Neutralization data were fit to Tx=1/1+(D/ID50)

Neutralization data were fit to Tx=1/1+(D/ID50). Here Tx is the number of foci normalized to the number of foci in the absence of plasma on the same plate at dilution D and ID50 is the plasma dilution giving 50% neutralization. may evolve immune escape of vaccines and enhanced escape of Delta immunity, with implications for vaccine breakthrough and reinfections. during expansion in VeroE6 cells and likely confers moderate neutralization escape (36). E484K was first detected on day 6 (Fig 2B). This mutation persisted at day 20 and 34 but was replaced with the F490S substitution starting on day 71 when the K417T mutation was also detected. The N501Y mutation was detected in the virus isolated on day 190 post-diagnosis. Mutations were clustered in the RBD, including K417T, F490S, and N501Y in the day 190 viral isolate (Fig 2C, see Fig S2 for mutations per timepoint). Among the RBD mutations in the day 190 Rosmarinic acid isolate, K417T is found in the Gamma variant, and F490S is found in the Lambda variant. Among NTD mutations, T95I is found in Mu, and R190K is at the same location as the R190S in Gamma. N501Y is found in Beta, among others. Interestingly the Omicron variant has emerged as this work was being revised and has mutations at many of the same sites as the evolving virus described here, also shared with some of the other variants (https://covdb.stanford.edu/page/mutation-viewer/#sec_b-1-351). This includes the D796Y mutation only found in Omicron among the major variants (Fig 2B). We tested three of the isolates for neutralization: viruses outgrown from day 6 and day 20 swabs (designated D6, D20) representing viruses from early infection, and virus outgrown from the Rosmarinic acid day 190 swab (D190), after substantial evolution. Neutralization of the D6, D20, and D190 isolates by self-plasma was low at the early timepoints (Fig 2D). However, neutralization of D6 and D20 was evident by plasma sampled from day 190 and was more pronounced in the plasma sampled from day 216. The D6 isolate was the most sensitive to neutralization by day 216 plasma. Neutralization declined for D20 and further for D190, suggesting sequential evolution of escape (Fig 2D). The ancestral virus and Beta and Delta variants were also tested for neutralization using day 216 plasma. Neutralization was lower for all three non-self strains relative to self-derived virus. The strongest neutralization was of ancestral virus. Delta was neutralized to a lesser degree, and Beta was not detectably neutralized (Fig 2D). We also tested the D6, D20, and D190 isolates against plasma from other convalescent participants infected with ancestral virus. Neutralization of D190 by ancestral infection elicited plasma was decreased dramatically relative to D6, with FRNT50 for D190 being 9.3-fold lower despite the presence of the E484K mutation in D6 (Figure 2E). The difference was smaller between D190 and D20 (5.1-fold, Figure 2F), consistent with evolution of some neutralization escape in D20 relative to D6. We also tested neutralization of D190 virus using Pfizer BNT162b2 vaccinated participants (Table S4 lists participant characteristics). BNT162b2-elicited plasma neutralization capacity was decreased 5-fold against D190 relative to ancestral virus with the D614G mutation (Fig 2G). We compared neutralization of Beta, D6, D20, and D190 on a subset of remaining BNT162b2 plasma samples from 5 participants 5C6 months post-vaccine, where neutralization declined to relatively low levels. Despite this limitation, neutralization showed a pattern consistent with the other results: D190 neutralization escape was very similar to Beta, while D6 and D20 showed no escape from Mmp9 BNT162b2 elicited neutralization (Fig S3). A 5-fold reduction is less than the fold-drop we previously obtained for the Beta variant with convalescent plasma from previous infection (9) and confirmed below. Moreover, BNT162b2 is effective against Beta, albeit with some reduction. Therefore, these results are consistent with substantial but incomplete escape of D190 from Rosmarinic acid BNT162b2. We next assessed the serological distance between D190, D6, the ancestral strain, Beta, Delta, and Alpha variants. We tested against convalescent plasma obtained from participants infected with ancestral strains or Beta or Delta variants. Neutralization by ancestral plasma immunity declined 8.8-fold relative to ancestral virus for the D190 isolate (Fig 2H), similar to the Beta variant. In contrast, there was only a 1.6-fold decline for the D6 isolate (Fig 2I). D190 virus was neutralized relatively well by Beta variant infection elicited plasma, with a 2.6-fold reduction (Fig 2J). This was similar to Rosmarinic acid D6 neutralization, which was reduce 2.3-fold relative to Beta.