| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 University of British Columbia Centre for Disease Control, Vancouver, BC V5Z 4R4, Canada
2 Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK S7N 5E3, Canada
3 Emerging Pathogens Department, Southern Research Institute, Birmingham, AL 35205, USA
4 Departments of Pathology and Molecular Medicine and Biology, McMaster University, Hamilton, ON L8N 3Z5, Canada
5 Departments of Molecular and Medical Genetics and Microbiology and Biochemistry, University of Toronto, Toronto, ON M5S 1A8, Canada
6 Michael Smith Foundation for Health Research, Vancouver, BC V6H 3X8, Canada
7 Vaccine Evaluation Centre, British Columbia Institute for Children's and Women's Health, BC Children's Hospital, Vancouver, BC V6H 3V4, Canada
8 Brody School of Medicine, Department of Microbiology and Immunology, East Carolina University, Greenville, NC 27834, USA
9 Michael Smith Laboratories and Departments of Biochemistry and Molecular Biology and Microbiology and Immunology, University of British Columbia, Vancouver, BC V6T 1Z3, Canada
Correspondence
B. Brett Finlay
bfinlay{at}interchange.ubc.ca
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
-propiolactone) SARS-CoV vaccine and a combination of two adenovirus-based vectors, one expressing the nucleocapsid (N) and the other expressing the spike (S) protein (collectively designated Ad S/N), were evaluated for the induction of serum neutralizing antibodies and cellular immune responses and their ability to protect against pulmonary SARS-CoV replication. The whole killed virus (WKV) vaccine given subcutaneously to 129S6/SvEv mice was more effective than the Ad S/N vaccine administered either intranasally or intramuscularly in inhibiting SARS-CoV replication in the murine respiratory tract. This protective ability of the WKV vaccine correlated with the induction of high serum neutralizing-antibody titres, but not with cellular immune responses as measured by gamma interferon secretion by mouse splenocytes. Titres of serum neutralizing antibodies induced by the Ad S/N vaccine administered intranasally or intramuscularly were significantly lower than those induced by the WKV vaccine. However, Ad S/N administered intranasally, but not intramuscularly, significantly limited SARS-CoV replication in the lungs. Among the vaccine groups, SARS-CoV-specific IgA was found only in the sera of mice immunized intranasally with Ad S/N, suggesting that mucosal immunity may play a role in protection for the intranasal Ad S/N delivery system. Finally, the sera of vaccinated mice contained antibodies to S, further suggesting a role for this protein in conferring protective immunity against SARS-CoV infection.
Present address: Departments of Anatomy and Radiology and Infectious Diseases, University of Georgia, Athens, GA 30602, USA. 
Present address: Animal Resources Center, Department of Surgery, University of Chicago, Chicago, IL 60637, USA.
Present address: Cross Cancer Institute, Department of Oncology, University of Alberta, Edmonton, AB T6G 1Z2, Canada.
||Present address: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.
¶Present address: Tulane University School of Medicine, Department of Microbiology and Immunology, New Orleans, LA 70112, USA.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Rota et al., 2003) and by experimental infection of macaques to fulfil Koch's postulates (Fouchier et al., 2003).
Currently, there is no effective treatment for SARS. Prevention through contact-reduction or transmission-blocking measures has been the only means available to modify the devastating impact of this illness. Prevention through vaccination would be an attractive alternative that is less reliant on individual case detection to be effective. No vaccines are currently licensed for any of the human CoVs, but effective vaccines have been produced for some animal CoVs, such as certain strains of Infectious bronchitis virus (poultry), Bovine coronavirus and Canine coronavirus (Cavanagh, 2003; Enjuanes et al., 1995; Pratelli et al., 2003; Saif, 2004; Takamura et al., 2002). Individuals convalescing from SARS develop high titres of neutralizing antibodies (Tan et al., 2004) and the appearance of antibodies coincides with the onset of resolution of SARS pneumonia (Liu et al., 2004; Woo et al., 2004). Thus, there is some optimism that an effective vaccine against SARS-CoV may also be possible.
Coronavirus spike (S) proteins have long been known to be a major determinant in coronavirus pathogenesis, given that this viral protein interacts with cellular receptors as well as contains determinants for eliciting a protective immune response (Enjuanes et al., 1995; Navas-Martin & Weiss, 2003). Consequently, the SARS-CoV S glycoprotein, shown to be responsible for receptor binding to the cellular angiotensin-converting enzyme 2 (ACE2), is an attractive target for both vaccine and therapeutics development (Li et al., 2003). This is strongly supported by the finding that a human mAb that binds to the N terminus of the S protein potently neutralizes SARS-CoV infection and inhibits syncytia formation through blocking of receptor binding (Berger et al., 2004). Moreover, the S protein has been shown to induce serum neutralizing antibodies and to confer protective immunity against SARS-CoV challenge in mice and African green monkeys (Bisht et al., 2004; Bukreyev et al., 2004; Yang et al., 2004). Studies from other animal CoV vaccines have also shown that the CoV nucleocapsid (N) protein, which encapsidates the viral genome, may represent another antigen candidate for vaccine development (Antón et al., 1996; Olsen, 1993). Although antibodies to CoV N proteins have no virus-neutralizing activity in vitro, there is evidence that the protein may provide in vivo protection by induction of cell-mediated immunity (Enjuanes et al., 1995; Stohlman et al., 1995; Wesseling et al., 1993). The N protein has been shown to generate CoV-specific CD8+ T cells (Boots et al., 1991; Seo et al., 1997; Stohlman et al., 1993, 1995) and to provide protection in animals following infection (Collisson et al., 2000; Seo et al., 1997).
Several potential strategies can be considered for vaccination against SARS-CoV, including a whole killed virus (WKV) vaccine, a live-attenuated SARS-CoV vaccine, a viral vector such as adenovirus or Vaccinia virus expressing SARS-CoV genes, recombinant SARS-CoV proteins and DNA-based vaccines (reviewed by See et al., 2005). In this report, two SARS vaccine approaches were developed in parallel and evaluated for their efficacy in a murine SARS model by the SARS Accelerated Vaccine Initiative described elsewhere (Finlay et al., 2004). We report the first direct comparison of a whole killed SARS-CoV vaccine and a combination of attenuated adenoviruses, one expressing SARS-CoV S protein and the other expressing the N protein (collectively called Ad S/N), for their ability to protect against live SARS-CoV challenge in vivo. Vaccine candidates were developed in parallel and evaluated for immunogenicity and efficacy against SARS-CoV infection in a murine model previously demonstrated to support virus replication (Hogan et al., 2004).
