Systematic Enhancement of Salmonella-Based Vaccines to Control Infectious Agents in Poultry

Non-Technical Summary:<br/>
There are numerous approaches to control infectious agents, including viral, bacterial, fungal or parasitic pathogens. Among others, vaccination is one of the most promising strategies to control them in poultry as well as in human. Effective control of a variety of infectious diseases is crucial for successful poultry production, because economic loss associated with infectious diseases is a significant burden for poultry production. When it comes to the pathogens that infect human through consumption of contaminated poultry products (foodborne pathogens), control of those pathogens in poultry is important for both poultry industry as well as human public health. The use of virulence-attenuated Salmonella as a vaccine vector has been established as a promising vaccination strategy. We have developed a novel vaccination strategy using a LamB protein for efficient surface-display of foreign epitopes in Salmonella. <br/>In this project, we will maximize the efficacy of this vaccination strategy by identifying and employing the most suitable carrier proteins, and epitopes. The candidate vaccine vectors will be tested and the most promising Salmonella vector system will be used for construction and evaluation of Salmonella-based vaccines for various infectious agents of importance in poultry as well as in human. Our strategy combines genetic tools, functional genomics, next-generation sequencing technology, bioinformatics, and immunology to maximize the potential of our current Salmonella-based vaccination strategy in a systematic way. We anticipate that this vector system, when combined with appropriate antigenic epitopes, will become a universal vaccine platform for efficient control of various infectious agents in poultry as well as in human. Our main focus is to develop vaccines for use in poultry, but the same approach will be also applied to mouse infection model for development of vaccines for human.
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Approach:<br/>
Aim 1. Genome scanning for genetic elements important for optimization of Salmonella-based vaccines. (1) We will conduct global scanning of Salmonella genome a) to define all genetic elements (coding & noncoding genes, their promoter regions) required for in vitro (LB, M9 media) and in vivo fitness (chicken and mouse), b) to identify all surface-exposed proteins under in vitro (LB and M9 media) and in vivo conditions (chicken and mouse), and c) to identify all immune-protective epitopes whose mutation would allow escaping of the mutant from adaptive immune defense mechanisms. (2) To accomplish the goals described in (1), we will use the method, termed Tn-seq, for deep profiling of all insertions in a transposon mutant library using Illumina sequencing in conjunction with a modified mariner transposon TnSC189 (1, 10). TnSC189 contains a KmR cassette flanked by two FRT sites with the same orientation. When a complex TnSC189 mutant library (original library; OL) of Salmonella is transformed with pCP20 expressing flippase enzyme, the library will be converted into the same insertion library that yet contains shortened TnSC189 (201 bp) encoding 3X FLAG tags (modified library; ML). (3) For identification of fitness factors, OL will be selected in vitro and in vivo. The resulting libraries before and after the selection will be subjected to Tn-seq analysis to identify all genetic elements required for fitness in vitro and in vivo. (4) For identification of surface-exposed proteins, ML will be grown in vitro and in vivo. Then, the mutants expressing 3X FLAG tags on the surface proteins will be captured and analyzed en mass by Tn-seq to identify all surface proteins. (5) For discovery of the immuno-protective epitopes, ML will be selected in vivo (chicken and mouse) vaccinated with wild type Salmonella strain. The resulting libraries will be analyzed by Tn-seq to identify all mutants in which an immuno-protective epitopes are disrupted by insertion.
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Aim 2. Optimization and evaluation of Salmonella-based vaccine vectors. (1) We will design improved Salmonella vaccine vectors expressing 6XHis tags as a fusion to the surface proteins that are shown to be expressed most abundantly in vivo in Aim 1 using the scarless and site-directed mutagenesis method (2). (2) After vaccination of the hosts (chicken and mouse), in vivo expression of His tags and host immune responses against the His tags will be determined to identify the most promising Salmonella vector. Aim 3. Development and evaluation of Salmonella-based vaccines for infectious agents. (1) The Salmonella vector chosen in Aim 2 will be used to develop vaccine strains expressing the promising epitopes identified in Aim 1 to construct Salmonella-based vaccines to control Salmonella strains. (2) The same vectors will be also used to develop vaccines for other infectious agents including Campylobacter jejuni.
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Progress:<br/>
2012/01 TO 2012/12<br/>
OUTPUTS: The use of virulence-attenuated Salmonella as a vaccine vector has been established as a promising vaccination strategy. We have developed a novel vaccination strategy using a LamB protein for efficient surface-display of foreign epitopes in Salmonella. We will maximize the efficacy of this vaccination strategy by identifying and employing the most suitable carrier proteins, and epitopes. The candidate vaccine vectors will be tested and the most promising Salmonella vector system will be used for construction and evaluation of Salmonella-based vaccines for various infectious agents of importance in poultry as well as in human.
<br/>PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. <br/>PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
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Impact:<br/> We developed a powerful functional genomics method called, Tn-seq (transposon-sequencing), for genome-wide and quantitative mapping of all insertions in a complex mutant library utilizing massively parallel Illumina sequencing. This method was applied to a genome-saturating Salmonella Typhimurium mutant library recovered from selection under various in vitro growth conditions (low-nutrients, bile salts, chicken carcass, refrigeration, and freezing) mimicking various aspects of environmental stressors found in poultry production and processing. Numerous genes have been identified, revealing previous unknown mechanisms Salmonella uses to survive against the stressors in poultry production and processing. With continuously increasing sequencing capacity of next generation sequencing technologies, this robust Tn-seq method will aid in revealing unexplored genetic determinants and the underlying mechanisms of various biological processes in Salmonella and the other important foodborne bacterial pathogens. These experimental approaches represent powerful means to study Salmonella gene functions, which is readily applicable to explore complex genetic mechanisms of this important foodborne pathogen to survive and persist in poultry production as well as human food chains in general. Understanding the genetic factors and survival mechanisms will provide valuable and practical insights for development of effective strategies to reduce Salmonella in poultry and poultry products. We also continued development of Salmonella-based vaccines targeting CfrA and CfrB protein epitopes to control Campylobacter species. Three candidate vaccines have been constructed - Live CfrA/CD154(H), Live CfrA/CD154(C), and Live CfrB. These vaccine candidates will be evaluated for protection against Campylobacter challenge using chicken model of cecal colonization.