Impacts / Baseline data Project title: A dual recombinant vaccine for brucellosis and immunocontraception in feral swine

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1 Impacts / data Project title: A dual recombinant vaccine for brucellosis and immunocontraception in feral swine Please use the table below to guide you on reporting your impacts or baseline data from your 2012/2013 SIPMC funded project. Append a brief narrative to explain any details needed to help us report these outcomes. Plan 1) What is the overall for your Overall Objective: To develop and perform preliminary testing of the dual recombinant immunocontraceptive vaccine VTRS2-mGnRHb in the mouse model. VTRS2-mGnRHb is intended to control both the disease brucellosis and population growth in feral swine. Objectives for this project include: In vitro characterization of the VTRS2-mGnRH Assessment of clearance characteristics of VTRS2-mGnRHb in the mouse model and protection against virulent Brucella suis challenge. These assessments were performed using funds from a cooperative research agreement with USDA- APHIS separate from the SRIPM enhancement funds however they are crucial to preliminary characterization of the candidate vaccine and therefore results of that effort are included herein. Assessment of the contraceptive effect of the immunogenic gonadotropin releasing hormone multimer mgnrh expressed by VTRS2-mGnRHb in the mouse model (NOTE: experiment is ongoing, start was delayed due to in vitro and in silico analysis results leading to changes in the molecular structure of the mgnrh construct, the additional required genetic engineering steps delayed the beginning of animal trials) 2) What are the measurement indicators to help you achieve your? In vitro and in silico analysis of strain VTRS2-mGnRH was performed. Measurements included analysis of growth-characteristics in culture, assessment of antibiotic susceptibility, and assessment of mgnrh expression by Western blotting. Initial testing of a live vaccine in vivo involves assessment of the clearance characteristics of the and the ability of the vaccine to protect against virulent B. suis challenge. To assess clearance, 10 female BALB/c mice were inoculated with approximately 5 x 10 5 CFU intraperitoneally (IP) of the candidate immunocontraceptive vaccine VTRS2-mGnRHb. An additional 20 mice received a virulent dose of wild-type B. suis 1330 as an infection control. At weeks 4 and 6 post-inoculation, 5 mice per group were assessed for clearance of bacteria from their spleens. To assess protection against virulent Brucella challenge by the vaccine, 20 mice were given either VTRS2-mGnRHb (~5 x 10 5 CFU) (n=10) or sterile saline IP. 8 weeks post-inoculation mice, were either boosted with ~5 x 10 4 CFU VTRS2-

2 mgnrhb (n=5) or sterile saline or challenged with ~5 x 10 4 CFU of virulent B. suis The boosted mice were challenged 2 weeks post-booster. All mice were euthanatized 2 weeks post-challenge and clearance of bacteria from spleens assessed. To assess the contraceptive effect of VTRS2-mGnRHb in the mouse model a 2x2 experiment is in process to assess litter size and mating behavior in and un male and female BALB/c mice. Male mice were IP with ~5 x 10 5 CFU VTRS2mGnRHb (n=3) or received sterile phosphate-buffered saline at 6 weeks of age. Likewise, 18 female mice received vaccine and 18 received saline. At 6-weeks postvaccination, all mice will receive a booster (5x10 4 CFU). 2 weeks post-booster 3 un females will be housed with each male ( and un) and checked daily for the presence of a sperm plug as evidence of mating. Females will be individually housed 2 days prior to expected date of parturition (19-21d post-mating) and pups counted. A second round of mating will measure the effect of vaccination on the female mice. 3 females will be placed with each male ( and un) and mating and counting of pups will be performed as above (see table for summary of breeding evaluations). By using two rounds of breeding in addition to the effect on males and females individually, the effect of using both males and females can be measured without requiring additional negative control animals. This is a variation from the original experimental design. Expected date of completion: In addition to counting pups, at the end of the experiment all animals will be euthanized, gonads and secondary sex organs will be submitted to the USDA-APHIS National Wildlife Disease Center, Fort Collins, CO, for histopathological analysis. 2x2 Breedings Vaccinated Males (Group N, n=3) Un Males (Group M, n=3) Un Females (n= 9+9) Group G 1-3 n=3 (3 females/male) Group E 1-3 n=3 (3 females/male) Vaccinated Females (n= 9+9) Group H 1-3 n= 3 (3 females/male) Group F 1-3 n=3 (3 females/male)

3 Methodology and Results: (measured indicators vs goals for each ) Objective: In vitro and in silico assessment of VTRS2-mGnRH No enhanced growth characteristics of strain VTRS2-mGnRHb in culture vs the wild-type Colony forming units (CFU) and Klett Units (light transmission) over time in TSA broth cultures No increased growth in the (no specific value) CFU and Klett Units between and wild-type 0, 8, 24, 48, 72, and 120 hours post-inoculation of culture No statistically significant difference observed between wild-type and in culture GS Outputs Increased growth in culture would suggest increased vigor and possible virulence of the candidate vaccine strain, decreased growth could suggest attenuation which is desirable Demonstrate likelihood of expression of the immunogenic mgnrh multimer in Brucella by in silico analysis No indication that the mutations in the increased vigor of the bacteria; this does not rule out that the is attenuated and agrees with previous experiments; results encourage further characterization The mgnrh DNA fragment was sequenced (Virginia Bioinformatics Institute) and the SIXFRAME program (seqtool.sdsc.edu) was used to predict translated peptide sequence; codon agreement with Brucella trna was performed using the JAVA Codon Adaptation Tool (JCAT, JCAT.de) was evidence of the DNA sequence being cloned in frame as is required for expression and for appropriate codon usage in the mgnrh multimer for expression in Brucella (See Indicators) Sequence was cloned in frame however codons were not optimized for use in Brucella in the original mgnrh fragment; mgnrhb was created (synthesized by GenScript) using codons optimized for Brucella with the same translation as the original mgnrh provided by USDA-APHIS GS Difficulty demonstrating expression in the original construct led to the in silico analysis, troubleshooting led to changes made in the project In silico analysis led to changes in the DNA sequence of mgnrh to adapt the construct to Brucella, the resulting mgnrhb was then analyzed for expression by Western blot

