Vaccines to prevent severe acute respiratory syndromecoronavirus-induced disease
Luis Enjuanes a,∗, Marta L. DeDiego a, Enrique Alvarez a,Damon Deming b, Tim Sheahan b, Ralph Baric b,∗∗
a Centro Nacional de Biotecnologıa (CNB), CSIC, Campus Universidad Autonoma, Cantoblanco, Darwin 3, 28049 Madrid, Spainb Department of Microbiology and Immunology, University of North Carolina at Chapel Hill,
802 Mary Ellen Jones Building, Chapel Hill, NC 27599-7290, USA
Available online 9 April 2007
bstract
An important effort has been performed after the emergence of severe acute respiratory syndrome (SARS) epidemic in 2003 to diagnose andrevent virus spreading. Several types of vaccines have been developed including inactivated viruses, subunit vaccines, virus-like particles (VLPs),NA vaccines, heterologous expression systems, and vaccines derived from SARS-CoV genome by reverse genetics. This review describes several
spects essential to develop SARS-CoV vaccines, such as the correlates of protection, virus serotypes, vaccination side effects, and bio-safeguardshat can be engineered into recombinant vaccine approaches based on the SARS-CoV genome. The production of effective and safe vaccines to
revent SARS has led to the development of promising vaccine candidates, in contrast to the design of vaccines for other coronaviruses, that ineneral has been less successful. After preclinical trials in animal models, efficacy and safety evaluation of the most promising vaccine candidatesescribed has to be performed in humans.
Severe acute respiratory syndrome (SARS) is an infec-ious disease caused by a coronavirus (SARS-CoV) (Marrat al., 2003; Rota et al., 2003). SARS was detected for therst time in the Guandong province of China in late 2002,pread rapidly around the World and resulted in more than000 cases, 10% of which resulted in death, in 33 countries,nd areas of five continents (http://www.cdc.gov/mmwr/mguidears.en.html). World Healh organization (WHO) declared thend of the SARS outbreak in July 5th 2003, nevertheless, severalsolated outbreaks subsequently occurred because of accidentalontaminations in laboratories of Taiwan, Singapore, and main-and China (http://www.who.int/csr/sars/en/). In late 2003 and
arly 2004, newly infected persons who had contact with animalsnfected with SARS-CoV strains significantly different fromhose predominating in the 2002–2003 outbreak were reported
n Guandong, China (Peiris et al., 2004). These events indi-ate that a SARS epidemic may recur at any time in the future,lthough this event most likely would require the generation ofew isolates evolving from SARS-CoV-like virus circulating innimals (Jiang et al., 2005). As a consequence of this possibility,nd because SARS-CoV could be used as a biological weapon,t has been declared as a category C priority pathogen by theational Institute of Allergy and Infectious Diseases Biodefense
http://www2.niaid.nih.gov/Biodefense/bandc priority.htm).SARS-CoV infection results in severe acute respiratory dis-
ase, pneumonia, diarrhea, and sometimes death (Peiris et al.,003). SARS-CoV is a zoonotic virus that crossed the speciesarrier, most likely originating from bats, and has been amplifiedn other species, preferentially civets (Lau et al., 2005; Li et al.,005; Woo et al., 2006). The SARS-CoV like viruses that existn animals do not cause typical SARS-like disease in the natu-al host and are not easily transmitted from animals to humans.nder certain conditions, the virus may have evolved into the
arly human SARS-CoV, with the ability to be transmitted fromnimals to humans and from humans to humans, resulting inocalized outbreaks and mild human disease (Jiang et al., 2005).n fact, the virus most likely crossed the species barrier before
he 2002 outbreak, as sera samples collected in 2001 were pos-tive in 1.8% of the cases (Zheng et al., 2004). Furthermore,0% of animal traders, whereas only a 5% of vegetable tradersn Guandong markets were seropositive for SARS-CoV withouthowing signs of disease (Guan et al., 2003).
