Characterization of a Lineage C.36 SARS-CoV-2 Isolate with Reduced Susceptibility to Neutralization Circulating in Lombardy, Italy

doi:10.3390/v13081514

. 2021 Jul 31;13(8):1514.

doi: 10.3390/v13081514.

Characterization of a Lineage C.36 SARS-CoV-2 Isolate with Reduced Susceptibility to Neutralization Circulating in Lombardy, Italy

Matteo Castelli¹, Andreina Baj^{2

3}, Elena Criscuolo¹, Roberto Ferrarese¹, Roberta A Diotti¹, Michela Sampaolo⁴, Federica Novazzi², Daniela Dalla Gasperina^{3

5}, Daniele Focosi⁶, Davide Ferrari⁷, Massimo Locatelli⁸, Massimo Clementi^{1

4}, Nicola Clementi^{1

4}, Fabrizio Maggi^{2

3}, Nicasio Mancini^{1

4}

Affiliations

¹ Laboratory of Microbiology and Virology, Vita-Salute San Raffaele University, 20132 Milan, Italy.
² Laboratory of Microbiology, ASST Sette Laghi, 21100 Varese, Italy.
³ Department of Medicine and Surgery, University of Insubria, 21100 Varese, Italy.
⁴ Laboratory of Microbiology and Virology, IRCCS Ospedale San Raffaele, 20132 Milan, Italy.
⁵ Internal Medicine Unit, ASST Sette Laghi, 21100 Varese, Italy.
⁶ North-Western Tuscany Blood Bank, Pisa University Hospital, 56127 Pisa, Italy.
⁷ SCVSA Department, University of Parma, 43121 Parma, Italy.
⁸ Laboratory Medicine Service, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy.

PMID: 34452380
PMCID: PMC8402759
DOI: 10.3390/v13081514

Characterization of a Lineage C.36 SARS-CoV-2 Isolate with Reduced Susceptibility to Neutralization Circulating in Lombardy, Italy

Matteo Castelli et al. Viruses. 2021.

. 2021 Jul 31;13(8):1514.

doi: 10.3390/v13081514.

Authors

Affiliations

¹ Laboratory of Microbiology and Virology, Vita-Salute San Raffaele University, 20132 Milan, Italy.
² Laboratory of Microbiology, ASST Sette Laghi, 21100 Varese, Italy.
³ Department of Medicine and Surgery, University of Insubria, 21100 Varese, Italy.
⁴ Laboratory of Microbiology and Virology, IRCCS Ospedale San Raffaele, 20132 Milan, Italy.
⁵ Internal Medicine Unit, ASST Sette Laghi, 21100 Varese, Italy.
⁶ North-Western Tuscany Blood Bank, Pisa University Hospital, 56127 Pisa, Italy.
⁷ SCVSA Department, University of Parma, 43121 Parma, Italy.
⁸ Laboratory Medicine Service, IRCCS San Raffaele Scientific Institute, 20132 Milan, Italy.

PMID: 34452380
PMCID: PMC8402759
DOI: 10.3390/v13081514

Abstract

SARS-CoV-2 spike is evolving to maximize transmissibility and evade the humoral response. The massive genomic sequencing of SARS-CoV-2 isolates has led to the identification of single-point mutations and deletions, often having the recurrence of hotspots, associated with advantageous phenotypes. We report the isolation and molecular characterization of a SARS-CoV-2 strain, belonging to a lineage (C.36) not previously associated with concerning traits, which shows decreased susceptibility to vaccine sera neutralization.

Keywords: COVID-19; L452R; R346K; SARS-CoV-2; spike; variant of concern.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

**Figure 1**
Phylogeny and distribution of lineage C.36. (A) Spike amino acid mutations associated with the currently most relevant VOC and VOI. Mutations associated with increased affinity are highlighted in red. (B) Phylogram of C.36 spike amino acid sequences deposited on GISAID before 18 May 2021, with sublineage 1, 2, and 3 highlighted in blue, red, and yellow, respectively. For each sublineage, the first sampling date is reported. (C) Geographic distribution of the three sublineages. (D) Spike amino acid mutations most frequently associated to each C.36 sublineage. (E) Sublineages global monthly incidence normalized by the total number of sequenced isolates over the same period.

**Figure 2**
Neutralization assay. Neutralizing activity was assessed using two sera dilutions (1:80 and 1:160) against 0.01 MOI of four SARS-CoV-2 variants: D614G (black), B.1.1.7 (blue), B.1.351 (orange), C.36 (green). Mean values + SD are reported, each condition was tested in triplicate. Sera are ordered according to the anti-RBD titer and reported in blue, light blue and grey for titer >2500 U/mL, 2500–1000 U/mL or <1000 U/mL, respectively.

