REDUCTION OF CAMPYLOBACTER IN POULTRY BY LIVE ORAL-VECTORED VACCINE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0215105
• Grant No.: 2008-35201-04683
• Project No.: ARK02201
• Proposal No.: 2008-01445
• Program Code: 32.0A
• Project Start Date: Sep 1, 2008
• Project End Date: Aug 31, 2011
• Grant Year: 2008

Project Director
Hargis, B. M.

Recipient Organization
UNIVERSITY OF ARKANSAS
(N/A)
FAYETTEVILLE,AR 72703

Performing Department
POULTRY SCIENCE

Non Technical Summary
Food-borne bacterial pathogens causes significant public health problems. The bacteria species Campylobacter is a leading cause of food-borne diarrhea, with the primary source being contaminated poultry. In addition, Campylobacter infections have been associated with chronic diseases such as the neurological disease Guillain-Barr? Syndrome. Presently, we are quite limited with regard to our repertoire of safe and cost-effective vaccines against Campylobacter. Therefore, we have a real need to find an effective and inexpensive Campylobacter vaccine to protect, decrease, or eliminate Campylobacter contamination in poultry. The long-term goal of this research is development of safe, rapidly produced, orally effective, and low cost vaccines to protect humans against food-borne bacteria such as Campylobacter. Vaccine vectors that are able to elicit a good immune responses against Campylobacter will offer a promising alternative to existing vaccine strategies. This project utilizes a novel approach in the development of Campylobacter vaccines by inserting multiple copies of selected potent Campylobacter antigens, in combination with the helper molecules, to be obligatorily expressed on the cell surface of a recombinant live harmless bacterial carrier. Specific objectives of this project are to: 1) identify candidate cell surface antigens associated with Campylobacter infection using proteomics; 2) construct several live safe Salmonella strains that express Campylobacter antigens and evaluate the immune response in chickens; 3) develop and evaluate other even safer bacteria expressing Campylobacter antigens and their ability to elicit immune responses in chickens; 4) evaluate the vaccine strains for protection against Campylobacter challenge.

Animal Health Component

(N/A)

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	4010	1040	50%
712	3220	1100	50%

Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3220 - Meat-type chicken, live animal; 4010 - Bacteria;

Field Of Science
1100 - Bacteriology; 1040 - Molecular biology;

Keywords

vectored-vaccines

campylobacter

poultry

Goals / Objectives
The leading bacterial cause of human gastrointestinal disease worldwide is Campylobacter. Bacterial gastroenteritis continues to pose a significant threat to the general public here in the United States and abroad for the foreseeable future. Campylobacter jejuni has also been associated with the neuropathological disease Guillain-Barre Syndrome. Increasing pressure to limit Campylobacter infection antemortem in Europe and increasing regulatory interest for this agent in the United States may signal future pressures for the U.S. industry. Vaccination against Campylobacter has had limited success using killed whole-cell or protein based vaccines because of the development of Guillain-Barre syndrome. There is a real need to find an effective and inexpensive Campylobacter vaccine to protect, decrease, or eliminate Campylobacter contamination in poultry. This project utilizes a novel approach in the development of vaccines by inserting multiple copies of linear epitopes, to be expressed on the cell surface of recombinant live attenuated bacterial vaccine vectors. HYPOTHESIS: Oral live attenuated Salmonella or Lactobacillus vaccine vectors expressing Campylobacter epitope(s) will stimulate systemic, mucosal, humoral, and cell-mediated immune responses against multiple serovars of Campylobacter, thereby reducing poultry sources of this food-borne organism. Specific objectives of this project are to: 1) identify candidate cell surface polypeptides associated with Campylobacter infection using proteomics; 2) construct several live attenuated Salmonella strains that express Campylobacter epitopes and evaluate the immune response in chickens; 3) develop and evaluate Lactobacillus vaccine vectors expressing Campylobacter epitopes and their ability to elicit immune responses chickens; 4) evaluate the vaccine strains for protection against Campylobacter challenge. An effective vectored vaccine developed in chickens is expected to provide a possible means to greatly reduce the frequency of poultry-mediated food borne illness in the United States. With a bacterial-vectored vaccine, it may not be necessary to stockpile large quantities of vaccine because large amounts of orally-effective bacterially-vectored vaccine could be amplified very quickly and at low cost. Importantly for potential application to poultry, this vectored vaccine approach is expected to be effective by oral administration, an essential attribute for potential commercial adoption due to the cost of handling and individually administering vaccines to poultry.