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) was kindly provided by Dr Tim Booth, Canadian Science Centre for Human and Animal Health, Winnipeg, Canada (Public Health Agency of Canada). SARS-CoV Tor2 strain was passaged in Vero E6 cells, purified by ultracentrifugation and subsequently inactivated with -propiolactone (BPL) in a biosafety-level 3 facility, as described previously (Zakhartchouk et al., 2005a). Inactivated virus was stored at –80 °C until use. BPL-inactivated virus (approx. 1 mg ml–1 by Coomassie blue dye assay) was diluted in 0·1 M PBS, pH 7·0 (without divalent cations), to a protein concentration of 250 µg ml–1. For animals receiving inactivated virus in alum adjuvant (2 % Alhydrogel; Accurate Chemical & Scientific Corp.), BPL-inactivated virus (500 µg ml–1) was diluted in PBS and mixed with 2 % Alhydrogel diluted 1 : 4 for 
6 h at 4 °C to give a final concentration of 250 µg virus and 2·5 mg Alhydrogel ml–1.
Adenovirus-vectored SARS vaccine.
The full-length SARS-CoV N gene (nt 28120–29482 of the SARS-CoV genome, GenBank accession no. NC_004718; Marra et al., 2003) was amplified from an isolate of the Tor2 strain by RT-PCR and inserted into the pCR-Blunt II-TOPO vector (Invitrogen) to generate pTOPO-N. The N gene fragment was then isolated from pTOPO-N and inserted into the Ad5 shuttle plasmid pDC516(io) (AdMax Hi-IQ kit J; Microbix) downstream of a modified murine cytomegalovirus immediate-early promoter and upstream of the simian virus 40 polyadenylation signal. The S gene (corresponding to nt 21492–25259 of the SARS-CoV genome) was PCR-amplified from the plasmid pIR-GP SARS-V5, obtained from Nabil Seidah (Institut de Recherches Cliniques de Montreal, Quebec, Canada) and inserted into pDC516(io) to generate the shuttle plasmid pDC516(io)SARS-S. 293-IQ cells (Microbix) (Matthews et al., 1999) were co-transfected with the genomic plasmid pBHGfrtdE1,3FLP (Ng et al., 2000) and the shuttle plasmid pDC516(io)SARS-N or pDC516(io)SARS-S to generate AdSARS-N complete (shortened to Ad N) and AdSARS-S (shortened to Ad S), respectively, by Flp recombinase-mediated site-specific recombination. These vectors were amplified, purified and titrated by plaque assay as described by Hitt et al. (2005) with no evidence of heterologous insert instability. The Ad N vector described here carried the same N gene as Ad5-N-V reported recently (Zakhartchouk et al., 2005b), but differed in the promoter (modified murine cytomegalovirus promoter in this Ad N construct) used in controlling N gene transcription. Expression of the SARS-CoV N or S protein was confirmed by Western blot analysis of lysates from human embryonic kidney (HEK293) or HeLa cells infected with the adenovirus-based vector for 24 h, using convalescent sera from SARS patients as a source of antibodies.
Molecular cloning, expression and purification of SARS-CoV N protein in Escherichia coli.
DNA fragments containing the SARS-CoV N gene were generated by RT-PCR using SARS-CoV genomic RNA as the template. In order to subclone the PCR product as an NdeI–EagI fragment into the pET30b(+) vector (Novagen), the following forward and reverse primers were used: 5'-GAATTCCATATGTCTGATAATGGACCCAATC-3' and 5'-GAAAGCCGGCCGATGCCTGAGTTGAATCAGCAG-3'. The PCR mixture contained 2 mM MgSO4, 200 µM dNTPs, 2·5 U Pfu DNA polymerase and 25 pmol each oligonucleotide primer. PCR cycling conditions were as follows: one cycle of 95 °C for 2 min, followed by 35 cycles of 94 °C for 15 s, 58 °C for 30 s and 72 °C for 2 min. Plasmids containing the N gene were then transformed into E. coli strain BL21 (DE3). SARS-CoV N protein expression was induced in transformed BL21 (DE3) host cells by adding 1 mM IPTG for 4 h. The expressed N protein, containing an N-terminal histidine tag, was subsequently purified by using the His Bind purification system (Novagen) and confirmed by SDS-PAGE.
S protein fragment (aa 318–510).
A mammalian expression vector consisting of a codon-optimized gene encoding the SARS-CoV S protein aa 318–510 fragment (kindly provided by David Kelvin, University of Toronto, Canada) downstream of the mammalian transin-secretion signal peptide, a protein A purification tag (Sanchez-Lopez et al., 1988), and a tobacco etch virus (TEV) protease-cleavage site was generated by using the pIRESpuro3 plasmid (Clontech). The secreted N-terminal fusion protein was purified from the culture medium of stably transformed HEK293T cells by IgG affinity chromatography. Following TEV cleavage, the S protein was further purified by hydrophobic-interaction chromatography, cation-exchange chromatography and gel filtration. Purity was assessed by Coomassie blue staining following SDS-PAGE.
Mice.
Six-week-old female 129S6/SvEv mice, free of adventitious rodent pathogens, ectoparasites and endoparasites, were purchased from Taconic Farms (Germantown, NY, USA). Mice were housed in groups of eight in polysulfone microisolator cages on Sani-Chip bedding (PJ Murphy Forest Products). They were provided with food and water ad libitum. All procedures were in accordance with the NRC (1996), the Animal Welfare Act and the CDC NIH (1999).
Mice immunizations.
