key: cord-0850465-r83wxh87
authors: Choudhary, M. C.; Chew, K. W.; Deo, R.; Flynn, J. P.; Regan, J.; Crain, C. R.; Moser, C.; Hughes, M.; Ritz, J.; Ribeiro, R. M.; Ke, R.; Dragavon, J. A.; Javan, A. C.; Nirula, A.; Klekotka, P.; Greninger, A. L.; Fletcher, C. V.; Daar, E. S.; Wohl, D. A.; Eron, J. J.; Currier, J. S.; Parikh, U. M.; Sieg, S. F.; Perelson, A. S.; Coombs, R. W.; Smith, D. M.; Li, J. Z.
title: Emergence of SARS-CoV-2 Resistance with Monoclonal Antibody Therapy
date: 2021-09-15
journal: medRxiv : the preprint server for health sciences
DOI: 10.1101/2021.09.03.21263105
sha: 99b6700722886230e96fc6188eee7a8363cd6395
doc_id: 850465
cord_uid: r83wxh87

Resistance mutations to monoclonal antibody (mAb) therapy has been reported, but in the non-immunosuppressed population, it is unclear if in vivo emergence of SARS-CoV-2 resistance mutations alters either viral replication dynamics or therapeutic efficacy. In ACTIV-2/A5401, non-hospitalized participants with symptomatic SARS-CoV-2 infection were randomized to bamlanivimab (700mg or 7000mg) or placebo. Treatment-emergent resistance mutations were significantly more likely detected after bamlanivimab 700mg treatment than placebo (7% of 111 vs 0% of 112 participants, P=0.003). There were no treatment-emergent resistance mutations among the 48 participants who received bamlanivimab 7000mg. Participants with emerging mAb resistant virus had significantly higher pre-treatment nasopharyngeal and anterior nasal viral load. Intensive respiratory tract viral sampling revealed the dynamic nature of SARS-CoV-2 evolution, with evidence of rapid and sustained viral rebound after emergence of resistance mutations, and worsened symptom severity. Participants with emerging bamlanivimab resistance often accumulated additional polymorphisms found in current variants of concern/interest and associated with immune escape. These results highlight the potential for rapid emergence of resistance during mAb monotherapy treatment, resulting in prolonged high level respiratory tract viral loads and clinical worsening. Careful virologic assessment should be prioritized during the development and clinical implementation of antiviral treatments for COVID-19.

Across a broad spectrum of viral infections, host immune pressure 1,2 and antiviral therapy 3-5 can 81 select for viral escape mutations. The detection and characterization of antiviral resistance 82 mutations has been critical for the selection of appropriate antiviral therapies and to advance our 83 understanding of viral adaptation against evolutionary pressures 6 . Monoclonal antibody (mAb) 84 therapy is the current treatment of choice for non-hospitalized persons with early SARS-CoV-2 85 infections and mild to moderate COVID-19 7,8 . Bamlanivimab was the first mAb to receive FDA 86 emergency use authorization (EUA) after it was demonstrated that treatment with bamlanivimab 87 decreased nasopharyngeal (NP) SARS-CoV-2 detection and the risk of hospitalization or death 88 when compared to placebo 9 . The emergence of SARS-CoV-2 sequence changes was reported 89

shortly after the introduction of mAbs 7,10,11 , but there has not been definitive evidence that the 90 emergence of SARS-CoV-2 resistance mutations can lead to altered in vivo intrahost viral 91 replication dynamics and loss of therapeutic efficacy. 92 ACTIV-2/A5401 is a platform trial to evaluate efficacy of antiviral agents to prevent 93 disease progression in non-hospitalized persons with symptomatic SARS-CoV-2 infection 94 (NCT04518410). Participants were randomized to receive either bamlanivimab or placebo, with 95 frequent NP swab and daily anterior nasal (AN) swab collection. We utilized quantitative viral 96 load testing and Spike (S) gene next-generation sequencing to assess the emergence of viral 97 resistance mutations to bamlanivimab and their impact on viral load dynamics. These results 98 provide a window into the dynamic nature of SARS-CoV-2 intrahost viral population shifts and 99 demonstrate for the first time that in non-immunosuppressed persons, the emergence of viral 100 resistance can alter viral decay kinetics and lead to loss of antiviral activity. 101 102 RESULTS 103 SARS-CoV-2 resistance mutations emerging with mAb treatment were associated with 104 changes in viral replication kinetics. A total of 94 participants were enrolled in the 7000mg 105 cohort (48 in the treatment arm and 46 in the placebo arm, enrolled between August 2020 and 106

