3.11  Molecular Dynamics Simulation
The stability and dynamics of the designed vaccine construct ensemble docked to innate immune receptors were disclosed through 100-ns of MD production run and interpreted through the root mean square deviation (RMSD) [81], root mean square fluctuation (RMSF) 82, the radius of gyration (Rg) [83] and beta factor (β-factor) [84] assays as depicted in Figure 10 .
Fig. 10 MD trajectories based calculation of Cα RMSD (top left), RMSD (top right), Rg (bottom left), and β-factor (bottom right).
The average Cα atomic distance over 10,000 frames of TLR3-MEPVC and TLR4-MEPVC was decoded through RMSD assay. An average RMSD of 3.44 Å (maximum, 6.30 Å) and 3.36 Å (maximum, 5.22 Å) were estimated for TLR3-MEPVC and TLR4-MEPVC, respectively. The TLR3 and TLR4 receptors are observed more compact than the MEPVC and as a result, continuous movements of the vaccine construct through its length are noticed at the exposed regions though rooted stable at the docked position. This seems to be responsible for bringing small structural deviation as probed by RMSD. Visual inspection of the trajectories illustrated no major global and local secondary structure conformation changes in the receptor TLR3 and TLR4 structure. The MEPVC movements with respect to the TLR3 are shown at different snapshots as illustrated in Figure 11 . The MEPVC seems responsible for the deviations in the receptor TLR4 at positions Glu1-Ser79, Hie540-Asn614, Gln1144-Gln1209, Gln1347-Pro1371, and Lys1489-Lys1491. Continuous flexibility of the loops of MEPVC exposed region is observed and is highly flexible responsible for the movements of the MEPVC at the docked site though rooted stably (Figure 12 ).
Fig. 11 Binding mode of MEPVC at different snapshots at the TLR3 docked side.
Fig. 12 Binding mode of MEPVC at different snapshots at the TLR4 docked side.
The second statistical parameter computed for both complexes was the root mean square fluctuations (RMSF) that demonstrate average dynamical residue fluctuations over a specific length of time. The mean RMSF concluded for TLR3-MEPVC system is 2.1 Å whereas, an average RMSF of 2.03 Å for TLR4-MEPVC was demonstrated. Majority of the receptors residues are showing less variability and are satisfactory stable with mean RMSF < 3 Å. The residues range mentioned above were reported to have higher RMSF values and revealed fluctuating throughout the simulation time. These fluctuations are in all likelihood as an outcome of the moving adjuvant of the MEPVC. This is supported by the fact that the mean RMSF of the MEPVC is comparatively very high than that of receptors. The RMSF of MEPVC in TLR3-MEPVC and TLR4-MEPVC is 1.86 Å (maximum, 10.16 Å) and 7.45 Ǻ (maximum, 13.04 Ǻ). The receptors stability in both complexes is reaffirmed by radius of gyration (Rg) analysis that depicted stable plot with mean value of 33.97 Å (maximum, 35.62 Å) and 41.01 Ǻ (maximum, 41.65 Ǻ) for TLR4-MEPVC. The Rg findings are coherent with that of RMSD in interpreting systems stable behavior and compact nature of the receptors. Lastly, thermal beta factor (β-factor) indicated the same pattern of residues dynamics as shown by RMSF. An average β-factor derived from TLR3-MEPVC and TLR4-MEPVC is 121.34 Ų and 123.47 Ų, respectively.