The ongoing evolution of variants of concern and interest of SARS-CoV-2 in Brazil revealed by convergent indels in the amino (N)-terminal domain of the Spike protein

TL;DR: In this article, the authors identified that SARS-CoV-2 lineages circulating in Brazil with mutations of concern in the receptor-binding domain (RBD) independently acquired convergent deletions and insertions in the NTD of the S protein.

Abstract: Mutations at both the receptor-binding domain (RBD) and the amino (N)-terminal domain (NTD) of the SARS-CoV-2 Spike (S) glycoprotein can alter its antigenicity and promote immune escape. We identified that SARS-CoV-2 lineages circulating in Brazil with mutations of concern in the RBD independently acquired convergent deletions and insertions in the NTD of the S protein, which altered the NTD antigenic-supersite and other predicted epitopes at this region. These findings support that the ongoing widespread transmission of SARS-CoV-2 in Brazil is generating new viral lineages that might be more resistant to neutralization than parental variants of concern.

Summary

Introduction

• Recurrent deletions in the amino (N)-terminal domain (NTD) of the spike (S) glycoprotein of SARS-CoV-2 have been identified during long-term infection of immunocompromised patients 1–4 as well as during extended human-to-human transmission 3.
• The VOCs B.1.1.7 and B.1.351 are resistant to neutralization by several anti-NTD monoclonal antibodies (mAbs) and NTD deletions at RDR2 and RDR4 are important for such phenotype 9–14.
• With the exception of N.10, none of the other variants described in Brazil displayed indels in the NTD.

Results and Discussion

• The authors genomic survey identified 35 SARS-CoV-2 sequences from seven different Brazilian states that harbor a variable combination of mutations in the RBD (K417T, E484K, N501Y) and indels in the NTD region of the S protein.
• The ML phylogenetic analyses further confirm that all sequences belonging to variants P.1-like ins214ANRN (Fig. 1A) and VOI N.10 (Fig. 1B) branched in highly supported (aLRT > 99%) monophyletic clades.
• Most P.1 𝚫144, 𝚫141-144 and 𝚫189-190 sequences were detected in the Amazonas or were recovered from individuals that were transferred from or that reported a travel history to the Amazonas state, like all P.1 𝚫144 sequences from the Bahia state described previously 21 and in the present study (Table 1).
• These findings suggest that NTD indels detected here probably abrogate the binding of NAb directed against the antigenic-supersite and other epitopes.
• Finally, a recent longitudinal analysis of intra-host SARS-CoV-2 evolution during acute infection in one immunocompetent individual revealed the emergence of virus haplotypes bearing deletions 𝚫144 and 𝚫141-144 in the NTD following the development of autologous anti-NTD specific antibodies 39.

Material and Methods

• The SARS-CoV-2 genomes were recovered using Illumina sequencing protocols as previously described 43,44.
• The alignment was refined using the InDels and Structural Variants module.
• Additionally, the same reads were imported in a different pipeline 45 based on Bowtie2 and bcftools 46 mapping and consensus generation allowing us to further confirm the indels supported by paired-end reads, removing putative indels with less than 10x of sequencing depth and with mapping read quality score below to 10 for all samples sequenced in this study.

Structural Modeling

• The resolved crystallographic structure of SARS-CoV-2 NTD protein bound to the neutralizing antibody 2-51 was retrieved from the Protein Databank (PDB) under the accession code 7L2C 33.
• This structure was then used as a template to model the NTD variants using the Swiss-Model webserver.
• The modeled structures of the NTDs variants were superimposed onto the coordinates of the PDB ID 7L2C to visualize the differences between the NTD-antibody binding interfaces.
• Image rendering was carried out using Visual Molecular Dynamics (VMD) software 51. 54 and the InterfaceAnalyzer protocol of the Rosetta package interfaced with the RosettaScripts scripting language 55.

Epitope prediction

• Epitopes in the NTD region were predicted by the ElliPro Antibody Epitope Prediction server 56.
• NTD are shown as predicted linear epitopes when using PDB accession codes 6VXX 57 and 6VSB 58, (structural coordinates corresponding to the entire S protein), along with a minimum score of 0.9, i.e., a highly strict criterion.

Figures

Table 1. SARS-CoV-2 Brazilian variants with indels at NTD of the Spike protein.

Figure 1. ML phylogenetic tree of whole-genome lineage P.1/P.1-like (A) and B.1.1.33 (B) Brazilian sequences showing the recurrent emergence of deletions at the NTD of the S protein. Tip circles representing the SARS-CoV-2 sequences with NTD indels are colored as indicated. The branch lengths are drawn to scale with the left bar indicating nucleotide substitutions per site. For visual clarity, some clades are collapsed into triangles.