Two groups of 129S6/SvEv mice were used in this study. Group 1 mice (n=5 per vaccine group) were immunized on day 0 and week 4 prior to sacrifice at week 7. These mice were examined for both humoral and cell-mediated immune responses as a result of immunization with the different SARS vaccines. Group 2 mice (n=8 per vaccine group) were immunized as above, but were subsequently challenged with live SARS-CoV Tor2 at week 7 (see below). For immunizations on day 0, mice were vaccinated either with a combination of adenoviruses, one expressing SARS-CoV S protein and the other expressing SARS-CoV N protein (collectively called Ad S/N), or with the WKV vaccine in the presence or absence of alum. Specifically, 129S6/SvEv mice were immunized in one the following manners: (i) subcutaneously with 0·2 ml 0·1 M PBS, pH 7·0 (without divalent ions); (ii) subcutaneously with WKV alone, consisting of 50 µg inactivated virus in 0·2 ml 0·1 M PBS; (iii) subcutaneously with WKV vaccine plus alum, consisting of 50 µg inactivated virus plus 500 µg Alhydrogel in 0·2 ml 0·1 M PBS; (iv) intranasally with adenovirus-based SARS-CoV S and N vectors (3x108 p.f.u. each) in a total volume of 30 µl (Ad S/N IN); (v) intramuscularly with adenovirus-based SARS-CoV S and N vectors (3x108 p.f.u. each) into the hind leg (Ad S/N IM); (vi) intranasally or intramuscularly with a control adenovirus 5 (6x108 p.f.u.) that lacks SARS-CoV genes (Ad-Ctrl). Before intranasal vaccination with the recombinant adenovirus SARS vaccines, each mouse was anaesthetized with isoflurane. Four weeks after the initial immunization, mice were reimmunized with the same vaccine at the same dose and monitored until week 7.
SARS-CoV challenge studies.
Following vaccination at 0 and 4 weeks, group 2 mice were challenged at 7 weeks with the SARS-CoV Tor2 strain. For challenge experiments, eight mice from each vaccine group were anaesthetized with isoflurane and infected via intranasal inhalation with 1x106 p.f.u. SARS-CoV Tor2 in a total volume of 30 µl. On day 3 post-challenge, half (n=4) of the animals were euthanized with CO2 and necropsied. The remaining four animals were sacrificed in the same way on day 7 post-challenge.
Collection of blood and lung tissues.
Blood was collected from each mouse via retro-orbital puncture during the following time periods: prior to the first immunization, week 4 (pre-boost) and week 7 (pre-challenge). Blood samples were processed by clotting for 30 min at room temperature and centrifuged to remove cellular debris, and the resulting sera were stored at –80 °C. The lungs from each mouse were removed aseptically and homogenized in PBS as described below. Fifty microlitres of the lung homogenate was mixed with 450 µl TRIzol reagent and stored at –80 °C for RNA analysis. The remaining lung homogenate was stored at –80 °C for virus titration.
Virus titration.
Frozen lung (previously unwashed) samples were homogenized in PBS containing penicillin (100 U ml–1), streptomycin (100 µg ml–1) and gentamicin (50 µg ml–1). Lung homogenates were then diluted serially (half-log) in Dulbecco's minimal essential medium containing 2 % heat-inactivated fetal bovine serum (Atlanta Biologicals) and antibiotics (penicillin and streptomycin) before addition to 90–95 % confluent Vero E6 monolayers in 96-well plates. Cells were monitored for cytopathic effect (CPE) in positive-control wells. After incubation for 48 h at 37 °C, 5 % CO2, CPE was measured by the addition of neutral red to the wells and measurement of A540. TCID50 was determined by a 50 % reduction in CPE as described previously (Guo et al., 2004; Schmidt, 1989; Smee et al., 2001). Viral titres were expressed as log10 TCID50 ml–1 for lung homogenates.
Virus neutralization assay.
Twofold dilutions of heat-inactivated serum were tested for the presence of antibodies that would neutralize the infectivity of 100 TCID50 of SARS-CoV in Vero E6 cell monolayers as described previously (Zakhartchouk et al., 2005a). The CPE of SARS-CoV on Vero E6 cell monolayers was read on day 3. The dilution of serum that completely inhibited CPE in 50 % of the wells was calculated as described previously (Reed & Muench, 1938).
Gamma interferon (IFN-
) ELISPOT assay.
MultiScreen-IP (Millipore) 96-well plates were coated overnight with rat anti-mouse IFN- (Pharmingen) at 4 °C. Plates were washed with sterile PBS and incubated in triplicate with 2x105 murine splenocytes per well in RPMI 1640. Recombinant N protein was isolated as described above and added to a final concentration of 10 µg ml–1. After incubation for 40 h at 37 °C, plates were washed six times with PBS containing 0·05 % Tween 20 and incubated overnight with 100 µl biotinylated rat anti-mouse IFN- (Pharmingen) per well. After washing, streptavidin–alkaline phosphatase was added and the plates were developed and read as described previously (Zakhartchouk et al., 2005a).
Western blot analysis using pooled mice sera.
Purified bacterial N protein (0·2 µg) and either 0·5 or 5 µg truncated mammalian S protein (aa 318–510) were prepared in SDS sample buffer, separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Specific proteins on the membranes were visualized as described previously (Zakhartchouk et al., 2005a).
SARS-CoV-specific ELISA.
Total SARS-CoV-specific IgG, IgG isotypes and IgA titres in sera from immunized mice were measured by ELISA. Each well of a 96-well plate was coated overnight with 0·1 ml purified inactivated SARS-CoV at a concentration of 1 µg ml–1. Washing of plates, addition of sera and colour development were performed as described previously (Zakhartchouk et al., 2005a).
RT-PCR assay for SARS-CoV RNA.
Fifty microlitres of lung homogenate was placed in TRIzol reagent and processed as described previously (Hogan et al., 2004). RNA was isolated with an RNeasy kit (Qiagen) and tissue RNA was quantified with RiboGreen (Molecular Probes). DNase treatment was performed to eliminate the remaining DNA in the RNA samples. The SARS-CoV genome was detected with LUX primer sets for the SARS-CoV N domain and the Superscript III Platinum One-step Quantitative RT-PCR System (Invitrogen) in a LightCycler (Roche) as described previously (Hogan et al., 2004). To confirm the RT-PCR results, RT-PCR products from some of the study samples were verified by gel electrophoresis. The results ranged from less than one copy to up to 10 000 copies per reaction.
