# Peptide Mimicrying Between SARS Coronavirus Spike Protein and Human Proteins Reacts with SARS Patient Serum

Authors: K.-Y. Hwa; W. M. Lin; Y.-I. Hou; T.-M. Yeh
Journal: Journal of Biomedicine and Biotechnology
 (2008)

---

## Abstract

Molecular mimicry, defined as similar structures shared by molecules from dissimilar genes or proteins, is a general strategy used by pathogens to infect host cells.  Severe acute respiratory syndrome (SARS) is a new human respiratory infectious disease caused by SARS coronavirus (SARS-CoV).  The spike (S) protein of SARS-CoV plays an important role in the virus entry into a cell.  In this study, eleven synthetic peptides from the S protein were selected based on its sequence homology with human proteins.  Two of the peptides D07 (residues 927–937) and D08 (residues 942–951) were recognized by the sera of SARS patients. Murine hyperimmune sera against these peptides bound to proteins of human lung epithelial cells A549. Another peptide D10 (residues 490–502) stimulated A549 to proliferate and secrete IL-8.  The present results suggest that the selected S protein regions, which share sequence homology with human proteins, may play important roles in SARS-CoV infection.


---

## Body

## 1. Introduction

Severe acute respiratory syndrome (SARS)
is a new emerging infectious disease,  which was first reported in China in 2002
and was rapidly spreading all over the world in 2003 [1, 2]. The disease was
transmitted by droplets and close contact. Patients develop persistent fever, dry cough, progressive
radiographic changes of chest, and lymphopenia once infected. Despite
treatment, about 10–15% of the patients would die due to the acute respiratory
distress [3–6]. A novel coronavirus (SARS-CoV) was isolated from
SARS patients [7–9]. SARS-CoV is a positive-stranded RNA virus with an envelop.
The genome of SARS-CoV is around 29,727 nucleotides in length. The sequence was
annotatedin silico [10].
Comparative genomic studies using the in silico annotated proteins have
suggested that SARS virus belongs to a new group of coronavirus.According to the genomic sequence of
SARS-CoV, it is predicted that there are several structural proteins can be
produced by SARS-CoV including spike (S), envelop (E), membrane (M), and
nucleocapsid. Spike protein is very important in the binding and fusion of
coronavirus to the host cells [11, 12]. The S protein of SARS-CoV has 1255 amino
acids in length and 23 potential N-linked glycosylation sites. The amino
terminus of the SARS-CoV S protein contains a short type 1 signal sequence
composed of hydrophobic amino acids that are presumably removed during
cotranslational transport through the endoplasmic reticulum. The carboxyl
terminus consists of a transmembrane domain and a cytoplasmic tail rich in
cysteine residues. The majority of protein (residues 12–1195) is outside the virus
particle, which can be divided into amino-terminal S1 and carboxyl-terminal S2
domain. The S1 domain (residues 12–672) binds to the host cell receptor, angiotensin-converting enzyme 2 (ACE2), while the
S2 domain is responsible for membrane fusion [13–15]. Monoclonal
antibodies against S1 domain can block the receptor binding and contain potent
neutralization activity against SARS-CoV [16]. However, peptides derived
from S2 domain can also inhibit SARS-CoV infection [12].Molecular mimicry, which is defined as similar structures shared by
molecules from dissimilar genes or by their protein products, is a
general strategy for pathogens to infect host cells and has been proposed as a pathogenic mechanism for autoimmune disease [17].
Therefore, identification of the molecular mimic regions of pathogen may be
helpful to understand the disease induced by that pathogen. At present, it is
unclear whether molecular mimicry occurs between SARS-CoV S proteins and human
peptides. We have approached this question using computer to analyze the
sequence of spike protein of SARS-CoV and select regions that share the sequence
homology with human proteins. The criteria for the selection of potential
regions include antigenic analysis and surface accessibility. In this study, we
find that several regions of the S protein share sequence homology with human
proteins. Synthetic peptides, which represent some of these regions, were
synthesized to understand their roles in SARS-CoV infection.