Figure 3. Representation of the Spike NTD 3D structure of wild type (pink) and NTD deleted variants (colored in blue) complexed to the NAb 2-51 heavy (gray) and light (green) chains. A) Relative position of the five NTD loops (red arrows) and the NTD deletions detected in our sample. B) Native interactions of mAb NAb 2-51 with N3 (left close-up) and N5 (right close-up) loops on the 3D structure of the wild type Spike NTD antigenic supersite. The N5 loop representation is also rotated 180° around its z-axis. C) Potential interactions of mAb NAb 2-51 with N3 and N5 loops on the 3D structure of the Spike NTD of N3 and N5 deleted variants. Residues making contact in the interface are depicted in the licorice representation, with carbon atoms in cyan, nitrogen atoms in blue and oxygen atoms in red. The dotted lines indicate the interacting residues-pair.

Table 2. Impact of indels on the binding between SARS-CoV-2 NTDs and NAb 2-51, expressed as loss of putative interactions.

Figure 2. Amino acid alignment of Sarbecovirus NTD Spike region up to amino acid 335 including representative sequences of SARS-CoV-2 lineages harboring indels in the NTD and SARS-CoV-2-related coronavirus (SC2r-CoV) from bats and pangolins. IRs and RDRs positions (gray and red shaded areas, respectively) are approximations due to the high genetic variability in these alignment positions. Dotted rectangles highlight the indels identified in this study. The relative identity level estimated for each position of the alignment is displayed at the top.

Full text

The ongoing evolution of variants of concern and interest of SARS-CoV-2
in Brazil revealed by convergent indels in the amino (N)-terminal domain of
the Spike protein
Paola Cristina Resende
1
*, Felipe G Naveca*
2
, Roberto D. Lins
3
, Filipe Zimmer
Dezordi
4,5
, Matheus V. F. Ferraz
3,6
, Emerson G. Moreira
3,6
, Danilo F. Coêlho
3,6
,
Fernando Couto Motta
1
, Anna Carolina Dias Paixão
1
, Luciana Appolinario
1
, Renata
Serrano Lopes
1
, Ana Carolina da Fonseca Mendonça
1
, Alice Sampaio Barreto da
Rocha
1
, Valdinete Nascimento
2
, Victor Souza
2
, George Silva
2
, Fernanda Nascimento
2
,
Lidio Gonçalves Lima Neto
7
, Fabiano Vieira da Silva
7
, Irina Riediger
8
, Maria do Carmo
Debur
8
, Anderson Brandao Leite
9
, Tirza Mattos
10
, Cristiano Fernandes da Costa
11
,
Felicidade Mota Pereira
12
, Cliomar Alves dos Santos
13
, Darcita Buerger Rovaris
14
Sandra Bianchini Fernandes
14
, Adriano Abbud
15
, Claudio Sacchi
15
, Ricardo Khouri
16
,
André Felipe Leal Bernardes
17
, Edson Delatorre
18
, Tiago Gräf
19
, Marilda Mendonça
Siqueira
1
, Gonzalo Bello**
20
, and Gabriel L Wallau**
4,5
on behalf of Fiocruz
COVID-19 Genomic Surveillance Network.
1. Laboratory of Respiratory Viruses and Measles (LVRS), Instituto Oswaldo Cruz,
FIOCRUZ-Rio de Janeiro, Brazil.
2. Laboratório de Ecologia de Doenças Transmissíveis na Amazônia (EDTA), Instituto
Leônidas e Maria Deane, FIOCRUZ-Amazonas, Brazil.
3. Department of Virology, Instituto Aggeu Magalhães, FIOCRUZ-Pernambuco, Brazil.
4. Departamento de Entomologia, Instituto Aggeu Magalhães, FIOCRUZ-Pernambuco,
Brazil.
5. Núcleo de Bioinformática (NBI), Instituto Aggeu Magalhães
FIOCRUZ-Pernambuco, Brazil.
6. Department of Fundamental Chemistry, Federal University of Pernambuco, Recife,
Brazil
7. Laboratório Central de Saúde Pública do Estado do Maranhão (LACEN-MA), Brazil.
8. Laboratório Central de Saúde Pública do Estado do Paraná (LACEN-PR), Brazil.
9. Laboratório Central de Saúde Pública do Estado do Alagoas (LACEN-AL), Brazil.
10. Laboratório Central de Saúde Pública do Amazonas (LACEN-AM), Brazil.
11. Fundação de Vigilância em Saúde do Amazonas, Brazil.
12. Laboratório Central de Saúde Pública do Estado da Bahia (LACEN-BA), Brazil.
13 Laboratório Central de Saúde Pública do Estado de Sergipe (LACEN-SE), Aracajú,
Sergipe, Brazil.
14. Laboratório Central de Saúde Pública do Estado de Santa Catarina (LACEN-SC),
Florianópolis, Santa Catarina, Brazil.
15. Instituto Adolfo Lutz, São Paulo, São Paulo, Brazil.
16. Laboratório de Enfermidades Infecciosas Transmitidas por Vetores, Instituto
Gonçalo Moniz, FIOCRUZ-Bahia, Salvador, Bahia, Brazil.
17. Laboratório Central de Saúde Pública do Estado de Minas Gerais (LACEN-MG).
. CC-BY 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted April 19, 2021. ; https://doi.org/10.1101/2021.03.19.21253946doi: medRxiv preprint
NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