Statistical analyses.
Statistical significance was assessed by using the non-parametric Wilcoxon rank sum test (Hollander & Wolfe, 1973). Differences between mean values for the vaccine groups were considered significant if the P value (two-tailed) was 
0·05.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The addition of alum to the WKV vaccine preparation did not increase serum neutralizing-antibody levels significantly at either time period (Fig. 1a). Mice given Ad S/N IM showed similar serum neutralizing-antibody titres at weeks 4 and 7, but overall titres were 25-fold lower than those observed in the sera of mice given WKV with or without alum (Fig. 1a). In contrast, the neutralizing-antibody titres in the sera of Ad S/N IN-vaccinated mice were low during the first 4 weeks, but increased 11-fold at the end of 7 weeks, albeit with overall titres that were lower than those found in Ad S/N IM sera (Fig. 1a). No serum neutralizing-antibody responses could be detected within the 7 week period in mice immunized with the control adenovirus (Ad-Ctrl) (Fig. 1a).
|
, no significant difference in total SARS-CoV-specific IgG levels was observed between WKV alone and WKV plus alum at either week 4 or week 7, supporting the data in Fig. 1(a). However, a 19-fold difference (P=0·02) in total SARS-CoV-specific IgG levels was observed at weeks 4 and 7 in the sera of mice vaccinated with WKV alone or with WKV plus alum (Fig. 1b). The total SARS-CoV-specific IgG levels measured from week 7 sera of either Ad S/N IM- or Ad S/N IN-vaccinated mice did not differ statistically from those of week 4 sera and were approximately sevenfold lower than those induced by the WKV plus alum vaccine at week 7 (Fig. 1b). No SARS-CoV-specific IgG was detected in the sera of mice vaccinated with either PBS or Ad-Ctrl at either time period. The SARS-CoV-specific IgG subclasses were also determined in week 7 sera. Fig. 1(c) shows that SARS-CoV-specific IgG2a levels in week 7 sera were significantly higher than IgG1 levels in response to WKV alone (P=0·008), WKV plus alum (P=0·03) or Ad S/N given either intramuscularly (P=0·002) or intranasally (P=0·0001). The addition of alum to WKV significantly augmented (P=0·008) the SARS-CoV-specific IgG1, but not the IgG2a, response (Fig. 1c). Our results showed that the WKV alone and Ad S/N IM vaccines induced similar levels of SARS-CoV-specific IgG isotypes at week 7. Additionally, we analysed the sera of all vaccinated mice by ELISA for the presence of SARS-CoV-specific IgA, which would indicate vaccine stimulation of mucosal immunity. The results in Fig. 1(d) indicated that sera from mice vaccinated with Ad S/N IN showed a greater than 3 log increase in SARS-CoV-specific IgA titres compared with Ad-Ctrl-vaccinated mice at weeks 4 and 7. No detectable SARS-CoV-specific IgA was found in the sera of mice immunized with either WKV (with or without alum) or Ad S/N IM for up to 7 weeks (Fig. 1d).
To determine the SARS-CoV proteins recognized by the sera of mice vaccinated with either WKV or Ad S/N at week 7, Western blot analysis was performed on purified recombinant S or N proteins. In contrast to mice receiving either PBS or Ad-Ctrl, bands corresponding to the molecular masses of the S fragment (Fig. 2a) and the N protein (Fig. 2b) were detected by pooled sera from mice vaccinated with WKV, WKV plus alum, Ad S/N IM or Ad S/N IN. Interestingly, a consistently stronger immunoreactivity towards both the S and N proteins was evident in the sera of mice vaccinated with WKV plus alum compared with WKV alone (Fig. 2a and b, lanes 2 and 3), suggesting that alum may either enhance the response of this class of antibody or increase its avidity. In addition, the immunoreactivity of sera from Ad S/N IM- or Ad S/N IN-vaccinated mice to recombinant S protein was much weaker than that observed with WKV sera and required 10-fold more antigen to visualize the immunoreactive S protein band (Fig. 2a). Collectively, our results indicate that the S and N proteins in the WKV and Ad S/N preparations were able to induce a readily quantifiable humoral response.
|
by mouse splenocytes stimulated in vitro with recombinant SARS-CoV N protein. Fig. 3 shows that, when stimulated with N protein, splenocytes from mice vaccinated with either WKV or Ad S/N secreted significantly higher levels (P=0·002) of IFN- compared with splenocytes from control mice. As indicated in Fig. 3, vaccination of mice with Ad S/N IM resulted in the highest number of IFN--secreting splenocytes, followed by WKV alone, Ad S/N IN and WKV plus alum, in descending order.
