Progress 08/15/08 to 08/14/11
Outputs
OUTPUTS: Mycobacterium avium subspecies paratuberculosis (MAP) is a facultative intracellular organism that resides inside of host macrophages. MAP causes a fatal wasting syndrome in ruminants, identified by granulomatous enteritis in the small intestine. In the effort of developing an effective vaccine candidate, Type III secretion system of Salmonella was tested to delivery efficacy and immunological response against MAP. Attenuated Salmonella (ssaV;yej) expressing fusion constructs of MAP antigen was use to vaccinate mice. intraperitoneal vaccination of mice with the Salmonella/ MAP antigen fusion constructs generated a potent TH1 response and significant IFN production. Challenging with wild type MAP 66115-98 show significant protection equivalent to standard positive controls which was lasting over a 16 weeks period after challenge. This was further supported by the improved liver and spleen histopathology of the immunized animals, which showed fewer granulomas and lower numbers of acid-fast bacilli as compare negative control .Additionally, this vaccination regimen was observed to be statistically equivalent in terms of protection to immunization with MAP316F. Thus, live priming with the recombinant Salmonella/MAP antigen and represents an effective approach to further advancement to develop effective vaccine candidates. PARTICIPANTS: Drs. Subhash Chandra, Jenn-Wei Chen and Syed Faisal have worked on this project. TARGET AUDIENCES: We will submit manuscripts for publication. This is important for the other scientists who are working on this field. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
In order to develop an effective vaccine for MAP using Salmonella as a vehicle to deliver MAP protective antigen, an attenuated Salmonella strain expressing MAP antigen and exporting antigen out via type III secretion is required. We have constructed and evaluated a attenuated Salmonella strain which contains a low copy plasmid pSU39 encoding Ag85A-SOD- Ag85B and 74F1-148+607-789 by sopE promoter and sopE 104 fragment as a fusion in order to deliver MAP fusion in cytosol of target cells to elicit T cell response. Animal experiments carried out using mice showed positive protective results. The cytokine response obtained from vaccinated and control groups showed generation of Cellular immunity against MAP. Mice vaccinated with Attenuated S. Typhimuirum, expressing fusion Ag85A-SOD- Ag85B and 74F1-6 mounted protective T-cell immunity against MAP infection. ELISA and ELISPOT showed IFN production by splenocytes obtained from vaccinated mice with test vaccine as compare negative control. The comparison of vaccinated mice with Salmonella expressing Ag85A-SOD- Ag85B alone and mice vaccinated with Ag85A-SOD-Ag85B plus 74F1-6 revealed that 74F1-6 enhance the immune response and protection against MAP infection . However, the protection level was equivalent to standard positive control. This result signify the result of our experiment that Salmonella antigen delivery system work pretty good to deliver heterologus antigen via type III secretion system to deliver in cytosol resulting cellular immunity. We will use goats as an animal model for further revaluation this vaccine candidate.
Publications
- Two manuscripts have submitted and are in review. 2011
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Progress 08/15/09 to 08/14/10
Outputs
OUTPUTS: we have constructed many clones using pSU39 expression vector and tested the delivery efficiency of Salmonella delivery system to deliver MAP antigens(Ag85A Ag85 B, SOD, and 74F) into culture medium via type III secretion system. To make the efficient delivery , we have used sopE promoter and sopE104 with MAP antigens. The sopE04 fragment direct the fusion protein to deliver through type III secretion system to enter in cytoplasm of antigen presenting cells. We found that Salmonella Typhimurium (Δ aroA ; Δ yej) was not able to deliver the whole antigen efficiently . So to make an efficient delivery , we truncated MAP antigens into small fragments and checked delivery efficiency of truncated antigens. Our data show that truncated fragments are delivered efficiently into culture medium. This indicated that some amino acid motif in antigens that could hinder the delivery of these antigen through type III secretion system. Further, a fusion construct of Ag85Ac, SOD72, Ag85Bc i.e -pSU39 sopE104Ag85Ac SOD72Ag85Bc ) and 74F 1-148 and 74F 669-786 i.e pSU39sopE10474F1-148+669-786, were found to be expressed and delivered very efficiently by type III secretion system of Salmonella. List of constructs - 1-pSU39 sopE104Ag85A43-347, 2-pSU39sopE104Ag85A43-207, 3-pSU39sopE104Ag85A85-347 4-pSU39sopE104Ag85A139-347, 5-pSU39sopE104Ag85A170-347, 6-pSU39sopE104Ag85A202-347 7-pSU39 sopE104Ag85B41-330, 8-pSU39sopE104Ag85B41-198, 9-pSU39sopE104Ag85A62-330 10-pSU39sopE104Ag85B94-330, 11-pSU39sopE104Ag85B122-330, 12-pSU39sopE104Ag85B173-330 13-pSU39sopE104SOD146, 14-pSU39sopE104SOD72-146, 15-pSU39sopE104SOD1-72 16-pSU39sopE104SOD1-100, 17-pSU39sopE104SOD1-120, 18-pSU39sopE104SOD1-140 19 -pSU39sopE104 74F1-789, 20-pSU39sopE104 74F142-261, 21-pSU39sopE104 74F255-415 22-pSU39sopE104 74F409-557 , 23- pSU39sopE104 74F552-676 , 24-pSU39sopE104 74F669-789 25-pSU39sopE104 74F1-148+669-786 , 26-pSU39 sopE104Ag85Ac SOD72Ag85Bc PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The histopathology analysis confirmed that mice group who were vaccinated with Salmonella expressing MAP antigen showed significant reduced granuloma as compare mice control( with out vaccination or Salmonella mutant containing only plasmid. Similar result were observed in spleen. Non vaccinated mice showed severe inflammation in liver and spleen while vaccinated mice group had no mild or sign of inflammation. In control group 11-25 MAP bacilli were counted per granulama and in vaccinated group it was zero or less than zero. Thus Salmonella MAP vaccine reduced granuloma and bacterial load similar to mice group that were vaccinated with Mycobacterium avium subsp. Paratuberculosis 316F a vaccine strain which has been shown to provide immunity against MAP infection.
