Combination antiviral therapy may be helpful in the treatment of SARS-CoV-2 infection; however, no clinical trial data are available, and combined use of direct-acting antivirals (DAA) and monoclonal antibodies (mAb) has been reported only anecdotally. To assess the cooperative effects of dual drug combinations in vitro, we used a VERO E6 cell-based in vitro system with the ancestral B.1 or the highly divergent BQ.1.1 virus to test pairwise combinations of the licensed DAA, including nirmatrelvir (NRM), remdesivir (RDV) and the active metabolite of molnupiravir (EIDD-1931) as well the combination of RDV with four licensed mAbs (sotrovimab, bebtelovimab, cilgavimab, tixagevimab; tested only with the susceptible B.1 virus). According to SynergyFinder 3.0 summary and weighted scores, all the combinations had an additive effect. Within DAA/DAA combinations, paired scores with the B.1 and BQ.1.1 variants were comparable. In the post hoc analysis weighting synergy by concentrations, several cases of highly synergistic scores were detected at specific drug concentrations, both for DAA/DAA and for RDV/mAb combinations. This was supported by in vitro confirmation experiments showing a more than a linear shift of a drug-effective concentration (IC₅₀) at increasing concentrations of the companion drug, although the effect was prominent with DAA/DAA combinations and minimal or null with RDV/mAb combinations. These results support the cooperative effects of dual drug combinations in vitro, which should be further investigated in animal models before introduction into the clinic.

Doravirine and islatravir have shown promising activity against multidrug resistant HIV-1. In this study we aimed to evaluate the in vitro susceptibility to doravirine and islatravir as well as their combinatorial activity in a panel of 38 recombinant viruses harboring multiple NRTI and NNRTI mutations. One additional recombinant virus had the M184V mutation alone. According to the IC₅₀ fold-change (FC) values calculated with respect to the NL4-3 wild-type strain, full susceptibility to doravirine was detected in 15/39 (38.5 %) samples, while high-level resistance was mainly associated with specific doravirine resistance mutations. Decreased susceptibility to islatravir was associated with the presence of the M184V/I mutation and increasing numbers of TAMs and NRTI resistance mutations. According to ZIP model of the SynergyFinder Plus tool, the combination of doravirine and islatravir showed additive activity in 37/40 (92.5 %) viruses (including the NL4-3 strain), while synergy and antagonism in one and two cases, respectively. The combination sensitivity score calculated by SynergyFinder Plus indicated a cooperative effect between doravirine and islatravir higher than that observed for the reference NL4-3 strain in 22 (56 %) recombinant viruses. The Multi-dimensional Synergy of Combinations (MuSyC) tool predicted synergy and antagonism in 25 (62.5 %, including NL4-3 virus) and 15 (37.5 %) cases, respectively. MuSyC scores showed a negative correlation with doravirine FC values, number of NRTI mutations and presence of M184V/I, but not with islatravir FC values. Doravirine and islatravir may cooperatively inhibit NRTI and NNRTI resistant viruses despite complex mutational profiles, however the accumulation of resistance mutations may reduce the combinatorial activity.

Background: Next-generation sequencing (NGS) kits are needed to finalise the transition from Sanger sequencing to NGS in HIV-1 genotypic drug resistance testing. Materials and Methods: We compared a homemade NGS amplicon-based protocol and the AD4SEQ HIV-1 Solution v2 (AD4SEQ) NGS kit from Arrow Diagnostics for identifying resistance-associated mutations (RAMs) above the 5% threshold in 28 plasma samples where Sanger sequencing previously detected at least one RAM. Results: The samples had a median 4.8 log [IQR 4.4–5.2] HIV-1 RNA copies/mL and were mostly subtype B (61%) and CRF02_AG (14%). Homemade NGS had a lower rate of samples with low-coverage regions (2/28) compared with AD4SEQ (13/28) (p < 0.001). Homemade NGS and AD4SEQ identified additional mutations with respect to Sanger sequencing in 13/28 and 9/28 samples, respectively. However, there were two and eight cases where mutations detected by Sanger sequencing were missed by homemade NGS and AD4SEQ-SmartVir, respectively. The discrepancies between NGS and Sanger sequencing resulted in a few minor differences in drug susceptibility interpretation, mostly for NNRTIs. Conclusions: Both the NGS systems identified additional mutations with respect to Sanger sequencing, and the agreement between them was fair. However, AD4SEQ should benefit from technical adjustments allowing higher sequence coverage.

Combination antiviral therapy may be helpful in the treatment of SARS-CoV-2 infection, however no clinical trial data are available and combined use of direct acting antivirals (DAA) and monoclonal antibodies (mAb) has been reported only anecdotally. To assess the cooperative effects of dual drug combinations in vitro, we used a VERO E6 cell based in vitro system with the ancestral B.1 or the highly divergent BQ.1.1 virus to test pairwise combinations of the licensed DAA, including nirmatrelvir (NRM), remdesivir (RDV) and the active metabolite of molnupiravir (EIDD-1931) as well the combination of RDV with four licensed mAbs (sotrovimab, bebtelovimab, cilgavimab, tixagevimab; tested only with the susceptible B.1 virus). According to SynergyFinder 3.0 summary and weighted scores, all the combinations had an additive effect. Within DAA/DAA combinations, paired scores with the B.1 and BQ.1.1 variants were comparable. In the post-hoc analysis weighting synergy by concentrations, several cases of highly synergistic scores were detected at specific drug concentrations, both for DAA/DAA and for RDV/mAb combinations. This was supported by in vitro confirmation experiments showing a more than linear shift of a drug effective concentration (IC50) at increasing concentrations of the companion drug, although the effect was prominent with DAA/DAA combinations but minimal or null with RDV/mAb combinations. These results support a cooperative effects of dual drug combinations in vitro, which should be further investigated in animal models before introduction into the clinic.

The authors wish to make the following corrections to this original publication