key: cord-351649-87g7g5au
authors: Haagmans, Bart L.; Osterhaus, Albert D.M.E.
title: SARS
date: 2009-01-30
journal: Vaccines for Biodefense and Emerging and Neglected Diseases
DOI: 10.1016/b978-0-12-369408-9.00036-6
sha: 
doc_id: 351649
cord_uid: 87g7g5au

Abstract Five years after the first severe acute respiratory syndrome (SARS) outbreak, several candidate SARS-coronavirus (CoV) vaccines are at various stages of preclinical and clinical development. Based on the observation that SARSCoV infection is efficiently controlled upon passive transfer of antibodies directed against the spike (S) protein of SARS-CoV, vaccines containing the S protein have been formulated. Animals immunized with inactivated whole virus vaccines or live-recombinant vaccines expressing the SARS-CoV S protein (e.g., using rabies virus, vesicular stomatitis virus, bovine parainfluenza virus type 3, adenovirus, or attenuated vaccinia virus MVA as a vector), as well as mice immunized with DNA vaccines expressing the S protein gene all developed neutralizing antibodies to SARS-CoV and were protected against SARS-CoV challenge. Although much effort has been focused on developing a SARS vaccine, the commercial viability of such a vaccine for SARS-CoV will ultimately depend on whether the virus re-emerges in the near future. This vaccine should induce highly cross-reactive neutralizing antibodies to protect against newly emerging viruses related to SARS-CoV and protect both the gastrointestinal and respiratory tract in the absence of significant side effects. Given the fact that in the previous outbreak mainly the elderly succumbed to the infection, special attention should be given to vaccines that are able to efficiently protect aged individuals.

was successfully re-isolated from the nasal swabs and lung lesions of these animals, and a specific antibody response to the virus was shown in the infected animals, SARS-CoV proved to be the causative agent of this infectious disease Kuiken et al., 2003 ) .

SARS-CoV is a single-stranded, positive-sense RNA virus, phylogentically related to coronaviruses from group 2 despite the fact that it does not encode a hemagglutinin-esterase protein ( Snijder et al., 2003 ) . The genome is packaged together with the nucleocapsid protein, at least five membrane proteins (M, E, 3a, 7a, and 7b) and the spike (S) protein ( Fig. 36.1 ) . The S1 region within the S protein, and more specifically a 193-amino acid fragment of the S protein (corresponding to residues 318-510), has been identified as the region that interacts with the cell receptor, angiotensin-converting enzyme 2 ( Li et al., 2005a ) . The majority of neutralizing antibodies are directed against this region of the S protein. Antibodies raised

Severe acute respiratory syndrome coronavirus (SARS-CoV) first emerged in the human population in November 2002. Phylogenetic analysis of SARS-CoV isolates from animals indicated that this virus most probably originated from bats, was transmitted first to palm civets and subsequently to humans at the wet markets in southern China. Subsequent outbreaks occurred early 2003 in Hong Kong, Hanoi, Toronto, and Singapore, and could be directly traced back to one index patient who acquired the infection in Guangdong and traveled to Hong Kong. A worldwide epidemic was halted through the efforts of the World Health Organization, which responded rapidly to this threat by issuing a global alert, rigorous local containment efforts, warning against unnecessary travel to affected areas, and by creating a network of international experts to combat this virus. In the end only 8096 people became ill, and 774 people died in this first SARS epidemic. Because SARS-CoV could re-emerge and cause another epidemic at any time, development of effective vaccines remains of vital importance.

Although several infectious agents, including chlamydia, influenza A subtype H5N1, and human metapneumovirus, were considered as a possible cause of SARS, three groups independently reported the isolation of a previously unrecognized CoV from clinical specimens of SARS patients ( Peiris et al., 2003 ; Rota et al., 2003 ; Drosten et al., 2003 ) .

Through electron microscopy, serology, and reversetranscription PCR with consensus-and randomprimers, and subsequent sequencing of the replicase gene, its identity could be revealed and consistently demonstrated in clinical specimens from patients with the disease but not in healthy controls. To conclusively establish a causal role for this CoV, cynomolgous macaques were inoculated with a SARS-CoV isolate. Because the disease in macaques caused by SARS-CoV infection was pathologically similar to that seen in human patients with SARS, and since the virus should induce highly cross-reactive neutralizing antibodies to protect against newly emerging viruses related to SARS-CoV and protect both the gastrointestinal and respiratory tract in the absence of significant side effects. Given the fact that in the previous outbreak mainly the elderly succumbed to the infection, special attention should be given to vaccines that are able to efficiently protect aged individuals. protein also inhibit SARS-CoV replication in vitro but their relevance in protection remains unclear. The genome also encodes two large poly-proteins with diverse enzymatic activities needed for efficient replication and several accessory proteins with unknown function (3b, 6, 8a, 8b, and 9b).