Publications
- No publications reported this period
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Progress 08/15/08 to 08/14/09
Outputs
OUTPUTS: In order to develop a vaccine against Mycobacterium Avium Subsp.Paratuberculosis , we are using salmonella delivery system. Since Salmonella can enter inside antigen presenting cells and deliver the antigen into cytosol via type III secretion system, it presents a noble delivery system for targeted antigen delivery . Salmonella delivery system has been applied to develop vaccine to many intracellular pathogens. Since MAP is an intracellular pathogen , we are applying attenuated salmonella to deliver Map antigens into cytosol aiming elicitation of CTL response. In our effort, first we have tested the delivery ability of Salmonella delivery system to deliver MAP antigens(Ag85A Ag85 B, SOD, and 74F) into culture medium via type III secretion system. For that we have used pSU39 expression vector to clone the above genes with sopE promoter and sopE104 to deliver via type III secretion system. We found that Salmonella Typhimurium mutant can not deliver the whole antigen efficiently , therefore to make efficient delivery , we truncated MAP antigens into small fragments and checked delivery efficiency of truncated antigens. Our data show that truncated fragments are delivered efficiently into culture medium. This indicates that there are some amino acid motif that cause inhibition of delivery of these antigen through type III secretion system. Further, we made a fusion construct of three genes which was delivered very efficiently by type III secretion system of Salmonella. We found that the fusion of Ag85Ac SOD72Ag85Bc was expressed and delivered efficiently into culture medium. After checking the secretion of antigens in into culture medium, we have started integration of MAP antigens into salmonella genome to constructed engineered Salmonella strain expressing MAP antigen. Up to now , we have integrated sopE promoter- sopE104Ag85Ac SOD72Ag85Bc into Salmonella genome in place of yej gene resulting attenuated Salmonella expressing Ag85Ac SOD72Ag85Bc . In next step we are trying to integrate sopE104 74F1-148+669-786 into genome of Salmonella. PARTICIPANTS: Yung-Fu Chang, PI. Kumanan Kathaperumal, Post-doctoral associate Sean P. McDonough, Co-I TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Clone of Map antigens were prepared in pSU39 vector using routine cloning techniques. After that Salmonella Typhimurium mutant was transformed with different clones and were grown in LB medium containing kanamycin 50ug/ml for 6hr at 37Ċ, after that culture were spun at high speed for 15 min. 5ml of each supernatant were filtered through 0.2 µm syringe filter and precipitated by methanol precipitation method as follows. 5 mL supernatant was mixed with 20ml methanol followed by 4ml of chloroform and 15 ml of water. Whole mixture was spun at 3000rpm for 20min. After that upper layer was removed carefully leaving interface of protein and chloroform. 15ml of methanol was added, mixed and centrifuged at 3000 rpm for 20 min. supernatant was decanted and pellet was dried by N2 gas. Pellet was dissolved in SDS loading buffer and used for SDS and western blot analyasis. Another sample were prepared by spinning 1ml transformed SalmonellaTyphimurium mutant culture followed by dissolving pellet in SDS loading buffer and boiling for 5 min. Protein sample were resolved on SDS PAGE and blotted on nitrocellulose membrane. After blotting, membrane were blocked by 5% skimmed milk in 1X PBS buffer for 30 min at RT and followed by washing by PBST buffer 3 times and incubation with anti-HA mouse IgG for 2hr at RT. After that secondary antibody goat anti-mouse labeled with alkaline phosphatase was added to incubate membranes for 1hr at RT, and then members were washed 3 time with PBST buffer. 1X solution of NBT and BCIP was added to membranes to develop color and when color was developed, membrane was washed to stop reactions. Membranes were analyzed for protein expression. The integration of sopE promoter- sopE104Ag85Ac SOD72Ag85Bc into genome of salmonella was carried out using lambda red system protocol as described. We have integrated sopE104Ag85Ac SOD72Ag85Bc construct at yej gene by homologous recombination resulting attenuation of SalmonellaTyphimurium. We have targeted yej for integration of sopE104Ag85Ac SOD72Ag85Bc because this gene have been reported to suppress CTL response , so disrupting this gene by knock in sopE104Ag85Ac SOD72Ag85Bc can enhance CTL response. The detection of expression of Ag85Ac SOD72Ag85Bc was carried as described above.
Publications
- Kathaperumal, K,, V. Kumanan, S. McDonough, L. H. Chen, S. U. Park, M. A. Moreira, B. Akey, J. Huntley, C. F. Chang, and Y. F. Chang. 2009. Evaluation of immune responses and protective efficacy in a goat model following immunization with a coctail of recombinant antigens and a polyprotein of Mycobacterium avium subsp. paratuberculosis. Vaccine 27:123-135.
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