SARS-CoV vaccines are urgently needed to prevent poten-ial SARS epidemics. As SARS-CoV disease severity has beeninked to age, with higher mortalities for ages over 45, spe-ial attention should be paid to vaccine development to protectlderly people. Several types of vaccines are being developed,ncluding inactivated viruses, subunit vaccines, virus-like parti-les (VLPs), DNA vaccines, heterologous expression systems,nd vaccines derived from SARS-CoV genome by reverseenetics. This paper will review previously published SARSaccination strategies and announce new accomplishments inARS-CoV based vaccination approaches while focusing on
he correlates of protection, detected serotypes, vaccination sideffects, and bio-safeguards that can be engineered into recom-inant vaccine approaches based on the SARS-CoV genome.
. Complexity of SARS-CoV serotypes
Knowledge on the diversity of serotypes is essential infor-ation for vaccine design. Phylogenetic analysis of SARS-CoV
solates from animals and humans strongly suggest that the virus
riginated from animals, most likely bats (Lau et al., 2005; Li etl., 2005; Poon et al., 2005), was amplified in palm civets, andransmitted to human population via live animal markets (Kant al., 2005).
ig. 1. Phylogenetic analysis of human, bat, and civet cat-racoon dog virus spike sivided into four groups. Group 1 includes viruses isolated from animals in southern C*) in 2003. Group 3 includes viruses from all three phases of the human SARS epide005–06. A multiple sequence alignment of the spike gene of each virus was createdith Mr. Bayes, with Markov chain Monte Carlo sampling of four chains for 500,00ith a burn in of 1000. Branch confidence values are shown as posterior probabilities
nd GZ0401) may represent infections where a human acquired the virus from animaonger line in the tree (∼10 times longer), thus the distance of the line is not represen
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The neutralization of a set of eight pseudoviruses expressinghe spike glycoprotein of eight SARS-CoV strains selected fromhe three phases of the SARS epidemic (early, middle and late),lus another human isolate collected at the end of 2003 (GD03),nd two civet cat isolates from 2003 (SZ16 and SZ3) has beentudied (Chinese-Consortium, 2004; Yang et al., 2005). Humanonoclonal antibodies against the S protein of the Frankfurt
solate (FRA-1), derived from Epstein-Barr virus transformedlymphocytes were used. The virus tested in the neutralization
ssays included pseudotypes made with the S protein from mem-ers of the four main genetic clusters defined with the Bayesiannalysis of the SARS-CoV glycoproteins (Fig. 1A) (Deming etl., 2006):
(i) Group 1, originating from animals isolated in 2003. A pro-totype of this group is the isolate SZ16 which primarilyuses civet but not human angiotensin-converting enzyme 2(ACE2) as a receptor.
(ii) Group 2, low pathogenic viruses originating from civets,raccoon dogs, or sporadic human cases, such as strainGD03 reported from a sporadic SARS case in December22, 2003. This virus represented an independent introduc-tion of a less pathogenic virus, having an S glycoproteinsequence that is the most divergent of all human strains(Chinese-Consortium, 2004). In general, group 2 isolates
appear to have a receptor binding domain (RBD) that iscapable of recognizing the human ACE2 receptor, and havebeen successfully cultured. GD03 S glycoprotein contains18 amino acid substitutions relative to group 3 Urbani S
equences. An unrooted Bayesian phylogenetic gene tree of 24 SARS viruseshina in 2003. Group 2 is a cluster of viruses isolated from animals and humans
mic of 2002–2003. Group 4 represents a cluster of viruses isolated from bats inusing ClustalX 1.83 with default settings. Bayesian inference was conducted
0 generations, and a consensus tree was generated using the 50% majority rule. The three human isolates that fall within the animal cluster (GZ0402, GD03,ls. The dashed line between Group 3 and Group 4 is used to represent a muchtative of the distance between bat and human SARS.
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hineutralizing epitopes are located within the spike protein of thevirus (Buchholz et al., 2004; Hogan et al., 2004). Two domainsare defined in the spike protein, the amino-terminus (S1) andthe carboxy-terminus (S2) halves. In some coronaviruses, but
L. Enjuanes et al. / Virus
protein, many of which map within neutralizing epitopesbetween amino acids 130–150 and 318–510, correspond-ing to the RBD. Recombinant viruses encoding the GD03S glycoprotein have been isolated using reverse genetics(Baric et al., 2006). Recombinant icGD03 virus replicatesabout 0.5–1.0 logs less efficiently in human airway epithe-lial cells (6.8 × 107 PFU/ml) as compared to wt Urbani(3.0 × 107 PFU/ml) at 42 h postinfection.
