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The Emergence of SARS CoV-2 Variants of Concern in Oklahoma

Authors:
  • Hale O'mana'o Biomedical Research

Abstract

The emergence of SARS-CoV-2 variants of concern were expected but with the recent surge of cases and hospitalizations associated with the B.1.617.2 (Delta) lineage, a strong response is necessary. This article compares the recent lineages with our collective experiences since the first case was recorded in March of 2020 in Oklahoma, USA. This article discusses the mutation frequencies in the entire proteome expressed by the virus with emphasis not only on transmission, but also the contribution of each viral peptide to COVID-19. Lastly, this article presents unique binding considerations in the Delta variant that requires further study.
Emergence SARS CoV-2 Variants of Concern in Oklahoma
Author
Robert Ricketson, BS, MD
Senior Research Scientist
Hale O’mana’o Research, Division of Emerging Pathogens
Edmond, Oklahoma
From the onset of this pandemic, the scientific community was overwhelmed by the
transmission rate of this new human coronavirus. Because of the rapid transmission
from Wuhan, China to the rest of the Western World, a crucial piece of Operation Warp
Speed (OWS) was focused on vaccine research and development necessary if we were
ever capable of controlling the rapid spread of this virus with the goal of accelerating
testing, supply, development, and distribution of safe and effective vaccines,
therapeutics, and diagnostics by January 2021 (Figure 1) [1].
Figure 1. Operation Warp Speed Accelerated Vaccine Process (Image
courtesy of Coronavirus Operation Warp Speed, Department of Defense, USA)
In the United States, both Moderna and Pfizer elected to bypass OWS funding and
began the development and testing of a mRNA-based vaccine, designed to block viral
1
entry at the surface glycoprotein-hACE2 interface by inducing the formation of
neutralizing antibodies (Figures 2 and 4). Both Pfizer and Moderna’s mRNA vaccines
encode for a form of the SARS-CoV-2 spike glycoprotein that incorporated a
pseudouridine base substitution for uridine so as to evade immune detection through
pathogen nucleic acid recognition Toll-like receptors [2].This allows for translation of the
S gene to the SARS-CoV-2 surface glycoprotein so and induce the formation of
neutralizing antibodies, effectively reducing transmission by blocking the receptor
binding capacity to the human ACE2 receptor critical interacting residues (Figure 2).
Figure 2. The critical SARS-CoV-2:hACE2 interacting residues Y489-N501.
The corresponding sequence from Wuhan-Hu1 is shown below the 3D structure
(PDB 6VW1.) Y489 (magenta arrow) at the end terminus and N501 (green arrow)
at the C-terminus of the surface glyoprotein:nACE2 interactome are shown. The
interacting pocket within the receptor binding domain of the SARS-CoV-2
glycoprotein is identified (thick blue arrow). Structure annotated and visualized
with UCSF Chimera [3-4].
The SARS CoV-2 S gene encodes for a 1273 amino acid surface glycoprotein that
contains the Receptor Binding Domain (RBD) from position 341-514 (accession number
2
N501
QHD434160). The critical interacting residues in the receptor binding domain (position
437-508) is located at the C-terminus. This region appears to be highly antigenic by
epitope analysis and was deemed to be a favorable target for vaccine development
(Figure 3).
Figure 3. B-cell epitope analysis of the SARS CoV-2 Spike glycoprotein.
Both the receptor binding domain (439-508) and the downstream PRRAR furin
cleavage motif epitopes were predicted to be highly antigenic (yellow=greater
than threshold). B-cell epitope prediction of the B.1 SARS-CoV-2 surface
glycoprotein was obtained via the IEDB interface [5-6].
The modified mRNA vaccine developed was sequence based, identified from the initial
B lineage Wuhan-Hu1 complete genome (accession number MN908947; collection date
December 2019; Wuhan, China) . The S gene CDS (position 21563-25384) encodes for
the spike glycoprotein and was used in the development in the initial vaccine for both
the Pfizer and Moderna mRNA constructs (Figure 4).