18. Departamento de Biologia. Centro de Ciências Exatas, Naturais e da Saúde,
Universidade Federal do Espírito Santo, Alegre, Brazil.
19. Plataforma de Vigilância Molecular, Instituto Gonçalo Moniz, FIOCRUZ-Bahia,
Brazil.
20. Laboratório de AIDS e Imunologia Molecular, Instituto Oswaldo Cruz,
FIOCRUZ-Rio de Janeiro, Brazil.
*, ** These authors contributed equally to this work.
. CC-BY 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted April 19, 2021. ; https://doi.org/10.1101/2021.03.19.21253946doi: medRxiv preprint

Abstract
Mutations at both the receptor-binding domain (RBD) and the amino (N)-terminal
domain (NTD) of the SARS-CoV-2 Spike (S) glycoprotein can alter its antigenicity and
promote immune escape. We identified that SARS-CoV-2 lineages circulating in Brazil
with mutations of concern in the RBD independently acquired convergent deletions and
insertions in the NTD of the S protein, which altered the NTD antigenic-supersite and
other predicted epitopes at this region. Importantly, we detected communitary
transmission of four lineages bearing NTD indels: a P.1 𝚫69-70 lineage (which can
impact several SARS-CoV-2 diagnostic protocols), a P.1 𝚫144 lineage, a P.1-like
lineage carrying ins214ANRN, and the VOI N.10 derived from the B.1.1.33 lineage
carrying three deletions (𝚫141-144, 𝚫211 and 𝚫256-258). These findings support that
the ongoing widespread transmission of SARS-CoV-2 in Brazil is generating new viral
lineages that might be more resistant to antibody neutralization than parental variants of
concern.
Keywords: COVID-19, pandemics, antibody escape, coronavirus, communitary
transmission
. CC-BY 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted April 19, 2021. ; https://doi.org/10.1101/2021.03.19.21253946doi: medRxiv preprint

Introduction
Recurrent deletions in the amino (N)-terminal domain (NTD) of the spike (S)
glycoprotein of SARS-CoV-2 have been identified during long-term infection of
immunocompromised patients
1–4
as well as during extended human-to-human
transmission
3
. Most of those deletions (90%) maintain the reading frame and cover four
recurrent deletion regions (RDRs) within the NTD at positions 60-75 (RDR1), 139-146
(RDR2), 210-212 (RDR3), and 242-248 (RDR4) of the S protein
3
. The RDRs that
occupy defined antibody epitopes within the NTD and RDR regions might alter
antigenicity
3
. Interestingly, the RDRs overlap with four NTD Indel Regions (IR - IR-2
to IR-5) that are prone to gain or lose short nucleotide sequences during sarbecoviruses
evolution both in animals and humans
5,6
.
Since late 2020, several more transmissible variants of concern (VOCs) and also
variants of interest (VOI) with convergent mutations at the receptor-binding domain
(RBD) of the S protein (particularly E484K and N501Y) arose independently in humans
7,8
. Some VOCs also displayed NTD deletions such as lineages B.1.1.7 (RDR2 𝚫144),
B.1.351 (RDR4 𝚫242-244), and P.3 (RDR2 𝚫141-143) that were initially detected in
the United Kingdom, South Africa, and the Philippines, respectively
3
. The VOCs
B.1.1.7 and B.1.351 are resistant to neutralization by several anti-NTD monoclonal
antibodies (mAbs) and NTD deletions at RDR2 and RDR4 are important for such
phenotype
9–14
. Thus, NTD mutations and deletions represent an important mechanism
of immune evasion and accelerate SARS-CoV-2 adaptive evolution in humans.
Several SARS-CoV-2 variants with mutations in the RBD have been described
in Brazil, including the VOC P.1
15
and the VOIs P.2
16
, N.9
17
and N.10
18
. With the
exception of N.10, none of the other variants described in Brazil displayed indels in the
NTD. Importantly, although the VOC P.1 displayed NTD mutations (L18F) that
abrogate binding of some anti-NTD mAbs
14
and further showed reduced binding to
RBD-directed antibodies, it is more susceptible to anti-NTD mAbs than other VOCs
9–14,19
. In this study, we characterized the emergence of RDR variants within VOC and
VOIs circulating in Brazil that were genotyped by the Fiocruz COVID-19 Genomic
Surveillance Network between November 2020 and February 2021.
Results and Discussion
Our genomic survey identified 35 SARS-CoV-2 sequences from seven different
Brazilian states that harbor a variable combination of mutations in the RBD (K417T,
. CC-BY 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted April 19, 2021. ; https://doi.org/10.1101/2021.03.19.21253946doi: medRxiv preprint