iii) Group 3, highly pathogenic viruses representing the2002–2003 epidemic strains associated with the early, mid-dle, or late phase. Prototypes of these viruses are the earlyisolate and middle isolates GZ02, and CUHK-W, respec-tively, and the late Urbani, FRA-1, or Toronto 2 (TOR-2)strains.
iv) Group 4, bat SARS-CoV strains have not been success-fully cultured but were sequenced from samples taken fromRhinolophus spp. like the Chinese horseshoe bat. Theseviruses differ from Urbani by about 12–22% in amino acidsequence and generally have about 3–4 out of 13 contactinterface residues with human ACE2 receptor. Using theS glycoprotein gene, an unrooted Bayesian analysis sug-gests that bat strains are most closely related to early phasehuman strains.
Basically, all virus pseudotyped with S proteins from dif-erent strains were neutralized to the same extent, except theuman GD03 and the two civet cat isolates SZ16 and SZ3, indi-ating that there were at least two human SARS-CoV serotypes,ost likely originated from two independent transmissions of
he virus from civet cat to human (Baric et al., 2006; Yangt al., 2005). It will be important to determine if recombi-ant viruses bearing zoonotic S glycoproteins display similareutralization kinetics as pseudotyped viruses bearing SARSpikes.
The SARS-CoV-like isolated from bats identified in Rhinolo-hus spp. has a nucleotide identity with TOR-2 strain higherhan 92% (Lau et al., 2005; Li et al., 2005). In addition, six noveloronaviruses from six different bat species have been describedWoo et al., 2006). Four of these coronaviruses belong to group 1,nd two of them to group 2. Based on sequence data these authorsave proposed the classification of bat CoV in three subgroups2a, 2b, and 2c). Subgroup 2b comprises both SARS-CoV andbat-SARS-CoV (Rp3 isolate). The sequences of these SARS-oV isolates differ in the S1 domain of the S protein, where
equence identity fell to 64%. This sequence divergence in the1 domain corroborated the serum neutralization studies, which
ndicate that although bat sera have a high level of cross-reactiventibodies, they failed to neutralize human or civet cat SARS-oV when tested in Vero E6 cells (Li et al., 2005). In contrast,ther authors (Lau et al., 2005) have reported that 42% of theat sera samples tested neutralized human SARS-CoV isolateHKU-39849) using FRhK-4 cells. The discrepancy could beue to the presence of a different SARS-CoV-like virus serotype
n the bats studied by the two different groups, or to the two testystems used in the evaluation. Consequently vaccine designhould take into account this antigenic diversity. Interestingly,ecent characterization of the antigenic structure of SARS-CoV
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protein with a large panel of Mabs has shown that at least onepitope providing susceptibility to SARS-CoV that maps in theBD for ACE-2 is highly conserved in most virus strains and
herefore, may confer protection to most SARS-CoV strains (Het al., 2006).
. Antigens involved in the protection against CoVnduced infections
SARS-CoV has at least seven structural proteins (S, 3a, E,, 7a, 7b, and N) (Fig. 2) (Huang et al., 2006; Schaecher and
ekosz, 2006; Weiss and Navas-Martin, 2005). SARS-CoV S,a, E, and M proteins are viral membrane proteins with domainsxposed to the external face of the virus (Ito et al., 2005; Shent al., 2005) that, in principle, could be involved in protec-ion by induction of neutralizing antibodies. It has been shownhat proteins S and 3a induce in vitro SARS-CoV neutralizingntibodies, with S protein being the main component of protec-ive immunity (Qiu et al., 2005; Saif, 2004). Although strongmmune responses are elicited against both S and N proteinsBuchholz et al., 2004; Subbarao et al., 2004; Wang et al., 2005;hu et al., 2004), passive transfer studies illustrate that only-specific antibodies confer protection from SARS-CoV repli-ation in the mouse model (Bisht et al., 2004; Subbarao et al.,004).