3
Figure 4. Interaction of the SARS-CoV-2 surface glycoprotein with ACE2
All RNA viruses have an expected mutation rate of around 106 to
104 substitution/nucleotide/cell infection, significantly higher than their human hosts
[7]. SARS-CoV-2 is no exception with the reported mutation rate estimated to be 0.80 –
2.38 × 10-3 nucleotide substitution per site per year [8]. Rates of spontaneous mutation
are critical to understanding the genetic structure of populations over time and thus to
understanding the course of evolution as to the subsequent appearance of
nonsynonymous mutations that may affect the interaction between viruses and their
human receptor. We noticed in December 2020, for example, that the rate of
nonsynonymous mutations downstream from the ORF1a/6 nonstructural protein was
significantly higher in the S protein leaving behind a concern as to how these may affect
any future intervention vaccine effectiveness (Figures 5,6). The likely cause of that is
probably due to less negative selection pressure (pressure for polymerase to remain
unchanged) and more positive selection pressure (for surface proteins to adapt to new
host and evade host immune response) [10].
4
Figure 5. Frequency of Unique Nonsynonymous Mutations in SARS-CoV-2.
5
Figure 6. Comparison of the various Viral Peptide Frequency of Unique,
Nonsynonymous Mutations in SARS-CoV-2 as of December 2020.
As such, the subsequence of variants following the development of the initial vaccine is
expected. How those subsequent mutations may affect transmission were a matter of
speculation.
Unfortunately, by the end of 2020, during the peak surge in this pandemic,
serosurveillance identified the emergence of Variants of Concern (VOC’s) that directly
impacted the surface glycoprotein-hACE2 interaction (Figure 7).
6
Figure 7. New US case data as of December 31, 2020
(Source: https://covid.cdc.gov/covid-data-tracker/#trends_dailytrendscases)
7
Figure 8. New Case Data in Oklahoma as of December 31, 2020 [10].
8
The Emergence of Variants of Interest (VOI), Concern (VOC), and High
Consequence
As these new variants began to emerge, Rambaut et al developed a rational and
dynamic method of SARS-CoV-2 nomenclature using a phylogenetic method of the
complete genome (~29,000 bp) necessary to identify lineages that contribute to active
spread of the virus. As of this date, all VOIs in Oklahoma are decreasing (See
Supplemental files) [11]. This method was termed PANGO (Phylogenetic Assignment of
Named Global Outbreak lineages (not based upon the pangolin sequence similarity
between the S protein of pangolin CoV’s and SARS-CoV-2) and has proven useful in
genomic surveillance of emerging SARS-CoV-2 variants [12-13]. Both the World Health
Organization and the US use this system to determine which emerging lineage might
escalate or deescalate the current pandemic.
Variant of Interest (VOIs) are characterized by:
Specific genetic markers that are predicted to affect transmission (mutations in
the receptor binding domain and/or furin cleavage motifs), laboratory diagnostics,
therapeutic intervention strategies (antivirals, interferon inhibitors, etc.), or
immune escape from diminished effectiveness of vaccine-induced neutralizing
antibodies
Evidence that it is the cause of an increased proportion of cases or unique
outbreak clusters shown through epidemiologic and genomic surveillance
Limited documented prevalence or expansion in the US or in other countries
These VOIs might necessitate further intensive public health actions and monitoring
over time to fully understand transmission dynamics, changes in disease severity, and
effectiveness of current treatment protocols.
PANGO WHO COUNTRY
FIRST
IDENTIFIED
SGP
SUBSTITUTIONS* USA-OK initial
collection
date/Trend
B.1.427 EPSILON USA-CA L452R,D614G 2021-02-21;
QRX54461;
Decreasing
B.1.429 EPSILON USA-CA S13I, W152C,
L452R, D614G
2021-01-09
QRX02846.;
Decreasing
B.1.525 ETA UK/Nigeria A67V, 69del, 70del,
144del, E484K,
D614G, Q677H,
F888L
2021-01-25
QTZ59889.1;
Decreasing
B.1.526 IOTA USA-NY L5F, (D80G), T95I,
(Y144-), (F157S),
D253G, (L452R),
2021-03-14
QTX86954.1;
Decreasing
9
(S477N), E484K,
D614G, A701V,
(T859N), (D950H),
(Q957R)
B.1.617.1 KAPPA India (T95I), G142D,
E154K, L452R,
E484Q, D614G,
P681R, Q1071H
2021-04-05
QUP25016.1
Decreasing (one
isolate obtained)
B.1.617.3 Unclassified India T19R, G142D,
L452R, E484Q,
D614G, P681R,
D950N
Not identified in
Oklahoma as of
2021-07-01
P.2 ZETA Brazil E484K, (F565L*),
D614G, V1176F
Not identified in
Oklahoma as of
2021-07-01
*SGP (surface glycoprotein nonsynonymous substitutions (reference sequence
accession number MN908947.2, Wuhan-Hu-1; translated S protein QHD43416.1). The
residues directly involved in the SARS-CoV-2:ACE2 receptor binding (Bold/Black)site
and furin cleavage motifs (Bold/Red)are identified.