## 2. Materials and Methods

### 2.1. Peptide Prediction and Synthesis

Publically available human and coronavirus genome sequences at the National Center for Biotechnology Information (Md, USA)
were used for in silicoprediction. Algorithms predicting immunogenicity, second
structure prediction, protein topology analysis, and hydrophobicity were
conducted to design the tested peptides. Immunogenic viral peptides were
calculated based on the algorithm developed by Kolaskar and Tongaonkar [18]. The
algorithm is based on a table constructed from the occurrence of amino acid
residues in experimentally known antigenic epitopes. The reported accuracy of the method is about
75% [18]. In silico secondary structural analyses of spike protein were
performed based on PHD [19] and PREDATOR [20] algorithms. Protein topology
prediction was based on the algorithm developed by TMHMM [21]. Hydrophobicity of the peptides was calculated
based on the algorithm HMOMENT [22]. Similarity searches between S protein and
human genome database were performed by using BLASTP [23] with BLOSUM 62. Extra amino acid residues were added at either
N- or C-terminus to keep the hydrophobic amino acid content below 50%.
Peptides with high hydrophobicity are difficult to be tested in biochemical experiments
since most of in vitro assays
are conducted in aqueous buffers. Also,
on average, one charged residue is added for every five amino acids. Multiple antigen peptides were synthesized by
CytoMol Corp (Mountain View, Calif, USA). In addition, bradykinin and
angiotensin I (Ang I) were purchased from Sigma (St. Louis, Mo, USA).


### 2.2. SARS Patient Sera

SARS patient sera were collected by the Center for Disease Control, Department of
Health (Taipie, Taiwan) from March to June, 2003. Diagnosis of SARS was based on the clinicalcriteria
established by the World Health Organization (WHO). Patients with
SARS-CoV were confirmed by laboratory methods, including viral antigen detection,
RT-PCR, and serologic methods. Ten SARS patient sera collected at the convalescent stage (≥20 days after disease onset)  were included in this study. Ten
normal sera from healthy
individuals were used as controls.


### 2.3. Enzyme-Linked Immunosorbent Assay (ELISA)

Antibodies against peptides in human sera were detected by solid-phase capture technique
using individual peptide-coated plates. ELISA plate was coated with or without 50μL
peptides (100 μg/mL) per well and blocked by 1% bovine serum albumin (BSA) in 0.05% Tween-20 in phosphate-buffered saline (PBS)
for 1 hour at room temperature. Test
serum samples were 1:100 diluted and added to the plate for 2 hours at room
temperature. After incubation, the ELISA plate was washed with 0.05% Tween-20 in PBS for three times. The
bound antibodies were detected by horseradish peroxidase- (HRP-) conjugated antihuman
immunoglobulin antibodies (Sigma Aldrich, St. Louis, Mo, USA) and peroxidase substrate,
TMB (Promega, Madison, Wiss, USA). The absorbance was measured using the Vmax microplate
reader (Molecular Devices Corporation, Sunnyvale, Calif, USA) at 450 nm.
Antibodies against peptides in mouse sera were assayed by ELISA as in human
sera except HRP-conjugated antimouse immunoglobulin antibodies (Sigma Aldrich)
was used to detect bound antibodies.


### 2.4. Cell Culture

Human lung adenocarcinoma cell line A549 and Vero cells were grown in DMEM supplemented
with 10% heat-inactivated FCS, 2 mM L-glutamate, and 50 ng/mL gentamycin. Cells were incubated in CO2 incubator at 37°C with 5% CO2 in a humidified atmosphere. For immunofluorescent microscopy
observation, monolayers of A549 cells were cultured on sterile glass slides
before the experiment.


### 2.5. Mice Immunization

Six- to eight-week-old female BALB/c mice were used in this study. These mice were
originally purchased from Jackson Laboratory (Bar Harbor, Me, USA) and bred in
the Laboratory Animal Center, National Cheng Kung University (Tainan, Taiwan).
Synthetic peptides (1 mg/mL) were emulsified with complete Freund's adjuvant
and injected intraperitoneally into BALB/c mice. Mice were boosted with the
same peptide in PBS (50μg/mouse) intraperitoneally two weeks
after priming. Sera were collected from the axially plexus of the mice at
different time intervals and tested for the presence of antibody against
peptides by ELISA as mentioned above. Significant increase of antibody titer (greater
than 4 folds) against immunized-peptide was found in mouse hyperimmune sera as
compared to normal sera after boosting.


