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Medical Countermeasures Analysis of 2019-nCoV and Vaccine Risks for Antibody-
dependent Enhancement (ADE)
Darrell O. Ricke, PhD1 & Robert W. Malone2, MD
1Biological and Chemical Technologies
Massachusetts Institute of Technology Lincoln Laboratory
Lexington, MA USA
2Chief'Medical'Officer,'Alchem'Laboratories'
13305'Rachel'Boulevard,'Alachua'FL'32615'USA'
rmalone@alchem.com'
ORCID:'0000-0003-0340-7490'
Corresponding author: Darrell O. Ricke, Ph.D.
Email: Darrell.Ricke@ll.mit.edu
ORCID for Darrell Ricke is 0000-0002-2842-2809
Summary
Background In 80% of patients, COVID-19 presents as mild disease1,2. 20% of cases develop
severe (13%) or critical (6%) illness. More severe forms of COVID-19 present as clinical severe
acute respiratory syndrome, but include a T-predominant lymphopenia3, high circulating levels
of proinflammatory cytokines and chemokines, accumulation of neutrophils and macrophages in
lungs, and immune dysregulation including immunosuppression4.
Methods All major SARS-CoV-2 proteins were characterized using an amino acid residue
variation analysis method. Results predict that most SARS-CoV-2 proteins are evolutionary
constrained, with the exception of the spike (S) protein extended outer surface. Results were
interpreted based on known SARS-like coronavirus virology and pathophysiology, with a focus
on medical countermeasure development implications.
Findings Non-neutralizing antibodies to variable S domains may enable an alternative infection
pathway via Fc receptor-mediated uptake. This may be a gating event for the immune response
dysregulation observed in more severe COVID-19 disease. Prior studies involving vaccine
candidates for FCoV5,6 SARS-CoV-17-10 and Middle East Respiratory Syndrome coronavirus
(MERS-CoV) 11 demonstrate vaccination-induced antibody-dependent enhancement of disease
(ADE), including infection of phagocytic antigen presenting cells (APC). T effector cells are
believed to play an important role in controlling coronavirus infection; pan-T depletion is present
in severe COVID-19 disease3 and may be accelerated by APC infection. Sequence and structural
conservation of S motifs suggests that SARS and MERS vaccine ADE risks may foreshadow
SARS-CoV-2 S-based vaccine risks. Autophagy inhibitors may reduce APC infection and T-cell
depletion12 13. Amino acid residue variation analysis identifies multiple constrained domains
suitable as T cell vaccine targets. Evolutionary constraints on proven antiviral drug targets
present in SARS-CoV-1 and SARS-CoV-2 may reduce risk of developing antiviral drug escape
mutants.
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© 2020 by the author(s). Distributed under a Creative Commons CC BY license.
Interpretation Safety testing of COVID-19 S protein-based B cell vaccines in animal models is
strongly encouraged prior to clinical trials to reduce risk of ADE upon virus exposure.
Introduction
COVID-19 is caused by the SARS-CoV-2 (2019-nCoV) betacoronavirus. The SARS-CoV-2 is a
novel betacoronavirus with sequenced genomes ranging from 29.8k to 29.9k RNA bases. The
SARS-CoV-2 genome encodes replicase proteins, structural proteins, and accessory proteins14
(Table 1). The ORF1a and ORF1ab polyproteins are proteolytically cleaved into 16 non-
structural proteins designated nsp1-1614 (Table 1). Like SARS, COVID-19 manifests as a
virulent zoonotic virus-mediated disease in humans with currently 81,191 confirmed cases and
2,768 deaths as of Feb. 26, 202015.
Zoonotic MERS-CoV, SARS-CoV-1, and SARS-CoV-2 are evolutionarily related, and share
many similarities in human disease characteristics and progression. The mild variant first phase
of viral progression generally presents with mild flu-like symptoms. Most patients never
progress beyond this phase, and typically recover quickly and uneventfully. In a mouse animal
model, phagocytic cells contribute to the antibody-mediated elimination of SARS-CoV-116, and
it may be that innate responses are sufficient to suppress MERS-CoV and SARS-CoV-2 in the
majority of patients. For some individuals (18.5%17), infection progresses to a second severe-
critical variant phase. Progression to the second phase often coincides with the typical timing of
onset of adaptive humoral immunity antibody response (approximately 7-14 days post infection).