At the end of 2004, 30 countries reported a total of 8096 probable cases of SARS ( Fig. 36.2 ) . Pathogenic SARS-CoVs do not circulate in the human population at the moment, but their re-emergence from animal reservoirs may likely occur in the future. Because many of the early SARS patients in Guandong had epidemiological links to the live-animal market trade, different animal species were tested for the presence of SARS-like viruses. Soon after the outbreak, a SARS-like coronavirus, which had more than 99% homology with human SARS-CoV, was detected by RT-PCR in the nasal and fecal swabs of palm civets ( Paguma larvata ) and a raccoon dog ( Nyctereutes procyonoides ) ( Guan et al., 2003 ) . More recent studies indicate that bats may potentially act as natural reservoirs for SARS-like CoVs ( Li et al., 2005b ; Lau et al., 2005 ) . However, sequence comparison of the S protein genes from bat SARS-like CoV and palm civet SARS-like CoV revealed only 64% genetic homology. Subsequent studies by Tang et al. (2006) have demonstrated that approximately 6% of bats sampled in China were positive for CoVs. Interestingly, these CoVs are genetically diverse and many bat CoVs clustered with existing group 1 viruses, while others formed a separate lineage that included only viruses from bats (putative group 5). Other SARS-CoV like viruses clustered in a putative group 4 consisting of two subgroups, one of bat CoVs and another of SARS-CoVs from humans and other mammalian hosts. However, from these studies the direct progenitor of the SARS-CoV isolated from palm civets has not been identified. Major genetic variations in the S protein gene of these viruses from civet cats, seemed essential for the transition from animal-to-human transmission EPIDEMIOLOGY FIGURE 36.2 Reported suspected SARS cases from November 1, 2002 to July 31, 2003 (data from the World Health Organization, http:// www.who.int/csr/sars/country/table2004_04_21/en/index.html ). Countries with Ͼ 5 suspected cases are indicated. patients. In fact, none of the SARS-CoV-infected children aged below 12 years in Hong Kong required intensive care or mechanical ventilation ( Ng et al., 2004 ) . This is not totally explained by comorbid factors but similar age dependence in mortality is seen in patients with other (nonviral) causes of acute respiratory stress syndrome ( Rubenfeld et al., 2005 ) . Second, virus transmission is low in the first days of illness and peaks around day 10 after disease onset ( Chu et al., 2004 ) . Finally, several studies revealed that high viral load in the nasopharyngeal aspirate was found to be an independent predictor of mortality ( Hung et al., 2004 ; Chu et al., 2004 ) . Therefore, vaccine strategies aimed at reducing the viral load may suffice to provide clinical benefit.

The first efforts to treat SARS patients were mainly based on the use of ribavirin and corticosteroids. Ribavirin, which targets IMP dehydrogenase, has been known a long time as a broad-spectrum antiviral agent. However, current data do not support the use of ribavirin for SARS treatment; in vitro studies did not show significant antiviral activity ( Cinatl et al., 2003 ) and ribavirin enhanced the infectivity of SARS-CoV in mice ( Barnard et al., 2006 ) . On the other hand, a protective effect of interferon (IFN)-α has been observed in a preliminary study during the SARS outbreak ( Loutfy et al., 2003 ) . These results are in concordance with several studies that noted antiviral activity in vitro ( Cinatl et al., 2003 ; Hensley et al., 2004 ) and animal studies showing that pegylated IFN-α effectively reduced SARS-CoV replication and excretion, viral antigen expression by type 1 pneumocytes and the pulmonary damage in cynomolgous macaques that were infected experimentally with SARS-CoV ( Haagmans et al., 2004 ) . However, despite an extensive literature reporting on SARS treatments, it is not possible to determine whether treatments benefited patients during the SARS outbreak. Because of variation in treatment regimens-particularly the wide range in doses, duration of therapy, and route of administration of ribavirin and corticosteroids, no clear conclusion can be drawn regarding the efficacy of the drugs tested ( Stockman et al., 2006 ) . In the event of a future outbreak of SARS-CoV or another novel agent, attempts should be made to develop treatment protocols, organize randomized trials and to collect and contribute information for a standardized minimum dataset that could facilitate analysis of treatment outcomes among different settings ( Stockman et al., 2006 ) . to human-to-human transmission, which eventually caused the SARS outbreak of 2002-2003.

There is at present no evidence for the virus persisting in the human population. Possible options for the re-emergence of SARS include the escape of the virus from laboratories, which has already occurred on three occasions. The re-emergence of the virus from its animal reservoir remains possible, given that the virus is detectable in the feces and respiratory secretions of some animals. Indeed, SARS-CoV re-emerged in four patients in Guangdong in December 2003, although these SARS-like CoVs caused milder clinical disease ( Liang et al., 2004 ) . The US National Institute of Allergy and Infectious Diseases Biodefense Network classified SARS-CoV as a category C priority pathogen pointing out that SARS-CoV could be a potential biothreat agent.