### 2.6. Immunofluorescent Stain

Mouse hyperimmune sera against peptide D08 were incubated with A549 cells
at 4°C for 1 hour. After washing
three times with PBS, cells were incubated with 1 mL of 1 μg/mL FITC-conjugated antimouse IgG (Jackson ImmunoResearch
Laboratories Inc., West Grove, Pa, USA) at 4°C for 1 hour and washed again with PBS. The immunofluorescent stain of cells was
observed by fluorescent microscopy.


### 2.7. SDS-PAGE and Western Blot Analysis

Proteins in the cell lysate of A549 were separated by 12% SDS-PAGE
and transferred to nitrocellulose sheets as described previously [24]. Proteins
recognized by normal or peptide D08 hyperimmune mice sera were detected using
HRP-conjugated antimouse immunoglobulin antibodies (Sigma Aldrich) and
substrate.


### 2.8. Cell Proliferation

Vero E6 (4×104) and A549 cells (5×103) were incubated with
different doses of synthetic peptides as indicated for 72 hours. Cell
proliferation was detected using commercial XTT assay (Roche Diagnostics,
Indianapolis, Ind, USA).


### 2.9. IL-8 Assay

The IL-8 production was assessed by commercial ELISA kits (R&D systems, Minneapolis,
Minn, USA) according to the manufacturer's instructions. Briefly, A549 cells (1×105) were cultured alone or with different doses of peptides for 48
hours. Culture supernatants were collected after incubation, added to precoated
ELISA plates, and incubated for 2 hours at 37°C. Plates were washed four times
with the washing buffer. The bound IL-8 was detected by HRP-conjugated
antibodies and substrate. The developed color was read by the Vmax microplate
reader (Molecular Devices, Calif, USA). The concentration of IL-8 was calculated
according to the standard curve.


### 2.10. Statistical Analyses

Data are expressed as mean ± standard deviation (SD). The levels of
significance for the differences between groups were analyzed using Student'st-test. A value of P < .05 was considered to be significant.


## 2.1. Peptide Prediction and Synthesis

Publically available human and coronavirus genome sequences at the National Center for Biotechnology Information (Md, USA)
were used for in silicoprediction. Algorithms predicting immunogenicity, second
structure prediction, protein topology analysis, and hydrophobicity were
conducted to design the tested peptides. Immunogenic viral peptides were
calculated based on the algorithm developed by Kolaskar and Tongaonkar [18]. The
algorithm is based on a table constructed from the occurrence of amino acid
residues in experimentally known antigenic epitopes. The reported accuracy of the method is about
75% [18]. In silico secondary structural analyses of spike protein were
performed based on PHD [19] and PREDATOR [20] algorithms. Protein topology
prediction was based on the algorithm developed by TMHMM [21]. Hydrophobicity of the peptides was calculated
based on the algorithm HMOMENT [22]. Similarity searches between S protein and
human genome database were performed by using BLASTP [23] with BLOSUM 62. Extra amino acid residues were added at either
N- or C-terminus to keep the hydrophobic amino acid content below 50%.
Peptides with high hydrophobicity are difficult to be tested in biochemical experiments
since most of in vitro assays
are conducted in aqueous buffers. Also,
on average, one charged residue is added for every five amino acids. Multiple antigen peptides were synthesized by
CytoMol Corp (Mountain View, Calif, USA). In addition, bradykinin and
angiotensin I (Ang I) were purchased from Sigma (St. Louis, Mo, USA).


## 2.2. SARS Patient Sera

SARS patient sera were collected by the Center for Disease Control, Department of
Health (Taipie, Taiwan) from March to June, 2003. Diagnosis of SARS was based on the clinicalcriteria
established by the World Health Organization (WHO). Patients with
SARS-CoV were confirmed by laboratory methods, including viral antigen detection,
RT-PCR, and serologic methods. Ten SARS patient sera collected at the convalescent stage (≥20 days after disease onset)  were included in this study. Ten
normal sera from healthy
individuals were used as controls.