|
). From the vaccination period to the time of SARS-CoV challenge, mice appeared normal in activity and appetite and showed no signs of adverse reaction to any of the vaccines at the administration site. Viral titres were assessed in lung homogenates during the early and late courses of infection by using a CPE assay. Pilot studies with PBS showed that SARS-CoV titres in the murine respiratory tract were highest at day 3 post-challenge and dropped dramatically to undetectable levels by day 7 post-challenge (Hogan et al., 2004). SARS-CoV replication in the lungs was assessed in the group 2 mice by examining viral titres and RNA levels in lung homogenates. Fig. 4(a) shows that lung viral titres in mice immunized with WKV in the absence or presence of alum were reduced by 4 logs to undetectable levels compared with titres observed in the PBS control on day 3 post-challenge (P=0·02). Lung viral titres in animals vaccinated with Ad S/N IN were also reduced significantly (P=0·03) compared with Ad-Ctrl-vaccinated animals harvested on day 3, albeit not as strongly as observed with WKV (Fig. 4a). A similar reduction trend, although not statistically significant, was observed when the lung viral titres of Ad S/N IM-vaccinated mice were compared with those of mice vaccinated with Ad-Ctrl (Fig. 4a). Viral titres in lung homogenates were below the assay detection limit for all vaccine groups on day 7 post-challenge. Levels of SARS-CoV RNA correlated well with the lung viral-titre data on day 3 post-challenge. Compared with the PBS control on day 3 post-challenge, levels of viral RNA were undetectable in lung homogenates of mice immunized with WKV alone or with WKV plus alum (Fig. 4b). A significant reduction (P=0·02) in the level of SARS-CoV RNA was also observed in day 3 post-challenge lung homogenates of mice given Ad S/N IN compared with animals vaccinated with Ad-Ctrl. SARS-CoV RNA was still detectable in lung homogenates at day 7 post-challenge for mice vaccinated with either PBS or Ad-Ctrl (Fig. 4b). By day 7 post-challenge, SARS-CoV RNA was undetectable for both groups of WKV-vaccinated animals (Fig. 4b). Residual low levels of SARS-CoV RNA were observed at day 7 post-challenge for mice immunized with Ad S/N IN or Ad S/N IM, but the levels were not significantly different from those of Ad-Ctrl-vaccinated mice (Fig. 4b).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
secretion by splenocytes of vaccinated mice following in vitro stimulation with recombinant N protein. The number of IFN--secreting splenocytes in Ad S/N IM-immunized mice was significantly higher (1·5–2·5-fold) than those of mice vaccinated with Ad S/N IN, WKV alone or WKV plus alum. However, when evaluating protection against pulmonary virus replication in 129S6/SvEv mice, the WKV vaccine was more effective than Ad S/N administered intramuscularly or intranasally in reducing pulmonary SARS-CoV replication following challenge. No significant protection was observed in mice vaccinated with Ad S/N IM compared with the control animals. Our results suggest that humoral immunity, but not cellular immune responses, correlates well with the ability of WKV to protect against pulmonary SARS-CoV replication.
Recipients of the Ad S/N IN vaccine also demonstrated significantly reduced levels of SARS-CoV replication in the lungs, although protection was not as effective as the WKV vaccine. In contrast, the intramuscular route of the Ad S/N vaccine had only a limited effect in reducing pulmonary SARS-CoV replication, despite demonstrating high serum neutralizing-antibody titres prior to SARS-CoV challenge and the ability to induce a robust cellular immune response. In our study, the two intramuscular injections of the Ad S/N vaccine produced a strong SARS-CoV-specific IgG1 and IgG2a antibody response in mice, with IgG2a titres significantly higher than IgG1. The serum neutralizing-antibody levels in Ad S/N IN-vaccinated mice were lower than in animals immunized with Ad S/N IM. This result suggests that other factors, such as mucosal immunity (e.g. secretory IgA and/or mucosal T cells), may play an important role in limiting lung virus replication by the Ad S/N IN vaccine. The detection of serum SARS-CoV-specific IgA in mice vaccinated with Ad S/N IN, but not with Ad S/N IM or Ad-Ctrl, suggests that mucosal SARS-CoV-specific IgA does play an important role in conferring protection against SARS-CoV pulmonary replication. Moreover, the IFN--secreting response observed in splenocytes of Ad S/N IN-vaccinated mice was more than twofold lower than that found in mice immunized with Ad S/N IM, indicating that cellular immune responses do not contribute to protection against SARS-CoV challenge as strongly as an antibody response, consistent with other studies (Yang et al., 2004). Our result points to a potential approach whereby SARS-CoV infection could potentially be blocked at the primary site of entry (e.g. respiratory tract) by using an intranasal adenovirus-based vaccine (Bukreyev et al., 2004; Peiris et al., 2003).
There are several possible explanations as to why the WKV vaccine conferred more protection against pulmonary SARS-CoV replication than the Ad S/N vaccine. Firstly, the WKV vaccine induced higher serum virus-neutralizing activity than mice immunized with either Ad S/N IN or Ad S/N IM. Secondly, the route of administration (subcutaneous for WKV vaccine, intranasal or intramuscular for Ad S/N) or the vaccine dose may contribute to the protective differences observed between the WKV and Ad S/N vaccines. Thirdly, expression of the S protein from the Ad S/N construct may be lower than that of WKV. This was evident in Fig. 2(a), where sera from WKV-vaccinated mice showed a much stronger immunoreactivity towards the S protein than sera from Ad S/N-immunized animals. Indeed, we have recently constructed a single adenovirus vector containing codon-optimized S and N genes and found that the serum neutralizing-antibody titres induced by this construct, when given intranasally/intramuscularly to mice, were comparable to levels found in animals vaccinated with WKV (data not shown). Lastly, unlike the Ad S/N vaccine, which contains only genes that encode the N and S proteins, the WKV vaccine contains the complete complement of viral proteins present in the natural virion conformation and thus may induce a broader immune response. In support of this, others have reported that sera from mice vaccinated with inactivated SARS-CoV contain antibodies to a number of proteins including S, N, M and 3CL of the SARS-CoV Tor2 strain (Xiong et al., 2004).
