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Introduction
Severe acute respiratory syndrome (SARS) emerged in
Southeast Asia in late 2002 and subsequently spread
internationally. The causative agent was quickly identified
as a previously unknown member of the Coronaviridae
family[1-3]. According to the World Health Organization, up
to 2004 Apr 21, SARS coronavirus infected more than 8000
people in various countries worldwide and caused
approximately 800 deaths[4]. Although SARS infection of human
beings has been contained through infection-control
measures, resurgence is still a threat because the causative
agent remaining in animal reservoirs is not fully understood,
and sporadic cases continue to be reported in
Singapore[5,6], Taiwan[7] and mainland
China[8,9].
There are no specific vaccines and effective drugs
currently available for
SARS-CoV[1,2,10,11]. Until an effective
vaccine is developed, the best hope for the treatment of
infection and the prevention and control of future outbreaks is
the development of passive immunotherapy with
SARS-CoV-specific antibodies[11]. Immunoglobulin is an effective method
used in protection against animal coronavirus: transmissible
gastroenteritis virus (TGEV)[12,13], mouse hepatitis
virus[14], and bovine
coronavirus[15]. There is clinical evidence that
serum from recovered patients is effective in infected
individuals[16,17]. These observations suggest that hyperimmune
serum could be developed for the passive treatment of SARS.
The use of equine antisera for emergent prevention and
treatment of infectious diseases has been proven to be an
effective and safe strategy, such as in rabies
virus[18,19]. Therefore, immunoprophylaxis and treatment of SARS coronavirus
infection with equine hyperimmune globulin might be a viable
strategy for controlling SARS.
Materials and methods
Virus strains Severe acute respiratory syndrome
corona-virus Z2-Y3 (AY394989) and F69 (AY313906), isolated
from the samples of 2 different Cantonese onset SARS
patients in 2003, were sequenced and compared, showing
certain differences (Table 1). Viral titres of SARS-CoV Z2-Y3
and F69 strains were determined to be
106.5 50% tissue-culture-infective doses
(TCID50)/mL and
106.7 TCID50/mL with the
Reed-Muench method, respectively[20-22].
Antigen preparation F69 strain was used as antigen for
immunization. African green monkey kidney (Vero-E6) cells,
infected with SARS CoV F69 strain, were cultivated in
serum-free minimum essential medium (MEM)
(GIBCO) and observed periodically for cytopathic effect (CPE). When
75%-100% cytopathy was reached, infected Vero-E6 cells
were frozen and thawed 3 times, which was subsequently
centrifuged at 8000×g for 30 min, and then the cell debris
was decanted. The supernatant was collected and stored at
-70 ºC until used. The viral supernatant was then
centrifuged at 30 000×g for 3 h. The precipitate was diluted with
phosphate buffered saline (PBS), which was used as antigen
for immunization.
Animal immunization Six 4-9 year-old healthy horses
were provided by the Quartermaster University of PLA.
Immunization of horses was performed according to the State
Food and Drug Administration (SFDA) standard operating
procedures. On d 0 and d 10, all horses were injected with
1.0 mL antigen intramuscularly (SARS-CoV F69) with
complete Freund’s adjuvant (FCA, Sigma). On d 21 and d 28,
horses were injected with the same antigen 2.5 mL im, with
incomplete Freund’s adjuvant (FIA, Sigma). Eight batches
of sera were collected from trachelo veins on
d 0, d 10, d 21, d 28, d 35,
d 42, d 49, and d 55 after the first immunization,
which were stored at -20 °C for the measurement of antibody
titers.
Enzyme-linked immunosorbent assay (ELISA) Severe acute respiratory syndrome coronavirus specific IgG was
measured using an indirect enzyme-linked immunosorbent
assay (ELISA) and whole purified SARS-CoV F69 as antigen.
In brief, polystyrene micro well plates were coated with
antigen (100 μL/well containing 1.0 μg/mL virus protein) in
carbonate-bicarbonate buffer (pH 9.6). The wells were washed
3 times with PBS and then blocked with 15% bovine
serum in PBS containing 0.05% Tween-20 (PBST)
at 37 °C for 1 h. After 3 washes with PBST,
serially twofold diluted serum samples (from 1:100 to a final
1:51200) were added to the plates and incubated at
37°C for 1 h. Horseradish peroxidase (HRP)-conjugated
goat anti-horse IgG (Sigma, USA) diluted 3000-fold in PBS was added, followed by 3 washes.
