Infectious disease accounts for approximately 35 percent of deaths worldwide, and is the world’s biggest killer of children and young adults. One focus of our laboratory is to utilize our expertise in genetics, pathogenesis and microbiology to modify Salmonella to serve as a flexible and inexpensive antigen delivery platform for developing vaccines against a variety of human and animal pathogens.
Recombinant attenuated Salmonella vaccines (RASVs) also termed “attenuated Salmonella vaccine vecrtors (ASVVs)” provide a number of unique and important advantages over traditional vaccines. Our technology allows for needle-free administration of vaccines. This is an advantage in the developing world, as its administration does not require a trained health care professional since it can be given orally on a paper strip that will dissolve in the mouth. It also does not need a “cold-chain” required by traditional vaccines to ensure the vaccine stays at a cold temperature from warehouse to final destination. Both of these advantages mean that our vaccines can be easily delivered and administered in remote regions of the developing world. Perhaps the most important advantage of RASVs is derived from the lifestyle of Salmonella, which interacts with immune cells to stimulate all three arms of the immune system: humoral, cellular and mucosal. Since most pathogens enter the body through a mucosal surface (e.g. gut, lungs, nasal), the presence of a strong mucosal defense against the pathogen invader is critical in preventing the initial infection. For pathogens clever enough to get past the mucosal defense, the strong humoral and cellular responses elicited by RASVs serve to clear the infection quickly. RASVs also stimulate immune memory cells, resulting in a strong, robust and long-lasting immunity.
Figure 1 – Three stages in the delivery of a recombinant attenuated Salmonella vaccine with regulated delayed attenuation, regulated delayed antigen synthesis and regulated delayed lysis in vivo. (In Vitro) The first panel shows a Salmonella bacterium as it is grown in vitro in medium supplemented with the sugars arabinose and mannose. LPS O-antigen is synthesized with exogenously provided mannose and is attached to the LPS core. Several key proteins are shown being produced with the exogenously provided arabinose (colored dots). Fur (iron uptake regulatory protein) and Crp (cAMP receptor protein) enable bacteria to display wild-type virulence. MurA and Asd are enzymes made in the presence of arabinose that are required for synthesis of the essential peptidoglycan cell wall components muramic acid and diaminopimelic acid, respectively, to maintain the integrity of the cell wall. The LacI repressor protein is also synthesized in the presence of arabinose and acts to repress protective antigen production. (In Vivo Initial) The middle panel shows the consequences of the vaccine entering the in vivo environment with the unavailability of the sugars arabinose and mannose. The absence of arabinose causes a reduction in the Fur and Crp proteins that are diluted out as a consequence of cell division, which gradually attenuates the vaccine. Reduced Fur protein results in increased synthesis of iron-regulated outer membrane proteins (IROMPs), which are immunodominant protective antigens and also result in iron uptake to toxic levels. Proteins MurA and Asd are also reduced which results in decreased bacterial cell wall peptidoglycan synthesis. The absence of the arabinose also causes a reduction in LacI concentration due to cell division and this initiates the production of the vaccine protective antigen (black dots). The absence of mannose ceases synthesis of new LPS O-antigen such that LPS cores with LPS O-antigen attached decrease as a consequence of cell division. (In Vivo Late) The final panel shows the complete lysis of the bacterium following some 5 to 10 cell divisions with the absence of Fur, Crp, LacI, MurA and Asd proteins resulting in the release of the protective antigen which has been synthesized in large amount prior to cell lysis. There is also release of IROMPS and LPS cores that are highly immunogenic and cross-protective against all Salmonella serotypes. Lysing of the bacterial vaccine strain also ensures no persistence of vaccine cells in vivo, and no survival if released into the environment through shedding.
Our lab is interested in developing live attenuated Salmonella strains as agents to deliver antigens to generate vaccines against a variety of human and animal pathogens. The reason we use Salmonella is that Salmonella is capable of efficiently invading and colonizing deep lymphoid tissues after oral delivery. Salmonella is the most efficient pathogen in accomplishing this at the lowest mucosally delivered (e.g. oral) immunizing dose. This ability is key to induction of long-lasting immune responses against both Salmonella and the additional antigen(s) we have engineered it to carry. Orally delivered Salmonella is able to stimulate the mucosal, cellular and humoral arms of the immune system, making it an ideal antigen delivery vector. In addition, Salmonella has excellent adjuvant qualities, which may enhance the immune response against the delivered antigen.
