Conversion of RpoS- Attenuated Salmonella enterica Serovar Typhi Vaccine Strains to RpoS+ Improves Their Resistance to Host Defense Barriers.

The vast majority of live attenuated typhoid vaccines are constructed from the Salmonella enterica serovar Typhi strain Ty2, which is devoid of a functioning alternative sigma factor, RpoS, due to the presence of a frameshift mutation. RpoS is a specialized sigma factor that plays an important role in the general stress response of a number of Gram-negative organisms, including Salmonella. Previous studies have demonstrated that this sigma factor is necessary for survival following exposure to acid, hydrogen peroxide, nutrient-limiting conditions, and starvation. In addition, studies with Salmonella enterica serovar Typhimurium and the mouse model of typhoid fever have shown that RpoS is important in colonization and survival within the infected murine host. We converted 4 clinically studied candidate typhoid vaccine strains derived from Ty2 [CVD908-htrA, Ty800, and χ9639(pYA3493)] and the licensed live typhoid vaccine Ty21a (also derived from Ty2) to RpoS+ and compared their abilities to withstand environmental stresses that may be encountered within the host to those of the RpoS- parent strains. The results of our study indicate that strains that contain a functional RpoS were better able to survive following stress and that they would be ideal for further development as safe, effective vaccines to prevent S. Typhi infections or as vectors in recombinant attenuated Salmonella vaccines (RASVs) designed to protect against other infectious disease agents in humans. The S. Typhi strains constructed and described here will be made freely available upon request, as will the suicide vector used to convert rpoS mutants to RpoS+. IMPORTANCE Recombinant attenuated Salmonella vaccines (RASVs) represent a unique prevention strategy to combating infectious disease because they utilize the ability of Salmonella to invade and colonize deep effector lymphoid tissues and deliver hetero- and homologous derived antigens at the lowest immunizing dose. Our recent clinical trial in human volunteers indicated that an RpoS+ derivative of Ty2 was better at inducing immune responses than its RpoS- counterpart. In this study, we demonstrate that a functional RpoS allele is beneficial for developing effective live attenuated vaccines against S. Typhi or in using S. Typhi as a recombinant attenuated vaccine vector to deliver other protective antigens.

Use of Ensure® nutrition shakes as an alternative formulation method for live recombinant Attenuated Salmonella Typhi vaccines.

BACKGROUND: To be effective, orally administered live Salmonella vaccines must first survive their encounter with the low pH environment of the stomach. To enhance survival, an antacid is often given to neutralize the acidic environment of the stomach just prior to or concomitant with administration of the vaccine. One drawback of this approach, from the perspective of the clinical trial volunteer, is that the taste of a bicarbonate-based acid neutralization system can be unpleasant. Thus, we explored an alternative method that would be at least as effective as bicarbonate and with a potentially more acceptable taste. Because ingestion of protein can rapidly buffer stomach pH, we examined the possibility that the protein-rich Ensure® Nutrition shakes would be effective alternatives to bicarbonate. RESULTS: We tested one Salmonella enterica serovar Typhimurium and three Salmonella Typhi vaccine strains and found that all strains survived equally well when incubated in either Ensure® or bicarbonate. In a low gastric pH mouse model, Ensure® worked as well or better than bicarbonate to enhance survival through the intestinal tract, although neither agent enhanced the survival of the S. Typhi test strain possessing a rpoS mutation. CONCLUSIONS: Our data show that a protein-rich drink such as Ensure® Nutrition shakes can serve as an alternative to bicarbonate for reducing gastric pH prior to administration of a live Salmonella vaccine.

A low gastric pH mouse model to evaluate live attenuated bacterial vaccines.

