Progress 05/01/12 to 04/30/17
Outputs
Target Audience:Veterinarians and research scientists are informed of the results from this project through scientific meeting presentations. Changes/Problems:There are two major changes: (1). The original PD was Dr. Elankumaran Subbiah, who passed away in September 2015 approxmately 8 months prior to the end of the project. After Dr. Subbiah's passing, with the approval of the USDA NIFA, the project was transfered to the new PD Dr. X.J. Meng. (2). The original vector proposed for the trivalent vaccine was swine parainfluenza virus 3 (sPIV3). However, prior to the passing of the original PD Dr. Subbiah, his lab was unable in three years to successfully develop the reverse genetics system of the sPIV3 for use as the proposed vaccine vector. Therefore, the new PD Dr. Meng utilized an altrenative approach in which an attenuated strain of PRRSV (instead of sPIV3) was used as the vector for the trivalent vaccine. This alternative approach did not alter the original objective or the ultimate goal which is to develop a trivalent vaccine against PRRSV, PCV2 and SIV. In fact, the alternative approach of using PRRSV-based live viral vector has unique advantages than the original sPIV3 vector (PRRSV vector can deliver foreign antigen via generation of additional separate subgenomic mRNAs. Also, the use of an attenuated PRRSV as live virus vaccine vector can provide protection against PRRSV in addition to the other intended pathogens). At the end, the original propsosed objective and project goal were achieved as we have now a candidate trivalent vaccine against all three pathogens as originally proposed. Furthermore, the project also produceda novel PRRSV-based viral vaccine vector for mulitvalent vaccine development. What opportunities for training and professional development has the project provided?Two graduate students, Qian Cao and Nicholas Catanzaro, and one postdoctoral associate, Dr. Debin Tian, have received training for PRRSV research in this project. Qian Cao has successfully defended her PhD in molecular virology on Sept 29, 2017, and she will start her postdoctoral training in December 2017 at Virginia Tech. Nicholas Catanzaro has now successfully applied and been awarded a NIFA pre-doctoral fellowship on PRRSV. Dr. Debin Tian, who is currently still a postdoctoral associate, has now submitted a NIFA AFRI research grant proposal on PRRSV as a co-project director and this proposal is currently under review. He is also the lead author of a recent publication in Journal of General Virology reporting the main finding from this project. How have the results been disseminated to communities of interest?Yes, we have presented the results of this project in scientific meetings. A major manuscript reporting the results from this project have now been published in the Journal of General Virology. Peer-reviewed publication: Tian D, Sooryanarain H, Matzinger SR, Gauger PC, Karuppannan AK, Elankumaran S, Opriessnig T, Meng XJ.Protective efficacy of a virus-vectored multi-component vaccine against porcine reproductive and respiratory syndrome virus, porcine circovirus type 2 and swine influenza virus.Journal of General Virology. 2017 Nov 1. doi: 10.1099/jgv.0.000964. [Epub ahead of print],PMID:29091579. Scientific Meeting presentations: (1). Elankumaran S, Meng XJ.A Multicomponent Virus Vectored Vaccine for PRRS, PCVAD and Swine Influenza.2013 USDAAFRI & NRI Annual Project Director's Workshop. Oct 19, 2013, Chicago, IL. (2). Opriessnig T, P.G. Halbur, P. Gauger, J.Q. Zhang, Q. Chen, D. Tian, Y.Y. Ni, X.J. Meng, M. Tan. 2016. Update on novel experimental pig vaccine approaches.Proceedings of the 2016 North American PRRS Symposium. #S4, Dec 3-4, 2016. Chicago, IL. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The original specific objectives were (1) Develop and characterize recombinant sPIV3 vector entirely from cloned cDNA and further attenuate and replication restrict the vector using reverse genetics. (2) Develop and test recombinant sPIV3 (rsPIV3) that express GP5 and M genes of PRRSV, capsid gene of PCV2 and hemagglutinin (HA) of SIV as a multi component vaccine. We have successfully accomplished the ultimate original goal of the project which was to develop a trivalent vectored vaccine. However, after the death of the original PI Dr. Elankumaran Subbiah and the transfer of the project to the new PI Dr. X.J. Meng, we modified the original proposed approaches to acheive the ultimate goal, although the ultimate goal of the project remains the same. Specifically, for the original objective 1 (100% completed), instead of developing a