The relevance of S protein in protection against SARS-CoVas been reinforced by the identification of neutralizing antibod-es from convalescent patients. The majority of the coronavirus
ig. 2. Structure and genome organization of SARS-CoV. (A). Schematic dia-ram of SARS-CoV structure. S, spike protein; M, membrane protein; E,nvelope protein; N, nucleoprotein; 3a, 7a, and 7b, structural proteins of SARS-oV. (B). Representation of a prototype SARS-CoV genome. Poly(A) tail is
ndicated by AAA. Numbers and letters indicate viral genes.
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pparently not in the case of SARS-CoV, the S protein is pro-essed in these two halves (Weiss and Navas-Martin, 2005;ong et al., 2004). Recent evidence has determined that SARS-oV neutralization is sensitive to deglycosylation of the spikerotein, suggesting that conformational epitopes are importantn antibody recognition (Song et al., 2004). The RBD locatedn the S1 subunit of S protein contains multiple conformationaleutralizing epitopes. This suggests that recombinant proteinsontaining RBD, and vectors encoding the RBD sequence, cane used to develop safe and effective SARS vaccines (Jiang etl., 2005). The continuous viral epitopes targeted by antibodiesn plasma samples from convalescent SARS patients have beendentified by biopanning with a M13 phage display dodecapep-ide library (Zhong et al., 2005). These epitopes converged toery short peptide fragments, one on each of the spike, nucle-capsid, 3a, 9b and nsp3 proteins. Immunoassays found thatost of the patients (82%) that recovered from SARS developed
ntibodies to the epitope-rich region on the spike S2 domain,ndicating that this domain also is an immunodominant siten the S protein. These S2-targeting antibodies were showno effectively neutralize SARS-CoV. Moreover, it is possiblehat S2-specific antibodies provided protective immunity to helphe patients recover from viral infection (Zhong et al., 2005).n fact, among the rabbit antibodies elicited by different frag-ents covering the entire S protein expressed in E. coli, some of
hem were specific for aa 1029–1192, which include the heptad-epeat sequence of the S2 domain that interacts to form S proteinrimers, and had neutralizing activities, indicating that this regionf the S protein also carries neutralizing epitopes.
SARS-CoV 3a protein consist of 274 amino acids, containshree putative transmembrane domains, and is expressed on theirus and cell surface (Ito et al., 2005; Tan et al., 2004b). Theopology of 3a protein on the cell surface was experimentallyetermined; the first 34 aa, located before the first transmem-rane domain, are facing the extracellular matrix (Akerstromt al., 2006), and its C-terminal, after the third transmembraneomain (aa 134–274), is facing the cytoplasm (Tan et al., 2004b).nterestingly, in two separate cohorts of SARS patients, one fromaiwan (Liu et al., 2004) and the other from Hong Kong (Zhongt al., 2005), B cells recognizing the N-terminal region of 3arotein were isolated from patients. Moreover, significant pro-ortion (40%) of the convalescent SARS patients examined in aot blot assay using a synthetic peptide with a sequence corre-ponding to amino acids 12–27 of the N terminus of the proteinere positive (Zhong et al., 2005). In addition, it was recently
eported that the N-terminal domain of 3a protein elicits strongnd potentially protective humoral responses in infected patientsZhong et al., 2006). Accordingly, rabbit polyclonal antibodiesaised against a synthetic peptide corresponding to aa 15–28 ofa protein inhibit SARS-CoV propagation in Vero E6 cells, inontrast to antibodies specific for the C-terminal domain of therotein (Akerstrom et al., 2006).
SARS-CoV E, M, and 7a proteins have shown low immuno-
enicity (Tan et al., 2004a). Sera from three convalescent phaseARS patients do not recognize these proteins expressed inammalian cells. Accordingly, SARS-CoV E protein peptidesere not recognized by convalescent patient antisera using a
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rotein microarray (Qiu et al., 2005). In a study using rabbitntibodies to 13 recombinant fragments associated with SARS-oV S, E, M, N, 3a, 3b, 6, 7a, and 9b proteins, strong neutralizingntibodies were only elicited by the S1 fragment (aa 241–591) ofprotein (Qiu et al., 2005). The incorrect folding of the proteins
xpressed in E. coli could be responsible for the lack of detectionf antibodies to other viral proteins, such as the 3a protein thats known to induce neutralizing antibodies (see above).