Several groups have developed SARS vaccines based on the SARS-CoV S protein as a target. A DNA-based vaccine (Buchholz et al., 2004), a modified Ankara vaccinia virus (Bisht et al., 2004) and a recombinant attenuated parainfluenza virus (Bukreyev et al., 2004) containing the SARS-CoV S gene have been shown to induce serum neutralizing antibodies and to inhibit pulmonary virus replication in animals. These animal models for SARS include macaques (Fouchier et al., 2003; Kuiken et al., 2003), African green monkeys (Bukreyev et al., 2004; McAuliffe et al., 2004), ferrets (Martina et al., 2003), mice (Glass et al., 2004; Subbarao et al., 2004) and hamsters (Roberts et al., 2005). Although all of the above animal models support virus replication, no single animal species has been shown to reproduce all of the clinical signs and lethality observed in humans infected with SARS-CoV. The importance of a humoral response to the S protein has been demonstrated by the protection of naïve mice from live SARS-CoV challenge after passive IgG transfer from immunized animals (Yang et al., 2004). In contrast, antiserum to N protein has not been shown to contain neutralizing antibodies (Pang et al., 2004). However, the N protein has been shown to be a vaccine candidate by inducing SARS-CoV antigen-specific T-cell and virus-neutralizing responses (Kim et al., 2004; Zhu et al., 2004). Several laboratories (He et al., 2004; Qu et al., 2005; Takasuka et al., 2004; Tang et al., 2004) have developed an inactivated whole SARS-CoV vaccine that can induce serum neutralizing antibodies when injected into mice, but whether these vaccines can confer protective immunity against live SARS-CoV challenge was not reported. Recently, a WKV vaccine has been shown to protect against pulmonary SARS-CoV replication in BALB/c mice, although characterization of the immune response was not reported (Stadler et al., 2005). In this study, we also demonstrated that a whole killed SARS-CoV vaccine can induce serum neutralizing antibodies and can protect against SARS-CoV challenge in mice. We showed that protection from SARS-CoV infection by the WKV vaccine is associated with the induction of both an IgG1 and IgG2a immune response. The addition of alum, which selectively stimulates IgG1 immune responses (Kenney & Edelman, 2003), only increased serum SARS-CoV-specific IgG1 but not IgG2a responses, which is consistent with the findings of others (Takasuka et al., 2004). In our study, total serum SARS-CoV-specific IgG and neutralizing-antibody levels were not enhanced by the addition of alum to the WKV vaccine preparation and protection from virus replication was not improved discernibly. In contrast, the immunoreactivity of pooled mouse sera towards both the full-length N protein and the S protein fragment was enhanced when the WKV vaccine formulation contained alum. These results suggest that the inclusion of alum in the WKV vaccine may lead to enhanced production of antibody to denatured protein. Interestingly, the S protein domain (aa 318–510) recognized by the sera of WKV-vaccinated mice in our studies is known to bind to the SARS-CoV functional receptor, ACE2 (Babcock et al., 2004; Wong et al., 2004; Xiao et al., 2003). Antibodies present against this S domain may explain why sera from our WKV-vaccinated mice were able to block SARS-CoV-mediated CPE in Vero E6 cells.
In summary, this is the first description of two SARS vaccines evaluated head-to-head for their ability to induce immunogenicity and to reduce viral load in the murine respiratory tract. Our results showed that the WKV vaccine was more effective than the Ad S/N vaccine in the reduction of viral load in the respiratory tract of vaccinated mice after live SARS-CoV challenge, with the intranasal route of Ad S/N also providing significant protection. Such a direct comparison of SARS vaccines in an animal model will not only determine which vaccine strategy is more effective, but will also shorten the time lines for moving the best candidate forward into human testing.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Babcock, G. J., Esshaki, D. J., Thomas, W. D., Jr & Ambrosino, D. M. (2004). Amino acids 270 to 510 of the severe acute respiratory syndrome coronavirus spike protein are required for interaction with receptor. J Virol 78, 4552–4560.
Berger, A., Drosten, Ch., Doerr, H. W., Stürmer, M. & Preiser, W. (2004). Severe acute respiratory syndrome (SARS) – paradigm of an emerging viral infection. J Clin Virol 29, 13–22.[CrossRef][Medline]
Bisht, H., Roberts, A., Vogel, L., Bukreyev, A., Collins, P. L., Murphy, B. R., Subbarao, K. & Moss, B. (2004). Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice. Proc Natl Acad Sci U S A 101, 6641–6646.
Boots, A. M., Kusters, J. G., van Noort, J. M., Zwaagstra, K. A., Rijke, E., van der Zeijst, B. A. & Hensen, E. J. (1991). Localization of a T-cell epitope within the nucleocapsid protein of avian coronavirus. Immunology 74, 8–13.[Medline]
Buchholz, U. J., Bukreyev, A., Yang, L., Lamirande, E. W., Murphy, B. R., Subbarao, K. & Collins, P. L. (2004). Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity. Proc Natl Acad Sci U S A 101, 9804–9809.
Bukreyev, A., Lamirande, E. W., Buchholz, U. J., Vogel, L. N., Elkins, W. R., St Claire, M., Murphy, B. R., Subbarao, K. & Collins, P. L. (2004). Mucosal immunisation of African green monkeys (Cercopithecus aethiops) with an attenuated parainfluenza virus expressing the SARS coronavirus spike protein for the prevention of SARS. Lancet 363, 2122–2127.[CrossRef][Medline]
Cavanagh, D. (2003). Severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus. Avian Pathol 32, 567–582.[CrossRef][Medline]
CDC NIH (1999). Biosafety in Microbiological and Biomedical Laboratories, 4th edn. Washington, DC: US Government Printing Office.
Collisson, E. W., Pei, J., Dzielawa, J. & Seo, S. H. (2000). Cytotoxic T lymphocytes are critical in the control of infectious bronchitis virus in poultry. Dev Comp Immunol 24, 187–200.[CrossRef][Medline]
Enjuanes, L., Smerdou, C., Castilla, J., Anton, I. M., Torres, J. M., Sola, I., Golvano, J., Sanchez, J. M. & Pintado, B. (1995). Development of protection against coronavirus induced diseases. A review. Adv Exp Med Biol 380, 197–211.[Medline]
Finlay, B. B., See, R. H. & Brunham, R. C. (2004). Rapid response research to emerging infectious diseases: lessons from SARS. Nat Rev Microbiol 2, 602–607.[CrossRef][Medline]
Fouchier, R. A. M., Kuiken, T., Schutten, M. & 7 other authors (2003). Aetiology: Koch's postulates fulfilled for SARS virus. Nature 423, 240.[CrossRef][Medline]
Glass, W. G., Subbarao, K., Murphy, B. & Murphy, P. M. (2004). Mechanisms of host defense following severe acute respiratory syndrome-coronavirus (SARS-CoV) pulmonary infection of mice. J Immunol 173, 4030–4039.
Guo, J.-P., Petric, M., Campbell, W. & McGeer, P. L. (2004). SARS corona virus peptides recognized by antibodies in the sera of convalescent cases. Virology 324, 251–256.[CrossRef][Medline]
He, Y., Zhou, Y., Siddiqui, P. & Jiang, S. (2004). Inactivated SARS-CoV vaccine elicits high titers of spike protein-specific antibodies that block receptor binding and virus entry. Biochem Biophys Res Commun 325, 445–452.[CrossRef][Medline]
Hitt, M. M., Ng, P. & Graham, F. L. (2005). Construction and propagation of human adenovirus vectors. In Cell Biology: a Laboratory Handbook, 3rd edn, vol. 1, pp. 435–443. Edited by J. E. Celis. San Diego: Academic Press.