## 2.3. Enzyme-Linked Immunosorbent Assay (ELISA)

Antibodies against peptides in human sera were detected by solid-phase capture technique
using individual peptide-coated plates. ELISA plate was coated with or without 50μL
peptides (100 μg/mL) per well and blocked by 1% bovine serum albumin (BSA) in 0.05% Tween-20 in phosphate-buffered saline (PBS)
for 1 hour at room temperature. Test
serum samples were 1:100 diluted and added to the plate for 2 hours at room
temperature. After incubation, the ELISA plate was washed with 0.05% Tween-20 in PBS for three times. The
bound antibodies were detected by horseradish peroxidase- (HRP-) conjugated antihuman
immunoglobulin antibodies (Sigma Aldrich, St. Louis, Mo, USA) and peroxidase substrate,
TMB (Promega, Madison, Wiss, USA). The absorbance was measured using the Vmax microplate
reader (Molecular Devices Corporation, Sunnyvale, Calif, USA) at 450 nm.
Antibodies against peptides in mouse sera were assayed by ELISA as in human
sera except HRP-conjugated antimouse immunoglobulin antibodies (Sigma Aldrich)
was used to detect bound antibodies.


## 2.4. Cell Culture

Human lung adenocarcinoma cell line A549 and Vero cells were grown in DMEM supplemented
with 10% heat-inactivated FCS, 2 mM L-glutamate, and 50 ng/mL gentamycin. Cells were incubated in CO2 incubator at 37°C with 5% CO2 in a humidified atmosphere. For immunofluorescent microscopy
observation, monolayers of A549 cells were cultured on sterile glass slides
before the experiment.


## 2.5. Mice Immunization

Six- to eight-week-old female BALB/c mice were used in this study. These mice were
originally purchased from Jackson Laboratory (Bar Harbor, Me, USA) and bred in
the Laboratory Animal Center, National Cheng Kung University (Tainan, Taiwan).
Synthetic peptides (1 mg/mL) were emulsified with complete Freund's adjuvant
and injected intraperitoneally into BALB/c mice. Mice were boosted with the
same peptide in PBS (50μg/mouse) intraperitoneally two weeks
after priming. Sera were collected from the axially plexus of the mice at
different time intervals and tested for the presence of antibody against
peptides by ELISA as mentioned above. Significant increase of antibody titer (greater
than 4 folds) against immunized-peptide was found in mouse hyperimmune sera as
compared to normal sera after boosting.


## 2.6. Immunofluorescent Stain

Mouse hyperimmune sera against peptide D08 were incubated with A549 cells
at 4°C for 1 hour. After washing
three times with PBS, cells were incubated with 1 mL of 1 μg/mL FITC-conjugated antimouse IgG (Jackson ImmunoResearch
Laboratories Inc., West Grove, Pa, USA) at 4°C for 1 hour and washed again with PBS. The immunofluorescent stain of cells was
observed by fluorescent microscopy.


## 2.7. SDS-PAGE and Western Blot Analysis

Proteins in the cell lysate of A549 were separated by 12% SDS-PAGE
and transferred to nitrocellulose sheets as described previously [24]. Proteins
recognized by normal or peptide D08 hyperimmune mice sera were detected using
HRP-conjugated antimouse immunoglobulin antibodies (Sigma Aldrich) and
substrate.


## 2.8. Cell Proliferation

Vero E6 (4×104) and A549 cells (5×103) were incubated with
different doses of synthetic peptides as indicated for 72 hours. Cell
proliferation was detected using commercial XTT assay (Roche Diagnostics,
Indianapolis, Ind, USA).


## 2.9. IL-8 Assay

The IL-8 production was assessed by commercial ELISA kits (R&D systems, Minneapolis,
Minn, USA) according to the manufacturer's instructions. Briefly, A549 cells (1×105) were cultured alone or with different doses of peptides for 48
hours. Culture supernatants were collected after incubation, added to precoated
ELISA plates, and incubated for 2 hours at 37°C. Plates were washed four times
with the washing buffer. The bound IL-8 was detected by HRP-conjugated
antibodies and substrate. The developed color was read by the Vmax microplate
reader (Molecular Devices, Calif, USA). The concentration of IL-8 was calculated
according to the standard curve.


## 2.10. Statistical Analyses

Data are expressed as mean ± standard deviation (SD). The levels of
significance for the differences between groups were analyzed using Student'st-test. A value of P < .05 was considered to be significant.


