Regulated programmed lysis of recombinant
            <i>Salmonella</i>
            in host tissues to release protective antigens and confer biological containment

Kong, Wei; Wanda, Soo-Young; Zhang, Xin; Bollen, Wendy; Tinge, Steven A.; Roland, Kenneth L.; Curtiss, Roy

doi:10.1073/pnas.0803801105

Cited by 129 publications

(190 citation statements)

References 43 publications

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“…To address these problems, work in our laboratory has led to the development of a regulated delayed attenuation system, applied first to Salmonella, in which the vaccine strains display features of the wild-type virulent pathogen at the time of immunization to enable the strain to effectively colonize lymphoid tissues and then become completely attenuated in vivo to preclude inducing disease symptoms (11). We have applied this new technology to the crp gene in Y. pestis by constructing a strain in which crp expression is dependent on the presence of arabinose, a sugar that is not present in host tissues (22,25). Arabinose is provided during in vitro growth so that the strain expresses crp, making it fully functional to interact with host tissues.…”

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confidence: 99%

Yersinia pestiswith Regulated Delayed Attenuation as a Vaccine Candidate To Induce Protective Immunity against Plague

et al. 2010

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Two mutant strains of Yersinia pestis KIM5؉, a ⌬crp mutant and a mutant with arabinose-dependent regulated delayed-shutoff crp expression (araC P BAD crp), were constructed, characterized in vitro, and evaluated for virulence, immunogenicity, and protective efficacy in mice. Both strains were highly attenuated by the subcutaneous (s.c.) route. The 50% lethal doses (LD 50 s) of the ⌬crp and araC P BAD crp mutants were approximately 1,000,000-fold and 10,000-fold higher than those of Y. pestis KIM5؉, respectively, indicating that both strains were highly attenuated. Mice vaccinated s.c. with 3.8 ؋ 10 7 CFU of the ⌬crp mutant developed high anti-Y. pestis and anti-LcrV serum IgG titers, both with a strong Th2 bias, and induced protective immunity against subcutaneous challenge with virulent Y. pestis (80% survival) but no protection against pulmonary challenge. Mice vaccinated with 3.0 ؋ 10 4 CFU of the araC P BAD crp mutant also developed high anti-Y. pestis and anti-LcrV serum IgG titers but with a more balanced Th1/Th2 response. This strain induced complete protection against s.c. challenge and partial protection (70% survival) against pulmonary challenge. Our results demonstrate that arabinose-dependent regulated crp expression is an effective strategy to attenuate Y. pestis while retaining strong immunogenicity, leading to protection against the pneumonic and bubonic forms of plague.Bubonic and pneumonic plague are zoonotic diseases endemic in many parts of the world, including the United States, and have resulted in over 200 million deaths over the course of human history (51). The etiological agent of plague is Yersinia pestis. Although the number of confirmed plague cases that occur worldwide has stabilized over the last 50 years at an average of about 1,700 per year, plague remains a serious public health threat in some regions of the world and outbreaks still occur (19). In addition to the potential for natural infections, Y. pestis is generally considered to be among the top five potential biological weapons (19). Recent efforts to create a safe and effective pneumonic plague vaccine have focused on the development of recombinant subunit vaccines that elicit antibodies against two well-characterized Y. pestis antigens, the F1 capsule and the virulence protein LcrV (2,8,40,53). A plague vaccine based on live attenuated Y. pestis provides the theoretical advantage of simultaneously priming against many antigens, thereby greatly enhancing the likelihood of broadbased protection. In the past, live attenuated strains were generated by selection, rather than precise genetic manipulation, thus raising concern about their genetic composition and stability. The live EV76 vaccine is an apparent pgm mutant that has been used in some countries (49). However, a concern is that the EV76 vaccine strain can cause disease in primates, raising questions about its suitability as a human vaccine (29). Nevertheless, as recently as 2002, USAMRIID researchers noted, "Despite their drawbacks, there is ample evidence that live-a...

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Yersinia pestiswith Regulated Delayed Attenuation as a Vaccine Candidate To Induce Protective Immunity against Plague

et al. 2010

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“…Previous works have created asd mutants in Salmonella enterica serovar Typhimurium (13) and Legionella pneumophila (17) and demonstrated a growth requirement for DAP. In addition, the S. Typhimurium ⌬asd strain has been extensively used in clinical studies with human subjects as a vaccine delivery strain (25,40). The pathway for synthesizing DAP from aspartate is absent in mammals; therefore, no DAP is present in mammalian hosts, including humans (13,36).…”