The M protein of transmissible gastroenteritis coronavirusTGEV) is required for virus assembly and budding, and Mrotein specific antibodies significantly, but weakly, neutral-ze TGEV and mediate complement-dependent lysis of TGEVnfected cells (Delmas et al., 1986; Risco et al., 1995; Woods etl., 1987). Consistent with the TGEV data described above, itas been shown that SARS-CoV M protein also induced viruseutralizing antibodies in the absence of complement (Buchholzt al., 2004). In addition, a mixture of S and M proteins showed aynergistic effect in the in vitro synthesis of TGEV neutralizingntibodies by immune leukocytes (Anton et al., 1995). In thease of SARS-CoV, immunization of hamsters with a parain-uenza virus vector has shown a differential role of S, M, E, orproteins in protection (Buchholz et al., 2004). Parainfluenza
irus expressing S protein alone provided complete protectiongainst SARS-CoV challenge in the lower respiratory tract andartial protection in the upper respiratory tract. This protectionas slightly augmented by co-expression with M and E proteins
Buchholz et al., 2004). Nevertheless, expression of M, E, or Nroteins in the absence of S protein did not confer detectablerotection. These results identify S as a main SARS-CoV neu-ralization and protective antigen among the structural proteins,nd confers a limited role to SARS-CoV M protein in protection.
SARS-CoV N protein specific antibodies do not neutralizehe virus in vitro as it could be expected for an internal virus pro-ein (Pang et al., 2004). However, SARS-CoV N protein induces-cell responses (Gao et al., 2003). Accordingly, DNA immu-ization using SARS-CoV N gene induces potent Th1 polarizedmmune responses in mice, as well as specific antibodies in thesenimals. In fact, the highest levels of humoral response and Tell proliferation activity were induced by the N gene constructJin et al., 2005). Analysis of the immune response to anotheroronavirus (TGEV), using an in vitro antibody synthesis sys-em, has shown that the optimum combination of viral proteinso stimulate the production of TGEV neutralizing antibodies initro was a mixture of S and N proteins, or a combination ofprotein oligomers (rosettes) and the N or N protein-derived
eptides (Anton et al., 1996; Anton et al., 1995). These data, inrinciple, suggest that N protein could be used in a vaccine toromote the synthesis of S-specific neutralizing antibodies.
9b protein (98 aa) elicits antibodies in SARS-CoV patients,ndicating that it is expressed in natural disease and that it ismmunogenic (Qiu et al., 2005; Zhong et al., 2005). In fact 100%f convalescent phase patients sera were positive for 9b protein.ased on this data, it has been speculated that 9b protein could
e structural (Qiu et al., 2005). Nevertheless, the presence of 9brotein in SARS-CoV virions needs to be further proved.
In summary, with the available data, SARS-CoV proteins Snd 3a elicit strong neutralizing antibody responses, whereas
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rotein M only induces a reduced neutralizing humoral immuneesponse. These antigens probably are relevant in the protectiongainst SARS-CoV. In addition, other structural proteins (suchs E, M, 7a, 7b, and N), and possibly protein 9b, could also playrole in protection.
. Role of humoral and cellular compartments inrotection against SARS
To study the role of the humoral immune response to SARS-oV, spike specific monoclonal antibodies that neutralize theirus have been developed (Berry et al., 2004; Subbarao et al.,004; Traggiai et al., 2004; Zhang et al., 2004). Passive transferf these monoclonal antibodies or immune serum into naıve micerotected them from infection with SARS-CoV (Subbarao et al.,004). Using Epstein-Barr virus transformed human B cells, theemory repertoire of a patient who recovered from SARS-CoV
nfection has been rescued (Traggiai et al., 2004). Some of theonoclonal antibodies exhibited neutralization activity in vitro,
nd some of them also conferred protection in a mouse model ofARS-CoV infection. In addition, human IgG monoclonal anti-odies neutralizing SARS-CoV developed using phage displayibraries protected ferret from lung disease and virus sheddingn pharyngeal secretions (ter Meulen et al., 2004). In both mousend ferret models, administration of human monoclonal antibod-es with in vitro neutralization activity reduced SARS-CoV titersn the lungs 3 to 6-log10-unit, protecting from lung pathologyn ferrets (ter Meulen et al., 2004). Overall, these data indi-ate that humoral immune responses alone can protect againstARS.