The clinical symptoms of SARS-CoV infection are those of lower respiratory tract disease and include fever, malaise, peripheral T cell lymphocytopenia, decreased platelet counts, prolonged coagulation profiles, and mildly elevated serum hepatic enzymes ( Peiris et al., 2004 ; Li et al., 2004 ) . Chest radiography reveals infiltrates with subpleural consolidation or " ground glass " changes compatible with viral pneumonitis. Around 20−30% of individuals with SARS require management in intensive care units and the overall case:fatality rate reached approximately 10%.

Although the main clinical symptoms are those of severe respiratory illness, SARS-CoV actually also causes a gastrointestinal and urinary tract infection; SARS-CoV can be detected in the feces and urine of patients, and electron microscopic studies of biopsies of the upper and lower intestinal mucosae of patients with SARS confirmed the presence of the virus in these tissues ( Peiris et al., 2004 ) . Fecal transmission proved to be important in at least one major community outbreak in Hong Kong (Amoy Gardens), in which over 300 patients were infected within a few days.

Three features of SARS may be relevant for intervention strategies. First, progressive age dependence in mortality and disease severity is observed in SARS

The major sources of transmission in humans are droplets that deposit on the respiratory epithelium. Unlike the situation in several other respiratory viral infections, viral load of SARS-CoV in the upper respiratory tract peaked around day 10 after disease onset ( Peiris et al., 2004 ) . Therefore, virus transmission may be less efficient in the first days of illness, a finding supported by epidemiological observations. Realtime PCR assays detect SARS-CoV during the first week in specimens of the lower respiratory tract (e.g., bronchoalveolar lavage, sputum, endotracheal aspirates), nasopharyngeal aspirate, throat swabs, and/or serum ( Chan et al., 2004 ) , whereas fecal samples may show very high viral loads toward the end of the first week and second week of illness. In typical cases, which were largely confined to adult and elderly individuals, SARS presented with acute respiratory distress syndrome, characterized by the presence of diffuse alveolar damage and multiorgan dysfunction upon autopsy ( Nicholls et al., 2003 ) . The pathological changes in lung alveoli most likely follow a common pathway characterized by an acute phase of proteinrich alveolar fluid influx into the alveolar lumina as a consequence of the injury to the alveolar wall. Subsequently type-2 pneumocyte hyperplasia takes place to replace the loss of infected type-1 pneumocytes and to cover the denuded epithelial basement membrane, resulting in restoration of the normal alveolar architecture. Severe alveolar injury may lead to fibrosis with loss of alveolar function in more protracted cases ( Fig. 36 .3 ).

It has been hypothesized that the pathological changes observed in the lungs are initiated by a disproportional innate immune response, illustrated by elevated levels of inflammatory cytokines and chemokines, such as CXCL10 (IP-10), CCL2 (MCP-1), IL-6, IL-8, IL-12, IL-1 β , and IFN-γ ( Huang et al., 2005 ; Wong et al., 2004 ) . These in vivo data have been confirmed in vitro, demonstrating that SARS-CoV infection induces a range of cytokines and chemokines in diverse cell types Law et al., 2005 ) . Although in vitro studies argued that production of type I IFNs is inhibited or delayed by SARS-CoV, in SARS-CoVinfected macaques, and also early during SARS in humans, type I IFNs can be readily demonstrated (De Lang et al., 2007) . Because prophylactic treatment of macaques with pegylated IFN-α reduces SARS-CoV replication in the lungs, regulation of the production of IFNs may be important in controlling SARS ( Haagmans et al., 2004 ) . 

Based on the observation that SARS-CoV infection is efficiently controlled upon passive transfer of antibodies directed against the S protein of SARS-CoV, a range of vaccines containing the S protein/gene has been developed. Animals immunized with inactivated whole virus vaccines or live-recombinant vaccines expressing the SARS-CoV S protein (e.g., using rabies virus, vesicular stomatitis virus, bovine parainfluenza virus type 3, adenovirus, or attenuated vaccinia virus MVA as a vector), as well as mice immunized with DNA vaccines expressing the S protein gene all developed neutralizing antibodies to SARS-CoV and were protected against SARS-CoV challenge. Table 36 .1 displays an overview of vaccines that have been tested for efficacy in animal models.

Inactivated SARS vaccines have been reported to elicit high titers of S protein-specific neutralizing antibodies. Few studies, however, have addressed whether inactivated whole SARS-CoV virions confer protection from virus challenge. Mice that were immunized twice with a candidate SARS-CoV vaccine, produced through a two-step inactivation procedure involving sequential formaldehyde and U.V. inactivation developed high-antibody titers against the SARS-CoV S protein and high levels of neutralizing antibodies ( Spruth et al., 2006 ) (see Chapter 11). Moreover, the vaccine conferred protective immunity as demonstrated by prevention of SARS-CoV replication in the respiratory tract of mice after intranasal challenge with SARS-CoV. Protection of mice was correlated to the antibody titer against the SARS-CoV S protein and neutralizing antibody titer. Similar results have been obtained using a beta-propiolactone inactivated SARS-CoV vaccine in mice . In addition, two Chinese groups have demonstrated protective efficacy of inactivated SARS vaccines in rhesus monkeys ( Qin et al., 2006 ; Zhou et al., 2005 ) . A soluble recombinant polypeptide containing the N-terminal segment of the S glycoprotein may suffice to induce neutralizing antibodies and protective immunity in mice ( Bisht et al., 2005 ) . In addition, a trimeric recombinant S protein was able to elicit an efficacious protective immune response in hamsters ( Kam et al., 2007 ) .