October 2020) and 223 participants were enrolled in the ACTIV-2/A5401 phase 2 bamlanivimab 107 700mg cohort (111 in the treatment and 112 in placebo arms, enrolled between October 2020 and 108

November 2020). The 7000mg dosing group was halted early due to the results of the BLAZE-1 109 study showing similar virologic efficacy between the bamlanivimab 7000mg and 700mg groups 9 . 110 Viral sequences were successfully obtained from 207 participants in the 700mg bamlanivimab 111 study and 78 in the 7000mg study from at least 1 respiratory sample with quantitative SARS-CoV-112 2 measurement ≥2 log10 RNA copies/mL at baseline or during 28 days of follow-up. Primary 113 resistance mutations (L452R, E484K, E484Q, F490S and S494P) 7,12 at ≥20% frequency were not 114 detected in any participants in the 7000mg bamlanivimab study, either at baseline or following the 115 single infusion. In the 700mg bamlanivimab arm, three participants had primary resistance 116 mutations at baseline (one L452R in the setting of B.1.427/429/Epsilon variant infection and two 117 participants with E484K) while the placebo arm had two cases of resistance mutations present at 118 baseline (both L452R in the setting of Epsilon infection, Figure 1 ). Treatment-emergent mutations 119 at ≥20% frequency (not detected at baseline) were significantly more likely to be detected after 120 bamlanivimab 700mg treatment than placebo (7% vs 0%, P=0.003); E484K was found in 5 of 8 121 cases of emergent resistance, E484Q in two cases, and S494P in one case (Figure 1b Quantitative SARS-CoV-2 viral loads were measured from NP swabs at days 0, 3, 7, 14, 127 21 and 28 of the trial, and from AN swabs daily at each of the first 14 days and at days 21 and 28. 128

We assessed differences in viral loads in those receiving bamlanivimab 700mg treatment by the 129 presence of emerging resistance. Pre-treatment NP and AN swab viral loads were higher for 130 participants with emerging resistance mutations compared to those with no mutations (emerging 131 vs no mutation viral loads at day 0, NP swab: median 7.6 vs 5.5 log10 copies/mL, P = 0.04; AN 132 swab: median 6.6 vs 4.3 log10 copies/mL, P = 0.02, Table 1 ). Those with emerging resistance also 133 had persistently elevated NP and AN viral loads throughout the first 14 days after study enrollment 134 ( Figure 2 ). Individuals with emerging resistance were older (emerging vs no mutation: median age 135 56 vs 45 years, P=0.01) and while not statistically significant, the median duration of symptoms at 136 study entry was modestly shorter in those with emerging resistance compared to those without any 137 mutations (emerging vs no mutations: median 4.5 vs 6.0 days, Table 1 Table 1 ) 13 . One participant (B2_6) did have a serum 143 concentration at day 28 below the limit of quantitation and an elimination half-life faster than 144 typical. 145 146 Evidence of dynamic SARS-CoV-2 viral population shifts and differential viral fitness after 147 mAb treatment. We observed that the emergence of SARS-CoV-2 resistance mutations was 148 closely associated with a relatively consistent change in viral load kinetics. This is exemplified in 149 Figure 3 with two examples of viral rebound in participants with emergence of escape variants. In 150 case B2_3, intensive S gene sequencing of virus isolated from the AN swabs revealed the 151 emergence of the E484K resistance mutation on study day 3 as a low frequency variant that rapidly 152 took over as the majority population by the next day ( Figure 3A , lower panel) and was associated 153 with a 3.6 log10 increase in AN swab viral loads over the subsequent 4 days to a peak of 7.8 log10 154 copies/mL on study day 7 before declining. For B2_2, the participant had evidence of baseline 155 E484K mutation and low-frequency E484Q in the NP swab ( Figure 3B ). The AN swab showed 156 low frequency E484K and Q mutations. After bamlanivimab treatment, there were rapid, dynamic 157 shifts in the viral population in the AN swab sample including both the E484K and Q mutations, 158

