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Letter to the Editor| Volume 86, ISSUE 3, e61-e63, March 2023

Genomic evidence for reinfection with different Omicron subvariants

Published:January 11, 2023DOI:https://doi.org/10.1016/j.jinf.2023.01.010

      Highlight

      • Of 4449 patients, 12 were reinfected with genetically distinct SARS-CoV-2 variants.
      • Five of these patients were infected with different Omicron subvariants.
      • L452R, F486V, and Q493R were unique at first and second infection in five patients.
      • These three mutations are associated with antigenic shift and Omicron reinfection.
      To the Editor,
      Reinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is occurring. Evidence shows that the reinfection rate has increased since the emergence of the Omicron strain
      • Pulliam J.R.C.
      • van Schalkwyk C.
      • Govender N.
      • et al.
      Increased risk of SARS-CoV-2 reinfection associated with emergence of Omicron in South Africa.
      ; however, whether genetically distinct subvariants are causing reinfections is unknown. Ideally, the viral genome of all infections should be sequenced to detect the variant. However, obtaining this genetic information is challenging owing to difficulties in collecting data and storing consecutive samples from the same patient.
      From February 12, 2020 to December 7, 2022, we classified SARS-CoV-2 variants by whole-genome and/or TaqMan analysis for 4,465 samples from 4,449 outpatients and hospitalized patients.
      • Hirotsu Y.
      • Maejima M.
      • Shibusawa M.
      • et al.
      Classification of Omicron BA.1, BA.1.1, and BA.2 sublineages by TaqMan assay consistent with whole genome analysis data.
      • Hirotsu Y.
      • Maejima M.
      • Shibusawa M.
      • et al.
      SARS-CoV-2 Omicron sublineage BA.2 replaces BA.1.1: Genomic surveillance in Japan from September 2021 to March 2022.
      • Hirotsu Y.
      • Omata M.
      Detection of the Omicron BA.2.75 subvariant in Japan.
      The variants were classified as D614G (n = 140), R.1 (n = 20), Alpha (n = 278), Gamma (n = 1), Delta (n = 590), Omicron BA.1* (n = 1,050), Omicron BA.2* (n = 788), Omicron BA.4* (n = 2), Omicron BA.5* (n = 1,366), Omicron BA.2.75* (n = 16), other Omicron variants (n = 6), and unknown (n = 208).
      Twelve of the 4,449 patients (0.27%; six men, six women) were infected with genetically distinct variants for their first and second infections (Fig. 1A). These patients had a median age of 52 years (range: 4–87 years). The average time between the first and second infection was 9.1 months (range: 3.4–23.2 months). The two infections showed no significant difference in the viral loads detected in nasopharyngeal swab samples (Fig. S1A in the Supplementary Appendix, paired Wilcoxon rank–sum test, P>0.05). The disease severity, symptoms, pneumonia incidence, and hospitalization rates also did not significantly differ (Table S1, P>0.05, Fisher's exact test).
      Fig 1
      Fig. 1SARS-CoV-2 genomic features in patients reinfected with different subvariants. (A) The characteristics of twelve reinfected patients. These patients were infected with different variants for the first and second infections. (B) The global phylogeny of subsampled isolates of the SARS-CoV-2 variants. The analyzed sample is indicated by arrows. The data were uploaded and visualized using Nextclade (https://clades.nextstrain.org). (C) The mutation profiles of the Omicron subvariants that caused reinfection. The mutations that are shared between the first and the second infection are defined as common, and the mutations that are different are defined as unique.
      The variants in the first and second infections of Cases #1–7 were D614G and Omicron in four cases, R.1 and Omicron in one case, and Delta and Omicron in two cases (Figs. 1A and S2). Cases #8–12 were infected with different Omicron subvariants: two were infected with Omicron BA.1* to Omicron BA.5*, and three were infected with Omicron BA.2* to Omicron BA.5* (Fig. 1A and B). Of these 12 patients, five were fully vaccinated (2x BNT162b2 [n = 2], 3x BNT162b2 [n = 1], 4x BNT162b2 [n = 1], 3x BNT162b2 / mRNA-1273 [n = 1]), and seven were unvaccinated at the time of the second infection (Fig. S3). The median time from the date of the last vaccination to infection was 7.4 months (range: 0.9–19.5 months).
