Ascetia Life

Introduction


The aim of this study is the assessment viral clearance and anti-inflammatory properties in the context of viral stimulation in and the determination of the possible inhibitory effects on viral replication.


The initial experimental plan included three distinct elements:

  • Determination of anti-inflammatory effects of JTX-M01 both +/- a viral background
  • Assessment of cell viability in the presence of JTX-M01
  • Assessment of a potential rationale for the mode of action

Anti-inflammatory properties of JTX-M01 (“Ascetia”)


Vero E6 Cells were cultured in DMEM + 10% FBS and incubated at 37°C and 5% CO2. When cells reached 80% confluence after the first passage, cell viability and cell density were measured by trypan blue exclusion assay. A cell suspension containing approximately 1 x 104 cells was transferred into each well of a 96-well plate and cultured with 150 uL of DMEM containing 50  uM of H2O2  and incubated at 37°C and 5% CO2 for two hours. After incubation, 225 ug of JTX-M01 was added to the culture for assessment at 24 hours. At the 24 hour time point, cell culture supernatant was aspirated and analysed on a ProteinSimple ELLA System. We measured IL-6, IL-8 and TNF-a cytokine responses in this way. 


Cell viability and anti-inflammatory effects of JTX-M01 within a viral background


A Vero E6 cell suspension containing approximately 1 x 104 cells was transferred into each well of a 96-well plate and cultured with 150 uL of DMEM containing different titrations of JTX-M01 (500, 250, 125 and 61.25 ug) and cultured for two hours at 37°C and 5% CO2


Following initial incubation, transport fluid that previously had been used to transport a nasopharyngeal swab was added to give a calculated MOI = 0.1. The plates were cultured for a further two hours after which the media was aspirated and replaced with DMEM containing the appropriate titrations of JTX-M01. At the 24 hour time point, cell viability was measured by trypan blue exclusion assay and cell culture supernatant was analysed for IL-1b, IL-6, IL-8 and TNF-a using the ProteinSimple ELLA System.


Figure 1: Experimental data showing significant differences between untreated controls and cells treated with 225 ug of JTX-M01. Graphs show mean ± standard deviation (* indicates p<0.05, student’s t-test).


Results


Anti-inflammatory properties of JTX-M01

At the 24-hour timepoint, we observed a significant attenuation of the analytes when compared to untreated controls.

Figure 1: Experimental data showing significant differences between untreated controls and cells treated with 225 ug of JTX-M01. Graphs show mean ± standard deviation (* indicates p<0.05, student’s t-test).


Anti-inflammatory effects with viral background

At the 24-hour timepoint, we observed a significant attenuation in IL-6, IL-8 with a marked difference in TNF-a. Significant differences were observed at 500 ug and 250 ug, while the remaining titrations showed no significant differences.

Figure 2: Experimental data showing significant differences between untreated controls and cells treated with 500ug of JTX-M01. Graphs show mean ± standard deviation (* indicates p<0.05, student’s t-test; # indicates p<0.1, students t-test).


Cell viability with viral background

At the 24-hour timepoint, we observed a significant increase in the number of viable cells when treated with JTX-M01 for the 500 ug and 250 ug titrations. The remaining titrations showed no difference in cell viability (Fig. 2).



Figure 3: Experimental data showing significant differences between untreated controls and cells treated with JTX-M01. Graphs show mean ± standard deviation (* indicates p<0.05, student’s t-test).


We have shown attenuation of key proinflammatory cytokines including IL-6, IL-8, TNF-a +/- a viral background. We have also shown a significant increase in the number of viable cells when compared to the untreated group. Results were comparable to healthy cells (Fig. 4: Control).


These results indicate that JTX-M01 has potential as a candidate therapeutic for SARS-CoV-2 and warrants further investigation.


Further work will include the potential mode of action and target as well as more detailed cytotoxicity experiments using a CCK-8 assay with further titrations of JTX-M01. 


References

Draft landscape of COVID-19 candidate vaccines (2020). Available at: https://www.who.int/publications/m/item/draft-landscape-of-covid-19-candidate-vaccines (Accessed: 21 July 2020).


Hoffmann M, Kleine-Weber H, Schroeder S, et al. SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell. 2020;181(2):271-280.e8. doi:10.1016/j.cell.2020.02.052


Kudoyarova-Zubavichene NM, Sergeyev NN, Chepurnov AA, Netesov SV. Preparation and use of hyperimmune serum for prophylaxis and therapy of Ebola virus infections. J Infect Dis. 1999;179 Suppl 1:S218-S223. doi:10.1086/514294

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