with the viral load peaking at 6.8 log10 copies/mL over 8 days concurrent with E484Q becoming 159 the dominant mutation. Among the 8 participants with emerging resistance, the median AN swab 160 viral load increase was 3.3 (range 0.3 -5.2) log10 RNA copies/mL over a median of 4.5 days 161 (Supplemental Figure 1) and this viral rebound is highlighted in the comparison of median viral 162 loads between those with and without emerging resistance mutations ( Figure 2 ). 163

To quantify the replicative fitness of the different strains, we developed a mathematical 164 model and fit it to both viral load data and variant frequency data collected from 6 participants in 165 the treatment arm with either E484K or Q resistance emergence. In this model, we assumed that 166 each variant is initially present and grows or declines exponentially at a constant rate (see Methods) 167 as was consistent with the data (Supplemental Figure 3) . We chose the first 8-13 time points for 168 model fitting covering the emergence of the resistance mutations but prior to the eventual viral 169 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 15, 2021. ; https://doi.org/10.1101/2021.09.03.21263105 doi: medRxiv preprint load declines. We estimated both the initial viral load and the rate of exponential increase or 170 decrease for each variant. From the estimation across the 6 individuals, the wild-type amino acid 171 (i.e., 484E), always declined under treatment, with the exponential rate varying from -0.2 to -3.2 172 per day (Supplemental Table 2 ). In contrast, the mutant 484K always increased under mAb 173 treatment, confirming it was a resistant mutant, with an exponential growth rate that varied over a 174 wide range (0.5 to 2.3 per day, Supplemental Figure 3G ). The 484Q mutant was found in two 175 participants, including low-frequency E484K and Q present at baseline in the AN swab sample of 176 participant B2_2. We estimated that virus harboring 484Q was more fit than 484K in the setting 177 of antibody treatment and grew at approximately twice the rate of the 484K variant (Supplemental 178 Table 2 ). In participant B2_5, viral loads in the setting of the 484Q mutant declined, but with a 179 rate much slower than the wildtype 484E, suggesting that it is more fit than 484E in the presence 180 of the mAb or developing host immune responses. 181 182 Emergence of additional Spike polymorphisms. In those with emerging bamlanivimab 183 resistance, we next assessed the emergence of additional S gene sequence changes outside of the 184 primary sites of resistance (L452R, E484K, E484Q, F490S and S494P). We found that emergence 185 of additional polymorphisms was common and could be detected in all participants with either 186 baseline or emerging bamlanivimab primary resistance mutations, although most were present at 187 low-frequencies ( Figure 4 ). One emergent polymorphism, Q493R, was detected in B2_7 and has 188 been described as a potential bamlanivimab resistance site 10 . Interestingly, a number of 189 polymorphisms were detected at sites distinct from the bamlanivimab site of activity and likely 190 reflect escape from host immune pressures. For example, deletions at amino acid positions 143 were detected to emerge in both participants B2_2 and B2_4. These N-terminal domain (NTD) 192 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. We used day 7 NP swab sequencing results to compare the rate of polymorphism 202 emergence across the participant groups as AN swab sequencing was performed only for 203 participants with evidence of resistance emergence. We detected no significant differences in the 204 number of emerging polymorphisms between those with emerging resistance, treated participants 205 without resistance and participants who received placebo (Supplemental Figure 4) . 206 207 Viral rebound during after bamlanivimab resistance emergence is associated with 208 worsened symptoms. To assess the clinical impact of the viral load resurgence seen in those 209 with emerging bamlanivimab resistance mutations, we compared longitudinal symptom scores 210 for bamlanivimab-treated participants with and without emerging resistance mutations. Total 211 symptom scores were calculated based on a 28 day diary completed by the participants for 13 212 targeted symptoms 19 . On an individual-level, higher symptom scores were frequently detected 213 after the emergence of resistance and increase in respiratory tract viral loads ( Figure 5A ). In the 214 population analysis, there was no significant differences in symptom scores between groups 215 before the emergence of resistance mutations. In participants of the bamlanivimab 700mg 216 treatment arm with emerging resistance mutations, median AN viral load increase began at the 217 time of resistance detection, with significantly higher subsequent viral loads and total symptom 218 scores compared to those in the treatment arm without resistance ( Figure 5B ). 219 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. In this virologic analysis of a randomized placebo-controlled clinical trial of non-hospitalized 221 persons with early COVID-19, we report the emergence of resistance mutations to the mAb 222 bamlanivimab and the effects of these mutations on viral decay and clinical symptoms. These 223 results represent the clearest evidence to date of several key principles: 1) the dynamic nature of 224 SARS-CoV-2 evolution and replication during mAb treatment, 2) treatment-emergent SARS-225

