Fig. 4: The immunization of Mpx-V3 immunogen confers protection to intranasal challenge of Vaccinia virus in BALB/c mice.

a Schematic of an intranasal challenge study with VACV Western Reserve strain (n = 8) for all the groups (n = 6 for body weight, survival, and n = 2 for titer). b, c The prime-boost immunized mice were intranasally challenged with 50 μL of 1 × 10⁷ PFU/mL of vaccinia virus. After the challenge, changes in b body weight and c survival were recorded every day for 2 weeks. The data shown here is mean ± SD from one challenge study, having n = 6 in each group’s and for survival, individual animal record was plotted. d Graphical representation of behavior scores of infected mice. Animals were scored based on the clinical symptoms exhibited by them into 1–10. Scoring with 0 = no alteration in behavior, 1 = no restriction of movement; blink frequently; no body stiffening; no hind limb paralysis; 2 = dull; 3 = piloerection; 4 = shivering; 5 = hunched posture; 6 = restriction of movement; 7 = blink frequently; no body stiffening; no hind limb paralysis; 8 = restriction of movement; body stiffening; no hind limb paralysis; 9 = restriction of movement; eyes closed; 10 = body stiffening; hind limb paralysis, sometimes tremor. The data shown here is each group’s mean ± standard deviation. e Heatmap of temperature changes observed after infection up to 14 days. All the groups, including normal control, adjuvant control, and Mpx-V3-immunized groups, were assessed for change in body temperature up to 14 days post-infection by the VACV. The data shown here is each group’s mean ± standard error of mean (SEM). f, g Vaccinia virus titer was determined in the lung and trachea samples harvested at 5 days post-infection, respectively (n = 2 mice). Upon harvesting, organ samples were weighed, homogenized, sonicated, and centrifuged. Later, aliquots of the supernatant from the samples were stored for titer calculation in Vero cells. Confluent Vero cells were infected with different dilutions of organ supernatant for 2 h, and later the cells were washed and layered with 2% CMC in 2% FBS containing DMEM for 48 h. Then, the media was removed, cells were fixed with 4% paraformaldehyde, and stained with 0.5% crystal violet. Plaques were counted from the plates to calculate virus titer in PFU/g. The values plotted are from biological replicates as mean ± SEM, statistical analysis between the two groups was performed by unpaired, two-tailed Student’s t-test.