Source: CDC SARS-CoV-2 Variant Classification and Definitions; [14]
Variant of Concern (VOCs) are additionally found by direct evidence to have:
An impact on diagnostics, treatments, or vaccines characterized by:
Widespread interference with diagnostic test targets
Evidence of substantially decreased clinical responsivity to one or more
class of therapies
Evidence of significant decreased neutralization by antibodies generated
during previous infection or vaccination
Evidence of reduced vaccine-induced protection from severe disease
(breakthrough infections)
Epidemiologic evidence of increased transmissibility
Evidence of increased disease severity (increased hospitalizations)
PANGO WHO COUNTRY
FIRST
IDENTIFIED
SGP
SUBSTITUTIONS
*
USA-OK initial
collection
date/Trend
B.1.1.7 Alpha United
Kingdom 69del, 70del,
144del, (E484K*),
(S494P*), N501Y,
A570D, D614G,
2021-01-25
QST04173.1;
Initial surge
through May
2021
10
P681H, T716I,
S982A, D1118H
(K1191N)
(predominant
variant).
B.1.351 Beta South Africa D80A, D215G,
241del, 242del,
243del, K417N,
E484K, N501Y,
D614G, A701V
2021-03-02
QTF10955.1;
Decreasing
P.1 Gamma Japan/Brazil L18F, T20N,
P26S, D138Y,
R190S, K417T,
E484K, N501Y,
D614G, H655Y,
T1027I
2021-01-11
QREO1802.1;
Increasing
B.1.617.2 Delta India T19R, (G142D*),
156del, 157del,
R158G, L452R,
T478K, D614G,
P681R, D950N
2021-05-21
QUG147861.1;
Significant
increase (April
2021 (0.9%)
through June
2021 (67%)
*SGP (surface glycoprotein nonsynonymous substitutions (reference sequence
accession number MN908947.2, Wuhan-Hu-1; translated S protein
QHD43416.1). The residues directly involved in the SARS-CoV-2:ACE2 receptor
binding (Bold/Black)site and furin cleavage motifs (Bold/Red)are identified.
Source: CDC SARS-CoV-2 Variant Classification and Definitions [14]
These VOC’s have subsequently been shown by genomic surveillance to increase
transmission, immune evasiveness, and reduce vaccine effectiveness to a variable
degree.
Variants of High Consequence
A variant of high consequence has clear evidence that prevention measures or medical
countermeasures (MCMs) have significantly reduced effectiveness of diagnostics,
vaccination (increase in breakthrough infection/low vaccination rates), approved
therapeutic, and an increase in severe clinical disease and/or hospitalization relative to
previously circulating variants.
At this time no current variant meet these criteria for a Variant of High Consequence.
11
2021: The Rise of VOCs in Oklahoma
Figure 9. Emergence of SARS-CoV-2 Variants of Concern in Oklahoma: March
2020 through July 1, 2021
The global concern of a shift from the less virulent variants seen in the early months of
the pandemic in 2020 to the potentially more virulent variants focused on increased
transmission (surface glycoprotein) and increased morbidity and mortality caused by the
many virulent factors present in SARS CoV-2 (Figure 5). Our early comparative data of
the nonsynonymous mutations across the multiple proteins produced by SARS-CoV-2
clearly demonstrated a higher frequency of nonsynonymous mutations in the structural
proteins (surface glycoprotein, ORF3a, envelope, membrane, ORF6, ORF7a/7b, ORF8,
nucleoprotein, and ORF10) downstream from the orf1a/b -1 frameshift. Those translated
viral peptides upstream from the frameshift remained highly conserved (>96%). As
such, it is unlikely that an increase in morbidity would by less likely to have resulted
from those viral peptides.
Of all the structural viral peptides that are known contribute to the pathogenesis of
COVID-19 (surface glycoprotein (ORF3a membrane protein, ORF6, ORF7a/7b, ORF8,
and nucleoprotein), the frequency of unique nonsynonymous mutations corrected for
peptide length in the surface glycoprotein (28%), ORF3a (7%), membrane protein
(74%), ORF6 (11%), ORF7a/7b (14-16%), ORF8 (10%), and nucleoprotein (13%)
12
require greater genomic surveillance. Since the vaccines were developed to reduce viral
entry through induction of neutralizing antibodies directed towards the receptor binding
site in the surface glycoprotein, it makes intuitive sense to monitor any significant
substitutions in covariant amino acids (change in function as a result a substitution to a
noncovariant amino acid; e.g. mutation to a charged amino acid leucine to arginine
(noncovariant substitution) in the binding pocket (e.g., L484K, N501Y) that may
increase transmission and viral load in the host and clinical outcomes (Figure 10) [15].