CoV-2 resistance mutations alter viral replication kinetics and extend the period of high viral loads, 226

3) drug resistance mutations can adversely affect both the virologic and clinical efficacy of a 227 COVID-19 antiviral medication, and 4) the emergence of resistance with mAb treatment is 228 dependent on the treatment dose. 229

In immunocompromised persons with COVID-19, viral evolution can lead to immune 230 escape and rapid emergence against even combination mAb therapy 11,15,20,21 . Whether these 231 findings are generalizable to the immunocompetent population has been unclear and there has not 232 been definitive evidence that the emergence of SARS-CoV-2 escape mutations impacts in vivo 233 viral replication dynamics and loss of therapeutic efficacy. In this study of bamlanivimab in a 234 general population of outpatients with mild to moderate COVID-19, we show that resistance 235 mutations to monoclonal antibody treatment can emerge quickly and are associated with rapid and 236 sustained increase in respiratory tract SARS-CoV-2 viral load. Importantly, this increase in viral 237 load was followed by significantly worsened clinical symptoms over the subsequent days. These 238 results are consistent with previous reports that during acute SARS-CoV-2 infection, high-level 239 respiratory tract viral loads often precede symptom onset by 1-2 days 22 . 240

We were able to identify several potential factors that may increase or decrease the risk of 241 mAb resistance. We found that older age and higher baseline respiratory tract viral load were 242 associated with higher risk of resistance emergence, while none of the 48 participants treated with 243 the higher dose bamlanivimab 7000mg therapy developed resistance. Studies of mAb treatments 244 have suggested that earlier initiation of therapy during periods of high respiratory tract viral load 245 is associated with a greater virologic response and likely improved therapeutic efficacy 23 . Our data 246 suggest that mAb treatment during periods of high-level viral loads may come at the cost of 247 increased risk of resistance emergence, although this effect may be mitigated by using higher doses 248 of mAbs. Interestingly, we also noted frequent increase in viral loads associated with resistance 249 emergence that lasted several days to more than a week before declining. Such prolonged rise in 250 viral loads is unusual, especially as these individuals had a median of 5 days of symptoms by the 251 time of study entry and it's expected that levels of respiratory viral loads should already be 252 declining 24 . While the exact cause is unclear, this finding raises several intriguing possibilities. 253

First, antiviral mAb therapies may have host immune modulating effects beyond their capacity to 254 bind and neutralize viral particles 25 . It is unknown whether mAb therapeutics may in some cases 255 interfere with host immune responses, especially in the setting of mAb resistance, leading to 256 suboptimal viral control. Alternatively, there have also been reports from in vitro studies that 257 certain SARS-CoV-2-specific antibodies may lead to antibody-dependent enhancement of 258 infection through an Fcγ receptor-dependent mechanism 26 , particularly at sub-neutralizing 259 concentrations, although in vivo confirmation has been challenging to obtain. 260