## 3. Results

### 3.1. Search for Molecular Mimic Regions in S Protein

The whole amino acid
sequence of spike protein was analyzed to find out the potential immunogenic
regions and the regions shared sequence homology with human proteins, which is defined
as the pathogenic regions. As shown in Figure1, there are 4 pathogenic
regions. Region 1 (residues 199–254), region 2 (residues 658–715), region 3
(residues 893–951), and region 4 (residues 1127–1184) have shared sequence homology
with hydroxyacid oxidase, human golgi autoantigen, Angrgm-52, and pallidin,
respectively. Among these regions, region 3 with homology to human Angrgm-52 has
the highest score (with 34% identities and 48% similarity of conservative
substitutions). Its sequence comparison with angrgm-52 is shown in Figure 2(a).
In addition, because des-Arg bradykinin and Ang I are the substrates for ACE2 [25], we also compared the sequence of S protein against bradykinin (RPPGFSPFR) and Ang I (DRVYIHPFHL) and
found that residues 490–502 (GYQPYRVVVLSFEE) of S protein showed sequence homology with bradykinin as
indicated by bold letters here and in Figure 2(b). The identity score is 27%; and
the similarity score from conservative substitutions is 36%.Figure 1
Sequence analysis of S protein. Putative S protein amino
acid sequence was analyzed to find immunogenic regions (yellow regions) and
pathogenic regions (regions shared sequence homology with human proteins, blue
regions). Purple regions are both immunogenic and pathogenic regions. The grey region
is the leader sequence and the brown region is the transmembrane region.Sequence homology between S protein, (a) human Angrgm-52, and (b) bradykinin. Spike protein
sequence (protein databank NP_828851) was compared with sequences from all nonredundant GenBank CDS translations, PDB,
SwissProt, PIR, and PRF of human, by using blastp (NCBI, NIH, USA). For Panel
(a), BLASTP was used to calculate the similarity between S protein and human
Angrgm-52, with matrix set at default. For an identical residue, one letter symbol of amino is shown between
the two sequences, and for a conservative substation, “+” is shown. For Panel (b), pairwise alignment was
calculated based on Smith-Water local alignment with matrix set at BLOSUM 45. “∣” is annotated for
identical residues; “:” and “.” are for similar residues.
(a)(b)


### 3.2. Screen for Peptides Recognized by SARS Patients' Sera

Eleven peptides (D01–D11, see Table1), which represent those pathogenic regions were
synthesized as well as bradykinin and Ang I were tested to see whether those peptides
can be recognized by SARS patients' sera.
The peptides were designed based on the algorithms predicting immunogenicity,
second structure, protein topology, and hydrophobicity. Our goal is to select for peptides with high
immunogenicity, with location on the protein surface, and with low hydrophobicity. The designed peptide sequences were
synthesized and tested with clinical samples of SARS patient sera. A significant increase of SARS patients' sera
binding to peptide D01, D07, and D08 was found as compared to the binding of
normal sera (see Figure 3).Table 1
Amino acid sequence of the eleven synthetic peptides*Extra amino acid residues which were indicated by italic letters were added at either N- or C-terminus to decrease the hydrophobicity.
PeptideAmino acid positionsAmino acid sequenceNo. of amino acidsD01199–210GYQPIDVVRDLG12D02658–669ASYHTVSLLRSTSQK15D03733–744EEGNLLLQYGSFCTQ15D04745–753EELNRALSGIAAGQ13D05763–770VFAQVKQM8D06911–919KAISQIQESLTTE13D07927–937GLGKLQDVVNQNGE14D08942–951ALNTLVKQLSSN12D091154–1162INASVVNIQK10D10490–502GYQPYRVVVLSFEE14D11306–317GFRVVPSGDVVRF13Figure 3
Antibody binding activity of SARS patients' sera to
different peptides. Sera of SARS patients at convalescent stage as well as
normal controls were collected as described in Section2. Antibodies binding to different peptides
were detected by ELISA as described in Section 2. BK represents bradykinin. “*”
indicates P < .05.