Thus, this article focuses on those mutations in VOCs that are observed to affect the
interacting residues within the receptor binding domain of SARS-CoV-2.
Lineage B.1.1.7 (Alpha)
From September to December of 2020, there were an increased proportion of cases
reported in Kent and Greater London in the United Kingdom despite social restrictions.
A new Variant of Concern was detected within the COVID-19 Genomics Consortium UK
surveillance set that represented a distinct phylogenetic cluster named lineage B.1.1.7
[17]. Within the spike glycoprotein, there were identified 2 deletions (HV69-70, Y144)
and 6 nonsynonymous mutations (N501Y, A570D, P681H, T716I, S982A, and D1118H.
Of these, N501Y is located in the critical binding pocket and P681H is located at the N-
terminus of the furin cleavage motif. Both sites are associated with increased infectivity
and virulence [18] (Figure 10).
13
Figure 10. SARS-CoV-2:hACE2 Wuhan-Hu1/B.1.1.7 Interactome Comparison
The 3D reconstruction comparing the critical binding residues of the SARS-CoV-
2 surface glycoprotein are demonstrated comparing the change in the surface
binding at position 501. [Template PDB 6VW1. Structure of SARS-CoV-2
chimeric receptor-binding domain complexed with its receptor human ACE2 [16].
Panel A. The spike glycoprotein – human ACE2 receptor binding region of the
wild-type Wuhan-Hu-1 (blue ribbon) to human ACE2 (tan ribbon). N501 surface
interaction with the human Q42 and L45 position are labeled.
Panel B. The B.1.1.7 tyrosine substitution (N501Y) is overlayed on the Wuhan-
Hu-1 structure demonstrating the comparatively larger surface binding interaction
region (red).
Panel C. Multiple sequence alignment of the 69-70HV deletions (top), critical
interacting residues (middle: positions 480-501), and the furin cleavage motif
(bottom) P681R substitution location.
[MSA was aligned with MUSCLE and visualized with Jalview. 3D reconstruction
performed with Modeller in UCSF Chimera].
The B.1.1.7 (Alpha) variant rapidly spread across the UK and Europe. It was first
identified in the US on 2020-12-24 (accession number MW422256; USA-CO) ). As of
July 1, 2021, a total of 417, 087 isolates of B.1.1.7 have been sequenced in the US
[Source: NCBI coronavirus datasets [19]. Despite the predominance of B.1.1.7 (Alpha)
here in the US, there has not been a significant increase in reported cases .
On 20201-21, our first case of the B.1.1.7 variant was identified (accession number
QST08028.1) here in Oklahoma. By the end of May 2021, this variant rapidly replaced
the B.1.2 clade as the predominant isolate (> 80%) (Figure 11).
Figure 11. Emergence of VOC B.1.1.7 in Oklahoma 2021. Data compiled from
all available surface glycoprotein sequences in the NCBI databank. All lineages
were confirmed by submission of the complete genome sequence for analysis
through the Pangolin COVID-19 Lineage Assigner web application [20-21]
14
Despite the emergence of B.1.1.7 as the predominant sequence identified in the NCBI
coronavirus databank, we did not observe an expected increase in cases as restrictions
were lifted across the state [Figure 12].
Figure 12. New case data in Oklahoma as of May 30, 2021
This could not be attributed to a majority of the population having received at least 1
vaccination (1,770,574) or being previously exposed to SARS-CoV-1 (cumulative cases
458,180) by the end of June 2021. With the predicted herd immunity (70% of 4
million=2.8 million) in our state, it is unlikely we had reached that threshold even if
asymptomatic transmissions were under consideration. The overall seropositivity was
quite high (18.9%) compared to other states here in the US, most likely because the
population did not present to clinics to be tested as frequently as it was by the end of
15
2020. Because of that, the actual number of new exposures were probably greatly
underestimated.
Vaccine hesitancy here in Oklahoma is a concern. Only 19 of Oklahoma’s 77 counties
are above 50% in one dose vaccinations with 20 counties between 30% and 40%.
