Clinical and molecular landscape of prolonged SARS-CoV-2 infection with resistance to remdesivir in immunocompromised patients

Abstract

Patients with hematologic diseases have experienced coronavirus disease 2019 (COVID-19) with a prolonged, progressive course. Here, we present clinical, pathological, and virological analyses of three cases of prolonged COVID-19 among patients undergoing treatment for B-cell lymphoma. These patients had all been treated with anti-CD20 antibody and bendamustine. Despite various antiviral treatments, high severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) levels persisted for >4 weeks, and two of them succumbed to COVID-19. The autopsy showed bronchopneumonia, interstitial pneumonia, alveolar hemorrhage, and fibrosis. Overlapping cytomegalovirus, fungal and/or bacterial infections were also confirmed. Sequencing of SARS-CoV-2 showed accumulation of mutations and changes in variant allele frequencies over time. NSP12 mutations V792I and M794I appeared independently in two cases as COVID-19 progressed. In vitro drug susceptibility analysis and an animal experiment using recombinant SARS-CoV-2 demonstrated that each mutation, V792 and M794I, was independently responsible for remdesivir resistance and attenuated pathogenicity. E340A, E340D, and F342INS mutations in the spike protein were found in one case, which may account for the sotrovimab resistance. Analysis of autopsy specimens indicated heterogeneous distribution of these mutations. In summary, we demonstrated temporal and spatial diversity in SARS-CoV-2 that evolved resistance to various antiviral agents in malignant lymphoma patients under immunodeficient conditions caused by certain types of immunochemotherapies. Strategies may be necessary to prevent the acquisition of drug resistance and improve outcomes, such as the selection of appropriate treatment strategies for lymphoma considering patients' immune status and the institution of early intensive antiviral therapy.

Keywords: COVID-19; SARS-CoV-2; drug resistance; malignant lymphoma; remdesivir.

Fig. 1.

Clinical courses and SARS-CoV-2 mutation analysis of three cases. On the top panel, the timeline of medications administered is shown with white blood cell count, hemoglobin level, platelet count, CRP, and viral copy number of SARS-CoV-2 since the day of onset (0W in A and C, and 0D in B). The numbers in square boxes are time points when mutation analyses were performed. In the middle panel, chest X-ray and CT scan at each time point are shown (a–f). On the bottom panel, additional mutations that were observed during treatment are shown. Diagram of SARS-CoV-2 coding region is on the left. Darkly painted boxes show acquired mutations. The boxes marked with amino acids (V792I, M794I, E340A, E340D, and F342INS) show mutations implicated in drug resistance. A: case 1, B: case 2, C: case 3. R/R, refractory/relapsed; WBC, white blood cell; Hb, hemoglobin; Plt, platelet; PSL, prednisolone; Dex, dexamethasone; PB, peripheral blood; W, weeks; D, days.

Fig. 2.

Pathological observations of the lung lesions at autopsy. A) Autopsy findings of case 1.a–d), gross findings on the lung tissues. b–d) Enlarged view of lesions of interstitial pneumonia (b), pulmonary hemorrhage (c), and bronchopneumonia (d). (e–i) Histological findings of the lungs: bronchopneumonia with neutrophil infiltration (e), squamous metaplasia (f), and interstitial fibrosis (g). h and i) Fibrin thrombi (×100). j and k) SARS-CoV-2-infected cells. l and m) CMV-infected giant cells with an owl's eye appearance. n) Coinfection of SARS-CoV-2 and CMV in the same cell demonstrated by immunostaining. Brown (3,3′-diaminobenzidine) and green (Vina Green) signals indicate SARS-CoV-2 and CMV, respectively. o) Coinfection of SARS-CoV-2 and CMV in the same cell demonstrated by fluorescent staining. Green (Alexa Fluor 488) and red (Alexa Fluor 568) signals indicate SARS-CoV-2 and CMV, respectively. Nuclear labeling was performed using DAPI (blue). B) Autopsy findings of case 2. a) The gross findings on the lung tissues. b and c), histological findings of the AFOP-like lesion (b) and the consolidation lesion (c) in the right lung (×40). d and e), presence of SARS-CoV-2-infected cells in areas of the AFOP-like lesion: hematoxylin and eosin stain (d), and IHC for SARS-CoV-2 nucleocapsid antigen (e) (×200). f and g) Presence of CMV-infected cells in areas of the AFOP-like lesion: hematoxylin and eosin stain (f), and IHC for CMV antigen (g) (×400). Detailed information about microscopes utilized is indicated in Supplementary Methods.

Fig. 3.

Crystal structure of NSP12 of SARS-CoV-2 and remdesivir. A) The positional relationship between remdesivir and the residues of V792 and M794 on the crystal structure of NSP12. B) Close-up view of the mutation positions V792I and M794I in NSP12. The structure of the NSP12–NSP7–NSP8 complex (PDB ID 7BV2 PMID: 32358203) is shown as a cartoon representation in cyan. The RNA structure is shown as a white stick representation, and the remdesivir residue is in magenta. The residues of V792 and M794 are shown as CPK representations in red. This figure was prepared using PyMOL (30) (http://www.pymol.org/pymol).

Fig. 4.

Virological features of V792I and M794I mutants. A) Evaluation of remdesivir susceptibility using recombinant SARS-CoV-2 carrying the GFP gene. HEK293-ACE2/TMPRSS2 cells or VeroE6/TMPRSS2 cells treated with a P-gp inhibitor were infected with either WT, V792I, or M794I mutant at a MOI of 0.05 containing a 2-fold serial dilution of remdesivir. After 72 h in HEK293-ACE2/TMPRSS2 cells or 48 h in VeroE6/TMPRSS2 cells, the inhibition of infection was quantified via observation of GFP signals from four biological experiments, and then the EC₅₀ of each virus was calculated by nonlinear regression analysis. B) Viral growth kinetics of WT, V792I, and M794I mutants using Vero cells in the absence (left panel) and presence (right panel) of remdesivir. Culture supernatants were collected at 12, 24, and 48 h postinfection, and infectious titers were determined and calculated as TCID₅₀/mL. C) Pathogenicity of the remdesivir-resistant viruses in Syrian hamsters. A group of 4-week-old hamsters (n = 6) were inoculated intranasally with either saline (uninfected control), WT, V792I, or M794I virus. The body weight was examined daily by 7 days postinfection. Data represent mean ± SD. Statistical analyses were performed using one-way ANOVA with multiple comparisons (the Dunnett's test) compared with WT as a control. Asterisks indicate significant differences (*P < 0.05, ** P < 0.01, *** P < 0.001).