Evolution of SARS-CoV-2 and escape of immunity in immunocompromised patients

To the editor:

Mutations in the thorn protein of severe acute coronavirus 2 syndrome (SARS-CoV-2), which provide an escape from neutralizing antibodies, may occur in immunocompromised patients with prolonged infection.1,2 Such viral avoidance is thought to contribute to of global variants of anxiety.3 In the absence of effective immune responses, selective pressures such as those from monoclonal antibody treatment can lead to immunologically important mutations.

To understand the selective pressures driving the evolution of SARS-CoV-2 in the host, we investigated the relationship between such evolution and endogenous immune responses and treatment with exogenous antibodies in convenient samples from five B-cell deficient patients. (Details of each patient’s clinical history are provided in the supplementary appendix, available in the full text of this letter to NEJM.org.) All patients had SARS-CoV-2 infections for 42 to 302 days after the first positive test (day 0) (Fig. S1 and table S1 in the supplementary annex). The study was approved by the Institutional Review Board at Emory University. Informed consent was obtained from patients who donated whole blood samples for testing (Patients 2, 4 and 5).

Figure 1. Figure 1. Neutralizing antibody titers, T-cell response effects, and spike mutations in five immunocompromised patients.

Panel A shows titers of neutralizing antibodies in the patient’s serum against Wuhan-Hu-1, the reference pseudovirus SARS-CoV-2, at various time points after infection. These titers represent the reciprocal serum dilution at which semi-maximal pseudoviral neutralization is observed. The data show the geometric means of two to five independent experiments; 𝙸 The bars show standard deviations. The dotted line represents the lower limit of detection. Panels B and C show background-excluded frequencies of CD4 + or CD8 + T cells expressing CD154, interferon-γ, tumor necrosis factor (TNF), or interleukin-2 as a percentage of naive (i.e., effector or memory) cells in response to stimulation of peripheral blood mononuclear cells with a peptide megapool containing 15-dimensional open reading frame (ORF) and a peptide megapool containing predicted CD8 + T-cell epitopes from ORF, 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 patients of the same age hospitalized with Covid-19 (Covid 1 and 2). Panel D shows mutations in the gene encoding the SARS-CoV-2 spike protein compared to the Wuhan-Hu-1 strain, according to the patient ID and time point. Shading indicates the frequency of mutations. For each mutation, the observed variant nucleotide is indicated above the graph, and the amino acid mutation is indicated below the graph.

Patient 1 did not receive antibody treatment and was negative for neutralizing antibodies on day 37. Patients 2 and 3 were treated with the bamlanivimab monoclonal antibody on days 4 and 8, respectively. Their serum strongly neutralized the reference pseudovirus (Wuhan-Hu-1) on day 33 (patient 2) and day 55 (patient 3) and maintained elevated neutralizing antibody titers on 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 titers of neutralizing antibodies on day 204. Binding of IgG titers to serum neutralizing protein is affected. titers (Fig. S2). All but one patient (patient 2) eventually recovered. Patients 2, 4 and 5 provided peripheral blood samples for immunophenotyping. All three of these patients had low lymphocyte counts and low to undetectable CD19 + B cell rates (0.19% in patient 2, 0.01% in patient 4, and 0.01% in patient 5) compared to healthy patients. controls and age-appropriate patients with coronavirus disease 2019 (Covid-19) (Fig. S3). Patient 3 had clinically low levels of T and B cells. Thus, responses to antibodies to the reference SARS-CoV-2 in patients 2, 3 and 5 are likely due to exogenous treatments. SARS-CoV-2-specific effector T cell responses were detected in patients 4 and 5, with CD8 + T cells secreting antiviral interferon-γ and tumor necrosis factor, but were detected only at the background level in patient 2 (Figure 1B and 1C and Figures S4, S5 and S6).

Sequencing of SARS-CoV-2 (Table S2 and Fig. S7 and S8) revealed the evolution of thorn protein in patients 2 and 3 (Figure 1D and Fig. S9); both patients treated with bamlanivimab had T and B cell deficiencies. Mutations at consensus level and single nucleotide variants within the sample were found in the receptor-binding domain (RBD) and N-terminal domain (NTD), regions that were associated with immune escape.4 Conversely, they were not RBD or NTD mutations were found in patient 1 who did not receive antibodies, or in patients 4 and 5 who received convalescent plasma and had intact 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 variant peaks (Fig. S10). Serum from patients 1, 2 and 3 did not neutralize pseudoviruses with variant peaks, although serum from patients 2 and 3 neutralized the reference pseudovirus (Fig. S11). Thus, thorn mutations in patients 2 and 3 confer resistance to neutralization of bamlanivimab.

Our results highlight the potential importance of selective pressure such as the use of monoclonal antibodies – combined with the lack of an effective endogenous immune response – to promote the emergence of mutations that have escaped SARS-CoV-2. These findings underscore the need for a better understanding of the implications of different therapies in immunocompromised patients. Our results also confirm the findings of previous studies that found that B-cell deficient patients produced effector T cells, a result that may signal an important role for T cells in controlling infection.

Erin M. Scherer, Ph.D., D.Phil.Ahmed Babiker, MB, BSMax W. Adelman, MDBrent Allman, BAAutum Key, MS Jennifer M. Kleinhenz, BSRose M. Langsjoen, Ph.D. Phuong-Vi Nguyen, BSIvy Onyechi, MS Jacob D. Sherman, BSTrevor W. Simon, MSHannah SoloffEmory University, Atlanta, Georgia [email protected]

Jessica Tarabey, MPHEmory Healthcare, Atlanta, Georgia

Jay Varkey, MDA Andrew C. Webster, MDEmory University, Atlanta, Georgia

Daniela Weiskopf, PhD La Jolla Institute of Immunology, La Jolla, CA

Daniel B. Weissman, Ph.D. Yongxian Xu, MDJesse J. Waggoner, MDKatia Koelle, Ph.D.Nadine Rouphael, MDS Stephanie M. Pouch, MDAnne Piantadosi, MD, Ph.D.Emory University, Atlanta, Georgia [email protected]

Supported by contract (75D30121C10084 under BAA ERR 20-15-2997, to Dr. Babiker, Waggoner, Koelle and Piantadosi) from the Centers for Disease Control and Prevention; through a grant (5UM1AI148576-02, by Dr. Rouphael and Scherer) from the National Institutes of Health (NIH); with the Simons Foundation Research Award for Mathematical Modeling of Living Systems (to Dr. Weissman); and from the Center for Pediatric Infections and Vaccines of the Alliance for Pediatric Research (to Dr. Piantadosi) and Children’s Health in Atlanta and the Emory Woodruff Health Science Center Covid-19 Center for Emergency Research (CURE) with the support of the O. Wayne Foundation Rollins and the William Randolph Hearst Foundation (to Dr. Piantadosi and Waggoner). The research reported in this letter was supported by a grant (K08AI139348, to Dr. Piantadosi) from the NIH National Institute of Allergy and Infectious Diseases and a contract (75N9301900065, to Dr. Weiskopf) from the NIH. The La Jolla Institute of Immunology has applied for patent protection for various aspects of the work on the design of T-cell epitopes and vaccines.

The disclosure forms provided by the authors are available with the full text of this letter to NEJM.org.

The views expressed in this letter are those of the authors and do not necessarily represent the official views of the National Institutes of Health.

This letter was published on June 8, 2022 in NEJM.org.

5 References

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