Our study also found that SARS-CoV-2 populations can turn over quickly, allowing for 261 quick selection of drug resistance associated mutations. In fact, viral populations were found to be 262 able to completely shift from fully sensitive to fully resistant viruses within 24 hours. The emerging 263 primary resistance mutations (e.g., E484K/Q) described in this report not only confer resistance to 264 mAb therapy but can also lead to decreased efficacy of vaccine-induced immune responses 27 . 265 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. While the rate of total polymorphism accumulation did not appear to be higher in those who 266 developed bamlanivimab resistance, many of the emerging polymorphisms are also key mutations 267 found in several variants of concern/interest (VOC/VOIs, including B. participants receiving the higher 7000mg dose of bamlanivimab, they were frequently detected in 280 the larger BLAZE-1 phase 2 trial of the 7000mg dose 7 . One difference between these studies was 281 the longer duration since symptom onset for the ACTIV-2 participants, who enrolled a median of 282 6 days since symptom onset versus 4 days for the BLAZE-1 participants. This likely led to higher 283 pretreatment viral loads, which we found to be a risk factor for resistance emergence. 284

Unfortunately, baseline viral loads could not be compared between studies as the BLAZE-1 study 285 did not use a quantitative SARS-CoV-2 viral load assay. These disparate results highlight the 286 importance of incorporating quantitative viral load testing and resistance testing for COVID-19 287 treatment trials of mAbs and other antiviral agents. 288

In summary, these results provide clear evidence that mAb treatment can rapidly select for 289 SARS-CoV-2 resistance, leading to dramatic viral rebound and worsened symptom severity. While 290 initiation of mAb treatment during early infection is recommended for optimal therapeutic benefit, 291 our results suggest that emerging resistance is a potential risk with treatment during periods of 292 high level viral replication. These findings have implications for the design and utilization of 293 SARS-CoV-2 antiviral therapeutics and provide insights into the prevention of SARS-CoV-2 294 resistance. Careful virologic and pharmacologic assessment of new treatments for COVID-19 295 should be prioritized. 296 297 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. 

The study participants were enrolled in the ACTIV-2/AIDS Clinical Trials Group (ACTG) A5401 300 phase 2 randomized, placebo-controlled trial of bamlanivimab 7000mg and 700mg mAb therapy. 301 Symptomatic adults ≥18 years of age with a documented positive SARS-CoV-2 antigen or nucleic 302 acid test were enrolled if the diagnostic sample was collected ≤7 days prior to study entry and 303 within 10 days of symptom onset. The 7000mg dosing group was halted early due to the results of 304 the BLAZE-1 study showing similar virologic efficacy between the bamlanivimab 7000mg and 305 700mg groups 9 . A total of 95 participants were randomized in the bamlanivimab 7000mg study 306 and 222 participants were randomized in the 700mg study and received an intervention (one 307 bamlanivimab or placebo intravenous infusion). All participants provided written informed 308 consent. Nasopharyngeal (NP) swab samples were collected by research staff at study days 0, 3, 309 7, 14 and 28, while anterior nasal (AN) swabs were self-collected by participants daily through 310 study day 14 and at days 21 and 28. Swabs were placed in 3ml of media (RPMI with 2% FBS). 311

Total symptom scores were calculated based on a 28 day diary completed by the 312 participants for 13 targeted symptoms 19 . The targeted symptoms are feeling feverish, cough, 313 shortness of breath or difficulty breathing, sore throat, body pain or muscle pain or aches, fatigue, 314 headache, chills, nasal obstruction or congestion, nasal discharge, nausea, vomiting, and diarrhea. 315

Each symptom is scored daily by the participant as absent (score 0), mild (1) moderate (2) and 316 severe (3). 317 318

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The copyright holder for this this version posted September 15, 2021. ; https://doi.org/10.1101/2021.09.03.21263105 doi: medRxiv preprint SARS-CoV-2 viral load from NP and AN swab samples were quantified using the Abbott m2000 320 system. SARS-CoV-2 quantitative Laboratory Developed Test (LDT) was developed utilizing 321 open mode functionality on m2000sp/rt (Abbott, Chicago, IL) by using EUA Abbott SARS-CoV-322 2 qualitative reagents 30 . Identical extraction and amplification protocols developed for RealTime 323 SARS-CoV-2 qualitative EUA assay were also used for the development of the RealTime SARS-324

CoV-2 quantitative LDT 31 . In this assay, 2 calibrator levels (3 log10 RNA copies/mL and 6 log10 325

