156 – Selection of Nucleocapsid Mutations With Virologic Failure of Tenofovir/Lamivudine/Dolutegravir

Background

Tenofovir-lamuvidine-dolutegravir (TLD) is highly effective as ART but the causes of virologic failure (VF) in individuals on TLD without integrase (IN) resistance mutations are not well understood. Off-target mutations in nucleocapsid (NC) have been selected in vitro and are associated with decreased DTG susceptibility in vitro. Samples from ACTG study A5381 were examined to assess the potential contribution of NC mutations to VF in individuals on TLD.

Methods

A5381 was an observational cohort study enrolling individuals aged >10y with viral load >1000 c/ml at start of TLD as 1st, 2^nd or 3rd-line ART. Paired study entry and VF samples from 57 participants with confirmed VF on ≥6 months of TLD were sequenced by whole genome Illumina MiSeq. Mutations were identified with HyDRA Web (IN) and a pipeline using reference alignment to HXB2 RefSeq (NC_001802.1) with BWA v0.7.17, LoFreq 2.1.5 and SNPeff 5.0 for putative variant calling (NC) with a mutation threshold of 20% frequency. Site directed NC and integrase (IN) mutants were phenotyped for DTG sensitivity with TZM-bl assays. 

Results

Mutations in NC at a frequency of ≥20% of the virus population at VF but not before TLD start were detected in 10 of 57 (17%) participants (Table).  Of these 10, two participants had both NC and IN mutations at VF but not at study entry, suggesting dual selection. Participant 1 selected A30T (57% frequency) and M46I (69% frequency) in NC and the accessory mutation H51Y (9% frequency) in IN. Participant 2 selected N27I (77% frequency) in NC and the major mutation R263K (100% frequency) and polymorphism M50I (100% frequency) in IN. Laboratory-generated infectious mutant clones of NC-N27I and IN-M50I/R263K show 1.8- and 2.4-fold resistance to DTG, respectively. When they are combined, the triple mutant showed 5.8-fold resistance. IN-M50I/R263K mutants show lower infectivity than wild-type (WT), but NC-N27I did not further affect viral infectivity in the context of IN-WT and or IN-M50I/R263K.

Conclusions

This work provides the first clinical evidence of selection of NC mutations with TLD failure and of decreased DTG susceptibility from the combination of NC and IN mutations. Our findings support further investigation of the potential contributions of mutations outside of IN to VF of TLD.

Table or Figure