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The Burkholderia pseudomallei Δ asd Mutant Exhibits Attenuated Intracellular Infectivity and Imparts Protection against Acute Inhalation Melioidosis in Mice

et al. 2011

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Burkholderia pseudomallei, the cause of serious and life-threatening diseases in humans, is of national biodefense concern because of its potential use as a bioterrorism agent. This microbe is listed as a select agent by the CDC; therefore, development of vaccines is of significant importance. Here, we further investigated the growth characteristics of a recently created B. pseudomallei 1026b ⌬asd mutant in vitro, in a cell model, and in an animal model of infection. The mutant was typified by an inability to grow in the absence of exogenous diaminopimelate (DAP); upon single-copy complementation with a wild-type copy of the asd gene, growth was restored to wild-type levels. Further characterization of the B. pseudomallei ⌬asd mutant revealed a marked decrease in RAW264.7 murine macrophage cytotoxicity compared to the wild type and the complemented ⌬asd mutant. RAW264.7 cells infected by the ⌬asd mutant did not exhibit signs of cytopathology or multinucleated giant cell (MNGC) formation, which were observed in wild-type B. pseudomallei cell infections. The ⌬asd mutant was found to be avirulent in BALB/c mice, and mice vaccinated with the mutant were protected against acute inhalation melioidosis. Thus, the B. pseudomallei ⌬asd mutant may be a promising live attenuated vaccine strain and a biosafe strain for consideration of exclusion from the select agent list.

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“…The asdA gene complements the lethal chromosomal ⌬asdA mutant allele of the RASV 9447 strain. The asdA and murA genes each contain a GTG start codon, which replaces the ATG codon to reduce their translation efficiency but increases their stability in the cell (35). The asdA selectable marker encodes an enzyme involved in lysine biosynthesis necessary for the synthesis of diaminopimelic acid (DAP), which is a basic component of the peptidoglycan layer of the bacterial cell wall and allows the maintenance of this antibiotic resistancefree plasmid.…”

Section: Resultsmentioning

confidence: 99%

“…It is therefore phenotypically wild type at the time of immunization and becomes attenuated and commences to synthesize protective antigen only after colonization of host tissues. To achieve this goal, we replaced the promoter sequences of genes involved in metabolic functions or virulence with the tightly arabinose-regulated araC P BAD activator-promoter cassette (14,35) and repressed the synthesis of protective antigen by the arabinose-regulated synthesis of LacI to prevent the expression of the lcrV196-carrying sequence on pYA4535 until after lymphoid tissue colonization. Strain 9447 harbors the deletion-insertion mutations ⌬asdA21::TT araC P BAD c2 and ⌬P mur7 ::TT araC P BAD murA.…”

Section: Resultsmentioning

confidence: 99%

Fine-Tuning Synthesis ofYersinia pestisLcrV from Runaway-Like Replication Balanced-Lethal Plasmid in aSalmonella entericaSerovar Typhimurium Vaccine Induces Protection against a LethalY. pestisChallenge in Mice

et al. 2010

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A balanced-lethal plasmid expression system that switches from low-copy-number to runaway-like highcopy-number replication (pYA4534) was constructed for the regulated delayed in vivo synthesis of heterologous antigens by vaccine strains. This is an antibiotic resistance-free maintenance system containing the asdA gene (essential for peptidoglycan synthesis) as a selectable marker to complement the lethal chromosomal ⌬asdA allele in live recombinant attenuated Salmonella vaccines (RASVs) such as Salmonella enterica serovar Typhimurium strain 9447. pYA4534 harbors two origins of replication, pSC101 and pUC (low and high copy numbers, respectively). The pUC replication origin is controlled by a genetic switch formed by the operator/ promoter of the P22 cro gene (O/P cro ) (P R ), which is negatively regulated by an arabinose-inducible P22 c2 gene located on both the plasmid and the chromosome (araC P BAD c2). The absence of arabinose, which is unavailable in vivo, triggers replication to a high-copy-number plasmid state. To validate these vector attributes, the Yersinia pestis virulence antigen LcrV was used to develop a vaccine against plague. An lcrV sequence encoding amino acids 131 to 326 (LcrV196) was optimized for expression in Salmonella, flanked with nucleotide sequences encoding the signal peptide (SS) and the carboxy-terminal domain (CT) of ␤-lactamase, and cloned into pYA4534 under the control of the P trc promoter to generate plasmid pYA4535. Our results indicate that the live Salmonella vaccine strain 9447 harboring pYA4535 efficiently stimulated a mixed Th1/Th2 immune response that protected mice against lethal challenge with Y. pestis strain CO92 introduced through either the intranasal or subcutaneous route.

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Regulated programmed lysis of recombinant Salmonella in host tissues to release protective antigens and confer biological containment

Cited by 129 publications

References 43 publications

Yersinia pestiswith Regulated Delayed Attenuation as a Vaccine Candidate To Induce Protective Immunity against Plague

Yersinia pestiswith Regulated Delayed Attenuation as a Vaccine Candidate To Induce Protective Immunity against Plague

The Burkholderia pseudomallei Δ asd Mutant Exhibits Attenuated Intracellular Infectivity and Imparts Protection against Acute Inhalation Melioidosis in Mice

Fine-Tuning Synthesis ofYersinia pestisLcrV from Runaway-Like Replication Balanced-Lethal Plasmid in aSalmonella entericaSerovar Typhimurium Vaccine Induces Protection against a LethalY. pestisChallenge in Mice

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