sPIV3 viral vector as originally proposed by the late PI Dr. Subbiah, we developed a PRRSV-based viral vector, which has many more advantages compared to sPIV3 and other viral vectors. First, PRRSV uses a set of subgenomic mRNAs to express its viral proteins, this making it possible to deliver foreign antigen via generation of additional separate subgenomic mRNAs. Second, since PRRSV itself is an economically-important pathogen in pigs, thus the use of an attenuated PRRSV as live virus vaccine vector can provide protection against PRRSV in addition to the other intended pathogens. we developed a PRRSV-based vaccine viral vector by utilizing an attenuated strain of PRRSV to express the protective antigens from PCV2 and SIV as a potential trivalent vaccine against PRRSV, SIV and PCV2. We chose the overlap region between ORF1b and ORF2a to insert the foreign genes, since insertion in this site showed more genetic stability of the inserted foreign genes. To further increase the genetic stability of the inserted foreign genes, we also included about 40 nt sequences of TRS 5 and TRS 6 from PRRSV genome as suggested in a previous study. This recombinant vectored trivalent vaccine virus was based on an attenuated strain of PRRSV as the vaccine vector to express the capsid protein of PCV2b as well as the truncated hemagglutinin (HA) of swine influenza virus (SIV). We demonstrated that the recombinant vectored vaccine virus DS722-SIV-PCV2b is replication-competent, viable, and can replicate to a relatively high infectious viral titer in MARC-145 cells although it has a reduced growth ability compared to the parental backbone vector virus DS722. Therefore, we have successfully completed theobjective 1in developinga recombinant PRRSV-based viral vector and use it to develop avectored trivalent vaccine. For the original objective 2 (100% completed), as originally proposed, we conducted a protective efficacy study in specific-pathogen-free pigs of the recombinant vectored trivalent vaccine virus DS722-SIV-PCV2b. A total of 48 piglets at 3 weeks of age were randomly divided into 6 groups of 8 piglets per group. Piglets in each group were vaccinated intramuscularly with the recombinant vectored trivalent vaccine virus DS722-SIV-PCV2b, or with PBS as control. At 42 days post-vaccination (dpv), the pigs were challenged with one of three different pathogenic viruses, PRRSV strain VR2385, PCV2b strain NC16845, and SIV strain A/swine/Minnesota/1145/2007(H3N2), respectively. The results revealed that pigs vaccinated with the vectored trivalent vaccine virus DS722-SIV-PCV2b had significantly reduced lung lesions and viral RNA loads in sera and lung tissues after PRRSV challenge, and had partially-reduced PCVAD-associated lymphoid lesions and lower viral DNA loads in sera and lymphoid tissues after PCV2b challenge. Also, compared to non-vaccinated pigs, the DS722-SIV-PCV2b-vaccinated pigs had significantly reduced acute respiratory symptoms (body temperature and respiratory scores) after SIV challenge. The data showed that the vectored trivalent vaccine candiadte DS722-SIV-PCV2b provided good protection against PRRSV and partial protection against PCV2b and SIV. Overal Impact:The vectored trivalent vaccine candiadte DS722-SIV-PCV2b developed in this project may serve as a potential trivalent vaccine for further advanced development against three of the most economically-important swine pathogens. Additionally, the results from this projectalso shed light on exploring PRRSV as a potential live virus-based vaccine vector for vaccine developmentr against other importnat swine pathogens. Therefore, this project produced a promising novel live vaccine virus vector as well as a novel candidate trivalent vaccine against three important swine pathogens (PRRSV, PCV2 and SIV). Both objective 1 (100%) and objective 2 (110%) have been successfully completed, although an imporved PRRSV-based viral vector system (instead of the original propsoed sPIV3 vector system) was developed for objective 1. A peer-reviewed scientific paper from the results of this project has been published in Journal of General Virology, and the results were also presented at two scientific meetings.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Tian D, Sooryanarain H, Matzinger SR, Gauger PC, Karuppannan AK, Elankumaran S, Opriessnig T, Meng XJ. Protective efficacy of a virus-vectored multi-component vaccine against porcine reproductive and respiratory syndrome virus, porcine circovirus type 2 and swine influenza virus. Journal of General Virology. 2017 Nov 1. doi: 10.1099/jgv.0.000964. [Epub ahead of print], PMID:29091579.