4 Output Demonstrate expression of mgnrhb in the VTRS2-mGnRHb Developed Western blot Western blotting of lysed protein samples of VTRS2- mgnrhb; VTRS2 pns4 (empty plasmid) was used as negative control Demonstrate a protein band at approximately 18kDa using anti-gnrh and/or anti-his antibodies Development of Western blot on radiology film, light emission from antibody binding indicates presence of peptide GS, HA Expression was demonstrated for mgnrhb, the original mgnrh transcript was not translated by Brucella Proof of expression precludes testing in animals Demonstration of protein expression allowed the project to enter the animal-testing phases; the change in DNA sequence caused a delay in animal experiments but greatly increased their chance of success Objective: Assessment of Clearance of VTRS2-mGnRHb in the mouse model Clearance of the between 4 and 6 weeks postinoculation CFU of bacteria collected from homogenized spleens from inoculated mice indicator ~5 x 10 5 CFU <2 log CFU in spleens after 6 weeks Serial dilution of mouse spleens and plating of diluted homogenate onto TSA plates at 4 and 6 weeks postinoculation GS, NS, HA, ED 99.99% bacterial load of mice that received VTRS2-mGnRH vs the wild-type B. suis 1330; 1.39 (± 0.21) LOG CFU remained after 6 weeks, the lower limit of detection is 1.30 (s) Clearance between 4 and 6 weeks postinoculation is ideal for a live bacterial vaccine By week 6 post-inoculation % of the inocula had cleared from the mice. This is in the target window for clearance of a live, beyond 6 weeks is undesirable as it demonstrates lack of attenuation, shorter than 4 weeks does not allow for enough exposure to the host immune system. These data precluded beginning challenge trials. Anti-mGnRH ELISA was also performed on inoculated mouse sera, there was a statistically significant difference in IgG antibody response in mice receiving VTRS2-mGnRH vs controls (P=.05) Objective: Assessment of protection against virulent Brucella challenge by VTRS2-mGnRHb in the mouse model ly lower bacterial burden in spleens of vs un mice following infectious challenge What was the indicator CFU of spleen homogenates in vs un mice ---- splenic CFU vs controls, ideal = >1 LOG reduction Spenic CFU were obtained by serial dilution of spleen homogenates NS, GS, HA In animals that did not receive a booster vaccination 0.5 LOG reduction (0.5 Units of Protection, determined

5 (s) Ideally protection should be greater than 1 LOG however a significant difference between and control animals is demonstration of protection by subtraction of mean of Log 10 converted CFU of un from mice) (P=0.018); surprisingly in animals receiving a booster there was no difference in clearance. This is thought to have happened due to the initial challenge dose being higher than predicted or as a result of a long period of time elapsing between original vaccination and challenge (8 weeks was used to allow for sufficient clearance). Protection was not as high as predicted however it was demonstrated sufficiently enough to warrant initiation of breeding experiments (in progress). Additionally, these trials are being repeated using a shorter time between vaccination and challenge/booster (in progress, experiment ends ) Objective: Assessment of contraceptive effect of VTRS2-mGnRHb in the mouse model breeding behavior or litter size; gonadal changes and reduction of secondary sex organs in animals number of females bred/male, number of pups/female (fecundity), and histopathological analysis of gonads and secondary sex characteristics in mice vs controls indicator What was the target value? breeding values in mice vs controls Experiment in progress, output will be number of females bred/male, number of pups/female (fecundity), and histopathological analysis of gonads and secondary sex characteristics. NS, GS (s) EXPERIMENT IN PROGRESS Final report will be submitted at the end of August, 2014 after completion of data collection and analysis. Successful breeding activity or litter size, combined with documented protection against Brucella challenge will merit further exploration of VTRS2-mGnRHb in the pig. Summary: Feral pigs are a major nuisance species in the United States, in particular in the Southern region, and have the potential to spread disease to both humans and livestock in addition to causing environmental and property damage. Further control methods are needed and the goal of these studies is to determine whether the novel vaccine VTRS2-mGnRHb has potential as a viable alternative to currently available methods of control in the United States. Preliminary assessment of the candidate vaccine described herein allows for cost-effective determination whether further evaluation in pigs is warranted. This study has demonstrated protection against virulent Brucella challenge in the mouse model and the immunocontraceptive effect of the vaccine is currently being assessed in breeding trials which are expected to conclude in August 2014 at which time this report will be updated.