RNA copies/mL) tested in triplicate were used to generate a calibration curve and 3 control levels 326 (negative, low positive at 3 log10 RNA copies/mL and high positive at 5 log10 RNA copies/mL) 327 were included in each run for quality management. In addition, batches of a matrix-specific control 328 ("external" swab control) with a target of 200 copies per mL were prepared and one unit was 329 included in every run. All controls were monitored using Levy-Jennings plots to monitor inter-330 run precision. Specimens that were greater than 7 log10 RNA copies/mL were diluted 1:1000 and 331 rerun to obtain an accurate viral load result. The lower limit of quantification was 2.0 log10 SARS-332

CoV-2 RNA copies/mL. 333 334 S gene sequencing was performed on NP swab samples at two time points for all participants: 335 baseline (study entry) and the last sample with a viral load (VL) ≥ 2 log10 SARS-CoV-2 RNA 336 copies/mL. In participants with evidence of slow viral decay (VL ≥ 2 log10 copies/mL at study day 337 14) or viral rebound (increase in NP swab VL), we performed S gene sequencing of all NP swab 338 samples with VL ≥ 2 log10 copies/mL. Sequencing of daily AN swab samples was performed for 339 participants with any emerging bamlanivimab resistance mutations detected on NP swab samples. 340 Viral RNA extraction was performed on 1 mL of swab fluid by use of the TRIzol-LS™ Reagent 341 (ThermoFisher), as previously described 32 . cDNA synthesis was performed using Superscript IV 342 All rights reserved. No reuse allowed without permission. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. were then merged with pear v0.9.6 aligned to the reference sequence using Bowtie2 v2.1.0). 350

Amino acid variants were then called at the codon level using perl code and used for resistance 351 interpretation with a 1% limit of detection. 352 353

We assessed the presence of previously confirmed bamlanivimab resistance mutations (L452R, 355 E484K, E484Q, F490S, and S494P) 7,12 . The detection of resistance mutations down to 1% 356 frequency was performed using Paseq 33 . Mutations detected by next-generation sequencing at <20% 357 of the viral population were labelled as "low frequency" variants as they would largely be missed 358 by traditional Sanger sequencing. A minimum average of 500x sequencing coverage per sample 359 was required for variant calling. The non-parametric Mann-Whitney U test was used to compare differences in viral loads between 386 groups. Chi-squared tests and Fisher's exact tests were used for analyses of proportions. All 387 loads and sequences were used for mathematical modeling. The mutational load was calculated by 389 multiplying resistance mutation frequency by the total viral load. In the model, we assumed that 390 the ith variant, Vi, has an initial load Vi,0 and its population size changes exponentially at a constant 391 rate, ri: 392

Then, the total viral load at time t, V(t) was calculated as: 394

The model predicted frequency of each variant was 396

We estimated the initial load, Vi,0 and the rate of exponential increase/decrease, ri, from the viral 399 load and viral frequency data fitted simultaneously. Note that in this model, we assumed that the 400 observed mutants were present at the time of antibody infusion or were produced quickly near that 401 time. 402

To calculate the goodness of fit of the model to the data, we first calculated the residual 403 sum of squares (RSS) between the model predicted viral load and the measured viral load on a 404 logarithmic scale using log10. The log-scale was used because viral loads were measured using 405 PCR and thus the measurement error was multiplicative, making the logarithm the natural scale to 406 use. We then calculated the RSS between model predicted frequencies and measured frequencies 407 for the mutants. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. ) 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. Viral loads between groups were compared at each time point using the Mann-Whitney U tests denoted by asterisks wherever significant. 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. preprint (which was not certified by peer review) is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.

The copyright holder for this this version posted September 15, 2021. ; https://doi.org/10.1101/2021.09.03.21263105 doi: medRxiv preprint 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.

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The copyright holder for this this version posted September 15, 2021. ; https://doi.org/10.1101/2021.09.03.21263105 doi: medRxiv preprint Figure 5 . Worsened COVID-19 symptoms with viral resurgence after emergence of resistance mutations. (A) Example of increased anterior nasal (AN) viral load and total symptom score 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 time of resistance emergence 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. 

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