The low pH of the stomach serves as a barrier to ingested microbes and must be overcome or bypassed when delivering live bacteria for vaccine or probiotic applications. Typically, the impact of stomach acidity on bacterial survival is evaluated in vitro, as there are no small animal models to evaluate these effects in vivo. To better understand the effect of this low pH barrier to live attenuated Salmonella vaccines, which are often very sensitive to low pH, we investigated the value of the histamine mouse model for this application. A low pH gastric compartment was transiently induced in mice by the injection of histamine. This resulted in a gastric compartment of approximately pH 1.5 that was capable of distinguishing between acid-sensitive and acid-resistant microbes. Survival of enteric microbes during gastric transit in this model directly correlated with their in vitro acid resistance. Because many Salmonella enterica serotype Typhi vaccine strains are sensitive to acid, we have been investigating systems to enhance the acid resistance of these bacteria. Using the histamine mouse model, we demonstrate that the in vivo survival of S. Typhi vaccine strains increased approximately 10-fold when they carried a sugar-inducible arginine decarboxylase system. We conclude that this model will be a useful for evaluating live bacterial preparations prior to clinical trials.

Salmonella as a vaccine delivery vehicle.

Attenuated Salmonella vaccines can be administered orally to deliver recombinant antigens to mucosal surfaces inducing a protective immune response against a variety of targeted pathogens. A number of exciting new approaches and technologies for attenuated Salmonella vaccines have been developed recently. However, a disconnect remains between results obtained with mice in preclinical studies and results obtained in human clinical trials. This is due to an incomplete understanding of Salmonella Typhi interactions with human hosts and inadequate animal models available for study. In this review, the authors describe recent progress in identifying important differences underlying S. Typhi-host interactions, the development of novel approaches to vaccine design and six recent clinical trials evaluating Salmonella-vectored vaccines.

A Phase I, dose-escalation trial in adults of three recombinant attenuated Salmonella Typhi vaccine vectors producing Streptococcus pneumoniae surface protein antigen PspA.

BACKGROUND: Live, attenuated, orally-administered Salmonella strains are excellent vectors for vaccine antigens and are attractive as vaccines based on previous use of S. Typhimurium in animals. A Phase I dose escalation trial was conducted to evaluate the safety and immunogenicity of three newly constructed recombinant attenuated Salmonella enterica serovar Typhi vaccine (RASV) vectors synthesizing Streptococcus pneumoniae surface protein A (PspA). METHODS: The 3 S. Typhi strains used as vectors to deliver PspA were S. Typhi ISP1820; S. Typhi Ty2 RpoS(-); and S. Typhi Ty2 RpoS(+). Sixty healthy adults (median age 25.2 years) were enrolled into 4 Arms (total 15 subjects per Arm); within each Arm, subjects were randomized 1:1:1 into 3 Groups of 5. All subjects in the same Group received the same vaccine vector, and all subjects in the same Arm received the same titer of vaccine (10(7), 10(8), 10(9) or 10(10)CFU). Adverse events, safety, shedding, and IgG and IgA titers against Salmonella outer membrane proteins (OMPs), lipopolysaccharide (LPS) and PspA were evaluated. RESULTS: In the highest dose group, no subject experienced severe reactions or serious adverse events. Most adverse events were mild; one subject had a positive blood culture. No subject shed vaccine in stool. No statistically significant differences for post vaccination ELISA or ELISPOT results between Groups were detected. However, a limited number of ≥ 4 fold increases from baseline for IgA anti-OMPs, IgA and IgG anti-LPS, and IgA anti-PspA occurred for a few individuals as measured by ELISA, and IgA anti-OMPs as measured by ELISPOT assay. CONCLUSIONS: All three S. Typhi vectored pneumococcal vaccines were safe and well-tolerated. Immunogenicity was limited possibly due to pre-existing high antibody titers prior to vaccination. Increases in IgA were most often observed.

Rapid, sensitive recovery of recombinant attenuated Salmonella enterica serovar Typhi vaccine strains from human blood.