Project Methods
In specific objective 1, candidate cell surface polypeptides associated with Campylobacter infection will be identified using proteomics or selected from the literature [Omp18/cjaD (cj0113), cjaA (cj0982c), and ACE393 (cj0420)]. We will identify candidate epitopes that are cell surface proteins that elicit an immune response to Campylobacter on the same blot. Using an aqueous biotinylation reagent to label outer membrane proteins then using two-dimensional electrophoresis to identify both cell surface molecules and immunogenic epitopes in a Western blot. Cell surface proteins that are immunogenic and outer membrane proteins will be identified using Mass Spectrometry. Samples will be run using the standard configuration of the Bruker Reflex III or Ultraflex II MALDI TOF (or TOF/TOF) In specific objective 2, we will construct several live attenuated Salmonella strains that express codon-optimized Campylobacter epitopes that were identified from the literature or specific objective 1. These constructs will be evaluated for their ability to invade, colonize, and persist in tissues and elicit immune responses against the Campylobacter epitopes in chickens. Attenuation of wild-type Salmonella strains will be achieved by deletion mutation of one or more virulence genes. Attenuated Salmonella strains will be transformed by using an overlapping and extension PCR product and the red recombinase system to chromosomally insert the chosen Campylobactor epitopes; a counter selection marker will be utilized to select for transformed clones. Cell surface expression of the Campylobacter epitopes and CD154 inserts will be confirmed with a simple antibody/antigen precipitation reaction and confirmed using DNA sequencing. Specific objective 3, we will develop and evaluate Lactobacillus vaccine vectors that present linear Campylobacter epitopes for their ability to elicit immune responses chickens. The most promising Campylobacter epitopes identified in specific objectives 1 and 2 will be chromosomally inserted into selected LAB candidates in the same fashion as for the Salmonella; slpS gene. The recombinant LAB vaccine strains will be utilized for in vivo challenge studies. In the final specific objective (objective 4), we will evaluate and compare Salmonella and Lactobacillus vector systems with selected epitope expression for the ability to protect chickens against infection from challenge with wild-type Campylobacter isolates. Chickens are highly susceptible to Campylobacter infections and the majority of commercial poultry are infected. In direct challenge studies, we will be able to directly compare the efficacy of these candidates for actually preventing infection in this highly susceptible model. Attenuated Salmonella or Lactobacillus candidate vaccine strains that have demonstrated the ability to elicit sufficient humoral and cell-mediated immune responses in specific objectives 2 and/or 3 will be selected for evaluation in challenge experiments to ascertain their ability to protect chickens against direct Campylobacter challenge.