We have developed a number of technologies designed to enhance the immunogenicity of recombinant attenuated Salmonella vaccines (RASVs) and to provide biological containment.
1. Regulated delayed attenuation.
To make Salmonella safe for use in humans and animals, it must first be rendered avirulent (attenuated) so that it does not cause disease. This is typically done by inactivating or deleting genes required for virulence, i.e. virulence genes. While this approach has resulted in some success, deleting virulence genes results in a greater sensitivity to the environmental stresses encountered upon entry into a host, in particular the GI tract. This can lead to a reduction in the ability of the attenuated strain to reach the immune tissues necessary for induction of strong immune responses.
Recognition of this led us to develop our regulated delayed attenuation technology. We have genetically designed and modified strains that display wild-type attributes at the time of immunization, allowing it to effectively deal with stresses imposed by the host. The strain is then able to colonize host tissues, but after colonization of mucosa-associated lymphoid tissues and internal lymphoid tissues, would gradually lose virulence traits as a function of cell division and do so in a timely manner so as to not cause disease symptoms.
Curtiss, R. III, S. Y. Wanda, B. M. Gunn, X. Zhang, S. A. Tinge, V. Ananthnarayan, H. Mo, S. Wang, and W. Kong. 2009. Salmonella enterica serovar Typhimurium strains with regulated delayed attenuation in vivo. Infect. Immun. 77:1071-1082.
Li, Y., S. Wang, G. Scarpellini, B. Gunn, W. Xin, S. Y. Wanda, K. L. Roland, and R. Curtiss III. 2009. Evaluation of new generation Salmonella entericaserovar Typhimurium vaccines with regulated delayed attenuation to induce immune responses against PspA. Proc. Natl. Acad. Sci. USA 106:593-598.
2. Regulated delayed antigen synthesis.
Achieving maximal immune responses against an antigen vectored by Salmonella is directly correlated with the amount of the antigen produced. Thus it is important that the immunizing vector strain produce adequate levels of antigen. However, for RASVs, this need must be weighed against the fact that high-level antigen production can be a drain on the nutrient and energy resources of the cell, leading to reduced growth rates and a compromised ability to colonize effector lymphoid tissues to induce an immune response. In addition, some antigens are inherently toxic to vaccine strains for other reasons, leading to a severe inhibition of growth rate and host colonizing potential and, in some cases, death of the RASVs. This often mandates alterations in antigen form by removing sequences causing toxicity while retaining Immunogenicity. Overexpression of foreign proteins can also result in mutations occurring in the antigen gene promoter or coding sequence, thus reducing or compromising the desired immune response. The advantage of delaying antigen synthesis is that it allows the Salmonella vaccine strain to direct its resources toward the initial host invasion, an important step for eliciting a robust immune response.
We have developed a regulated delayed antigen synthesis system in which the expression of the vectored gene(s) is shut off at the time of vaccination. Similar to the regulated delayed attenuation technology, after colonization of host lymphoid tissues, antigen synthesis gradually increases as a function of cell division and produce adequate levels of the antigen to induce a robust immune response.
Wang, S., Y. Li, G. Scarpellini, W. Kong, H. Shi, C. Baek, B. Gunn, S. Y. Wanda, K. L. Roland, X. Zhang, P. Senechal-Willis, and R. Curtiss III. 2010. Salmonella vaccine vectors displaying delayed antigen synthesis in vivo to enhance immunogenicity. Infect. Immun. 78:3969-3980.
Wang, S., Y. Li, H. Shi, W. Sun, K. L. Roland, and R. Curtiss III. 2011. Comparison of a regulated delayed antigen synthesis system with in vivo-inducible promoters for antigen delivery by live attenuated Salmonella vaccines. Infect. Immun. 79:937-949.
Xin, W., S. Y. Wanda, Y. Li, S. Wang, H. Mo, and R. Curtiss III. 2008. Analysis of type II secretion of recombinant pneumococcal PspA and PspC in aSalmonella enterica serovar Typhimurium vaccine with regulated delayed antigen synthesis. Infect. Immun. 76:3241-3254.
