Progress 10/01/20 to 09/30/21
Outputs
Target Audience:Scientists conducting research on PRRSV vaccine development. Biologics companies interested in PRRSV vaccines. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?
Nothing Reported
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?As mentioned above, our plan for the next reporting/funding period is to examine expression of the viral chimeric protein by immunoblotting with epitope-specific antibodies. Once the expression of the GP4-GP3-GP2 chimeric protein expression is confirmed, we will then purify the protein/nanoparticle by anion-exchange chromatography followed by size-exclusion chromatography. The purity of the protein preparation will be checked by coomassie blue staining of the gels and the protein will be quantified biochemically. Subsequently, we will plan to conduct animal immunization studies to examine if neutralization antibody responses are elicited in the animals. Again, both GP4-GP3-GP2 and GP5-M chimeric proteins will be purified and animal immunization studies will be conducted simultaneously.
Impacts
What was accomplished under these goals?
Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most economically important pathogen of swine. The swine industry in the USA and around the world suffers significantly due to the diseases caused by this virus. The currently available PRRSV vaccines are not very efficacious and do not confer protection to pigs infected with heterologous PRRSV. One of our major goals of this project is to develop an innovative vaccine platform that would allow higher levels of vaccine efficacy and confer homologous as well as heterologous protection to infected animals. Towards achieving this goal, we proposed to first use the well-characterized ferritin-based nanoparticles to display multiple copies of the surface glycoproteins of PRRSV. The expectation from this ferritin-based nanoparticle construct is that it would induce higher levels of antibodies that would neutralize PRRSV and therefore would serve as an excellent PRRSV vaccine candidate. In the previous funding period, we constructed such a nanoparticle but expression of the viral surface glycoprotein constructs could not be clearly established due to lack of a specific antibody that we could use to probe the viral glycoprotein expression. Although we were able to detect some nanoparticle structures under transmission electron microscope, biochemical confirmation of the viral protein expression was of paramount importance to further our studies for vaccine development. Therefore, we have now constructed another set of vaccine candidates in which the viral glycoproteins were fused in-frame with at least two different epitope tags to allow us to detect expression of the viral glycoproteins by epitope-specific antibodies. Specifically, we commercially synthesized a gene encoding the sequences of FLAG and His-8 epitope tags at the amino-terminus of the sequences of the viral glycoproteins GP4 ectodomain, followed by flexible linker amino acid sequences, followed by the ectodomain of the viral glycoprotein GP3, followed by another flexible linker amino acid sequences, followed by the ectodomain sequences the viral glycoprotein GP2. This construct is now being cloned into the ferritin nanoparticle construct. Once the authenticity of the construct is confirmed by nucleotide sequencing, we will examine expression of the chimeric viral glycoprotein construct and then proceed for studies proposed in the project to examine of neutralizing antibody responses are generated when animals are injected with the vaccine candidate. Our expectation is that these studies will be conducted in the next funding period. Since we were unable to detect expression of the chimeric viral glycoprotein construct with the previous construct, we have also commerically synthesized a new GP5-M gene containg the FLAG and His-8 epitope tags. The ectodomain of GP5 was appended to the ectodomain of M protein separated by flexible amino acid linker sequences This gene will be cloned into the ferritin-expressing vector in a manner similar to the GP4-GP3-GP2 chimeric gene construct described above. We plan to examine the expression of this GP5-M chimeric protein expression in the enxt funding period and conduct immunization studies following that.
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Progress 10/01/19 to 09/30/20
Outputs
Target Audience:Scientists conducting research on PRRSV vaccine developmemt. Changes/Problems:Due to SARS-CoV-2/COVID-19 pandemic, we experiencedsignificant disruptions in our planned studies and as a result, our progress has been significantly slowed down. But it is our expectation that we will be able to catch up with our progress in the coming andsubsequent project funding years. Currently, no major changes to the project are expected/planned. What opportunities for training and professional development has the project provided?The student was trained in this novel approach to generate nanocages for PRRSV vaccine development. 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?For the next reporting period, we plan to use the constructs to first examine expression of the PRRSV glycoproteins. Subsequently, we will examine if nanocages are generated and secreted into the culture supernatants of transfected cells. In the subsequent years, wewillpurify the nanocages to conduct animalinoculation studies to examine if immune response are generated against the PRRSV protein.
Impacts
What was accomplished under these goals?
Porcine reproductive and respiratory syndrome virus (PRRSV) is a major swine pathogen that inflicts significant economic burden to the swine industry in the USA and around the world. Currently, although some PRRSV vaccines are available, they are not efficacious and the swine industry still suffers losses of hundreds of millions of dollars annually. Our maingoal here is to use an innovative approach to develop a novel PRRSV vaccine that would be safe and highly efficacious. To achieve this broad goal, we proposed two objectives. In the firstobjective, we proposed to generate nanocages (spherical hollow protenaceous particles) containing various surface glycoproteins of PRRSV fused to the bacterial ferritin protein, which is known to form the nanocages. The expectation is that the viral glycoproteins will be displayed on the surface of the ferritin nanocages. When these nanocages are injected into pigs, immune response against the PRRSV glycoproteins will be generated that will protect the animals from PRRSV-induced diseases in challenge experiments. Toward this goal, in our first objective, we have generated several plasmidconstructs that encodethe three minor glycoproteins of PRRSV (GP2a, GP3, and GP4) fused in frame at the amino-terminus of the bacterial ferritin protein. The authenticity of these plasmids have now been confirmed by nucleotide sequencing. These constructs are currently being examined for expression of the encoded proteins. However, due to the SARS-CoV-2/COVID-19 pandemic, our progress in this project has been slow. Our expectation is that we will move faster in the second year of the project to examine the expression of the proteins and subsequent studies as proposed in our project. These experiments were conducted by one graduate studentwith 0.4 FTE. Thestudentwasintroduced to thisnew approach to generate a novel vaccine against PRRSV.
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