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Progress 05/01/15 to 04/30/16
Outputs
Target Audience:
Nothing Reported
Changes/Problems:The Project Director, Dr. Elankumaran Subbiah, passed away in Sepetmber 2015. Dr. Subbiah's postdoc associate who was working on the project also left Virginia Tech immediately after Dr. Subbiah's passing. In November 2015, Dr. X.J. Meng assumed the role of the new Project Director for this project with only 6 months left before the end of the project. Becasue of the transfer of the project to a new PD and the departure of Dr. Subbiah's postdoc working on the project, the immediate work the new PD's team did was to reproduce the SPIV reverse genetic system generated from Dr. Subbiah's previous NIFA AFRI annual report. Unfortunetly, the new PD's team found that the viable progeny viruses SPIV and SPIV-eGFP previosuly reported in the NIFA AFRI annual report by the former PD are likely an artifact. In lieu of this finding, we now have to start the project essentially from the scratch. We have requested a 1-year no-cost extension from NIFA AFRI so that we can continue to work on the project. We will continute the effort of rescuing the SPIV from a new reverse genetic system by reconstructing the three support plasmids and verifying the full-length clone by sequencing. The new PD realizes that he does not have much time left during the 1-year no-cost extension of the project, and therefore, if the SPIV reverse genetic system cannot be established, the new PD plans to execute an alternative strategy to achieve the propsoed objective which is to develop a trivalent vaccine against PRRSV, PCV2, and SIV. The alternative strategy is to use PRRSV as a vector to express antigens from PCV2 and SIV. PRRSV reverse genetic system is well established in the new PD's lab, and foreign antigens have been expressed in PRRSV vector successfully in the PD's lab. This alternative strategy represents a change from the original proposal but the end result woudl still be the same, whcih is to develop a trivalent vaccine candidate. What opportunities for training and professional development has the project provided?A graduate student and a postdoc associate are being trained in association with this project. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?Since the original PD passed away untimely in Sepetmber 2015, since there was only 6 months left for the project after the transfer of the project to the new PD, and since the new PD failed to reproduce the critical reagent of the project, the SPIV reverse geentic system, we now must start everything from the scratch. The plan for the new PD during the 1-year no-cost extension period is to reconstruct the three support plasmids and verify the full-length clone by sequencing, and hopefully rescue the SPIV so that the vaccien candidates can be constructed. First we plan to re-construct all three current support plasmids in pcDNA3.1. Although we found that failure to sufficiently express target proteins from support plasmids was the one reason leading to the inability to rescue the SPIV reverse genetic systems, we can not exclude other possible causes such as the authenticity of the full-length clones. Therefore, we plan to will determine the complete genomic sequences of pBR322SPIV. The ultimate goal is to establish a SPIV reverse genetic systems so that the vaccine candidates can be made. We realize that it is possible we may not be able to successfully rescue SPIV from the reverse geentics system at all since the former PD had tried for nearly 4 years with no apparent success. Therefore, we are planning an alternative strategy to achieve the propsoed objective in the project which is to develop a trivalent vaccine against PRRSV, PCV2, and SIV. So our alternative strategy is to use PRRSV as a vector to express antigens from PCV2 and SIV. PRRSV reverse genetic system is well established in the new PD's lab, and foreign antigens have been expressed in PRRSV vector successfully in the PD's lab. Therefore, we plan to use this alternative strategy if we are unable to successfully rescue SPIV system.
Impacts
What was accomplished under these goals?