Oklahoma County is #1 at 65.3% of its adult population having had at least one dose,
followed by Caddo County, at 63.9%, and Noble County, at 61.9% and Tulsa County at
61.2%. The lowest vaccine saturation is 28.8% in Dewey County (Source: CDC data
tracker, 2021-06-28).
Figure 13. Seropositivity in Oklahoma as of June 1, 2021.
Lineage B.1.351
On October 8, 2020, a new variant was detected to have significant nonsynonymous
mutations in the receptor binding domain of SARS-CoV-2 (K417N, E484K and N501Y)
on the east coast of Eastern Cape, South Africa. Both N501Y and E484K have been
identified to confer some degree of resistance to neutralizing antibodies [22]. (Figure
14).
16
Figure 14. SARS-CoV-2:hACE2/B.1.351 Interactome Comparison
Panel A: PDB 6VW1 (Structure of SARS-CoV-2 chimeric receptor-binding
domain complexed with its receptor human ACE2). The surface glycoprotein is
shown (blue ribbon) receptor binding domain bound to its haCE2 receptor (tan
ribbon). The primary SARS-CoV-2 residues in the N-terminus of the critical
binding domain are as labeled (S477, E484, Q493, and N501). Minimal surface
binding at N501 (green surface/atoms) with ACE2 is visualized.
Panel B: B.1.351 with hACE2 modeled using 6VW1 as template with Modeller in
USCF Chimera. Both the E484K and N501 nonsynonymous mutations provide
significant surface binding interactions with ACE2 compared to the B lineage
(Wuhan-Hu-1).
Panel C. Multiple sequence alignment of the Wuhan-Hu-1 and B.1.351 lineage
glycoprotein receptor binding domain used in the structure model. E484K and
N501Y nonsynonymous mutations are as indicated above the MSA in red. The
downstream PRRAR furin cleavage motif is conserved.
This lineage spread rapidly and became the dominant variant in that region. As with
B.1.1.7, this new lineage spread rapidly across the globe [23]. By the end of April 2021,
there were 1783 B.1.351 lineages detected here in the US (Figure 15).
17
.
Figure 15. Global Emergence of B.1.351 lineage
Image obtained from the PANGO network [24-25]
In Oklahoma, lineage B.1.351 was first detected (accession number MW795351) on
March 28, 2021 (See supplemental files). Over the next 3 months, only 6 occurrences of
this lineage have been detected in the NCBI database for Oklahoma. This lineage does
not appear to have gained a foothold here in our state (Figure 16).
18
Figure 16. Detection of B.1.351 lineage in Oklahoma, March through June 2021.
All lineages were confirmed by submission of the complete genome sequence for
analysis through the Pangolin COVID-19 Lineage Assigner web application [20]
Lineage P.1 (Gamma)
In November of 2020, Manaus, Brazil began to experience a rapid increase in
hospitalizations. Genomic surveillance identified a new lineage, P.1, detected in a local
isolate on October 1, 2020. 10 significant nonsynonymous mutations were identified in
the surface glycoprotein, three of which were identified in the receptor binding domain
(K417T, E484K, and N501Y). As with B.1.1.7, the furin cleavage motif was 100%
conserved (Figure 17) [26] .
Figure 17. SARS-CoV-2:hACE2/P.1 Interactome Comparison
19
Panel A: PDB 6VW1 (Structure of SARS-CoV-2 chimeric receptor-binding
domain complexed with its receptor human ACE2). The surface glycoprotein is
shown (cyan ribbon) receptor binding domain bound to its haCE2 receptor (tan
ribbon). The primary SARS-CoV-2 residues in the N-terminus of the critical
binding domain are as labeled (S477, E484, Q493, and N501). Minimal surface
binding at N501 (green surface/atoms) with ACE2 is visualized.
Panel B: P.1 surface glycoprotein with hACE2 modeled using 6VW1 as template
with Modeller in USCF Chimera. Both the E484K and N501 nonsynonymous
mutations provide significant surface binding interactions with ACE2 compared
to the B lineage (Wuhan-Hu-1). The K417T mutation does not appear to provide
any significant interaction with hACE2.
Panel C. Multiple sequence alignment of the Wuhan-Hu-1 and P.1 glycoprotein
receptor binding domain used in the structure model. E484K and N501Y
nonsynonymous mutations are as indicated above the MSA in red. The
downstream PRRAR furin cleavage motif is conserved.
The first detection of this new lineage in the US was collected on January 9, 2021, in
Minnesota and spread rapidly on the mainland with 13,060 isolates identified as of the
end of June 2021 (Source: NCBI SARS-CoV-2 Resources (Figure 18) [27].