Prior to initiating a phase 1 dose escalation trial of the safety and immunogenicity of live, oral, recombinant, attenuated Salmonella enterica serovar Typhi vaccine strains in human subjects, the suitability of conventional blood culture procedures to rapidly and reliably detect the organisms in human blood was investigated. Blood culture specimens, with and without added growth supplements, were inoculated with study organism concentrations ranging from approximately 300 to as few as 1 to 2 CFU/10 ml culture and processed in a Bactec 9240 fluorescent series aerobic blood culture system. All cultures seeded with >6 CFU and 93% of cultures seeded with ∼1 to 2 CFU were identified as positive for microbial growth within 44 h of incubation. The results were within the performance standard of ≤5 days to detection that is expected for Gram-negative cultures seeded at 10 to 50 CFU/vial. Recovery of test organisms from blood culture was not improved by the addition of supplements, but cultures with added supplements were identified positive an average of 5 h sooner than those without added supplements. Reliable detection of the investigational vaccine strains at <1 CFU/ml of blood within 2 days in conventional blood culture without added supplements allowed for shortened confinement time of study volunteers without compromising subject safety.

Low-pH rescue of acid-sensitive Salmonella enterica Serovar Typhi Strains by a Rhamnose-regulated arginine decarboxylase system.

For Salmonella, transient exposure to gastric pH prepares invading bacteria for the stresses of host-cell interactions. To resist the effects of low pH, wild-type Salmonella enterica uses the acid tolerance response and the arginine decarboxylase acid resistance system. However, arginine decarboxylase is typically repressed under routine culture conditions, and for many live attenuated Salmonella vaccine strains, the acid tolerance response is unable to provide the necessary protection. The objective of this study was to enhance survival of Salmonella enterica serovar Typhi vaccine strains at pHs 3.0 and 2.5 to compensate for the defects in the acid tolerance response imposed by mutations in rpoS, phoPQ, and fur. We placed the arginine decarboxylase system (adiA and adiC) under the control of the ParaBAD or PrhaBAD promoter to provide inducible acid resistance when cells are grown under routine culture conditions. The rhamnose-regulated promoter PrhaBAD was less sensitive to the presence of its cognate sugar than the arabinose-regulated promoter ParaBAD and provided tighter control over adiA expression. Increased survival at low pH was only observed when adiA and adiC were coregulated by rhamnose and depended on the presence of rhamnose in the culture medium and arginine in the challenge medium. Rhamnose-regulated acid resistance significantly improved the survival of ΔaroD and ΔphoPQ mutants at pHs 3 and 2.5 but only modestly improved the survival of a fur mutant. The construction of the rhamnose-regulated arginine decarboxylase system allowed us to render S. Typhi acid resistant (to pH 2.5) on demand, with survival levels approximately equivalent to that of the native arginine decarboxylase system.

Use of RapidChek® SELECT™ Salmonella to detect shedding of live attenuated Salmonella enterica serovar Typhi vaccine strains.

Identification of individuals shedding Salmonella enterica serovar Typhi in stool is imperative during clinical trial safety evaluations. Recovery of live attenuated S. Typhi vaccine strains can be difficult because the mutations necessary for safety in humans often compromise survival in stringent selective enrichment media. RapidChek® SELECT™ Salmonella is a highly sensitive detection method for S. enterica species which utilizes a bacteriophage cocktail designed to reduce the growth of competitor microbes in mildly selective enrichment medium. Detection of Salmonella is enhanced by means of a Salmonella-specific antibody strip targeted to lipopolysaccharide. The RapidChek® SELECT™ Salmonella method was compared to conventional enrichment and plating methods to determine the most sensitive method for detecting attenuated S. Typhi strains in human stool samples. Although traditional enrichment strategies were more sensitive to the presence of wild-type S. Typhi, RapidChek® SELECT™ Salmonella was superior at detecting attenuated strains of S. Typhi. Strains containing a wide variety of attenuating mutations were detected with equal sensitivity as the wild type by RapidChek® SELECT™ Salmonella. The presence of Vi capsule or mutations which affected O-antigen synthesis (Δpmi, ΔgalE) did not decrease the sensitivity of the RapidChek® SELECT™ Salmonella assay.