MERS-CoV can infect monocyte-derived macrophages (MDMs), monocyte-derived dendric
cells (MoDCs), and T-cells18,19, but the infectivity of SARS-CoV-2 in these cell populations
(with or without non-neutralizing antibody) has not been characterized. For patients with severe
and critical symptoms, the pathophysiology is consistent with increased infection of phagocytic
immune cells (immature MDMs and MoDCs); see Figure 1 for a diagram of the postulated
cascade mechanism. Chemokines released from infected cells may attract additional dendritic
cells and immature macrophages that are susceptible to infection, leading to a possible infection
amplifying cascade of immune cell infection and dysregulation. For some patients with severe
symptoms, excessive activation of macrophages may contribute to a chemokine and cytokine
storm20-22. Individuals with SARS have pronounced peripheral T-cell lymphocytopenia with
reduced CD4+ and CD8+ T-cells23,24, just as is observed with COVID-193. MERS-CoV and
SARS-CoV are also associated with T-cell apoptosis25,26. Infection of macrophages and some T-
cells along with viral dysregulation of cellular pathways result in compromised innate and
humoral immunity in patients during this second and more severe phase of infection 27. High
virus titer in blood plus the possibility of infected immune cell migration throughput the body
may account for the additional disease pathophysiologic and clinical observations observed with
these viruses. MHC I and interleukin (IL)-12 receptor B1 (IL-12RB1) genetic differences
associated with disease progression has been characterized for SARS28-30. Patients with low or
deficient serum levels of the innate immune response pattern recognition molecule mannose-
binding lectin (MBL) have increased frequency in SARS patients versus controls31. MHC
downregulation by epigenetic modifications seen with MERS-CoV infections may enhance
avoidance of T-killer cell responses, and direct infection of some T-cells18 may play a role in
increased mortality rate seen for MERS32. Other disease differences may simply be the different
population of cells with target host receptors angiotensin I converting enzyme 2 (ACE2) for
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 March 2020 doi:10.20944/preprints202003.0138.v1
SARS-CoV-1 and SARS-CoV-233, and dipeptidyl peptidase IV (DPP4) for MERS-CoV. ACE2
is expressed in high density in lungs34.
Characterizing variability and evolution of viral proteins must inform medical countermeasure
(MCM) design and development strategies for RNA viruses such as SARS-CoV-2. For viral
progeny, deleterious mutations are rapidly selected against35. Neutral mutations36 provide a
framework for antigenic drift to facilitate escape from immune responses; these residues will
continue to mutate over time. The critical-spacer model proposes that proteins have either amino
acid residue side-chains critical for function or have variable side-chains which may function for
positioning/folding of critical residues37. The divergence model of protein evolution proposes
that the number of critical residues for a protein is consistent for evolutionarily closely related
proteins38. Herein, these concepts are applied to SARS-CoV-2 proteins by leveraging closely
related coronavirus protein sequences to provide insights into viral vulnerabilities that can be
exploited when designing MCMs. The majority of the SARS-CoV-2 proteins exhibit very high
proportions of critical residues to total residues (see Table 2 and Figure 2); hence, these viral
enzymes are excellent small molecule targets. Such small molecule drug therapeutics or
prophylactics have good chances of being effective against SARS-CoV, SARS-CoV-2, and
SARS-like CoVs if they target these highly conserved domains. Non-exposed replicase and
accessory proteins have abundant highly conserved long peptide targets for selecting continuous
segments of critical residues for T-cell epitope vaccines39. In contrast, the extracellular domain
of the S protein exhibits exposed surface areas with high amino acid residue variability.
Increased risk for antibody-dependent enhancement (ADE) from vaccines targeting SARS-CoV-
2, SARS-CoV-1, and MERS-CoV exposed residues is indicated by observed ADE in animal
models and the antibody facilitated infection of phagocytic immune cells frequently observed
with coronaviruses16,40. Peptides and antibodies targeting HR2 and cell fusion have been shown
to block SARS-CoV-1 and MERS-CoV infections in cell lines41-47 and animal models48-50.
Based on the conservation of these domains observed with divergence-based modeling, testing of
similar peptides and antibodies to these targets for SARS-CoV-2 may yield new insights and
opportunities for MCM development. Likewise, drugs that target the phagocytic pathway
associated with Fc-receptor mediated endocytosis are promising candidates for blocking the
cascade of immune cell infections that results in immune dysregulation in COVID-19 patients.