One of the most promising vaccine candidates is based on the combination of recombinant S protein with the Protollin adjuvant. In both young and aged mice, an intranasal Protollin-formulated S protein

Seroconversion usually occurs in weeks 2 or 3 of illness and virus-neutralizing antibodies can be detected in convalescent human serum. In patients who had recovered from SARS-neutralizing antibody titers peaked at month 4 but were undetectable in 16% of patients at month 36 ( Cao et al., 2007 ) . Neutralizing antibodies may be directed against different regions of the S protein (S1 and S2) as monoclonal antibodies against these epitopes exert potent neutralization of SARS-CoV in vitro ( Sui et al., 2004 ; Lip et al., 2006 ) . Conversely, peptides which are located in these regions were able to induce neutralizing antibodies ( Bisht et al., 2005 ; Keng et al., 2005 ; Zhang et al., 2004 ) .

Although experiments in diverse animal models have revealed that relatively low levels of neutralizing antibodies exert potent protection against lower respiratory tract infection, the neutralizing antibody titer necessary to achieve protection in humans exposed to SARS-CoV is not known. A concern in case of reemergence of SARS is the possible absence of cross protection against these viruses. However, recent studies by He et al. (2006) have shown that the some neutralizing epitopes of SARS-CoV have been maintained during cross-species transmission, suggesting that receptor binding domain-based vaccines may induce broad protection against both human and animal SARS-CoV variants.

In most SARS autopsies, extensive necrosis of the spleen and atrophy of the white pulp with severe lymphocyte depletion have been observed. On the other hand, rapid phase in peripheral lymphocyte recovery usually coincided with improved clinical conditions of SARS patients (Peiris et al., 2004) . Long-lived memory T cell responses against SARS-CoV nucleocapsid and S protein have been demonstrated in recovered SARS patients, although their relevance in antiviral protection is not well understood Peng et al., 2006 ; Li et al., 2006 ) . However, despite potent immune responses and clinical recovery, peripheral lymphocyte counts in the recovered patients were not restored to normal levels .

Interestingly, mice that lack NK-T cells, or NK cells, or T and B cells all cleared the virus by day 9 after infection ( Glass et al., 2004 ) . These data argue that cell-mediated immune responses are not essential to control virus clearance. vaccine elicited high levels of antigen-specific IgG in serum and significant levels of antigen-specific lung IgA ( Hu et al., 2007 ) . In contrast, mice immunized intramuscularly with Alum absorbed S protein did not develop detectable IgA responses. Following virus challenge of the aged mice, no virus was detected in the lungs of mice vaccinated intranasally, whereas intramuscularly immunized mice did not show significant control of virus replication compared to controls ( Hu et al., 2007 ) .

A DNA vaccine encoding the S glycoprotein of the SARS-CoV induces T cell, neutralizing antibody responses, and protective immunity in a mouse model . These authors also demonstrated that antibody responses in mice vaccinated with an expression vector encoding a form of S protein that includes its transmembrane domain elicited neutralizing antibodies. Viral replication was reduced by more than six orders of magnitude in the lungs of mice vaccinated with these S protein plasmid DNA expression vectors, and protection was mediated by a humoral, but not a T-cell-dependent, immune mechanism. Subsequent studies using a prime-boost combination of DNA and whole killed SARS-CoV vaccines elicited higher antibody responses than DNA or whole killed virus vaccines alone . Apart from this study, several other groups have analyzed the immunogenicity of SARS DNA vaccines but none of these challenged the vaccinated animals with SARS-CoV.

Adenovirus-vector based vaccination strategies against SARS-CoV were employed early on after the SARS outbreak to demonstrate that vaccinated rhesus macaques developed virus-neutralizing antibody responses against fragment S1 of the S protein and T cell responses against the nucleocapsid ( Gao et al., 2003 ) . More recently, See et al. (2006) demonstrated that vaccination of C57B/L6 mice with adenovirus type 5-expressing S and nucleocapsid administered intranasally, but not intramuscularly, significantly limited SARS-CoV replication in the lungs. VACCINES reduction in pulmonary SARS-CoV titer compared with control animals ( DiNapoli et al., 2007 ) . Finally, Venezuelan equine encephalitis virus based vaccines have been tested extensively in young and aged mice. Most importantly, different recombinant SARS-CoV bearing epidemic and zoonotic S protein variants were used to challenge the vaccinated mice. Venezuelan equine encephalitis virus replicon particles expressing the 2003 epidemic Urbani SARS-CoV strain S glycoprotein but not particles containing the nucleocapsid protein from the same strain provided complete short-and long-term protection against homologous strain challenge in young and senescent mice ( Deming et al., 2006 ) . Although the S protein encoding vaccine provided complete short-term protection against heterologous (strain GD03) challenge in young mice, only limited protection was seen in vaccinated senescent animals. Interestingly, nucleocapsid-encoding vaccines not only failed to protect from homologous or heterologous challenge but also resulted in enhanced immunopathology with eosinophilic infiltrates within the lungs of SARS-CoVchallenged mice ( Deming et al., 2006 ) .

