Emergence of SARS-CoV-2 escape mutations during Bamlanivimab therapy in a phase II randomized clinical trial

Abstract

SARS-CoV-2 mutations that cause resistance to monoclonal antibody (mAb) therapy have been reported. However, it remains unclear whether in vivo emergence of SARS-CoV-2 resistance mutations alters viral replication dynamics or therapeutic efficacy in the immune-competent population. As part of the ACTIV-2/A5401 randomized clinical trial (NCT04518410), non-hospitalized participants with symptomatic SARS-CoV-2 infection were given bamlanivimab (700 mg or 7,000 mg) or placebo treatment. Here¸ we report that treatment-emergent resistance mutations [detected through targeted Spike (S) gene next-generation sequencing] were significantly more likely to be detected after bamlanivimab 700 mg treatment compared with the placebo group (7% of 111 vs 0% of 112 participants, P = 0.003). No treatment-emergent resistance mutations among the 48 participants who received 7,000 mg bamlanivimab were recorded. Participants in which emerging mAb resistant virus mutations were identified showed significantly higher pretreatment nasopharyngeal and anterior nasal viral loads. Daily respiratory tract viral sampling through study day 14 showed the dynamic nature of in vivo SARS-CoV-2 infection and indicated a rapid and sustained viral rebound after the emergence of resistance mutations. Participants with emerging bamlanivimab resistance often accumulated additional polymorphisms found in current variants of concern/interest that are associated with immune escape. These results highlight the potential for rapid emergence of resistance during mAb monotherapy treatment that results in prolonged high-level respiratory tract viral loads. Assessment of viral resistance should be prioritized during the development and clinical implementation of antiviral treatments for COVID-19.

Extended data Figure 1.. Viral load and primary resistance mutation frequencies.

Viral loads and frequencies of primary resistance mutations from nasopharyngeal swab (NP) and anterior nasal swab (AN) samples for participants displaying primary bamlanivimab resistance mutations.

Extended data Figure 2:

SARS-CoV-2 specific IgG antibody profiling at baseline in different study groups. Horizontal bars represent median antibody titer. Dashed lines represent antibody positivity detection threshold. RBD denotes Receptor binding domain, NTD N-terminal domain, N Nucleocapsid.

Extended data Figure 3:. Fitting of the mathematical model to viral load and viral frequency data in individuals with resistance mutations.

(A-F) In each panel, the upper plot shows the viral load kinetics in the individual with the ID shown in the title; the lower plot shows the frequencies over time for the mutants under analysis. Data used for model fitting are shown as ‘o’ and data not used for model fitting are shown as ‘x’. Simulation results using the best-fit parameters (Supplemental Table 2) are shown as lines. (G) Comparison of growth rates of the E484 and the 484K strains estimated from mathematical models for 5 individuals. P-value is calculated using a Wilcoxon signed rank test for paired data.

Extended data Figure 4:. Emerging polymorphisms in different participant counts.

Counts of emerging polymorphisms (including primary resistance sites) in NP samples on day 7 in three study groups: participants with emerging primary resistance mutations, treatment group participants without emerging primary resistance mutations, and the placebo group. Box plots show median and interquartile range.

Figure 1:. Prevalence of SARS-CoV-2 primary resistance mutations.

(A) Percent of participants harboring primary resistance mutations L452R, E484K, E484Q, F490s and S494P at ≥20% frequency in the bamlanivimab 7000mg and 700 mg treatment and placebo arms at baseline, emergent, and at any time-point. Participants without quantifiable viral load at baseline and/or follow-up time points were grouped with those without resistance. P-values were calculated using Fisher’s exact test. * P <0.05, ** P<0.01. (B) Pie-charts showing distribution of baseline and emergent resistance mutations in treatment arm. One participant had E484K at baseline with emerging E484Q mutation.

Figure 2:. SARS-CoV-2 viral kinetics in the bamlanivimab 700mg treatment arm.

SARS-CoV-2 viral loads from (A) Nasopharyngeal (NP) swabs (collected at day 0, 3, 7, 14, 21 and day 28) and (B) from anterior nasal (AN) swabs (collected daily through day 14 followed by day 21 and day 28) plotted against study day. Lines show median viral load. The lower limit of quantification was 2.0 log₁₀ SARS-CoV-2 RNA copies/mL while the lower limit of detection was 1.0 log₁₀ copies/mL. Viral loads between groups were compared at each time point using the two-sided Mann-Whitney U tests denoted by asterisks wherever significant. (NPH Day 0 P=0.0369, NPH Days 3, 7, 14 P<0.001, AN Day 0 P=0.0135, AN Day 1 P=0.0402, AN Day 2 P=0.0066, AN Day 3 P=0.0145, AN Day 4 P=0.0013, AN Days 5–8 P<0.001, AN Day 9 P=0.0018, AN Days 10–14 P<0.001).

Figure 3:. Evidence of viral rebound and/or slow viral decay coupled with dynamic viral population shift and potential compartmentalization.

Viral RNA from nasopharyngeal (NP) swabs and anterior nasal (AN) swabs were sequenced and results from two example participants from the bamlanivimab 700mg treated group are shown. (A) Participant B2_3 showed emergence of E484K and viral rebound between study days 3 and 7. (B) Participant B2_2 showed emergence of a mixed population of E484K and E484Q viruses along with multiple rebounds and slow viral decay. Alignments of consensus sequences from both compartments show position of primary escape and other consensus-level mutations at each time point. CP denotes cytoplasmic domain, FP fusion peptide, HR1 heptad repeat 1, HR2 heptad repeat 2, NTD N-terminal domain, RBD receptor binding domain, S1 subunit 1, S2 subunit 2, and TM transmembrane domain.

Figure 4:. Heat map showing distribution of Spike polymorphisms from AN swab samples in bamlanivimab 700mg treated participants with emerging primary resistance mutations.

(A) Panel A shows polymorphism in context of near-full length Spike gene. Y-axis shows participants’ ID followed by day of sample collection, while x-axis shows amino-acid positions in Spike gene. Different domains of Spike are shown at the top while color indicates frequency of polymorphisms starting with blue indicating lowest value while red indicates highest value in the scale. (B) Zoomed-in heat-map showing sites which harbors polymorphisms at least one of the samples across different participants. The order of samples is same that in panel A while x-axis denotes amino-acid sites with number indicating position of amino-acids while letter before and after the numbers indicate wild-type and polymorphic amino-acid respectively. SP denotes signal peptide, NTD N-terminal domain, RBD receptor binding domain, RBM Receptor binding domain, S1 subunit 1, S2 subunit 2, and FP fusion peptide.

Figure 5.. Worsened COVID-19 symptoms with viral resurgence after emergence of resistance mutations.

(A) Example of increased anterior nasal (AN) viral load (VL) and total symptom score (TSS) trend for one participant, B2_7, with emerging E484K resistance after bamlanivimab 700mg treatment. (B) Median AN viral load (solid line) and total symptom score (dashed line) plotted from the days since symptom emergence (DSSE) to ≥20% of the viral population (day 0) for participants in the bamlanivimab 700mg treatment group with (red) and without (green) emerging resistance mutations. For participants without emerging resistance, day 0 was equivalent to study day 4, which represented the median day of resistance emergence for those with emerging resistance. AN viral load and total score symptom score between the emerging resistance and no emerging resistance groups is compared at each day by two-sided Mann Whitney U tests VL: −3 DSSE P=0.0054, −1 DSSE P=0.0325, 0–10 DSSE P<0.001; TSS: 11 DSSE P=0.0368, 12 DSSE P=0.0339, 14 DSSE P=0.056, 15 DSSE P=0.0240.