To the editor:
Mutations can arise in the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein that confers escape from neutralizing antibodies in immunocompromised patients with long-standing infection.1,2 It is assumed that this viral evasion contributes to the emergence of global variables of concern.3 In the absence of effective immune responses, selective pressures such as those from treatment with monoclonal antibodies may give rise to immunologically important mutations.
To understand the selective pressures driving SARS-CoV-2 evolution within the host, we examined the relationship between this evolution and autoimmune responses and exogenous antibody treatment in appropriate samples obtained from five B-cell-deficient patients. (Details regarding each patient’s clinical history are provided in the Supplementary Appendix, available with the full text of this letter at NEJM.org.) All patients had SARS-CoV-2 infection that lasted 42-302 days after their first positive test (day) 0 ) (Fig. S1 and Table S1 in the Supplementary Appendix). The study was approved by the Institutional Review Board at Emory University. Informed consent was obtained from patients who donated whole blood samples for research (patients 2, 4 and 5).
Panel A shows the neutralizing antibody titer in patient serum against Wuhan-Hu-1 virus, the pseudo-reference SARS-CoV-2, at different time points after infection. These titers represent the reciprocal serum dilution where half-maximal pseudovirus neutralization was observed. The data show the geometric means of one to five independent experiments; 𝙸 bars indicate standard deviations. The dotted line represents the lower limit of detection. Panels B and C show the background elicited frequencies of CD4+ or CD8+ T cells expressing CD154, interferon-γ, tumor necrosis factor (TNF), or interleukin-2 as a percentage of non-naïve (ie effector or memory) cells in response to a stimulus. Peripheral blood mononuclear cells using megapool peptide containing 15 m serrated open reading frame (ORF) and megapool peptide containing CD8+ T-cell epitopes predicted from ORFs including spike, respectively. Frequencies were determined by flow cytometry in patients 4 and 5, as well as in a healthy control donor (HC2) and two age-matched patients hospitalized with Covid-19 (Covid 1 and 2). Panel D shows the mutations in the gene encoding the SARS-CoV-2 protein compared to the Wuhan-Hu-1 strain, according to patient ID and time point. Shading indicates mutation frequency. For each mutation, the observed variable nucleotide is listed above the plot and the amino acid mutation is listed below the plot.
Patient 1 did not receive antibody treatment and was negative for neutralizing antibody on day 37. Patients 2 and 3 were treated with the bamlanivimab monoclonal antibody on days 4 and 8, respectively. Their serum effectively neutralized the reference pseudovirus (Wuhan-Hu-1) on day 33 (in patient 2) and day 55 (in patient 3) and maintained elevated neutralizing antibody titers during days 77 and 83, respectively (Figure 1a). Patient 4 received convalescent plasma on days 0 and 104 and had undetectable neutralizing antibodies on days 82 and 101. Patient 5 received convalescent plasma on day 200 and had low neutralizing antibody titers on day 204. The titers (Fig. S2). All but one of the patients (patient 2) eventually recovered. Patients 2, 4, and 5 provided samples of peripheral blood for immunophenotyping. All three of these patients had lower lymphocyte counts and low to undetectable CD19+ B-cell frequencies (0.19% in patient 2, 0.01% in patient 4, and 0.01% in patient 5) compared to healthy controls and matched hospital residents. Age of COVID-19 patients (Fig. S3). Patient 3 had clinically low levels of T and B cells. Thus, the antibody responses against the reference SARS-CoV-2 in patients 2, 3 and 5 were probably due to exogenous treatments. Specific effector responses to SARS-CoV-2 were detectable in patients 4 and 5, with CD8+ T cells secreting interferon-γ and tumor necrosis factor, but were only detectable at the background level in patient 2 (Figure 1b and 1c and figs. S4, S5, and S6).
SARS-CoV-2 sequencing (Table S2 and Figures S7 and S8) revealed elevated protein development in patients 2 and 3 (Figure 1d and Figure S9); Each of these bamlanivimab-treated patients had T and B cell deficiencies. Consensus-level mutations and intra-sample single-nucleotide variants were found in the spike receptor-binding domain (RBD) and N-terminal domain (NTD), regions that have been associated with immune escape.4 In contrast, no RBD or NTD mutations were found in patient 1, who did not receive the antibodies, or in patients 4 and 5, who received convalescent plasma and had healthy T-cell responses to SARS-CoV-2.
To assess whether viruses obtained from patients 1, 2, and 3 were neutralized by autologous serum, we constructed infectious pseudoviruses expressing altered mutations (Fig. S10). Serum from patients 1, 2, and 3 did not neutralize the pseudoviruses with variable mutations, although the serum of patients 2 and 3 neutralized the reference pseudovirus (Fig. S11). Thus, abrupt mutations in patients 2 and 3 confer equivalency resistance to pamlanifemab.
Our results underscore the potential importance of selective pressures such as the use of monoclonal antibodies—in combination with the lack of an effective endogenous immune response—in promoting the emergence of escape mutants of SARS-CoV-2. These findings highlight the need to better understand the implications of different treatments in immunocompromised patients. Our results also corroborate the findings of previous studies showing that patients with B-cell deficiency induce effector T cells,5 The finding may indicate an important role for T cells in controlling infection.
Erin M. Shearer, Ph.D., Phil D.
Ahmed Babiker, MB, BS
Max W. Adelman, MD
Brent Allman, BA
Autum key, MS
Jennifer M. Kleinens, BA
Rose M. Languin, Ph.D.
Phuong-Vi Nguyen, B.S.
Ivy Onichi, MS
Jacob d. Sherman, BA
Trevor W. Simon, MS
Emory University, Atlanta, Georgia
Jessica Tarabay, MPH
Emory Healthcare, Atlanta, Georgia
Jay Varkey, MD
Andrew S. Webster, MD
Emory University, Atlanta, Georgia
Daniela Weskov, Ph.D.
La Jolla Institute of Immunology, La Jolla, California
Daniel P. Wiseman, Ph.D.
Yongxian Shu, MD
Jesse J. Wagner, MD
Katya Coyle, Ph.D.
Nadine Raphael, MD
Stephanie M. Bach, MD
Ann Piantadosi, MD, PhD.
Emory University, Atlanta, Georgia
backed by contract (75D30121C10084 under BAA ERR 20-15-2997, to Drs Babiker, Wagoner, Koelle, and Piantadosi) of
Disclosure forms provided by the authors are available with the full text of this letter at NEJM.org.
The opinions expressed in this letter are those of the authors and do not necessarily represent the official views of the National Institutes of Health.
This message was posted on June 8, 2022, at NEJM.org.
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