A new generation of vaccines may be obtained from manipulating the full-length infectious cDNA clone of SARS-CoV. One approach would be to delete the ORFs 3a, 3b, 6, 7a, 7b, 8a, 8b, or 9b similar to other coronavirus mutants generated previously; some mouse hepatitis or feline infectious peritonitis deletion viruses replicate to the same extent as wild-type viruses in vitro but are severely attenuated in vivo making them potential vaccine candidates. However, SARS-CoV deletion mutants lacking ORFs 3a, 3b, 6, 7a, or 7b, grew similar to that of the parental wild-type virus in the mouse model ( Yount et al., 2005 ) . On the other hand, a recombinant SARS-CoV that lacks the E gene was attenuated both in vitro and in vivo . Viable recombinant virus with the E gene deleted was recovered in Vero cells with a titer around 10 6 pfu/ml but titers in the respiratory tract of hamsters were 100-1000-fold reduced compared to wild-type SARS-CoV replication, suggesting that this mutant is attenuated. Multiplication of these viruses in packaging cell lines would provide the missing protein in trans and would make a promising SARS-CoV vaccine candidate that has the E gene deleted.

Live attenuated virus vaccines may revert to wildtype and recombine with other circulating human or The highly attenuated modified vaccinia virus Ankara (MVA) has been used to express the S glycoprotein of SARS-CoV in vaccination experiments using mouse, ferret, and rhesus monkey models ( Bisht et al., 2004 ; Chen et al., 2005 ; Weingartl et al., 2004 ) . Intranasal and intramuscular administration of MVA encoding the SARS-CoV S protein led to the induction of a humoral immune response in BALB/c mice, as well as reduced viral titers in the respiratory tract ( Bisht et al., 2005 ) .

Recombinant bovine-human parainfluenza virus type 3 vector (BHPIV3) is being developed as a live attenuated, intranasal pediatric vaccine against human parainfluenza virus type 3. Immunization of African green monkeys with a single dose of BHPIV3 expressing SARS-CoV S protein administered via the respiratory tract induced the production of SARS-CoV neutralizing antibodies . A recombinant BHPIV3 expressing SARS-CoV structural protein (S, M, and N) individually or in combination has been evaluated for immunogenicity and protective efficacy in hamsters . In the absence of S protein, expression of M, N, or E did not induce a detectable serum SARS-CoV-neutralizing antibody response and no protection against SARS-CoV challenge in the respiratory tract, whereas the vectors expressing the S protein induced neutralizing antibody responses and protection.

Recombinant rabies virus expressing the S protein of SARS-CoV induced a neutralizing antibody response in mice ( Faber et al., 2005 ) . Similarly, an attenuated vesicular stomatitis virus vector that encodes the SARS-CoV S protein may be used to induce neutralizing antibody responses ( Kapadia et al., 2005 ) . Mice vaccinated with recombinant vesicular stomatitis virus expressing S protein developed SARS-CoV-neutralizing antibody and were able to control a challenge with SARS-CoV performed at either 1 month or 4 months after a single vaccination. In addition, by passive antibody transfer experiments these authors demonstrated that the antibody response induced by the vaccine was sufficient to control SARS-CoV infection.

The efficacy of these vectors was further demonstrated in studies using aged mice. In aged mice, vaccinated with recombinant vesicular stomatitis virus expressing S protein, antibody titers induced were sufficient to protect them against subsequent challenge with SARS-CoV ( Vogel et al., 2007 ) .

African green monkeys immunized via the respiratory tract with two doses of a recombinant Newcastle disease virus encoding the S protein developed a relatively high titer of SARS-CoV neutralizing antibodies and upon challenge demonstrated a 1000-fold zoonotic coronaviruses. In order to prevent this, it has been proposed to delete an essential gene, located in a position distant from gene E, and the relocation of the deleted gene to the position previously occupied by gene E ( Enjuanes et al., 2008 ) . A potential recombination leading to the rescue of gene E would lead to the loss of the essential gene. Alternatively, the transcriptional regulatory sequences (TRS) of a vaccine virus could be genetically manipulated to a sequence incompatible with the TRS of any known circulating coronavirus as described by Yount et al. (2006) . This virus could be further modified by building attenuating mutations on the genetic backbone of the recombination resistant TRS rewired virus either for use as a safe high titer seed stock for making killed vaccines or as a live virus vaccine. One such attenuating mutation could be targeted to the nonstructural protein 1. Recombinant Mouse Hepatitis Virus (MHV) encoding a deletion in the nsp1-coding sequence grew normally in tissue culture but was severely attenuated in vivo ( Züst et al., 2007 ) . Low doses of nsp1 mutant MHV elicited potent cytotoxic T cell responses and protected mice against homologous and heterologous virus challenge. This attenuation strategy provides a new paradigm for the development of highly efficient coronavirus vaccines.