      We investigated whether differences in spike protein mutations were associated with reinfection. In patients infected with different Omicron subvariants, 254 mutations were common and 81 mutations were unique between the first and second infections. Of the 81 unique mutations, three were unique in all five patients (Fig. 2). Spike Q493R was present in only the first infection, whereas L452R and F486V were present in only the second infection. Spike L452R, F486V, and Q493R are located in the receptor-binding motif that interacts directly with angiotensin-converting enzyme 2 (ACE2) (Fig. 2), suggesting that these mutations are involved in antigenic shift and are associated with Omicron subvariant reinfection.
      • McCallum M.
      • Czudnochowski N.
      • Rosen L.E.
      • et al.
      Structural basis of SARS-CoV-2 Omicron immune evasion and receptor engagement.
      Fig 2
      Fig. 2Spike protein mutations in patients infected twice with different Omicron subvariants. (A) In each patient (n = 5, Cases #8–12), spike protein mutations found in the first and second infections were compared, and mutations that occurred in only one infection were considered unique mutations. The lollipop plot shows the number of patients with unique mutations: in all five patients, the mutation that occurred in only the first infection was Q493R, and the mutations that occurred in only the second infection were F486V and L452R. (B) Protein structure of SARS-CoV-2 spike and angiotensin-converting enzyme 2 (ACE2).
      • Benton D.J.
      • Wrobel A.G.
      • Xu P.
      • et al.
      Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion.
      Spike L452R, F486V, and Q493R residues are in close proximity to ACE2.
      This study provides the first robust proof of infection with genetically distinct Omicron subvariants. Our data suggest that reinfection can occur after vaccination and after infection-acquired immunity. In all 12 cases, the second infections were Omicron variants. Waves of Omicron infection may increase the number of reinfected cases, but the associations between reinfection and the symptoms, disease severity, and hospitalization rates are unclear.
      People who have had a previous post-vaccination infection with Omicron have shown persistent levels of antibodies that are highly cross-reactive with BA.5,
      • Malato J.
      • Ribeiro R.M.
      • Leite P.P.
      • et al.
      Risk of BA.5 infection among persons exposed to previous SARS-CoV-2 variants.
      and epidemiological evidence has shown low rates of reinfection.
      • Altarawneh H.N.
      • Chemaitelly H.
      • Ayoub H.H.
      • et al.
      Protective effect of previous SARS-CoV-2 infection against Omicron BA.4 and BA.5 subvariants.
      However, numerous mutations present in the Omicron spike protein show marked antigenic changes and are associated with SARS-CoV-2 immune evasion.
      • Tuekprakhon A.
      • Nutalai R.
      • Dijokaite-Guraliuc A.
      • et al.
      Antibody escape of SARS-CoV-2 Omicron BA.4 and BA.5 from vaccine and BA.1 serum.
      The unique mutations in reinfected cases suggest that the L452R and F486V mutations and the restoration of Q493R to wild type contribute to the antigenic shift of BA.5. Therefore, the Omicron BA.4/BA.5-adapted bivalent COVID-19 vaccines are expected to prevent reinfection.
      Our data were limited to those who attended an outpatient clinic or were hospitalized, leading to bias with respect to more severe symptoms. Reinfection is likely to have also occurred in people with minor symptoms and asymptomatic individuals.

      Declaration of Competing Interest

      No potential conflicts of interest for this study

      Funding

      This study was supported by a Grant-in-Aid for the Genome Research Project from Yamanashi Prefecture (to M.O. and Y.H.), the Japan Society for the Promotion of Science (JSPS) KAKENHI Early-Career Scientists JP18K16292 (to Y.H.), a Grant-in-Aid for Scientific Research (B) 20H03668 (to Y.H.), a Research Grant for Young Scholars (to Y.H.), the YASUDA Medical Foundation (to Y.H.), the Uehara Memorial Foundation (to Y.H.), and Medical Research Grants from the Takeda Science Foundation (to Y.H.).

      Appendix. Supplementary materials

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