Hogan, R. J., Gao, G., Rowe, T. & 10 other authors (2004). Resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires Stat1. J Virol 78, 11416–11421.
Hollander, M. & Wolfe, D. A. (1973). Nonparametric Statistical Methods. New York: Wiley.
Kenney, R. T. & Edelman, R. (2003). Survey of human-use adjuvants. Expert Rev Vaccines 2, 167–188.[CrossRef][Medline]
Kim, T. W., Lee, J. H., Hung, C.-F. & 9 other authors (2004). Generation and characterization of DNA vaccines targeting the nucleocapsid protein of severe acute respiratory syndrome coronavirus. J Virol 78, 4638–4645.
Kuiken, T., Fouchier, R. A. M., Schutten, M. & 19 other authors (2003). Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome. Lancet 362, 263–270.[CrossRef][Medline]
Li, W., Moore, M. J., Vasilieva, N. & 9 other authors (2003). Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus. Nature 426, 450–454.[CrossRef][Medline]
Liu, X., Shi, Y., Li, P., Li, L., Yi, Y., Ma, Q. & Cao, C. (2004). Profile of antibodies to the nucleocapsid protein of the severe acute respiratory syndrome (SARS)-associated coronavirus in probable SARS patients. Clin Diagn Lab Immunol 11, 227–228.
Marra, M. A., Jones, S. J. M., Astell, C. R. & 56 other authors (2003). The genome sequence of the SARS-associated coronavirus. Science 300, 1399–1404.
Martina, B. E. E., Haagmans, B. L., Kuiken, T., Fouchier, R. A. M., Rimmelzwaan, G. F., van Amerongen, G., Peiris, J. S. M., Lim, W. & Osterhaus, A. D. M. E. (2003). Virology: SARS virus infection of cats and ferrets. Nature 425, 915.[CrossRef][Medline]
Matthews, D. A., Cummings, D., Evelegh, C., Graham, F. L. & Prevec, L. (1999). Development and use of a 293 cell line expressing lac repressor for the rescue of recombinant adenoviruses expressing high levels of rabies virus glycoprotein. J Gen Virol 80, 345–353.[Abstract]
McAuliffe, J., Vogel, L., Roberts, A. & 8 other authors (2004). Replication of SARS coronavirus administered into the respiratory tract of African Green, rhesus and cynomolgus monkeys. Virology 330, 8–15.[CrossRef][Medline]
Navas-Martin, S. & Weiss, S. R. (2003). SARS: lessons learned from other coronaviruses. Viral Immunol 16, 461–474.[CrossRef][Medline]
Ng, P., Parks, R. J., Cummings, D. T., Evelegh, C. M. & Graham, F. L. (2000). An enhanced system for construction of adenoviral vectors by the two-plasmid rescue method. Hum Gene Ther 11, 693–699.[CrossRef][Medline]
NRC (1996). Guide for the Care and Use of Laboratory Animals. Washington, DC: National Research Council.
Olsen, C. W. (1993). A review of feline infectious peritonitis virus: molecular biology, immunopathogenesis, clinical aspects, and vaccination. Vet Microbiol 36, 1–37.[CrossRef][Medline]
Pang, H., Liu, Y., Han, X., Xu, Y., Jiang, F., Wu, D., Kong, X., Bartlam, M. & Rao, Z. (2004). Protective humoral responses to severe acute respiratory syndrome-associated coronavirus: implications for the design of an effective protein-based vaccine. J Gen Virol 85, 3109–3113.
Peiris, J. S. M., Yuen, K. Y., Osterhaus, A. D. M. E. & Stöhr, K. (2003). The severe acute respiratory syndrome. N Engl J Med 349, 2431–2441.
Pratelli, A., Tinelli, A., Decaro, N., Cirone, F., Elia, G., Roperto, S., Tempesta, M. & Buonavoglia, C. (2003). Efficacy of an inactivated canine coronavirus vaccine in pups. New Microbiol 26, 151–155.[Medline]
Qu, D., Zheng, B., Yao, X., Guan, Y., Yuan, Z.-H., Zhong, N.-S., Lu, L.-W., Xie, J.-P. & Wen, Y.-M. (2005). Intranasal immunization with inactivated SARS-CoV (SARS-associated coronavirus) induced local and serum antibodies in mice. Vaccine 23, 924–931.[CrossRef][Medline]
Reed, L. J. & Muench, H. (1938). A simple method of estimating fifty percent endpoints. Am J Hyg 27, 493–497.
Roberts, A., Vogel, L., Guarner, J., Hayes, N., Murphy, B., Zaki, S. & Subbarao, K. (2005). Severe acute respiratory syndrome coronavirus infection of golden Syrian hamsters. J Virol 79, 503–511.
Rota, P. A., Oberste, M. S., Monroe, S. S. & 32 other authors (2003). Characterization of a novel coronavirus associated with severe acute respiratory syndrome. Science 300, 1394–1399.
Saif, L. J. (2004). Animal coronavirus vaccines: lessons for SARS. Dev Biol (Basel) 119, 129–140.
Sanchez-Lopez, R., Nicholson, R., Gesnel, M. C., Matrisian, L. M. & Breathnach, R. (1988). Structure-function relationships in the collagenase family member transin. J Biol Chem 263, 11892–11899.
Schmidt, N. (1989). Cell culture procedures for diagnostic virology. In Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, 6th edn, pp. 51–100. Edited by N. Schmidt & R. Emmons. Washington, DC: American Public Health Association.
See, R. H., Roper, R. L., Brunham, R. C. & Finlay, B. B. (2005). Rapid response research – SARS coronavirus vaccines and application of processes to other emerging infectious diseases. Curr Immunol Rev 1, 185–200.