Although several types of vectored vaccines have been developed, several companies favored the classical approach using inactivated whole virus to develop a vaccine to be used for preclinical testing (see Chapter 11). Methods in place for the production of available vaccines could be easily used using wellestablished technologies. In the preclinical development stage, it is preferable that different animal species are used to evaluate the safety and efficacy of candidate vaccines.

Overall, a wide range of animal species, including rodents (mice and hamsters), carnivores (ferrets and cats), and nonhuman primates (cynomolgus and rhesus macaques, common marmosets, and African green monkeys) can be experimentally infected with SARS-CoV Roberts et al., 2005b ; Martina et al., 2003 ; Kuiken et al., 2003 ; McAuliffe et al., 2004 ; Haagmans and Osterhaus, 2006 ) . Most species show no clinical signs of disease, although the virus replicates efficiently in respiratory tissues. Aged mice and ferrets on the other hand, show signs of clinical disease, albeit in the absence of the typical lung lesions seen in humans with SARS ( Roberts et al., 2005a ) . In contrast, inoculation of SARS-CoV in the respiratory tract of cynomolgus macaques causes infection of bronchial epithelial cells and type-1 pneumocytes 1-4 days postinfection, followed by extensive type-2 pneumocyte hyperplasia in the lungs at 4-6 days postinfection Haagmans and Osterhaus, 2006 ) . The lesions, consisting of multiple foci of acute diffuse alveolar damage and characterized by flooding of alveoli with protein-rich edema fluid mixed with variable numbers of neutrophils, are quite similar to those observed in humans in the acute stages of SARS.

Remarkably, vaccine candidates tested in ferrets showed reduced efficacy. Vaccination with MVA encoding the S protein induced only moderate antibody responses and consequently did not protect against intranasal SARS-CoV infection and even resulted in an inflammatory response in the livers of the vaccinated ferrets (Weingartl et al., 2004) . Whether these aberrant responses resulted from immunopathological mechanisms, like antibody-dependent enhancement of infection, or represented recall responses to viral antigen in the liver is not clear at the moment but deserves further investigation. In addition, limited protection from SARS-CoV challenge was observed in ferrets vaccinated with inactivated whole virus ( Darnell et al., 2007 ) . Two out of four ferrets showed little or no neutralizing antibody even after the second immunization and none of the vaccinated ferrets were able to reduce virus excretion at day 2 after challenge but subsequently cleared the virus more rapidly compared to control animals. Adenovirus-based vaccines tested in ferrets seemed more powerful as they protected the lower respiratory tract efficiently but had less effect on virus excretion in the upper respiratory tract ( Kobinger et al., 2007 ) .

Five years after the first SARS outbreak a range of candidate vaccines have been developed. Early in 2006 some companies in China and the US initiated phase 1 trials. The first clinical trial has been initiated by a Chinese company, Sinovac Biotech of Beijing in collaboration with the Chinese academy of Medical Sciences using an inactivated whole virus vaccine. To evaluate the safety and immunogenicity of this vaccine, 36 subjects received two doses of vaccine or placebo control. On day 42, all individuals showed seroconversion and peak titers of neutralizing antibodies were reached 2 weeks after the second vaccination followed CLINICAL TRIALS III. VIRUS VACCINES with a type I coronavirus, feline infectious peritonitis virus (FIPV), when subsequently exposed to FIPV infection ( Weiss and Scott, 1981 ) . Macrophages are able to take up feline coronavirus-antibody complexes more efficiently causing the virus to replicate to higher titers. Interestingly, one study also demonstrated that antibodies against human SARS-CoV isolates enhance entry of pseudo-typed viruses expressing the civet cat SARS-like CoV S protein into cells, but not replication ( Yang et al., 2005 ) . To date, there is no evidence for enhanced replication following SARS-CoV challenge in previously immunized animals.

One other problem which may arise after vaccination with whole inactivated virus when absorbed with certain adjuvants such as alum, could relate to the induction of skewed Th2 recall responses similar to what has been observed in children vaccinated with inactivated respiratory syncytial and measles virus vaccines.

Although much effort has been focused on developing a SARS vaccine, the commercial viability of developing a vaccine for SARS-CoV will ultimately depend on whether the virus re-emerges in the near future. It is questionable whether possible future outbreaks will cause major outbreaks but vaccines, antivirals, or passive immunization would be relevant in the context of protecting high-risk individuals such as laboratory and health-care workers.

• Five years after the first SARS outbreak, several candidate SARS-CoV vaccines are at various stages of preclinical and clinical development.

• Based on the observation that SARS-CoV infection is efficiently controlled upon passive transfer of antibodies directed against the S protein of SARS-CoV, vaccines encoding this protein have been developed.

• Animals immunized with diverse vaccines containing the S protein or S protein gene developed SARS-CoV-neutralizing antibodies and were protected against SARS-CoV challenge.