Progress 09/01/08 to 08/31/11

Outputs
OUTPUTS: Bacterial gastroenteritis continues to pose a significant threat to the general public here in the United States and abroad. The leading bacterial cause of human gastrointestinal disease worldwide is Campylobacter. The actual burden of illness of Campylobacter gastroenteritis nationwide is 500-850 infections/100,000 persons. Not only is Campylobacter the leading cause of bacterial gastroenteritis, but C. jejuni has been associated with the neuropathological diseases in humans such as Guillain-Barre Syndrome and reactive arthritis. Presently, we are quite limited with regard to our repertoire of safe and cost-effective vaccines for generation of mucosal immunity against a variety of agents including Campylobacter. A successful vaccine would need to be cost-effective, safe, orally effective, and be produced in large quantities in a very short time-period. There have been several successful vaccines using a live bacterial vector. This project utilizes a novel approach in the development of Campylobacter vaccines by inserting multiple copies of selected immunodominant Campylobacter epitopes in combination with the immunostimulatory molecule CD154. Vaccination against Campylobacter has had limited success using killed whole-cell or protein based vaccines. In addition, there are concerns regarding the development of Guillain-Barre syndrome or other sequelae from killed whole-cell vaccination. Therefore, there is a real need to find an effective and inexpensive Campylobacter vaccine to protect, decrease, or eliminate Campylobacter contamination in poultry. A patent application has been submitted for the unique peptides that were selected for candidate vaccines. The epitopes selected were unique and effective peptide vaccine candidates. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Vaccine researchers PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The long-term goal of this research is to reduce the risk of Campylobacter contamination by developing vaccines that can effectively protect poultry against Campylobacter colonization. We have constructed live attenuated Salmonella strains that express three selected immunodominant peptides along with the immunopotentiation insert CD154. Recombinant S. enteritidis vaccine candidates containing stable integrated copies one of the three candidate epitopes, Omp18/cjaD (cj0113), cjaA (cj0982c), and ACE393 (cj0420), were constructed (Objective 1). The Sce-I/Km mutation was made in Loop 9, this region will be replaced by a codon-optimized candidate epitope-CD154 DNA sequence. For Objective 2, we evaluated these strains for their ability to invade into the liver and spleen (L/S) and to colonize in the cecal tonsil (CT) and persist in these tissues. Three animal experiments were performed with similar results. The second part of Objective 2 was to determine the immune response to Campylobacter following vaccination. All three vaccine candidates (cj0420, cj0113, cj0982) had significant antibody levels at all time points when compared to controls. Vaccine vector cj0113 caused a significant increase in the levels of sIgA when compared to the saline group and the two groups receiving either cj0420 or cj0982 and repeat experiments were similar with no effect by a backbone strain. Specific Objective 4 was to evaluate the most effective candidate vaccine for protection of chickens against infection following challenge with wild-type Campylobacter strains. We publish a manuscript showing the protective effect of vaccination with the Salmonella-vectored Cj0113 epitope in an American Society for Microbiology journal. In this publication we showed that the vaccine was very protective against a Campylobacter challenge. We have recently utilized a novel Bacillus subtilis (BS) strain, which expresses the Cj0113 epitope. We utilized this vector in a challenge study and found that similar to the Salmonella vectored Cj0113 there was a dramatic decrease in campylobacter colonization to below detectable levels following vaccination with the BS-Cj0113 vaccine. During the last year, several U.S. and international patent applications were generated from work that was stimulated by this project, and this project has been commercially licensed jointly by a US company and their international partner (Arkansas Biosciences/Pacific Gene Tech). Recently, an exclusive sublicense to a major US biologics company was granted for commercial development of a vaccine intended to protect poultry from infection with Campylobacter spp., based on these original data. This project was deemed highly successful by the investigators.