3. Regulated programmed lysis.
The release of genetically modified organisms into the environment can be of concern. In the case of RASVs, vaccine organisms shed by vaccines into the environment may lead to unintentional immunizations and the possible transfer of cloned genes to other bacterial species that might represent virulence attributes in some cases. A number of mutations have been identified in S. Typhimurium, including shdA, misL and ratB, that reduce environmental shedding in mice without negatively influencing immunogenicity. While these mutations lead to a reduction in fecal shedding, it is not clear how long these strains will persist in the environment. Therefore, more effective systems need to be developed.
To address this problem, we have developed a biological containment system that will allow the RASV time to colonize the host lymphoid tissues, a requirement for inducing a robust immune response and eventually lead to cell death by lysis, thus preventing spread of the vaccine strain into the environment. Strains engineered with this system will lyse after several rounds of replication in the host or in the environment.
Kong, W., S. Y. Wanda, X. Zhang, W. Bollen, S. A. Tinge, K. L. Roland, and R. Curtiss III. 2008. Regulated programmed lysis of recombinant Salmonella in host tissues to release protective antigens and confer biological containment. Proc. Natl. Acad. Sci. USA 105:9361-9366.
Ameiss, K., S. Ashraf, W. Kong, A. Pekosz, W. H. Wu, D. Milich, J. N. Billaud, and Roy Curtiss III. 2010. Delivery of woodchuck hepatitis virus-like particle presented influenza M2e by recombinant attenuated Salmonella displaying a delayed lysis phenotype. Vaccine 28:6704-6713.
Ashraf, S., W. Kong, S. Wang, J. Yang, and R. Curtiss III. 2011. Protective cellular responses elicited by vaccination with influenza nucleoprotein delivered by a live recombinant attenuated Salmonella vaccine. Vaccine 29:3990-4002.
Kong, W., M. Brovold, B. A. Koneneman, J. Clark-Curtiss, and R. Curtiss III. 2012. Turning self-destructing Salmonella into a universal DNA vaccine delivery platform. Proc. Natl. Acad. USA 109:19414-19419.
4. Production of detoxified endotoxin in RASVs.
Live attenuated Salmonella vaccines have the ability to stimulate all three branches of the immune system, mucosal, cellular and humoral-mediated immunity, making them ideal candidates for use as carriers to deliver antigenic proteins or DNA vaccines to protect against a wide variety of pathogens. One problematic issue in this field has been that while candidate vaccines are attenuated in animal models, when administered to humans they can be reactogenic, producing unwanted side effects, including diarrhea, abdominal pain, gastrointestinal disorder, nervous system disorders, and fever. Non-reactogenic strains often do not generate a strong immune response against vectored antigens in humans. Many of the symptoms associated with reactogenic Salmonella vaccines are consistent with known reactions to lipid A, the endotoxin component of the Salmonella lipopolysaccharide (LPS). We have developed methods for detoxifying the lipid A component of LPS in living cells without compromising the ability of the vaccine to stimulate a desirable immune response.
Kong, Q., D. A. Six, K. L. Roland, Q. Liu, L. Gu, C. M. Reynolds, X. Wang, C. R. H. Raetz, and R. Curtiss III. 2011. Salmonella synthesizing 1-dephosphorylated lipopolysaccharide exhibits low endotoxic activity while retaining its immunogenicity. J. Immunol. 187:412-423.
Kong, Q., D. Six, Q. Liu, L. Gu, S. Wang, P. Alamuri, C. R. H. Raetz, and R. Curtiss III. 2012. Phosphate groups of lipid A are essential for Salmonella entericaserovar Typhimurium virulence and affect innate and adaptive immunity. Infect. Immun. 80:3215-3224.
- Campylobacter jejuni publications
- Giardia lamblia publications
- Mycobacterium tuberculosis publications
- Salmonella enterica serovar Paratyphi A publications
- Salmonella enterica serovar Typhi publications
- Shigella species publications
- Streptococcus mutans publications
- Streptococcus pneumoniae publications
- Streptococcus sobrinus publications
- Yersinia pestis publications
- Hepatitis B virus publications
- influenza virus publications
- Salmonella Typhi
- Edwardsiella ictaluri and E. tarda publications
For additional information on our Biofuels work please see our Vaccine Development Program