The early humoral immune response to Bacillus anthracis toxins in patients infected with cutaneous anthrax.

Bacillus anthracis, the causative agent of anthrax, produces a tripartite toxin composed of two enzymatically active subunits, lethal factor (LF) and edema factor (EF), which, when associated with a cell-binding component, protective antigen (PA), form lethal toxin and edema toxin, respectively. In this preliminary study, we characterized the toxin-specific antibody responses observed in 17 individuals infected with cutaneous anthrax. The majority of the toxin-specific antibody responses observed following infection were directed against LF, with immunoglobulin G (IgG) detected as early as 4 days after the onset of symptoms in contrast to the later and lower EF- and PA-specific IgG responses. Unlike the case with infection, the predominant toxin-specific antibody response of those immunized with the US anthrax vaccine absorbed and UK anthrax vaccine precipitated licensed anthrax vaccines was directed against PA. We observed that the LF-specific human antibodies were, like anti-PA antibodies, able to neutralize toxin activity, suggesting the possibility that they may contribute to protection. We conclude that an antibody response to LF might be a more sensitive diagnostic marker of anthrax than to PA. The ability of human LF-specific antibodies to neutralize toxin activity supports the possible inclusion of LF in future anthrax vaccines.

Live recombinant Salmonella Typhi vaccines constructed to investigate the role of rpoS in eliciting immunity to a heterologous antigen.

We hypothesized that the immunogenicity of live Salmonella enterica serovar Typhi vaccines expressing heterologous antigens depends, at least in part, on its rpoS status. As part of our project to develop a recombinant attenuated S. Typhi vaccine (RASTyV) to prevent pneumococcal diseases in infants and children, we constructed three RASTyV strains synthesizing the Streptococcus pneumoniae surface protein PspA to test this hypothesis. Each vector strain carried ten engineered mutations designed to optimize safety and immunogenicity. Two S. Typhi vector strains (chi9639 and chi9640) were derived from the rpoS mutant strain Ty2 and one (chi9633) from the RpoS(+) strain ISP1820. In chi9640, the nonfunctional rpoS gene was replaced with the functional rpoS gene from ISP1820. Plasmid pYA4088, encoding a secreted form of PspA, was moved into the three vector strains. The resulting RASTyV strains were evaluated for safety in vitro and for immunogenicity in mice. All three RASTyV strains were similar to the live attenuated typhoid vaccine Ty21a in their ability to survive in human blood and human monocytes. They were more sensitive to complement and were less able to survive and persist in sewage and surface water than their wild-type counterparts. Adult mice intranasally immunized with any of the RASTyV strains developed immune responses against PspA and Salmonella antigens. The RpoS(+) vaccines induced a balanced Th1/Th2 immune response while the RpoS(-) strain chi9639(pYA4088) induced a strong Th2 immune response. Immunization with any RASTyV provided protection against S. pneumoniae challenge; the RpoS(+) strain chi9640(pYA4088) provided significantly greater protection than the ISP1820 derivative, chi9633(pYA4088). In the pre-clinical setting, these strains exhibited a desirable balance between safety and immunogenicity and are currently being evaluated in a Phase 1 clinical trial to determine which of the three RASTyVs has the optimal safety and immunogenicity profile in human hosts.

Immune response to two different dosing schedules of the anthrax vaccine precipitated (AVP) vaccine.

A pilot study compared the immune response of regular (0, 3, 6, 32 weeks) and extended (0, 10, 13, 32 weeks) schedules of the UK anthrax vaccine (anthrax vaccine precipitated, AVP). Concentrations of antibodies to protective antigen (PA) were higher (p<0.05) among recipients of the extended (n=7) versus regular schedule (n=6) at week 32, and 2 weeks after the second and third vaccinations. Toxin neutralisation assay levels and anti-lethal factor antibodies followed patterns similar to anti-PA antibodies. Extending the interval between the first two AVP vaccinations may produce a stronger immune response, but persistence of this effect needs further study.