E484K, N501Y) and indels in the NTD region of the S protein. These genomes were
classified within lineages N.10 (n = 16), P.1 (n = 14), P.2 (n = 1) and B.1.1.28 (P1-like,
n = 4) (Table 1). Seven VOC P.1 sequences displayed deletion 𝚫69-70 in the RDR1,
three sequences (two VOC P.1 and one VOI P.2) displayed deletion 𝚫144 in the RDR2,
two P.1 sequences showed a four amino acid deletion 𝚫141-144 in the RDR2, two P.1
sequences harbors a two amino acid deletion 𝚫189-190, and one P.1 sequence displayed
a three amino acid deletion 𝚫242-244 in the RDR4. We also detected four B.1.1.28
P.1-like genomes bearing an ins214ANRN insertion upstream to RDR 3 and sharing six
out of 10 P.1 lineage-defining mutations in the Spike protein (L18F, P26S, D138Y,
K417T, E484K, N501Y) as well as P.1 lineage-defining mutations in the NSP3
(K977Q), NS3 (S253P) and N (P80R) proteins
20
. The VOI N.10 displayed NTD indels
𝚫141-144 at RDR2, 𝚫211 at RDR3 and 𝚫256-258 close to RDR4
18
. Inspection of
sequences available at EpiCoV database in the GISAID (https://www.gisaid.org/) at
March 1st, 2021, retrieved three P.1 sequences from the Bahia state
21
and one B.1.1.28
sequence from the Amazonas state
20
with deletion 𝚫144 (Table 1).
The Maximum Likelihood (ML) phylogenetic analysis of lineage P.1 supports
recurrent emergence of variants 𝚫141-144 and 𝚫69-70 and the monophyletic origin of
variants 𝚫144 and 𝚫189-190 (Fig. 1A). Both P.1 𝚫141-144 sequences recovered from
patients from Amazonas and Rondônia states, all P.1 𝚫69-70 sequences from Santa
Catarina state and the P.1 𝚫242-244 sequence from Sergipe state appeared as singletons
intermixed among non-deleted P.1 sequences. The remaining P.1 variants with NTD
deletions were distributed in two sub-clades that also include non-deleted P.1 sequences.
One sub-clade (aLRT = 86%) was characterized by the mutations ORF1a:T951I and
A18945G and comprises nine sequences: the five P.1 𝚫144, the two P.1 𝚫189-190 and
two P.1 from Amazonas and Goiás states. The other sub-clade (aLRT = 85%) was
characterized by the synonymous mutations G29781A and T29834A and comprises
seven sequences: the three P.1 𝚫69-70 from São Paulo state plus four P.1 sequences
from São Paulo, Amazonas and Tocantins states. The ML phylogenetic analyses further
confirm that all sequences belonging to variants P.1-like ins214ANRN (Fig. 1A) and
VOI N.10 (Fig. 1B) branched in highly supported (aLRT > 99%) monophyletic clades.
These findings revealed that NTD deletions characteristic of VOCs B.1.1.7 (𝚫69-70 and
𝚫144) and B.1.351 (𝚫242-244) occurred at multiple times during the evolution of
lineage P.1 and also sporadically arose in lineages B.1.1.28, B.1.1.33 (N.10) and P.2.
. CC-BY 4.0 International licenseIt is made available under a
is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review)
The copyright holder for this preprint this version posted April 19, 2021. ; https://doi.org/10.1101/2021.03.19.21253946doi: medRxiv preprint

Frequently asked questions

Q1. What are the contributions in "The ongoing evolution of variants of concern and interest of sars-cov-2 in brazil revealed by convergent indels in the amino (n)-terminal domain of the spike protein" ?

In this paper, the authors characterized the emergence of SARS-CoV-2 lineages circulating in Brazil with mutations of concern in the receptor-binding domain ( RBD ) independently acquired convergent deletions and insertions in the NTD of the S protein. 