• Future challenges for the development of SARS-CoV vaccines are linked to the potential re-emergence of this virus. Vaccines able to induce highly cross-reactive antibodies which efficiently protect the gastrointestinal and respiratory tract are needed. Given the fact that in the previous outbreak mainly elderly humans succumbed to the infection, special attention should be given to protect specifically these individuals. by a significant decline 4 weeks later ( Lin et al., 2007 ) . Several other candidate SARS vaccines are at various stages of preclinical and clinical development.

In SARS patients who recover, high levels of neutralizing antibody responses are observed, suggesting that antibody responses play a role in determining the ultimate disease outcome of SARS-CoV-infected patients . Although attempts have been made to test the efficacy of serum preparations from seroconvalescent SARS patients in the acute phase of SARS, no conclusive evidence has been obtained regarding their efficacy. In mice, on the other hand, SARS-CoV infection is efficiently controlled upon passive transfer of convalescent immunoglobulines . The concept that antibodies protect against SARS has been further explored through the generation of human monoclonal antibodies against SARS-CoV. Prophylactic administration of a human monoclonal antibody reduced replication of SARS-CoV in the lungs of infected ferrets by 1000-fold, completely prevented the development of SARS-CoV-induced macroscopic lung pathology, and abolished shedding of virus in pharyngeal secretions ( Ter Meulen et al., 2004 ) . In subsequent studies, several other monoclonal antibodies were evaluated for their efficacy in mouse and hamster models ( Sui et al., 2005 ; Traggiai et al., 2004 ) .

The importance of assessing immunogenicity of candidate SARS-CoV vaccines using virus neutralization assays is well acknowledged, but the variety of these tests in use is a significant problem since there is at this time no consensus on the most sensitive, specific, and reproducible assay system. To compare data from each of the candidate vaccines requires international standardization of the immunological assays and the availability of an antibody standard used for the evaluation of these vaccines. To test cross reactivity of antibodies generated by vaccination, murine leukemia virus was used to generate infectious particles containing different S proteins ( Giroglou et al., 2004 ) .

Enhanced disease and mortality have been observed in kittens immunized against or infected

Enhancement of the infectivity of SARS-CoV in BALB/c mice by IMP dehydrogenase inhibitors, including ribavirin

Severe acute respiratory syndrome coronavirus spike protein expressed by attenuated vaccinia virus protectively immunizes mice

Neutralizing antibody and protective immunity to SARS coronavirus infection of mice induced by a soluble recombinant polypeptide containing an N-terminal segment of the spike glycoprotein

Contributions of the structural proteins of severe acute respiratory syndrome coronavirus to protective immunity

Mucosal immunisation of African green monkeys ( Cercopithecus aethiops ) with an attenuated parainfluenza virus expressing the SARS coronavirus spike protein for the prevention of SARS

Disappearance of antibodies to SARS-associated coronavirus after recovery

Detection of SARS coronavirus in patients with suspected SARS

Nucleocapsid protein as early diagnostic marker for SARS

Recombinant modified vaccinia virus Ankara expressing the spike glycoprotein of severe acute respiratory syndrome coronavirus induces protective neutralizing antibodies primarily targeting the receptor binding region

Cytokine responses in severe acute respiratory syndrome coronavirus-infected macrophages in vitro: possible relevance to pathogenesis

Initial viral load and the outcomes of SARS

Treatment of SARS with human interferons

Severe acute respiratory syndrome coronavirus infection in vaccinated ferrets

A severe acute respiratory syndrome coronavirus that lacks the E gene is attenuated in vitro and in vivo

Functional genomics highlights differential induction of antiviral pathways in the lungs of SARS-CoV-infected macaques

Vaccine efficacy in senescent mice challenged with recombinant SARS-CoV bearing epidemic and zoonotic spike variants

Newcastle disease virus, a host range-restricted virus, as a vaccine vector for intranasal immunization against emerging pathogens

Identification of a novel coronavirus in patients with severe acute respiratory syndrome

Vaccines to prevent severe acute respiratory syndrome coronavirus-induced disease

A single immunization with a rhabdovirus-based vector expressing severe acute respiratory syndrome coronavirus (SARS-CoV) S protein results in the production of high levels of SARS-CoV-neutralizing antibodies

Aetiology: Koch's postulates fulfilled for SARS virus

Effects of a SARS-associated coronavirus vaccine in monkeys

Retroviral vectors pseudotyped with severe acute respiratory syndrome coronavirus S protein

Mechanisms of host defense following severe acute respiratory syndromecoronavirus (SARS-CoV) pulmonary infection of mice

Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China

Pegylated interferon-alpha protects type 1 pneumocytes against SARS coronavirus infection in macaques

Nonhuman primate models for SARS

Cross-neutralization of human and palm civet severe acute respiratory syndrome coronaviruses by antibodies targeting the receptor-binding domain of spike protein

Interferon-beta 1a and SARS coronavirus replication

Intranasal Protollinformulated recombinant SARS S-protein elicits respiratory and serum neutralizing antibodies and protection in mice