### 3.3. Hyperimmune Sera Against D08 Crossreacted with A549 Cells

To test whether synthetic peptides D01, D07, and D08 can induce antibodies crossreacted with
human proteins, we immunized mice with these peptides to generate hyperimmune
sera against these peptides. We found hyperimmune sera against D08 can bind to
the cytoplasmic region of human A549 cells as demonstrated by immunofluorescent
stain (see Figure4). Using Western blot analysis, hyperimmune sera against D08
could recognize more bands in A549 cell lysate as compared to normal mice sera
(see Figure 5). In addition, hyperimmune sera against D07, but not D01, showed
similar crossreactivity to A549 cells as hyperimmune sera against D08 did (data
not shown).Immunofluorescent staining of mouse hyperimmune sera against
A549 cells. A549 cells were grown on the slides and stained with secondary
antibody alone (a) or mouse hyperimmune sera against D08 peptide (b) as
described in Section2.
(a)(b)Mouse hyperimmune sera against D08 peptide recognize
proteins in the A549 cell lysate. Proteins in the cell lysate of A549 cells
were separated by SDS-PAGE and transferred to membrane as described in Section2.
Western blots against this membrane using normal mouse sera (a) or hyperimmune
sera against D08 (b) are shown.
(a)(b)


### 3.4. Hyperimmune Sera Against D10 Crossreacted with Bradykinin

To test whether synthetic peptides D10, indeed, can induce antibodies crossreactive with
bradykinin and Ang I, we immunized mice with D10 peptides to generate
hyperimmune sera against this peptide. Significant increase of antibodies
against D10 was found in D10 hyperimmune sera, which could crossreact with
bradykinin, but not with Ang I-coated plates (see Figure6).The crossreactivity of D10 antibody with bradykinin and
Ang I. Hyperimmune sera from D10 immunized mice (▪) or normal mice sera (▴)
were diluted as indicated and reacted with D10-, bradykinin-, or Ang I-coated
ELISA plates as indicated. Bound antibodies were detected as described in Section 2.
Data represents the mean ± SD of triplicates.
(a)
D10(b)
Bradykinin(c)
Angiotensin I


### 3.5. Peptide D10 Induced IL-8 Secretion and Cell Proliferation of A549 Cells

To understand whether D10 has similar biological activity as Ang I, we incubated
Vero cells and lung epithelial A549 cells with D10, Ang 1, or control peptide D11.
Both Vero and A549 cells were induced to proliferate in the presence of D10 and
Ang I but not the control peptide (see Figure7). In addition, D10 and Ang I could
also induce chemokine IL-8 production of A549 cells (see Figure 8).Cell proliferation induced by D10 peptide and Ang I. Vero
and A549 cells were incubated with different doses of peptides as indicated. Cell
proliferation was detected after 72 hours of incubation by XTT assay as described
in Section2. Data represents the mean ± SD of triplicates.
(a)(b)Figure 8
IL-8 production of A549 cells induced by D10 peptide and Ang
I. A549 cells (1×105) were incubated with or without peptides for
48 hours. The levels of IL-8 in the culture supernatants were assayed as described in Section 2. Data represents the
mean ± SD of triplicates.


## 3.1. Search for Molecular Mimic Regions in S Protein

The whole amino acid
sequence of spike protein was analyzed to find out the potential immunogenic
regions and the regions shared sequence homology with human proteins, which is defined
as the pathogenic regions. As shown in Figure1, there are 4 pathogenic
regions. Region 1 (residues 199–254), region 2 (residues 658–715), region 3
(residues 893–951), and region 4 (residues 1127–1184) have shared sequence homology
with hydroxyacid oxidase, human golgi autoantigen, Angrgm-52, and pallidin,
respectively. Among these regions, region 3 with homology to human Angrgm-52 has
the highest score (with 34% identities and 48% similarity of conservative
substitutions). Its sequence comparison with angrgm-52 is shown in Figure 2(a).
In addition, because des-Arg bradykinin and Ang I are the substrates for ACE2 [25], we also compared the sequence of S protein against bradykinin (RPPGFSPFR) and Ang I (DRVYIHPFHL) and
found that residues 490–502 (GYQPYRVVVLSFEE) of S protein showed sequence homology with bradykinin as
indicated by bold letters here and in Figure 2(b). The identity score is 27%; and
the similarity score from conservative substitutions is 36%.Figure 1
Sequence analysis of S protein. Putative S protein amino
acid sequence was analyzed to find immunogenic regions (yellow regions) and
pathogenic regions (regions shared sequence homology with human proteins, blue
regions). Purple regions are both immunogenic and pathogenic regions. The grey region
is the leader sequence and the brown region is the transmembrane region.Sequence homology between S protein, (a) human Angrgm-52, and (b) bradykinin. Spike protein
sequence (protein databank NP_828851) was compared with sequences from all nonredundant GenBank CDS translations, PDB,
SwissProt, PIR, and PRF of human, by using blastp (NCBI, NIH, USA). For Panel
(a), BLASTP was used to calculate the similarity between S protein and human
Angrgm-52, with matrix set at default. For an identical residue, one letter symbol of amino is shown between
the two sequences, and for a conservative substation, “+” is shown. For Panel (b), pairwise alignment was
calculated based on Smith-Water local alignment with matrix set at BLOSUM 45. “∣” is annotated for
identical residues; “:” and “.” are for similar residues.
(a)(b)