The Project Director, Dr. Elankumaran Subbiah, passed away in Sepetmber 2015 with 6 months remaining before the end of this project. Dr. Subbiah's postdoc associate who was working on the project left Virginia Tech immediately after Dr. Subbiah's passing. In November 2015, Dr. X.J. Meng assumed the role of Project Director for this project. Becasue of the transfer of the project to a new project director and the departure of Dr. Subbiah's postdoc working on the project, the immediate objective after Dr. Meng took over the project in November 2015 was to reproduce the SPIV reverse genetic system generated from Dr. Subbiah's group before constructing the vaccine candidates. (1). Failure to rescue viable progeny viruses SPIV and SPIV-eGFP from full-length clones. The full-length clones pBR322SPIV (containing synthesized full antigenome sequence of wild type SPIV strain Tx-81), and pBR322SPIV-eGFP (pBR322SPIV inserted with eGFP ORF between P and M genes) were individually co-transfected with three supported plasmids (pGEMT-N, pGEMT-P, pcDNA3.1-L) into permissive cells according to Dr. Subbiah's former postdoc's protocol, however, no SPIV specific CPE or IFA or GFP signals was observed. We also tried repeatly with other published rescue protocols but could not resuce the viruses. The results showed that although the "SPIV-eGFP" causes cell death quickly, no GFP signal was observed. SPIV viral RNA was not detected by RT-PCR assay. These data indicated that the "SPIV-eGFP" was not viable, and the cell deaths ("CPE") previosuly observed was likely due to residual MVA virus as it was used as the helper virus to express T7 polymerase during transfection. (2). The three support plasmids did not efficiently express target proteins as verified by IFA assay with specific antibodies. After we fialed to confirm the viability of the SPIV reverse genetic system as shown in previous NIFA annual report, we began to dissect what causes the failure to rescue the SPIV clone. We first produced the peptide-specific antibodies against N, P, L proteins respectively, and demonstarted that the produced antibodies recognized its respective proteins well. Subsequently, by using the peptide-specific antibodies, we attempted to verify the three support plasmids (pGEMT-N, pGEMT-P, pcDNA3.1-L) used for the reverse genetic system rescue by IFA. However, after transfection into BSR or BHK cells, none of the three plasmids showed efficient expression of target proteins. Therefore, based on the results we obtained after the transfer of the project to the new project director Dr. X.J. Meng since November 2015, we concluded that the viable progeny viruses SPIV and SPIV-eGFP rescued from the reverse genetic systems as stated in previous annual report by the former project director are likely an artifact. In lieu of this finding, we now have to start the project essentially from the scratch. We have requested a 1-year no-cost extension from NIFA AFRI so that we can continue to work on the project.
Publications
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Progress 05/01/14 to 04/30/15
Outputs
Target Audience:Swine farmers of USA. Changes/Problems:During the course of our research, the immunogenicity of PRRSV GP3 and GP4 over GP5 of PRRSV was demonstrated by the Co-PI (Meng) and other groups. So, we have revised the strategy to clone GP3/4 instead of GP5. The relative merit of this approach needs to be determined by animal studies. What opportunities for training and professional development has the project provided?Training of a full-time research scientist was accomplished under this project and is continuing. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?The results of the research will be published in peer reviewed journals.
Impacts
What was accomplished under these goals?
Project Title: A Multicomponent Virus Vectored Vaccine for PRRS, PCVAD and Swine Influenza Objectives: The main objective of the present study is to generate a non-pathogenic recombinant swine parainfluenza virus (sPIV3) by incorporating porcine circovirus (PCV2a) capsid gene, porcine reproductive and respiratory syndrome virus (PRRSV) M, GP3 & GP4 genes and HA gene of swine influenza through reverse genetics approach. The recombinant virus generated in the present study is anticipated to protect pigs against four most economically important viral diseases in the global swine industry. Although effective vaccines are available for porcine circovirus 2 (PCV2), the vaccines for PPRS virus (PRRSV) and swine influenza virus (SIV) suffer from lack of efficacy against heterologous strains for multiple reasons including antigenic variation. Genetically engineered temperature sensitive (ts) mutation in the polymerase gene of recombinant sPIV3 with altered fusion protein cleavage site to that of elastase will make the virus replication restricted to the respiratory tract. Due to the modular nature of the genome, we can insert foreign genes in 5 positions in the genome. The live virus-vectored vaccine will provide a DIVA (differentiation of vaccinated and infected) platform and will be evaluated for efficacy in a triple challenge model in pigs. Accomplishments: We have successfully developed an infectious clone of sPIV3 (Tx-81) antigenome with unique restriction sites for insertion of foreign genes. We have inserted the enhanced green fluorescent protein (eGFP) gene between P and M genes sPIV3 (Tx-81) virus using a unique Not I site. The recombinant sPIV3-eGFP virus expressing eGFP protein was rescued through reverse genetics using the infectious clone. The pBR322sPIV3cDNA full-length infectious clone (pBR322sPIV M/GP3/GP4/Cap/HA) is engineered to carry PCV-2b Cap, PRRSV M, GP3 & GP4, swine influenza virus HA gene. The inserted heterologous genes for their reading frames and matched rule of 6 were verified through nucleotide sequencing. The subclones with L gene ts mutation and F-Elastase cleavage have been constructed and the replacement of the original sequences within these mutations on the full-length recombinant cDNA infectious clone pBR322sPIV M/GP3/GP4/Cap/HA is in progress. Outcomes and Impacts: A sPIV3 infectious cDNA clone expressing enhanced green fluorescent protein has been developed. The recombinant sPIV3-eGFP was rescued entirely from a cloned cDNA copy of the viral genome. We have also rescued the wild type sPIV3 without any foreign gene inserts using the full-length infectious clone. The successful rescue of recombinant sPIV3-eGFP demonstrates the feasibility of inserting heterologous genes into sPIV3 and expressing them without affecting the virus replication. Any unresolved challenges: During the course of our research, the immunogenicity of PRRSV GP3 and GP4 over GP5 of PRRSV was demonstrated by the Co-PI (Meng) and other groups. So, we have revised the strategy to clone GP3/4 instead of GP5. The relative merit of this approach needs to be determined by animal studies. Elevator pitch: A system to recover swine parainfluenza virus expressing eGFP protein from a cloned cDNA copy of the virus has been developed using a technique called reverse genetics. The rescue of sPIV-eGFP virus demonstrates the feasibility of expressing other heterologous genes through sPIV3. This recombinant virus serves as the marker for active virus replication. This will help us to deliver multiple vaccines in a single platform, thereby reducing the cost of vaccination in swine industry. Also, this vector platform can be used for delivering multiple vaccines to different species of animals and humans as well.