**Figure 3**
In silico spike characterization. (A) Structure of RBD-ACE2 complex with the relevant RBD mutations highlighted in yellow. The calculated affinity of wild type and mutated RBDs for ACE2 is reported. (B) C36_3 spike ectodomain representative conformation in the 1–up state (RBD colored in red), with SARS-CoV-2 1–up and closed RBDs highlighted in green and cyan, respectively (upper panel). C36_3 RBD opening angle over the MD simulation (lower panel, the three spikes follow the same coloring scheme).

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References

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[1] Maier J.A., Martinez C., Kasavajhala K., Wickstrom L., Hauser K.E., Simmerling C. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J. Chem. Theory Comput. 2015;11:3696–3713. doi: 10.1021/acs.jctc.5b00255. - DOI - PMC - PubMed

[2] Maier J.A., Martinez C., Kasavajhala K., Wickstrom L., Hauser K.E., Simmerling C. ff14SB: Improving the Accuracy of Protein Side Chain and Backbone Parameters from ff99SB. J. Chem. Theory Comput. 2015;11:3696–3713. doi: 10.1021/acs.jctc.5b00255. - DOI - PMC - PubMed

[3] Abraham M.J., Murtola T., Schulz R., Páll P., Smith J.C., Hess B., Lindahl E. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1:19–25. doi: 10.1016/j.softx.2015.06.001. - DOI

[4] Abraham M.J., Murtola T., Schulz R., Páll P., Smith J.C., Hess B., Lindahl E. GROMACS: High performance molecular simulations through multi-level parallelism from laptops to supercomputers. SoftwareX. 2015;1:19–25. doi: 10.1016/j.softx.2015.06.001. - DOI

[5] Henderson R., Edwards R.J., Mansouri K., Janowska K., Stalls V., Gobeil S.M.C., Kopp M., Li D., Parks R., Hsu A.L., et al. Controlling the SARS-CoV-2 spike glycoprotein conformation. Nat. Struct. Mol. Biol. 2020;27:925–933. doi: 10.1038/s41594-020-0479-4. - DOI - PMC - PubMed

[6] Henderson R., Edwards R.J., Mansouri K., Janowska K., Stalls V., Gobeil S.M.C., Kopp M., Li D., Parks R., Hsu A.L., et al. Controlling the SARS-CoV-2 spike glycoprotein conformation. Nat. Struct. Mol. Biol. 2020;27:925–933. doi: 10.1038/s41594-020-0479-4. - DOI - PMC - PubMed

[7] Huang K., Luo S., Cong Y., Zhong S., Zhang J.Z.H., Duan L. An accurate free energy estimator: Based on MM/PBSA combined with interaction entropy for protein–ligand binding affinity. Nanoscale. 2020;12:10737–10750. doi: 10.1039/C9NR10638C. - DOI - PubMed

[8] Huang K., Luo S., Cong Y., Zhong S., Zhang J.Z.H., Duan L. An accurate free energy estimator: Based on MM/PBSA combined with interaction entropy for protein–ligand binding affinity. Nanoscale. 2020;12:10737–10750. doi: 10.1039/C9NR10638C. - DOI - PubMed

[9] McCallum M., Bassi J., De Marco A., Chen A., Walls A.C., di Iulio J., Tortorici M.A., Navarro M.J., Silacci-Fregni C., Saliba C., et al. SARS-CoV-2 immune evasion by variant B.1.427/B.1.429. Science. 2021 doi: 10.1126/science.abi7994. - DOI - PMC - PubMed

[10] McCallum M., Bassi J., De Marco A., Chen A., Walls A.C., di Iulio J., Tortorici M.A., Navarro M.J., Silacci-Fregni C., Saliba C., et al. SARS-CoV-2 immune evasion by variant B.1.427/B.1.429. Science. 2021 doi: 10.1126/science.abi7994. - DOI - PMC - PubMed

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Characterization of a Lineage C.36 SARS-CoV-2 Isolate with Reduced Susceptibility to Neutralization Circulating in Lombardy, Italy

Affiliations

Characterization of a Lineage C.36 SARS-CoV-2 Isolate with Reduced Susceptibility to Neutralization Circulating in Lombardy, Italy

Authors

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Abstract

Conflict of interest statement

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