Difference between revisions of "Part:BBa K3504009"

Revision as of 17:58, 26 October 2020

Alphavirus replicon NSPs- Equine Encephalitis virus

NOTICE: Parts in our range for this season have been created as a part of our Phase I design of our project. These parts HAVE NOT been tested or characterized in the lab due to COVID-19-related precautionary measures. We have enriched our new parts pages with data from literature and results from our modeling and simulations. If you are intending on using this part or others in our range, please keep in mind these limitations and update these parts with data from your experimentation. Feel free to reach us at: igem.afcm@gmail.com for further inquiries.

Part Description

A composite of parts (BBa_K3504000,BBa_K3504001,BBa_K3504002,BBa_K3504003) and CMV promoter which form ,as a unit, the main constituent of alphavirus replicon which as a whole can give the circuit self-replicating ability

Usage

Alphavirus genomes encode four non-basic proteins, nsP1–4, which are translated from the genomic RNA and interact with host factors to form replicative enzyme complexes. These non-structural proteins are translated as a polyprotein that self-cleaves into separate proteins, which assemble into a replicase complex. The replicase then directs replication and amplification of our vaccine inside the Myocytes.

Characterization

Figure 1.in vitro replicon enhancement method: Using transfected Jurkat cells with replicon RNA which was encoded for mCherry and grown in cell culture under the SGP. Then during serial passage as shown in the flow cytometry histograms, the top 20 percent of mCherry were sorted around every 10 days. this has resulted that cells expressing higher levels of repoter gene were enriched. finally the 5th sort cells were seperated from the rest for replicon sequencing.

Figure 2.Mutation identification:Positive mCherry cells was reverse transcribed to cDNA,after that Nsp1 to 4 and the SGP were magnified by seven pairs of primers and amplicons and later on engineered into DNA of the plasmid and changed into E.coli to be amplified. The lower left part schematic illustrates roughly the locations of point mutations in the 5th sort in NSP2 & NSP3.

We have made simulations using mathematical modelling techniques to characterize the increase in expression when using replicons over traditional methods while also providing simulations that Characterize the function of replicon by eliciting an increased response in both Dendritic Cell population and T-Helper Population.

We also provide Functional characterization of replicons from literature. As This figure shows HIVA-specific T-cell responses after a single immunization with clinical-grade plasmid DNA vaccines between DREP.HIVA and pTHr.HIVA in individual mice immunized by 10 μg of them all of which complies with our mathematical modelling & simulations

Figure 3. Functional characterization of replicons from literature. This figure shows HIVA-specific T-cell responses after a single immunization with clinical-grade plasmid DNA vaccines between DREP.HIVA and pTHr.HIVA in individual mice immunized by 10 μg of them.

Figure 4. Mathematical modelling simulation of Number of positive strand RNA in traditional vaccination presented by the graph to the left vs with the use of self amplifying replicon on the right.

Figure 5. Mathematical modelling simulation of T-helper cells population response according to logfc in response to DREP vaccine on the left vs traditional DNA vaccine on the right.

Figure 6. Mathematical modelling simulation of Dendritic Cells population response according to logfc in response to DREP vaccine on the left vs traditional DNA vaccine on the right.

Improvements

Using information in literature we were able to increase the replicon cloning and functional ability by adding G110G, G763R Mutation to NSP2 and adding K94E, S243G,E255D,V305M Mutation to NSP3

Figure 1. Nsp2 G110G Amino acid mutation.

Figure 2. Nsp2 G763R Amino acid mutation.

Figure 3. Nsp3 K94E Amino acid mutation.

Figure 4. Nsp3 S243G Amino acid mutation.

Figure 5. Nsp3 E255D Amino acid mutation.

Figure 6. Nsp3 V305M Amino acid mutation.

References

Maruggi, Giulietta, et al. “Engineered Alphavirus Replicon Vaccines Based on Known Attenuated Viral Mutants Show Limited Effects on Immunogenicity.” Virology, vol. 447, no. 1–2, Dec. 2013, pp. 254–264, 10.1016/j.virol.2013.07.021. Accessed 25 Sept. 2020.

Li, Y., Teague, B., Zhang, Y., Su, Z., Porter, E., Dobosh, B., ... & Weiss, R. (2019). In vitro evolution of enhanced RNA replicons for immunotherapy. Scientific reports, 9(1), 1-10.

Nordström, E. K., Forsell, M. N., Barnfield, C., Bonin, E., Hanke, T., Sundström, M., ... & Liljeström, P. (2005). Enhanced immunogenicity using an alphavirus replicon DNA vaccine against human immunodeficiency virus type 1. Journal of general virology, 86(2), 349-354.

openarchive.ki.se/xmlui/bitstream/handle/10616/42305/Thesis_Maria_Lisa_Knudsen.pdf;sequence=3

Sequence and Features