Methods
2019-nCoV protein sequences from GenBank entry MN908947.3 were searched against the non-
redundant (nr) and PDB database using the NCBI BLASTP web interface. Hit protein sequences
were downloaded. Protein multiple sequence alignments were created with the Dawn program51.
Additional 2019-nCoV sequences were added to existing alignments with the Jalview program52.
Identified protein structures were downloaded from RCSB PDB database53. Dawn variation
results were visualized with the Jmol program54.
Role of the funding source
The funder of the study had no role in study design, data collection, data analysis, data
interpretation, or writing of the report. The corresponding authors had full access to all the data
in the study and had final responsibility for the decision to submit for publication.
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Results
Dawn variation results for 2019-nCoV amino acid residues were classified into residues with no
observed variability (candidate critical residues; colored dark green in Figure 2) and to residues
with 5 or more amino acid substitutions (candidate spacer residues; colored dark blue in Figure
2). Amino acids residues colored yellow are considered constrained, allowing only a subset of
possible amino acid substitutions. Amino acid residues with conservative substitutions are also
considered critical residues, and are colored light green in Figure 2; positions with > 95%
conservation of a single residue were included in this category to accommodate potential
sequencing errors and possible adaptative mutations. Twelve of the nsp replicase proteins have a
ratio of critical to total residues of 0.9 or higher (Table 1); this is illustrated in Figure 2 for 2019-
nCoV proteins with high proportions of critical residues in Figure 2 (dark green residues). In
sharp contrast, the S protein exhibits regions of extensive variability of exposed surface residues
(Figure 2).
Discussion
Variation Results
The observed amino acid variations in SARS-CoV-2 proteins are consistent with expected
natural variations in the context of random mutations and selection in the context of host immune
responses. For the nonstructural replicase proteins, the majority have fractions of critical
residues above 88% (Table 2). Long continuous stretches of invariant residues are excellent
candidates for T-cell vaccines epitope selection, and also for exploratory anti-viral small
inhibitory RNA (siRNAs)55 development. With a large RNA genome, the virus has evolved over
time by deleting unnecessary spacer residues. The S protein S1 extended domain shows the
highest number of exposed surface highly variable residues, in sharp contrast to the replicase
enzymes (Figure 2). These spacer residues may function as exposed antigens for antibody
responses with the possible adaptive benefit of suppressing immune responses to less
immunogenic surface antigens. Many of these S protein antigens may lead to non-neutralizing
antibodies. Alternately, evolutionary selection for mutations to these residues may facilitate
antigenic drift to escape immune responses. It seems unusual to have the excessive number of
spacer residues on the S1 extended domain, unless it provides 2019-nCoV with an additional
selective advantage associated with non-neutralizing antibodies bound to this domain.
Coronaviruses have Multiple Options for Cell Infection
The 2019-nCoV S protein contains receptor-binding domains (RBD) targeting human
angiotensin I converting enzyme 2 (ACE2)56,57; this is the initial route for infecting host cells.
To take advantage of antibody responses, coronaviruses also leverage antibody Fc uptake to
infect immune cells58. Coronaviruses use the S protein subunit 2 FP, HR1, and HR2 to infect
immune cells upon proteolytic cleavage of S within endosomes. HR1 and HR2 form a canonical
6-helix bundle involved in membrane fusion41. Jaume et al.58 found that antibody-mediated
infection was dependent on Fc receptor II and not the endosomal/lysosomal pathway utilized by
ACE2 targeting. Viral infection of complement receptor (CR) cells is an additional possible
route of infecting cells59. This multi-pronged approach provides coronaviruses like SARS-CoV-
1, MERS-CoV, and SARS-CoV-2 with more than one mechanism for infecting host cells. This
leads to the hypothesis that antibody mediated uptake of virus is the potential mechanism that
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 March 2020 doi:10.20944/preprints202003.0138.v1
induces ADE to vaccines and can also be mediated by maternally transferred antibodies
(matAbs)60-63.
