mass vaccination programmes against various infectious diseases. Therefore, it is crucial to gather knowledge about the possible impact of preexisiting immunity to carrier antigens on the immunogenicity of recombinant vaccines. Thirteen volunteers were preimmunized with Salmonella typhi Ty21a in order to evaluate the effects of prior immunization with the carrier strain. Then, they received three doses of 1–2×1010 viable organisms of either the vaccine strain S. typhi Ty21a (pDB1) expressing subunits A and B of recombinant Helicobacter pylori urease (n=9), or placebo strain S. typhi Ty21a (n=4). Four volunteers were preimmunized and boosted with the vaccine strain S. typhi Ty21a (pDB1). No serious adverse effects were observed in any of the volunteers. Whereas none of the volunteers primed and boosted with the vaccine strain responded to the recombinant antigen, five of the nine volunteers preimmunized with the carrier strain showed cellular immune responses to H. pylori urease (56%). This supports the results of a previous study in non-preimmunized volunteers where 56% (five of nine) of the volunteers showed a cellular immune response to urease after immunisation with S. typhi Ty21a (pDB1).