## 3.2. Screen for Peptides Recognized by SARS Patients' Sera

Eleven peptides (D01–D11, see Table1), which represent those pathogenic regions were
synthesized as well as bradykinin and Ang I were tested to see whether those peptides
can be recognized by SARS patients' sera.
The peptides were designed based on the algorithms predicting immunogenicity,
second structure, protein topology, and hydrophobicity. Our goal is to select for peptides with high
immunogenicity, with location on the protein surface, and with low hydrophobicity. The designed peptide sequences were
synthesized and tested with clinical samples of SARS patient sera. A significant increase of SARS patients' sera
binding to peptide D01, D07, and D08 was found as compared to the binding of
normal sera (see Figure 3).Table 1
Amino acid sequence of the eleven synthetic peptides*Extra amino acid residues which were indicated by italic letters were added at either N- or C-terminus to decrease the hydrophobicity.
PeptideAmino acid positionsAmino acid sequenceNo. of amino acidsD01199–210GYQPIDVVRDLG12D02658–669ASYHTVSLLRSTSQK15D03733–744EEGNLLLQYGSFCTQ15D04745–753EELNRALSGIAAGQ13D05763–770VFAQVKQM8D06911–919KAISQIQESLTTE13D07927–937GLGKLQDVVNQNGE14D08942–951ALNTLVKQLSSN12D091154–1162INASVVNIQK10D10490–502GYQPYRVVVLSFEE14D11306–317GFRVVPSGDVVRF13Figure 3
Antibody binding activity of SARS patients' sera to
different peptides. Sera of SARS patients at convalescent stage as well as
normal controls were collected as described in Section2. Antibodies binding to different peptides
were detected by ELISA as described in Section 2. BK represents bradykinin. “*”
indicates P < .05.


## 3.3. Hyperimmune Sera Against D08 Crossreacted with A549 Cells

To test whether synthetic peptides D01, D07, and D08 can induce antibodies crossreacted with
human proteins, we immunized mice with these peptides to generate hyperimmune
sera against these peptides. We found hyperimmune sera against D08 can bind to
the cytoplasmic region of human A549 cells as demonstrated by immunofluorescent
stain (see Figure4). Using Western blot analysis, hyperimmune sera against D08
could recognize more bands in A549 cell lysate as compared to normal mice sera
(see Figure 5). In addition, hyperimmune sera against D07, but not D01, showed
similar crossreactivity to A549 cells as hyperimmune sera against D08 did (data
not shown).Immunofluorescent staining of mouse hyperimmune sera against
A549 cells. A549 cells were grown on the slides and stained with secondary
antibody alone (a) or mouse hyperimmune sera against D08 peptide (b) as
described in Section2.
(a)(b)Mouse hyperimmune sera against D08 peptide recognize
proteins in the A549 cell lysate. Proteins in the cell lysate of A549 cells
were separated by SDS-PAGE and transferred to membrane as described in Section2.
Western blots against this membrane using normal mouse sera (a) or hyperimmune
sera against D08 (b) are shown.
(a)(b)