Macrophages and Immune Dysregulation
Lymphopenia is a common feature in patients with SARS23,64 or COVID-1965,66. Two receptors
have been identified for SARS-CoV-1 including ACE267 and C-type lectin domain family 4
member M (CLEC4M, CD209L, CD299, DC-SIGN2, DC-SIGNR, HP10347, and L-SIGN)68
with CLEC4M expressed in human lymph nodes69. Individuals homozygous for CLEC4M
tandem repeats are less susceptible to SARS infection70. In a mouse model, depletion of CD4+ T
cells resulted in an enhanced immune-mediated interstitial pneumonitis when challenged with
SARS-CoV-171. In contrast, depletion of CD4+ and CD8+ T cells as well as antibodies enabled
innate defense mechanisms to control the SARS-CoV-1 virus without immune dysregulation71.
Similar results were also observed in mice with SARS-CoV-1 challenge, but treatment with
liposomes containing clodronate, which deplete alveolar macrophages (AM), prevented immune
deficient virus-specific T cell response72. In a macaque model, anti-spike IgG causes acute lung
injury by skewing macrophage response towards proinflammatory monocyte/macrophage
recruitment and accumulation during acute SARS-CoV-1 infection73. These observations are
likely linked by antibody-dependent enhancement of coronavirus infection of macrophages58,74.
In SARS patients, severe SARS was associated with a more robust IgG response75; early
responders (antibody detectable within 2 weeks) had a higher death rate76,77. The
pathophysiology of severe and critical SARS and COVID-19 diseases fits a proposed model of
antibody-dependent infection of macrophages as the key gate step in disease progression from
mild to severe and critical symptoms, and may explain the observed dysregulated immune
responses78 including apoptosis contributing to development of pan-T cell lymphopenia,
proinflammatory cascade with macrophage accumulation, and cytokine and chemokine
accumulations in lungs with a cytokine storm in some patients.
Vaccine Risks for Antibody-dependent Enhancement (ADE)
Many of the viruses associated with ADE have cell membrane fusion mechanisms61. For
influenza A H1N1, vaccine-induced anti-HA2 antibodies promote virus fusion causing vaccine-
associated enhanced respiratory disease (VAERD)79. ADE was observed for the respiratory
syncytial virus (RSV) in the Bonnet monkey model60. Van Erp et al.60 recommends avoidance of
induction of respiratory syncytial virus (RSV) non-neutralizing antibodies or subneutralizing
antibodies to avoid ADE. In a mouse model, attempts to create vaccines for SARS-CoV-1 lead
to pulmonary immunopathology upon challenge with SARS-CoV-19; these vaccines included
inactivated whole viruses, inactivated viruses with adjuvant, and a recombinant DNA spike (S)
protein vaccine in a virus-like particle (VLP)-based vaccine. Enhanced hepatitis was observed in
a ferret model with a vaccine with recombinant modified vaccinia virus Ankara (rMVA)
expressing the SARS-CoV-1 S protein80. Jaume et al.58 point out the potential pitfalls associated
with immunizations against SARS-CoV-1. This leads to the prediction that new attempts to
create either SARS-CoV-1 vaccines81, MERS-CoV vaccines11, or SARS-CoV-2 vaccines have
potentially higher risks for inducing ADE in humans facilitated by antibody infection of
phagocytic immune cells. This potential ADE risk is independent of the vaccine technology82 or
targeting strategy selected due to predicted phagocytic immune cell infections upon antibody
uptake.
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Convalescent plasma therapy has been provided to SARS83 and COVID-1984 patients. Candidate
patients for convalescent plasma therapy are already experiencing advanced clinical disease
symptoms, potentially mitigating ADE risk. For Hong Kong SARS patients, convalescent
plasma therapy had improved outcomes (6.4% mortality rate) when it was provided before day
14 versus after (21.9% mortality rate) compared to the overall SARS-related mortality rate in of
17%. This is also being seen for initial COVID-19 patients treated with convalescent plasma
therapy84.
Antibody Targets
Analyzing the Cryo-EM structures of MERS-CoV and SARS-CoV-1 spike (S) glycoproteins,
Yuan et al. 85 suggest that the fusion peptide (FP) and the heptad repeat 1 region (HR1) are
potential targets for eliciting broadly neutralizing antibodies based on exposure on the surface of
the stem region, lack of N-linked glycosylation sites in this region, and sequence conservation.