Publications

• No publications reported this period

Progress 09/01/09 to 08/31/10

Outputs
OUTPUTS: Bacterial gastroenteritis continues to pose a significant threat to the general public here in the United States and abroad. The leading bacterial cause of human gastrointestinal disease worldwide is Campylobacter. The actual burden of illness of Campylobacter gastroenteritis nationwide is 500-850 infections/100,000 persons. Not only is Campylobacter the leading cause of bacterial gastroenteritis, but C. jejuni has been associated with the neuropathological diseases in humans such as Guillain-Barre Syndrome and reactive arthritis. Presently, we are quite limited with regard to our repertoire of safe and cost-effective vaccines for generation of mucosal immunity against a variety of agents including Campylobacter. A successful vaccine would need to be cost-effective, safe, orally effective, and be produced in large quantities in a very short time-period. There have been several successful vaccines using a live bacterial vector. This project utilizes a novel approach in the development of Campylobacter vaccines by inserting multiple copies of selected immunodominant Campylobacter epitopes in combination with the immunostimulatory molecule CD154. Vaccination against Campylobacter has had limited success using killed whole-cell or protein based vaccines. In addition, there are concerns regarding the development of Guillain-Barre syndrome or other sequelae from killed whole-cell vaccination. Therefore, there is a real need to find an effective and inexpensive Campylobacter vaccine to protect, decrease, or eliminate Campylobacter contamination in poultry. A patent application has been submitted for the unique peptides that were selected for candidate vaccines. The epitopes selected were unique and effective peptide vaccine candidates. The outputs are disseminated through presentations at scientific meetings (see below in Publications) and through submitted manuscripts to be reported in next year's annual report. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Vaccine researchers PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The long-term goal of this research is to reduce the risk of Campylobacter contamination by developing vaccines that can effectively protect poultry against Campylobacter colonization. We have constructed live attenuated Salmonella strains that express three selected immunodominant peptides along with the immunopotentiation insert CD154. Recombinant S. enteritidis vaccine candidates containing stable integrated copies one of the three candidate epitopes, Omp18/cjaD (cj0113), cjaA (cj0982c), and ACE393 (cj0420), were constructed (Objective 1). The Sce-I/Km mutation was made in Loop 9, this region will be replaced by a codon-optimized candidate epitope-CD154 DNA sequence. For Objective 2, we evaluated these strains for their ability to invade into the liver and spleen (L/S) and to colonize in the cecal tonsil (CT) and persist in these tissues. Three animal experiments were performed with similar results. The second part of Objective 2 was to determine the immune response to Campylobacter following vaccination. All three vaccine candidates (cj0420, cj0113, cj0982) had significant antibody levels at all time points when compared to controls. Vaccine vector cj0113 caused a significant increase in the levels of sIgA when compared to the saline group and the two groups receiving either cj0420 or cj0982 and repeat experiments were similar with no effect by a backbone strain. Specific Objective 4 was to evaluate the most effective candidate vaccine for protection of chickens against infection following challenge with wild-type Campylobacter strains. We adapted a qPCR method to quantitate Campylobacter in the tissues with excellent correlation with conventional microbiological enumeration techniques. Chickens were challenged with C. jejuni on day 21 post vaccination. Ileal mucosal samples were used for DNA sample preparation to enumerate C. jejuni within the gut using qPCR. Using the cj0113 vaccine candidate, there was a marked 8 log reduction (P<0.05) of C. jejuni in the ileum compared to the control birds. In experiment 2, qPCR data there was an approximate 5 log reduction of C. jejuni in cj0113 SE-vectored vaccine when compared to saline. Additionally, in experiment 3 vaccination with the cj0113 vector caused an approximate 5 log reduction, to below detectable levels, of C. jejuni as compared with the saline or Salmonella parent strain (backbone) which contained no epitope insert. We have recently found that vaccination of turkeys with the Cj0113 epitope protected against C. coli challenge. For Objective 3 we proposed to develop a vaccine vector in a generally regarded as safe organism such as a Bacillus subtilis or a Lactobacillus. We have recently utilized a novel Bacillus subtilis (BS) strain which expresses the Cj0113 epitope. We utilized this vector in a challenge study and found that similar to the Salmonella vectored Cj0113 there was a dramatic decrease in campylobacter colonization to below detectable levels following vaccination with the BS-Cj0113 vaccine.

Publications

• N.R. Pumford, S.L. Layton, M.J. Morgan and B.M. Hargis. Vaccination with Subunit Epitopes of Campylobacter Expressed in two different Bacterial Vector Systems Reduces Campylobacter jejuni in Chickens. Proceedings of the 59th Western Poultry Disease Conference: pp. 78-79, 2010. April 2010. Vancouver, BC, Canada.
• S.L. Layton, K. Cole, M.J. Morgan, Y.M. Kwon, D.J. Donoghue, B.M. Hargis, and N.R. Pumford. Evaluation of selected Salmonella-vectored Campylobacter epitopes for reduction of Campylobacter jejuni in broiler chickens. International Poultry Scientific Forum January, 2010. Georgia World Congress Center. Atlanta, Georgia.
• N.R. Pmford, S. L. Layton, M.J. Morgan, Y. M. Kwon, and B.M. Hargis. Reduction of Campylobacter in Poultry by Live Oral-Vectored Vaccine. Institute of Food Technologists Annual Meeting July 2010. Chicago IL.
• C.J. Kremer, S.L. Layton, M.J. Morgan, A.D. Wolfenden, K.M. OMeara, K. Cole, B. M. Hargis, and N.R. Pumford. Evaluation of recombinant Salmonella expressing an immunoprotective epitope of cj0113 for protection against Campylobacter coli in commercial turkeys. Southern Poultry Science Society. January 24-25, 2011. The International Poultry Scientific Forum. Georgia World Congress Center, Atlanta.