## 3.4. Hyperimmune Sera Against D10 Crossreacted with Bradykinin

To test whether synthetic peptides D10, indeed, can induce antibodies crossreactive with
bradykinin and Ang I, we immunized mice with D10 peptides to generate
hyperimmune sera against this peptide. Significant increase of antibodies
against D10 was found in D10 hyperimmune sera, which could crossreact with
bradykinin, but not with Ang I-coated plates (see Figure6).The crossreactivity of D10 antibody with bradykinin and
Ang I. Hyperimmune sera from D10 immunized mice (▪) or normal mice sera (▴)
were diluted as indicated and reacted with D10-, bradykinin-, or Ang I-coated
ELISA plates as indicated. Bound antibodies were detected as described in Section 2.
Data represents the mean ± SD of triplicates.
(a)
D10(b)
Bradykinin(c)
Angiotensin I


## 3.5. Peptide D10 Induced IL-8 Secretion and Cell Proliferation of A549 Cells

To understand whether D10 has similar biological activity as Ang I, we incubated
Vero cells and lung epithelial A549 cells with D10, Ang 1, or control peptide D11.
Both Vero and A549 cells were induced to proliferate in the presence of D10 and
Ang I but not the control peptide (see Figure7). In addition, D10 and Ang I could
also induce chemokine IL-8 production of A549 cells (see Figure 8).Cell proliferation induced by D10 peptide and Ang I. Vero
and A549 cells were incubated with different doses of peptides as indicated. Cell
proliferation was detected after 72 hours of incubation by XTT assay as described
in Section2. Data represents the mean ± SD of triplicates.
(a)(b)Figure 8
IL-8 production of A549 cells induced by D10 peptide and Ang
I. A549 cells (1×105) were incubated with or without peptides for
48 hours. The levels of IL-8 in the culture supernatants were assayed as described in Section 2. Data represents the
mean ± SD of triplicates.


## 4. Discussion

In this study, we have identified four
pathogenic regions of SARS-CoV S protein which share sequence homology with
different human proteins. Among them, pathogenic region 3 (residues 893–941),
which shares sequence homology with Angrgm-52 (GenBank accession no. AAL62340), a
novel gene upregulated in human mesangial cells stimulated by angiotensin II, may
deserve further investigation. Peptides D07 and D08 of this region were
recognized by the sera of SARS patient indicating that this region is
immunogenic and can be recognized by the immune system during SARS-CoV
infection. Murine hyperimmune sera against peptides D07 or D08 were able to
bind to recombinant S2 but not S1 domain of S protein (data not shown). In
addition, hyperimmune sera against D07 or D08 also bounded to the cytoplasmic region of A549 cells
and recognized several proteins in the A549 cell lysate. These results indicate
that regions represented by D07 and D08 are immunogenic and may induce
autoantibodies. However, further study is required to understand the biological
function of these regions and the role of their antibodies in the pathogenesis
of SARS-CoV infection.In addition to D07 and D08 peptides, we also noticed that D10
peptide which represents residues 490–502 of S1 domain contained some
interesting activities. The D10 peptide, which shared sequence homology with
bradykinin, was able to generate antibodies crossreactive with bradykinin. In addition,
D10 peptide could stimulate A549 to produce IL-8 and proliferation as Ang I did.
These results suggest that the region of D10 in S protein may bind to Ang I receptor, ACE2, and
may be involved in the binding of SARS-CoV to ACE2. This is consistent with the
previous report, which indicates that residues 318–510 of S1 domain can bind to ACE2 [25] and is similar to
the receptor binding domain of the HCoV-229E, which is within a fragment
containing residues 407 to 547 [26]. Therefore, region 490–502 of S1 domain may
be involved in the receptor binding domain of SARS-CoV.In summary, our results suggest that molecular mimicry occurs between
SARS-CoV and host proteins. Motifs shared sequence homology with host proteins of
SARS-COV may be involved in the binding and fusion of SARS-CoV to host cells. Antibody against these motifs may contain
neutralization activity against SARS-CoV infection or participate in the
immunopathogenesis induced by SARS-CoV.
As reported previously, SARS-CoV, like influenza, can inhibit the host's
corticosteroid stress response via a molecular mimicry strategy [27]. Our studies on the mimicry motifs of S protein, which is involved in the virus, entry may provide alternative approaches to disrupt the infection of SARS-CoV, similar to the previous studies on the virus entry
[28, 29].



---

*Source: 326464-2008-02-19.xml*