Antibodies that interrupt virus-cell fusion will likely block the infection of immune cells using
Fc-mediated uptake of virus58. This has been demonstrated for SARS-CoV-1 for antibodies to
the HR2 region86-88. Likewise, 2019-nCoV antibodies that block cell fusion are predicted to not
share the same ADE risk of other 2019-nCoV antibodies. Antibodies that target the S RBD89
may have an ADE risk unless combined with a second cell fusion blocking antibody.
Targeting Cell Fusion
In addition to antibodies, peptides targeting HR2 have been shown to effectively block infection
in cell and animal models. Multiple peptides based on the heptad repeat regions (HR1 and HR2)
have been shown to suppress SARS-CoV-1 cell entry42-46. Specific combinations of two
peptides show synergistic viral inhibition43. An HR2 peptide was effective in a mouse model
administered intranasally against human coronavirus 229E (HCoV-229E)48. An HR2 peptide
combined with human interferon-a (IFN-a) also have significant synergistic antiviral effect
against feline coronavirus (FCoV)90. Based on anti-HIV-1 peptide, T-2091, Lambert et al.
demonstrate that analogous peptides inhibit respiratory syncytial virus (RSV), human
parainfluenza virus type 3 (HPIV-3), and measles virus (MV)92. An HR2 peptide can effectively
inhibit MERS-CoV replication49. Gao et al.41 identified an HR2 peptide that inhibits MERS-
CoV fusion in their pseudotyped-virus system. MERS-CoV HR1 entry inhibitor peptides have
been modified to form intra-molecular salt-bridges and increase peptide solubility50. The peptide
MERS HP2P-M2 protected C57BL/6 mice and mice deficient for VDJ recombination-activating
protein 1 (RAG1); this protection was enhanced by combing this peptide with interferon-b50.
Similar results are demonstrated for additional mouse models93,94. Lipopeptides have been
design to target cell fusion peptides47. An analogous fusion inhibitor, enfuvirtide (T-20), has
been approved for treatment of HIV-1 infections91. This provides a path forward for peptide-
based MCMs for 2019-nCoV. A set of SARS-CoV-1 inhibitory peptides that could be adapted
or directly tested on SARS-CoV-2 are illustrated in Figure 3. The SARS-CoV-1 HR2 peptides
can be directly tested on 2019-nCoV without modification due to sequence identity in this region
of the S protein.
B cell Vaccine Designs
B cell vaccines that target the S protein cell fusion mechanisms have the highest chance of
raising neutralizing antibodies with minimal or no ADE risk. Antibodies targeting other portions
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 March 2020 doi:10.20944/preprints202003.0138.v1
of the S protein or other 2019-nCoV exposed proteins may enable infection of phagocytic
immune cells even if they are neutralizing.
T cell Vaccine Designs
Variation results identified multiple continuous linear segments of critical residues from which T
cell epitopes can be selected in SARS-CoV-2 replicase enzymes and accessory proteins (Figure
2). Antibodies developed against these epitopes are highly unlikely to enable antibody enhanced
infection of phagocytic immune cells because they are not exposed on the surface of 2019-nCoV.
Targeting Autophagy
Coronavirus replication exploits aspects of normal cellular autophagy95. SKP2 attenuates
autophagy through Beclin1-ubiquination; its inhibition by the licensed drug niclosamide, a
treatment for tapeworms, drastically reduced the replications of MERS-CoV in cell culture13.
Compounds that block autophagy are worth investigating as SARS-CoV-2 MCM.
Targeting Viral Enzymes
2019-nCoV enzyme proteins are highly conserved with minimal spacer residues (Table 2 and
Figure 2). The variation results indicate that available SARS-CoV-1 protein structures (Table 2)
can be directly used for in silico docking and high throughput compound screens. SARS-CoV-2
protein structures are becoming rapidly available96 for compound screening approaches. The
high conservation around enzyme pockets holds promise that compound inhibitors against
SARS-CoV-2 will also be effective against SARS-CoV-1 and SARS-like CoV enzymes.
Summary
Given past data on multiple SARS-CoV-1 and MERS-CoV vaccine efforts which have failed due
to ADE in animal models9,11, it is reasonable to hypothesize a similar ADE risk for SARS-CoV-2
vaccine efforts unless they specifically target domains which will block virus-immune cell
fusion. MCMs based on vaccines, antibodies, or peptides that block cell fusion could minimize
predicted ADE risks. Synergy has been observed for combinations of CoV countermeasures
including interferon-a and -b. Small molecules targeting viral enzymes should also be pursued.