An interferon gammarelated cytokine storm in SARS patients

Viral loads in clinical specimens and SARS manifestations

Antibodies against trimeric S glycoprotein protect hamsters against SARS-CoV challenge despite their capacity to mediate FcgammaRII-dependent entry into B cells in vitro

Long-term protection from SARS coronavirus infection conferred by a single immunization with an attenuated VSV-based vaccine

Amino acids 1055 to 1192 in the S2 region of severe acute respiratory syndrome coronavirus S protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents

Adenovirus-based vaccine prevents pneumonia in ferrets challenged with the SARS coronavirus and stimulates robust immune responses in macaques

Aged BALB/c mice as a model for increased severity of severe acute respiratory syndrome in elderly humans

Severe acute respiratory syndrome coronavirus infection of golden Syrian hamsters

Characterization of a novel coronavirus associated with severe acute respiratory syndrome

Incidence and outcomes of acute lung injury

Comparative evaluation of two severe acute respiratory syndrome (SARS) vaccine candidates in mice challenged with SARS coronavirus

Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage

A doubleinactivated whole virus candidate SARS coronavirus vaccine stimulates neutralising and protective antibody responses

SARS vaccine protective in mice

SARS: systematic review of treatment effects

Prior infection and passive transfer of neutralizing antibody prevent replication of severe acute respiratory syndrome coronavirus in the respiratory tract of mice

Potent neutralization of severe acute respiratory syndrome (SARS) coronavirus by a human mAb to S1 protein that blocks receptor association

Evaluation of human monoclonal antibody 80R for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants

Prevelance and genetic diversity of coronaviruses in bats from China

Human monoclonal antibody as prophylaxis for SARS coronavirus infection in ferrets

An efficient method to make human monoclonal antibodies from memory B cells: potent neutralization of SARS coronavirus

Utility of the aged BALB/c mouse model to demonstrate prevention and control strategies for severe acute respiratory syndrome coronavirus (SARS-CoV)

Immunization with modified vaccinia virus Ankara-based recombinant vaccine against severe acute respiratory syndrome is associated with enhanced hepatitis in ferrets

Antibody-mediated enhancement of disease in feline infectious peritonitis: comparisons with dengue hemorrhagic fever

Plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome

Modulation of the immune response to the severe acute respiratory syndrome spike glycoprotein by gene-based and inactivated virus immunization

Newly discovered coronavirus as the primary cause of severe acute respiratory syndrome

Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats

Chemokine up-regulation in SARS-coronavirus-infected, monocyte-derived human dendritic cells

Structure of SARS coronavirus spike receptor-binding domain complexed with receptor

Significant changes of peripheral T lymphocyte subsets in patients with severe acute respiratory syndrome

Long-term persistence of robust antibody and cytotoxic T cell responses in recovered patients infected with SARS coronavirus

Bats are natural reservoirs of SARS-like coronaviruses

Laboratory diagnosis of four recent sporadic cases of community-acquired SARS, Guangdong province

Safety and immunogenicity from a phase I trial of inactivated severe acute respiratory syndrome coronavirus vaccine

Monoclonal antibodies targeting the HR2 domain and the region immediately upstream of the HR2 of the S protein neutralize in vitro infection of severe acute respiratory syndrome coronavirus

Natural mutations in the receptor binding domain of spike glycoprotein determine the reactivity of cross-neutralization between palm civet coronavirus and severe acute respiratory syndrome coronavirus

Interferon alfacon-1 plus corticosteroids in severe acute respiratory syndrome: a preliminary study

Virology: SARS virus infection of cats and ferrets

Replication of SARS coronavirus administered into the respiratory tract of African Green, rhesus and cynomolgus monkeys

SARS in newborns and children

Lung pathology of fatal severe acute respiratory syndrome

Severe acute respiratory syndrome

Coronavirus as a possible cause of severe acute respiratory syndrome

Long-lived memory T lymphocyte responses against SARS coronavirus nucleocapsid protein in SARS-recovered patients

Immunogenicity and protective efficacy in monkeys of purified inactivated Vero-cell SARS vaccine

Long-lived effector/central memory T-cell responses to severe acute respiratory syndrome coronavirus (SARS-CoV) S antigen in recovered SARS patients

A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice

Evasion of antibody neutralization in emerging severe acute respiratory syndrome coronaviruses

Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: engineering a recombination-resistant genome

Severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice

Identification of an antigenic determinant on the S2 domain of the severe acute respiratory syndrome coronavirus spike glycoprotein capable of inducing neutralizing antibodies

Antibody responses against SARS coronavirus are correlated with disease outcome of infected individuals

Immunogenicity, safety, and protective efficacy of an inactivated SARS-associated coronavirus vaccine in rhesus monkeys

Prognostic factors for severe acute respiratory syndrome: a clinical analysis of 165 cases

Coronavirus nonstructural protein 1 is a major pathogenicity factor: implications for the rational design of coronavirus vaccines