20
Figure 18 Global Emergence of P.1 lineage (Image obtained from the
PANGO network)
In Oklahoma, the P.1 lineage was first detected on January 11, 2021 (accession
number MW559120). From January through June of 2021, 5 additional isolates of P.1
were collected and identified with the highest number of isolates found in May (6
isolates, 7.4% of 68 isolates). As with B.1.351, there is minimal evidence of extensive
local transmission of this lineage (Figure 19).
21
Figure 19. Emergence of P.1 lineage (Gamma) in Oklahoma.
All lineages were confirmed by submission of the complete genome sequence for
analysis through the Pangolin COVID-19 Lineage Assigner web application [20]
Lineage B.1.617.2 (Delta)
On 2020-09-07, this lineage was detected in India . There are several nonsynonymous
mutations observed (T19R, G142D, L452R, T478K, D614G, P681R, D950N0) with L452R
and T478K located within and close proximity to the critical binding residues in the receptor
binding domain. This variant also contains the P681R in the furin cleavage motif found to
enhance viral infectivity (Figure 20) [28].
Figure 20. SARS-CoV-2:hACE2/B.1.617.2 and B.1.617.1 Interactome
Comparison
Panel A: PDB 6VW1 (Structure of SARS-CoV-2 chimeric receptor-binding
domain complexed with its receptor human ACE2). The surface
glycoprotein is shown (cyan ribbon) receptor binding domain bound to its
haCE2 receptor (tan ribbon). The primary SARS-CoV-2 residues in the N-
terminus of the critical binding domain are as labeled (S477, E484, Q493,
and N501). Minimal surface binding at N501 (green surface/atoms) with
ACE2 is observed.
22
Panel B: B.1.617.2 (surface glycoprotein with hACE2 modeled using
6VW1 as template with Modeller in USCF Chimera. The L452R and
T478K is unique in this Oklahoma variation of the canonical B.1.617.2
variant as there does not appear to contain the E 484Q nonsynonymous
mutation as observed with the B.1.617.1 lineage. This variant of B.1.617.2
does not provide significant surface binding interactions with ACE2. A
shift upstream in the critical binding residues due to the L452R mutation
needs to be confirmed.
Panel C. B.1.617.1 (L452R/E484Q model). Oklahoma isolate
QUP250162.1 (MN908942.1 complete genome). The E484Q substitution
appears to have extensive surface interaction with K31 of the ACE2
residue (6VWI) as compared to E484. This appears to provide a potential
reduction in the neutralizing antibodies compared to variation a (Panel B,
predominant B.1.617.2 in Oklahoma).
Panel D. Multiple sequence alignment of the Wuhan-Hu-1, B.1.617.2 and
B.1.617.1 glycoprotein receptor binding domain used in the structure
model. E484K and N501Y nonsynonymous mutations are as indicated
above the MSA (variation b) in red. In comparison. The downstream
PRRAR furin cleavage motif is conserved in B.1.617.2 and B.1.617.1 in
Oklahoma: [Data: Hale O’mana’o Research, Edmond Oklahoma].
Sheik et al reported that the Delta variant was 50-60% more transmissible than the Alpha
variant (B.1.1.7) and the hospitalization rate among those infected with the Delta variant was
85% higher than those infected with the Alpha variant [29]. Bernal et al reported that vaccine
effectiveness was reduced with the Pfizer BNT162b2) and Azteca (ChAdOx1) vaccines, by
33.5% after 1 dose. Additionally, the authors reported there was a higher risk of infection with
the Delta variant compared to unvaccinated cases (OR 1.40; 95%CI:1.13-1.75). [30] . Similar
reductions in neutralizing antibody titers in B.1.617.2 were also reported by Wall et al.
[31].
By February 2021, this variant was detected in Australia (2021-02-07) and Singapore
(2021-02-06) followed closely in the US. By the end of May 2021, the Delta variant had
spread globally to 93 countries (Source: Rambaut Group (Figure 21) [32].
23
Figure 21. Global Expansion of B.1.617.2 lineage [Image source: https://cov-
lineages.org/global_report_B.1.617.2.html]
In the US, the first isolate was recorded as collected on 2021-02-15 in Massachusetts.
(USA-Massachusetts, Release date 2021-03-15, accession number MW750080 [33].
As of July 14, 2021, there were recorded 72,520 isolates in the US and 18 in Oklahoma
as of 2021-07-04 [34].