Data Availability
Protein multiple sequence alignments and associated variation files are included in Ricke,
Darrell, 2020, "Medical Countermeasures Analysis of 2019-nCoV / SARS-CoV-2 for COVID-
19", https://doi.org/10.7910/DVN/XWVOA8, Harvard Dataverse, V1.
Acknowledgements
The author acknowledges Nora Smith for literature search assistance, Irene Stapleford for
graphic art assistance, and Dr. Casandra Philipson for proof reading feedback.
Conflicts of Interest
Dr. Ricke and Dr. Malone have nothing to disclose.
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 March 2020 doi:10.20944/preprints202003.0138.v1
DISTRIBUTION STATEMENT A. Approved for public release. Distribution is unlimited.
This material is based upon work supported under U.S. Air Force Contract No. FA8702-15-D-
0001. Any opinions, findings, conclusions or recommendations expressed in this material are
those of the author(s) and do not necessarily reflect the views of the Under Secretary of Defense
for Research and Engineering.
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Tables
Table 1. 2019-nCoV proteins*.
Protein
Function
Cofactors
References
nsp1
cellular mRNA degradation, inhibiting type I
interferon (IFN) expression
97,98
nsp2
unknown
nsp3
multidomain protein
nsp3a
interacts with single-stranded RNA
99
nsp3b
ADP-ribose 1"-phosphatase
100
nsp3d
papain-like protease (Plpro), deubiquitinating
enzyme (DUB)
101
nsp4
double-membrane vesicles (DMV) formation
102
nsp5
3C-like protease (3CLpro)
103
nsp6
restricting autophagosome expansion, DMV
formation
104,105
nsp7
RNA binding
nsp8:nsp12
106,107
nsp8
RNA binding; primase
nsp7:nsp12, nsp9
106,107
nsp9
RNA binding, dimerization
nsp8
108
nsp10
scaffold cofactor
nsp10, nsp16
109,110
nsp11
unknown
nsp12
RNA-dependent RNA polymerase (RdRp)
nsp7:nsp8, nsp14
106
nsp13
RNA helicase, 5' triphosphatase
111
nsp14
3'-5' exoribonuclease (ExoN), guanine-N7
methyl transferase (N7-Mtase) for mRNA
capping, nsp12:nsp14 RNA synthesis and
proofreading
109
nsp15
endoribonuclease
112
nsp16
nsp16:nsp10 RNA cap 2'-O-methyltransferase,
negatively regulates innate immunity
110,113
E
forms homopentameric ion channels (IC) with
poor ion selectivity, Golgi complex-targeting
signal, PDZ-binding motif (PBM)
114-120
M
membrane protein
121
N
packages viral RNA
122
ORF3a
ORF6
ORF7a
Ig-like domain, ER retention signal
123-126
ORF7b
ORF8
ORF10
unknown
S
receptor binding, cell fusion
85
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*The E protein IC releases calcium from the endoplasmic reticulum intermediate compartment
(ERGIC), leading to NLRP3 inflammasome activation117,118. The E protein has a PDZ-binding
motif (PBM)116 that interacts with syntenin PDZ motifs to activate p38 mitogen-activated protein
kinase (MAPK) pathway and promotes an acute proinflammatory response119 and a virus PBM
domain is required for virulence120. The E protein PDZ-binding motif binds to PALS1 and alters
tight junction formation and epithelial morphogenesis127. The envelope (E) protein includes two
pathways to promote inflammation; these may contribute to the ADE response. ORF7a protein
has Ig-like domain123. Hänel et al.124 suggest that this ORF7a possess binding activity for aL
integrin I domain of LFA-1 suggesting that this might block newly synthesized LFA-1 molecules
from reaching the cell surface because ORF7a contains an ER retention signal125. Loss of LFA-1
negatively impacts immune responses126. This suggests possible interference of ORF7a with
immune surveillance mechanisms.
