Figure 7.

Genetic Stability of nOPV2 in Human Volunteers

(A) Top panel: schematic of vaccine administration and analysis in humans. Fifteen volunteers were inoculated with nOPV2 by oral route with 10⁶ cell-culture infectious doses 50% (CCID₅₀). Viruses were isolated from stool samples at 8–18 days post-vaccination. Mean and range of variant frequencies observed in samples from days 8 and 18 for nOPV2 in this trial are compared with data obtained previously for Sabin2 at 14 days post-vaccination with trivalent OPV (tOPV in polio vaccine naive human subjects; Stern et al., 2017). Bottom panel: to highlight regions of diversity across individuals, we used full-genome sequences obtained from all EES samples to calculate the sum of Shannon’s entropy across vaccines along the genome in 30-nucleotide windows. 5′Cre^a (U123C or G179, mean: 0.68; range: 0.19–1.00), domIV^b (nt459, mean: 0.07; range: 0.00–0.40), domV^c (any mutation UA to CG that increases domV structural stability, mean: <0.005, the limit of detection); 3D^{e & f} (any mutation in 3D^pol at positions HiFi K38R or Rec D53N, mean: <0.005). ^g95% extreme value test from 10,000 randomly sampled 30mers. We also observed significant increase in Shannon’s entropy in capsid proteins VP4 (position 41) and VP1 (positions 33 and 143).

(B) Positions for the most frequent mutations identified from shed viruses: domV A481G in Sabin2 (Evans et al., 1985); U123C (5′ cre), U459C (domIV), and A2969G (VP1-I143V) in nOPV2.

(C) Schematic representation for virulence test of reconstructed viruses (top panel). Mutations accumulating at high frequencies in virus isolated from vaccinees (B and Stern et al., 2017) were engineered into infectious molecular clones of Sabin2 or nOPV2 to generate Sabin2 (A481G) and nOPV2 (U123C, U459C, and I143V), respectively. Virulence (PD₅₀) of reconstructed viruses was determined with the Tg66-CBA mouse model after intraspinal inoculation (bottom panel).