Publications
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Progress 05/01/13 to 04/30/14
Outputs
Target Audience:
Nothing Reported
Changes/Problems: We may make additional immunizing antigens for PRRSV and SIV based on published literature and insert into sPIV3 for vaccination. What opportunities for training and professional development has the project provided? A postdoctoral research scientist was trained in teh project since inception until middle of 2013. A second postodtoral research scientist is being trained in teh project at present. How have the results been disseminated to communities of interest? Results are yet be communciated to the communities of interest. However, two book chapters on bovine and swine parainfleunzavirus 3 have been published by the PI. What do you plan to do during the next reporting period to accomplish the goals? In the next reporting period, teh following goals will be accomplished: 1. The elastase and ts mutants of sPIV3-Tx81 will be recovered by reverse genetics and characterized 2. The wild type and elastase and ts mutants of sPIV3-Tx81 will be characterized in vitro and in vivo. The patheogenicty and stability studies will be completed in pigs. 3. The immunizing antigens will be cloned into the full-length infectious clone and expression verified 4. ELISA methods for DIVA will be standardized.
Impacts
What was accomplished under these goals?
We have sucesssfully completed AIm 1 of teh project. A full length infectious clone of sPIV3 strain Tx81 (sPIV3-Tx81) has been made . The vector has been successfully recovered by reverse genetics. Attenuating mutations are engineered in teh vector and vector is made available for engineering foreign genes. Additionally, we are inporcess of developing an additional sPIV3 vector using strain ISU92. In aim 2, we have completed teh subcloning of respective immunizing antignes of PRRSV, PCV2 and Swine infleunza. We have alos made additional antigen constructs for SIV and PRRSV using other genes. Construction of PRRSV hypoglycosylated GP5 of PRRSV has been completed. We are in the process of commencing our pathogenicity and stability studies of teh sPIV3 vactor. Aim 3: We have successfully recovered teh sPIV3-Tx81 vector by reverse genetics. Accomplished deliverables: 1. A full-length infectious clone of sPIV3-Tx81 has been developed 2. Methods for recovering ifnectious virus from complementary DNA copuy of full-length genome of sPIV-3 has been developed 3. Subclones of immunizing antigens for PRRSV, PCV-2 and SIV were made.
Publications
- Type:
Book Chapters
Status:
Published
Year Published:
2013
Citation:
Elankumaran, S., 2013. Bovine Parainfluenza virus 3. In: Mononegaviruses of Veterinary Importance, Volume 1. Pathobiology and Molecular Diagnosis, (Munir, M., Ed.), CABI, England, Nov, 2013, pp, 117-140.
4. Elankumaran, S., 2013. Swine Parainfluenza virus 3. In: Mononegaviruses of Veterinary Importance, Volume 1. Pathobiology and Molecular Diagnosis, (Munir, M., Ed.), CABI, England, Nov, 2013, pp, 141-159
Kumar, S. R. P., M. Biswas and S. Elankumaran. 2014. Pandemic H1N1 influenza A virus induces a potent innate immune response in human chorionic cells. Viral Immunol. 27: 129-137.
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