Q2. What can be altered by the SARS-CoV-2 Spike?

Mutations at both the receptor-binding domain (RBD) and the amino (N)-terminal domain (NTD) of the SARS-CoV-2 Spike (S) glycoprotein can alter its antigenicity and promote immune escape. 

Q3. Where do some bats SC2r-CoV lineages display deletions?

Deletions 𝚫242-244 and 𝚫256-258 occur immediately upstream and downstream to IR-5, respectively, where some bat and pangolin SC2r-CoV lineages also displayed deletions. 

Q4. What is the mechanism of evolution of SARS-CoV-2?

Several studies of SARS-CoV-2 evolution in vitro and ex vivo also support thatNTD indels here observed in Brazilian SARS-CoV-2 VOC and VOI represent a mechanism of ongoing adaptive evolution to escape from dominant neutralizing antibodies directed against the NTD. 

Q5. What is the role of the NTD in the evolution of SARS-CoV-2?

In vitro co-incubation of SARS-CoV-2 with highly neutralizing plasma form COVID-19 convalescent patient, has revealed an incremental resistance to neutralization followed by the stepwise acquisition of indels at N3/N5 loops 35. 

Q6. What is the role of the NTD in the transmission of SARS-CoV-2?

The authors identified that SARS-CoV-2 lineages circulating in Brazil with mutations of concern in the RBD independently acquired convergent deletions and insertions in the NTD of the S protein, which altered the NTD antigenic-supersite and other predicted epitopes at this region. 

Q7. What was the sequence alignment used to determine the sequences of SARS-CoV-2?

The S amino acid sequences from selected SARS-CoV-2 and SC2r-CoV lineages available in the EpiCoV database were also aligned using Clustal W 49 adjusted by visual inspection. 

Q8. What are the main findings of this study?

These findings support that the ongoing widespread transmission of SARS-CoV-2 in Brazil is generating new viral lineages that might be more resistant to antibody neutralization than parental variants of concern. 

Q9. What is the role of the Spike gene in the evolution of SARS-CoV-2?

Studies of intra-host SARS-CoV-2 evolution in immuno-compromised hosts revealed the emergence of viral variants with NTD deletions at RDR1 (𝚫69-70), RDR2 (𝚫144 and 𝚫141-144) and RDR4 (𝚫243-244) following therapy with convalescent plasma 1,3,4,36,37. 

Q10. What is the criterion for the prediction of NTD?

NTD are shown as predicted linear epitopes when using PDB accession codes 6VXX 57 and 6VSB 58, (structural coordinates corresponding to the entire S protein), along with a minimum score of 0.9, i.e., a highly strict criterion. 

Q11. What is the sequence of the SARS-CoV-2?

Their search of SARS-CoV-2 sequences available at EpiCoV database in the GISAID (https://www.gisaid.org/) on March 1st retrieved only 146 SARS-CoV-2 sequences of lineages A.2.4 (n = 52), B (n = 3), B.1 (n = 7), B.1.1.7 (n = 1), B.1.177 (n = 1), B.1.2 (n = 1), B.1.214 (n = 80) and B.1.429 (n = 1) that displayed an insert motif of three to four amino acids (AKKN, KLGB, AQER, AAG, KFH, KRI, and TDR) in position 214 (Appendix Table 1). 

Q12. What is the aLRT of the other sub-clade?

The other sub-clade (aLRT = 85%) was characterized by the synonymous mutations G29781A and T29834A and comprises seven sequences: the three P.1 𝚫69-70 from São Paulo state plus four P.1 sequencesfrom São Paulo, Amazonas and Tocantins states. 

Q13. What is the significance of the lack of monophyletic clustering of P.1 ?

The lack of monophyletic clustering of P.1 𝚫69-70 sequences from Santa Catarina, however, shouldbe interpreted with caution due to the paucity of synapomorphic mutations within diversity of lineage P.1. 

Q14. What are the main characteristics of the SARS-CoV-2 genome?

Keywords: COVID-19, pandemics, antibody escape, coronavirus, communitary transmissionRecurrent deletions in the amino (N)-terminal domain (NTD) of the spike (S)glycoprotein of SARS-CoV-2 have been identified during long-term infection of immunocompromised patients 1–4 as well as during extended human-to-human transmission 3. 

Q15. What is the hypothesis that drives the evolution of SARS-CoV-2?

One hypothesis is that such a major selection pressure shift on the virus genome is driven by the increasing worldwide human population immunity acquired from natural SARS-CoV-2 infection that might also select for convergent deletions at NTD.