Following incubation at 37 °C for 1 h, the plates were washed
as above and the substrate tetramethylbenzidine (TMB)
solution (Sigma) was added to the wells. After incubation at
37 °C for 15 min, the reaction was stopped by adding
2.0 mmol/L sulfuric acid, and the absorbance value at 450 nm (A450)
was measured with a microplate reader (Model 550, BioRad).
IgG antibody titer was defined as the highest dilution of
serum when the A450 ratio (A450 of negative serum) was
greater than 2.0.
Microneutralization assay The neutralization assay was
performed according to modified operating procedures of
the Manual for the virological investigation of polio
formulated by WHO/EPI/GEN/97.01.
(http://www.who.ch/programmes/gpv/gEnglish/avail/gpvcatalog/catlog.htm).
Each serum sample was serially diluted in twofold 1:10
dilution in MEM maintenance medium to a final dilution of 1:20
480 and incubated with an equal volume of 100
TCID50/25 μL of purified F69 or Z2-Y3 strain for 3 h at 36
°C. The virus-antibody mix was then inoculated onto Vero-E6 cell
(3×105 cells/mL) monolayers in 96-well plates at 37
°C for approximately 6 d. Wells for normal cell control and virus control
were added to 100 μL maintenance medium and unneutralized
active virus dilution, respectively. The plates were
incubated until CPE developed in all the virus controls but the
cell control remained normal. Neutralizing antibody titer was
the highest dilution of serum, which protected 50% of the
cultures against 100TCID50 of the challenge virus, when the
virus control (no serum) showed complete CPE.
Purification of immunoglobulin Horse antiserum was
thawed in 37 °C water bath, added to 45% saturated
ammonium sulphate solution, then mixed gently at 22
°C for 30 min, centrifuged at
10 000×g, and the precipitation generated was
collected and stored at 4 °C overnight. The ammonium
sulphate precipitation was diluted using an equal volume of
0.9% NaCl solution and dialyzed to remove the salt. Then
pH was adjusted to 3.5 with 0.36 mol/L HCl. Then horse
serum was added to 2% pepsin (Sigma) solution and digested
at 37 °C for 8 h, 24 h, 36 h, 48 h, 60 h, and 72 h, respectively.
The reaction was stopped by adjusting the pH to 8.0 using
1.0 mol/L NaOH. Then the digested material was
ultrafiltrated. Anion-exchange separations of ultrafiltrated
material were further performed using
diethylaminoethyl(DEAE) Sepharose Fast Flow (Pharmacia) Column,
pre-equilibrated with 200 mL of buffer A (50 mmo/L Tris-HCl, pH 8.0).
The ultrafiltrated material was pumped down the column,
while the A280 nm of the eluted material was monitored,
followed by pumping fresh buffer A until the
A280 nm returned to the baseline. All the unbounded material, corresponding
to the F(ab’)2 fragments was collected and stored at 4
°C. Bound contaminants can then be eluted to regenerate the
column using a gradient of buffer B (containing 50 mmo/L
Tris-HCl, pH 8.0, 1 mmol/L NaCl, pH 8.0). The product
obtained using anion-exchange chromatography was
ultrafiltrated, concentrated and added to 0.3 mol/L
aminoacetic acid to obtain stock solution. According to the demanded
standards of biological product, the product characteristics
(eg, pH, the protein concentration and bacterial endotoxin
content) were detected using serial procedures.
Sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE) Non-reducing SDS-PAGE gels, using the
buffer system described by Laemmli
(1970)[23], were used to monitor the digestion process and to check for traces of
undigested IgG and other unwanted materials.
Results
Identification of SARS coronavirus The virus
(SARS-CoV F69) was electron microscopically visualized, and the
characteristic coronavirus particle form was observed
(Figure 1).
Level of the specific IgG antibody Six health horses were
immunized with purified SARS-CoV F69 strain. The titers of
total anti-SARS-CoV IgG was measured using an indirect
ELISA. The dynamic changes of specific anti-SARS-CoV
IgG antibody titers are shown in Figure 2. On d 10, all sera
distinctly showed positive reactions, with the range of
specific IgG antibody titers from 1:160 to 1:980. Titers of specific
IgG antibodies increased rapidly from week 4 and peaked at
week 7 after the first immunization; the maximum value was
1:14210.