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Table 2. 2019-nCoV Variance Analysis
Protein
V1: Critical
V2
V3
V4
V5+: Spacers
Residues
Fraction
Structure
nsp1
112
40
19
3
7
181
0.84
2GDT:A128
nsp2
279
187
101
46
25
638
0.73
nsp3
996
514
239
115
92
1,956
0.77
2GRI:A99
nsp3a
82
35
20
22
12
171
0.68
2ACF:A100
Plpro
212
68
24
10
5
319
0.88
5Y3E:A129
nsp4
337
112
34
13
4
500
0.90
nsp5
254
46
4
2
0
306
0.98
6LU796
nsp6
209
64
15
2
0
290
0.94
nsp7
69
13
1
0
0
83
0.99
2AHM:A10
7
nsp8
170
26
2
0
0
198
0.99
2AHM:G10
7
nsp9
95
16
2
0
0
113
0.98
1UW7:A10
8
nsp10
109
27
3
0
0
139
0.98
3R24:B130
nsp12
5,226
1374
346
105
50
7,101
0.93
nsp13
538
61
2
1
0
602
1.00
6JYT:A111
nsp14
442
78
7
0
0
527
0.99
5C8T:B131
nsp15
246
76
17
6
1
346
0.93
2GTH:A132
nsp16
230
55
8
1
2
296
0.96
3R24:A130
E
24
33
17
5
3
82
0.70
5X29:A112
M
178
29
11
4
0
222
0.93
N
294
76
33
15
4
422
0.88
2OFZ:A133
ORF3a
107
79
54
20
15
275
0.68
ORF6
17
21
22
3
0
63
0.60
ORF7a
55
28
30
10
4
127
0.65
1XAK:A123
ORF7b
5
33
11
4
1
54
0.70
ORF8
59
39
15
8
0
121
0.81
5O32:I134
ORF10
38
0
0
0
0
38
1.00
S
650
263
123
107
152
1,295
0.71
6CRZ:A135
Figures
Figure 1. Disease progression model with normal immune responses during the initial mild
symptoms phase (see 1-3). Antigen presenting cells migrate to the lymph nodes to activate T-
cells (2a). The progression gate to severe and critical disease is the infection of phagocytic
immune cells (3a) leading to immune dysregulation (4b). In the lungs, chemokines attract
additional dendritic cells and immature macrophages that are subsequently infected in an positive
feedback-loop infection cascade (4b). Virus and infected phagocytic immune cells disseminate
throughout the body infecting additional organs (5 & 6). Levels of chemokine and cytokines in
the lungs from infected cells can create a cytokine storm (6).
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 March 2020 doi:10.20944/preprints202003.0138.v1
Figure 2. 2019-nCoV variation results. Amino acid residue color code: dark green (critical
residues), light green (critical residues with conservative substitutions or variant in less than 10
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 March 2020 doi:10.20944/preprints202003.0138.v1
Figure 3. SARS-CoV-1 Inhibitory Peptides N4643, HR1-142, HR2-1842, WW-III136, WW-IV136, sHR2-245, sHR2-
845, HRC187, HRC287, CP-1137, SR9138, P643, and CB-11944. SARS-CoV-2 residues different from SARS-CoV-
1 are underlined for adapting SARS-CoV-1 inhibitory peptides.
SARS2 907-NGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQ-965
SARS1 889-NGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQ-947
N46 QKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQ
HR1-1 NGIGVTQNVLYENQKQIANQFNKAISQIQESLTTTSTA
SARS2 1046-GYHLMSFPQSAPHGVVFLHVTY-1067
SARS1 1028-GYHLMSFPQAAPHGVVFLHVTY-1049
WW-III GYHLMSFPQAAPHGVVFLHVTW
SARS2 1093-GVFVSNGTHWFVTQRNFYE-1111
SARS1 1075-GVFVFNGTSWFITQRNFFS-1093
WW-IV GVFVFNGTSWFITQRNFFS
SARS2 1144-ELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-1211
SARS1 1126-ELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK-1193
sHR2-8 ELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIK
sHR2-2 PKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYE
HR2-18 IQKEIDRLNEVAKNLNESLIDLQELGK
HRC2 QKEIDRLNEVIKNLNESIIDLQEL
HRC1 NASIVNLQKEIDRLNEVIKNLNES
CP-1 GINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYE
P6 GINASVVNIQKEIDRLNEVAKNL
SR9 ISGINASVVNIQKEIDRLNEVAKNLNESLIDLQEL
CB-119 SPDVDLGDISGINAS
Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 8 March 2020 doi:10.20944/preprints202003.0138.v1