The first isolated B.1.167.2 lineage identified in Oklahoma was collected on April 5,
2021 (accession number MZ028778.1, SARS-CoV-2/human/USA/OK-CDC-2-
4195490/2021) . The Delta variant has been increasingly identified in the northeast
portion of the state (Ottawa County) where less that 30% had at least one vaccination.
Since the first case was identified, the B.1.617.2 lineage represented 79% of the
isolates collected in the month of June 2021 (Figure 22).
24
Figure 22. Emergence of B.1.617.2 lineage (Delta) in Oklahoma.
All lineages were confirmed by submission of the complete genome sequence for
analysis through the Pangolin COVID-19 Lineage Assigner web application [20].
As these cases increased to 64.3/110K in one week (7-day positivity 10-14.9%) , there
was observed an increase in hospitalizations due to COVID-19. The overall vaccination
rate was also dismal where only 45.4 % of our total population has received at least 1
dose of the Pfizer or Moderna vaccines compare to 55.6% nationwide. Additionally, we
also observed an increase in hospitalizations across the state, up 28% compared to the
previous week [35-36].
Chronologic Summary
Both the Pfizer and Moderna vaccines appear to be very effective (Figure 22).
Remember that the vaccine was developed using the spike glycoprotein sequence in
the B lineage and its initial descendants. So, when the rollout began around the end of
December, we were in a major surge. Not from any new variants of concern (at least
here in the US-Alpha wasn't detected until January). After all we had been in a steady
decline of new cases from January through the first of May, despite the presence of
B.1.1.7 (Alpha), B.1.351 (Beta), and P.1 (Gamma) in our state population.
On April 5, 2021, we identified the B.1.617.2 (Delta) lineage here in Oklahoma. In
contrast to the other VOCs, we observed an uptick in cases and hospitalizations that we
really did not see with any of the previously mentioned VOCs. That could be due to
several factors: reduced neutralizing antibody titers, waning immunity, immune evasion,
vaccine hesitancy, and a variety of individual societal factors (Figure 23).
25
Figure 22. Timeline of the Emergence of Variants of Concern in Oklahoma
State-mandated Intervention
As the nation began its major surge following the July 4, 2020, weekend, it clearly
became necessary to legislate mandates on social gatherings and social isolation.
26
These measures nationwide included bar closures, gathering bans, mask mandates,
restaurant closures, and stay at home orders.
In Oklahoma, these measures failed to curb the increasing number of cases, Governor
Stitt announced the Seventh Amended Executive Order (EO 2020-20) [37] which stated:
All restaurants will be required to space tables at least six feet apart,
unless tables, booths and bar areas are separated by sanitized dividers
All bars and restaurants will be required to close by 11 p.m. No in-
person service of food or alcohol will be allowed after 11 p.m. except
for restaurant drive-thru windows or curbside pickup
All 33,000 state employees under the executive branch will be required
to wear a mask in common areas or when they’re around other people.
All visitors to state agency buildings will also be required to wear a
mask
The State’s mandates, however, did not include a mask mandate for the general public.
These measures were largely left to the discretion of local city government. Recently,
only 34% of Americans polled remain socially distanced. 25% continue to wear a mask
when out in public and less than 50% are willing to self-quarantine, down 13 points from
June of 2021 [38].
Despite the widespread availability of vaccines since the end of December, only two-
thirds of adults had received at least one dose of a COVID-19 vaccine. Unfortunately,
vaccine hesitancy continued to be a significant issue. In the population that remained
unvaccinated, less than 25% acknowledged they would be willing to be immunized
despite the outreach. Some of reasons for vaccine hesitancy included:
Safety and efficacy concerns. Myths and conspiracy theories fueled this
debate. These included:
oCOVID-19 vaccine can affect women’s fertility. It was suggested that
the surface glycoprotein of SARS-CoV-2 was similar to syncitin-1 encoded
by the ERVW-1 gene that is involved with the growth and attachment of
the placenta during pregnancy. Even with the possible molecular mimicry
consideration, the SARS-CoV-2 has only a 33.3% overall sequence
identity with Syncitin-1 and in no instance is there heptad identity greater
than 50% across the alignment. Additionally, no clinical trial has
demonstrated a loss of pregnancy that had received the Pfizer vaccine.
oIf you had COVID-19, a vaccine isn’t necessary. Sustained immunity
from exposure to SARS-CoV-2 does wane over time (6-8 months).