Titers of neutralizing antibodies The antiserum was
measured using a micro-neutralization test. The kinetics of
formation of neutralizing antibodies following immunization
for horses were observed (Figure 3). The neutralizing
antibodies were partially detectable on d 10 (from 1:10 to 1:60).
After the third immunization, the neutralizing antibody titers
of all the immunized horses increased rapidly on d 28. On
d 48 after the first immunization, the neutralizing antibody
titers of 4 of 6 equines reached the highest level. The other
2 continued increasing and reached the highest titer at 1:14240.
Cross neutralization response Z2-Y3 strain was used in
micro-neutralization test in vitro to measure the hyperimmune
sera from the SARS-CoV F69 strain. The results indicate
that the horse antiserum induced by the inactivated
SARS-CoV F69 strain is capable of neutralizing the SARS-CoV
Z2-Y3 strain completely.
F(ab’)2 preparation Digestion with pepsin at different
time points was assessed using SDS-PAGE (Figure 5). The
results indicate that IgG could be digested completely at
pH 3.5 within 48 h and unwanted protein bands
(eg albumin and transferrin) could be eliminated as well. The reaction
was stopped by adjusting the pH to 8.0 using 1.0 mol/L
NaOH. The anion-exchange chromatography with a salt
gradient was performed to further remove high molecular weight
aggregates and pepsin. Digested antisera in buffer A are
separated into 3 peaks (Figure 6). Material from peak I was
then concentrated. Finally, approximately15 g
F(ab’)2 fragments were obtained from 1 litre antiserum with the purity
above 90%. The titer of neutralizing antibodies after
purification was detected as 1:5120.
The product obtained above was dissolved in a suitable
volume of 0.9% NaCl to adjust the protein concentration to
10 g/L. Then pH determined at 20 °C was 7.0. The bacterial
endotoxin content was also detected at no more than
200 EU/mL. The terminal product according with the standard of
SFDA [(2003) No.267], was stored at 4 °C.
Discussion
Severe acute respiratory syndrome has resulted in
important challenges for the medical community. There are no
available specific vaccines and effective drugs for use against
SARS-CoV[11]. Control depends on prompt detection and
isolation of cases, good infection control in hospitals, and
the tracing and quarantine of
contacts[24]. The widespread clinical successful application of immunoglobulins derived
from heterogenous animals against rabies has a long
history[25]. The passive administration of neutralizing antibodies could
be an effective strategy for emergency prophylaxis and the
treatment of SARS[26].
The results of our research indicate that healthy horses
immunized with the SARS-CoV F69 strain can be induced to
generate effective, specific and neutralizing antibodies.
Analysis indicates that sequence difference existed among
SARS-CoVs[27]. The sequence of the SARS-CoV F69 strain
is different from that of the SARS-CoV Z2-Y3 strain (Table 1).
Immunoglobulin prepared from SARS-CoV F69 strain
isolated in April, 2003 could neutralize another SARS-CoV
Z2-Y3 strain, which was isolated from SARS patient in February,
2003. This showed that SARS-CoV F69 and Z2-Y3 strain
owned identical or similar neutralizing epitopes.
Heterogenous antisera used for treatment possibly
result in anaphylactoid severe acute
side-effects[28]. To avoid the side-effects caused by horse antiserum, IgG against
SARS-CoV was digested with pepsin and purified with
anion-exchange separations to exclude the immunogenicity of
Fc fragments and to retain the special activity of binding the
antigen of F(ab’)2 fraction. The titers of neutralizing
F(ab’)2 against SARS-CoV was detected at higher level (1:5120). And
approximately 15 g F(ab’)2 fragments were obtained from
1 litre antiserum, with the purity above 90%.
Until we have an efficacious vaccine and have
implemented effective epidemiologic infection control measures,
and given the presence of effective anti-SARS-CoV agents,
SARS is likely to remain a major health threat to the world. In
this article, we provide an alternative pathway of prevention
and treatment of SARS, with the purpose of combating any
resurgence of SARS. The profile of the antibody titer was
observed, while an effective, specific and neutralizing
hyperimmunoglobulin was prepared. The results indicate
that the kinetics of the induced specific IgG and neutralizing
antibodies are similar (Figure 4). This data paves the way for
the development of an inactivated SARS vaccine.
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