Progress 09/01/08 to 08/31/09

Outputs
OUTPUTS: Bacterial gastroenteritis continues to pose a significant threat to the general public here in the United States and abroad. The leading bacterial cause of human gastrointestinal disease worldwide is Campylobacter. The actual burden of illness of Campylobacter gastroenteritis nationwide is 500-850 infections/100,000 persons. Not only is Campylobacter the leading cause of bacterial gastroenteritis, but C. jejuni has been associated with the neuropathological diseases in humans such as Guillain-Barre Syndrome and reactive arthritis. Presently, we are quite limited with regard to our repertoire of safe and cost-effective vaccines for generation of mucosal immunity against a variety of agents including Campylobacter. A successful vaccine would need to be cost-effective, safe, orally effective, and be produced in large quantities in a very short time-period. There have been several successful vaccines using a live bacterial vector. This project utilizes a novel approach in the development of Campylobacter vaccines by inserting multiple copies of selected immunodominant Campylobacter epitopes in combination with the immunostimulatory molecule CD154. Vaccination against Campylobacter has had limited success using killed whole-cell or protein based vaccines. In addition, there are concerns regarding the development of Guillain-Barre syndrome or other sequelae from killed whole-cell vaccination. Therefore, there is a real need to find an effective and inexpensive Campylobacter vaccine to protect, decrease, or eliminate Campylobacter contamination in poultry. A patent application has been submitted for the unique peptides that were selected for candidate vaccines. The epitopes selected were unique and effective peptide vaccine candidates. The outputs will be disseminated through presentations at scientific meetings and through a submitted manuscript to be reported in next year's annual report. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Vaccine researchers PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
The long-term goal of this research is to reduce the risk of Campylobacter contamination by developing vaccines that can effectively protect poultry against Campylobacter colonization. We have constructed live attenuated Salmonella strains that express three selected immunodominant peptides along with the immunopotentiation insert CD154. Previously, we have created aroA and htrA deletion mutants from a single candidate Salmonella vector for vaccine development. Recombinant S. enteritidis vaccine candidates containing stable integrated copies one of the three candidate epitopes, Omp18/cjaD (cj0113), cjaA (cj0982c), and ACE393 (cj0420), were constructed (Objective 1). The Sce-I/Km mutation was made in Loop 9, this region will be replaced by a codon-optimized candidate epitope-CD154 DNA sequence. For Objective 2, we evaluated these strains for their ability to invade into the liver and spleen (L/S) and to colonize in the cecal tonsil (CT) and persist in these tissues. Three animal experiments were performed with similar results. We observed significant levels of colonization by the three candidate vectored vaccines within the cecal as well as significant invasion of the internal organs by the cj0113 expressing vector at the same time point. The second part of Objective 2 was to determine the immune response to Campylobacter following vaccination. Serum samples were used to determine C. jejuni- specific IgG and sIgA antibodies. In the first experiment all three vaccine candidates (cj0420, cj0113, cj0982) had significantly higher antibody levels at all time points when compared to the group which received only saline. Vaccine vector cj0113 caused a significant increase in the levels of sIgA when compared to the saline group and the two groups receiving either cj0420 or cj0982 and repeat experiments were similar with no effect by a backbone strain. Specific Objective 4 was to evaluate the most effective candidate vaccine for protection of chickens against infection following challenge with wild-type Campylobacter strains. We adapted a qPCR method to quantitate Campylobacter in the tissues with excellent correlation with conventional microbiological enumeration techniques. Chickens were challenged with C. jejuni on day 21 post vaccination. Ileal mucosal samples were used for DNA sample preparation to enumerate C. jejuni within the gut using qPCR. Vaccination with vector candidates cj0420 and cj0982 caused an approximate 2 log and 3 log reduction (P<0.05), respectively, in the level of C. jejuni present in the ileal samples. Using the cj0113 vaccine candidate, there was a marked 8 log reduction (P<0.05) of C. jejuni in the ileum compared to the control birds. In experiment 2, qPCR data there was an approximate 5 log reduction of C. jejuni in cj0113 SE-vectored vaccine when compared to saline. Additionally, in experiment 3 vaccination with the cj0113 vector caused an approximate 5 log reduction, to below detectable levels, of C. jejuni as compared with the saline or Salmonella parent strain (backbone) which contained no epitope insert.

Publications

• No publications reported this period