Although the instance of reported breakthrough infections is uncommon
(10262 SARS-CoV-2 vaccine breakthrough infections), the incidence of
27
breakthrough infections with the increasing number of variants was not
significantly different from 2020 before vaccines were available [39].
oGetting the COVID-19 vaccine gives you COVID-19. The vaccine does
not contain an intact virion, only the mRNA sequence that encodes for the
spike glycoprotein.
oThe COVID-19 vaccine can alter human DNA. Although Zhang et al
reported identifying target site duplications flanking the viral sequences
and consensus LINE1 endonuclease recognition sequences at the
integration sites, consistent with a LINE1 retrotransposon-mediated,
target-primed reverse transcription, no replication competent viral genome
had been identified in vitro. Moreover, these isolated integrations were at
LINEs, SINEs, or long-terminal repeats (LTRs). Because the mRNA in
the vaccine is rapidly degraded intracellularly, an integration event would
be extremely unlikely. [40].
Preference for physiologic immunity. There is some concern that the risk of
vaccination is greater than the risk of infection. Some minimize the 1.6% case
fatality rate in Oklahoma as being inconsequential (friends and family didn’t have
negative side effects; reported cases of negative side effects from vaccines).
Distrust in government and health organizations. This was the result of social
media and conservative media exposure increasing public doubt on
transmission, prevention, and a variety of conspiracy theories on the origin of
SARS-CoV-2.
Autonomy and personal freedom. There are those that react in opposition to
any infringement on personal autonomy and freedom despite the overall public
health risk. That feeling of coercion to comply with any mandate increases
anxiety and fear, leading to more reluctance to vaccinate.
Denial of Disease. Based on fear, anosognosia has long been identified in both
mental illness and, more importantly, a coping mechanism with a lack of willing
insight into their condition. Misinformation further compounds their willingness to
conform to shifting recommendations and authoritative recommendations.
Discussion
With the surge of human-human transmission from June 2020 through early January
2021 as the new vaccine was rolled out, the emergence of these variants of concern
were not unexpected given the known mutation rate of SARS-CoV-2 (0.80 – 2.38 × 10-
3 nucleotide substitution per site per year). We have not yet addressed the potential
impact of zooanthroponotic transmission despite the known occurrences in both
domestic and mammalian species of wildlife, both realized and potential. How that
reverse transmission and subsequent zoonotic chain of reintroduction to humans
remains to be defined in the interest of One Health [43, 44].
28
Transmission has been the focus of prevention in our public heath response. Morbidity
is not solely identified through the surface glycoprotein. As discussed earlier, there are
additional SARS-CoV-2 peptides with documented pathways in the interferon
agonist/antagonist pathways involved in the pathogenesis of COVID-19 (NSP1/Leader),
NSP2, NSP3, helicase, 3’-5’ exonuclease, endoRNAse, ORF3a, membrane
glycoprotein, ORF6, ORF7b, ORF8, and nucleocapsid) which need attention if we are to
make any significant therapeutic advances other than vaccination (Figure 23) (See
Supplemental files).
Transmission has been the focus of prevention in our public heath response. Morbidity
is not solely identified through the surface glycoprotein. As discussed earlier, there are
additional SARS-CoV-2 peptides with documented pathways in the interferon
agonist/antagonist pathways involved in the pathogenesis of COVID-19 (NSP1/Leader),
NSP2, NSP3, helicase, 3’-5’ exonuclease, endoRNAse, ORF3a, membrane
glycoprotein, ORF6, ORF7b, ORF8, and nucleocapsid) which need attention if we are to
make any significant therapeutic advances other than vaccination (Supplemental files).
29
30
Figure 23. Conservation between B.1 and Current Variants of Concern.
It has been suggested that a booster of the current formulation may be effective. Given
the current rate of transmission in the unvaccinated and the lack of corroborated
information on breakthrough infections in vaccinated individuals, the presence of new
variants may very well require new design of the mRNA vaccine, particularly if we see a
shift downstream of the receptor binding interacting residues in B.1.612.2 lineage
(Figure 20 discussion).
Ultimately, as with our collective experience with the West Africa Ebola outbreak, our
response needs to incorporate cultural perspectives, reaching out to community
leaders, thoughtful transparency in media coverage, improved therapeutics towards
reduction of morbidity and mortality in established COVID other than transmission
dynamics, improved epidemiologic surveillance and genomic surveillance in both
human and other mammalian species capable of ongoing geographic transmission, as
well as identifying other preventative measures to reduce the impact of this pandemic
now and future epidemics and pandemics when they arise in the future. Which they will.
31
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