Seo, S. H., Wang, L., Smith, R. & Collisson, E. W. (1997). The carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic T lymphocytes and protects chickens from acute infection. J Virol 71, 7889–7894.[Abstract]
Smee, D. F., Huffman, J. H., Morrison, A. C., Barnard, D. L. & Sidwell, R. W. (2001). Cyclopentane neuraminidase inhibitors with potent in vitro anti-influenza virus activities. Antimicrob Agents Chemother 45, 743–748.
Stadler, K., Roberts, A., Becker, S. & 8 other authors (2005). SARS vaccine protective in mice. Emerg Infect Dis 11, 1312–1314.[Medline]
Stohlman, S. A., Kyuwa, S., Polo, J. M., Brady, D., Lai, M. M. C. & Bergmann, C. C. (1993). Characterization of mouse hepatitis virus-specific cytotoxic T cells derived from the central nervous system of mice infected with the JHM strain. J Virol 67, 7050–7059.
Stohlman, S. A., Bergmann, C. C., van der Veen, R. C. & Hinton, D. R. (1995). Mouse hepatitis virus-specific cytotoxic T lymphocytes protect from lethal infection without eliminating virus from the central nervous system. J Virol 69, 684–694.[Abstract]
Subbarao, K., McAuliffe, J., Vogel, L. & 7 other authors (2004). Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice. J Virol 78, 3572–3577.
Takamura, K., Matsumoto, Y. & Shimizu, Y. (2002). Field study of bovine coronavirus vaccine enriched with hemagglutinating antigen for winter dysentery in dairy cows. Can J Vet Res 66, 278–281.[Medline]
Takasuka, N., Fujii, H., Takahashi, Y. & 13 other authors (2004). A subcutaneously injected UV-inactivated SARS coronavirus vaccine elicits systemic humoral immunity in mice. Int Immunol 16, 1423–1430.
Tan, Y.-J., Goh, P.-Y., Fielding, B. C. & 9 other authors (2004). Profiles of antibody responses against severe acute respiratory syndrome coronavirus recombinant proteins and their potential use as diagnostic markers. Clin Diagn Lab Immunol 11, 362–371.
Tang, L., Zhu, Q., Qin, E. & 15 other authors (2004). Inactivated SARS-CoV vaccine prepared from whole virus induces a high level of neutralizing antibodies in BALB/c mice. DNA Cell Biol 23, 391–394.[CrossRef][Medline]
Wesseling, J. G., Godeke, G.-J., Schijns, V. E. C. J., Prevec, L., Graham, F. L., Horzinek, M. C. & Rottier, P. J. M. (1993). Mouse hepatitis virus spike and nucleocapsid proteins expressed by adenovirus vectors protect mice against a lethal infection. J Gen Virol 74, 2061–2069.
Wong, S. K., Li, W., Moore, M. J., Choe, H. & Farzan, M. (2004). A 193-amino acid fragment of the SARS coronavirus S protein efficiently binds angiotensin-converting enzyme 2. J Biol Chem 279, 3197–3201.
Woo, P. C. Y., Lau, S. K. P., Wong, B. H. L., Tsoi, H.-W., Fung, A. M. Y., Chan, K.-H., Tam, V. K. P., Peiris, J. S. M. & Yuen, K.-Y. (2004). Detection of specific antibodies to severe acute respiratory syndrome (SARS) coronavirus nucleocapsid protein for serodiagnosis of SARS coronavirus pneumonia. J Clin Microbiol 42, 2306–2309.
Xiao, X., Chakraborti, S., Dimitrov, A. S., Gramatikoff, K. & Dimitrov, D. S. (2003). The SARS-CoV S glycoprotein: expression and functional characterization. Biochem Biophys Res Commun 312, 1159–1164.[CrossRef][Medline]
Xiong, S., Wang, Y.-F., Zhang, M.-Y. & 11 other authors (2004). Immunogenicity of SARS inactivated vaccine in BALB/c mice. Immunol Lett 95, 139–143.[CrossRef][Medline]
Yang, Z.-Y., Kong, W.-P., Huang, Y., Roberts, A., Murphy, B. R., Subbarao, K. & Nabel, G. J. (2004). A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice. Nature 428, 561–564.[CrossRef][Medline]
Zakhartchouk, A. N., Liu, Q., Petric, M. & Babiuk, L. A. (2005a). Augmentation of immune responses to SARS coronavirus by a combination of DNA and whole killed virus vaccines. Vaccine 23, 4385–4391.[CrossRef][Medline]
Zakhartchouk, A. N., Viswanathan, S., Mahony, J. B., Gauldie, J. & Babiuk, L. A. (2005b). Severe acute respiratory syndrome coronavirus nucleocapsid protein expressed by an adenovirus vector is phosphorylated and immunogenic in mice. J Gen Virol 86, 211–215.
Zhu, M.-S., Pan, Y., Chen, H.-Q., Shen, Y., Wang, X.-C., Sun, Y.-J. & Tao, K.-H. (2004). Induction of SARS-nucleoprotein-specific immune response by use of DNA vaccine. Immunol Lett 92, 237–243.[CrossRef][Medline]
Received 2 October 2005;
accepted 16 November 2005.
This article has been cited by other articles:
|
|
 |
|
  F. Luo, Y. Feng, M. Liu, P. Li, Q. Pan, V. T. Jeza, B. Xia, J. Wu, and X.-L. Zhang Type IVB Pilus Operon Promoter Controlling Expression of the Severe Acute Respiratory Syndrome-Associated Coronavirus Nucleocapsid Gene in Salmonella enterica Serovar Typhi Elicits Full Immune Response by Intranasal Vaccination Clin. Vaccine Immunol., August 1, 2007; 14(8): 990 - 997. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Rockx, T. Sheahan, E. Donaldson, J. Harkema, A. Sims, M. Heise, R. Pickles, M. Cameron, D. Kelvin, and R. Baric Synthetic Reconstruction of Zoonotic and Early Human Severe Acute Respiratory Syndrome Coronavirus Isolates That Produce Fatal Disease in Aged Mice J. Virol., July 15, 2007; 81(14): 7410 - 7423. [Abstract] [Full Text] [PDF]
|
|
|
<