Immune responses to SARS-CoV, elicited by a DNA vaccinencoding a codon optimized SARS spike protein (Yang et al.,004), or the S1 fragment, induced neutralizing antibodies (Zengt al., 2004), as well as T-cell responses. Nevertheless, protectionrom SARS-CoV challenge was mediated by a humoral immuneesponse but not by a T-cell-dependent mechanism (Yang et al.,004).
Surprisingly, immunodeficient mice can clear a SARS-CoVnfection, showing the role of innate immune responses in theefenses against SARS-CoV. C57BL/6 mice that lack NK-Tells (CD1−/−), or NK cells, or those that lack T and B cellsRagl−/−) cleared the virus by day 9 after infection (Glass etl., 2004) and displayed high induction of proinflammatoryytokines. These data suggest that the NK cells and the adapta-ive immune response were not essential for virus clearance in
ice. Possibly, interferon pathways were relevant in viral clear-nce. The importance of interferon response was reinforced bynfecting Stat1-deficient mice with SARS-CoV (Hogan et al.,004). Stat1 is important to the regulation of interferons, andtat1-deficient mice produced one hundred-fold increase in viral
iter over control mice. Additionally, the mutant mice devel-ped interstitial pneumonia, not seen in control mice (Hogan etl., 2004) but not alveolar damage, as seen in lungs of human
atients. It is unclear at this time if the observed pathologicalifferences between human and Stat 1-deficient mouse lungsere due to time of sampling or to differences in host responses
Hogan et al., 2004).
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. Development of non-replicating SARS-CoV vaccines
.1. Inactivated virus vaccines
Neutralizing antibodies were detected 2–3 weeks after thenset of disease in immunocompetent SARS patients, and0% of patients recovered without hospitalization (Ksiazek etl., 2003). These data indicate that most patients successfullyespond to SARS-CoV infection. Although many types of vac-ines for SARS-CoV have been attempted such as expression ofecombinant proteins, or the use of virus vectors, these vaccinesequire considerable research set-up time (Bradbury, 2003).herefore, the classical approach using inactivated, cell-cultureased SARS-CoV is likely to be the easiest way for SARSaccine development, based on the experience with availableaccines including inactivated or live polio and rabies vaccinesMontagnon, 1989; Zhou et al., 2005). This is the case of com-anies that favored the development of an inactivated candidatehole virus vaccine, based on the well-established technologies
or the development of such vaccines (Spruth et al., 2006).The fast spreading of SARS initially prompted a Chinese
ompany (Sinovac Biotech of Beijin) to develop a vaccine in col-aboration with the Chinese Academy of Medical Sciences thattarted tests of the vaccine, an inactivated form of SARS-CoVs early as 2004, using 30 volunteers (Marshall and Enserink,004). No side effects were reported for the first patients thatere injected with the inactivated virus.SARS-CoV inactivation to produce the killed vaccines has
een performed using different methods. For instance, SARS-oV purified by ultracentrifugation has been inactivated with-propiolactone and administered with or without Alhydrogels adjuvant (See et al., 2006). The virus subcutaneously adminis-ered to mice was more efficient than recombinant adenovirusesxpressing either the S or N proteins, provided by the intranasalr intramuscular routes. An alternative method was the inacti-ation of SARS-CoV by �-propiolactone before initiating theurification step. Immunization with this virus was comparedith vaccination using DNA or adenovirus vectors. The humoral
mmune response was most effective using inactivated virus withdjuvants such as MF59 (Chiron Vaccines) or Alum (Pierce,ockkford, IL), and was associated with stimulation of the CD4ut not the CD8 response, supporting the use of inactivatedARS-CoV as vaccine (Kong et al., 2005).
SARS-CoV has also been purified up to 98% homogeneityy ultrafiltration, gel filtration, and exchange chromatography,nd inactivated with �-propiolactone. Cynomolgus macaquesere immunized with different amounts of the purified virus
n the absence or in the presence of adjuvant. Monkeys werehallenged by the nasal route 30 days post-immunization. Highevels of neutralizing antibodies that prevented the replicationf SARS-CoV and interstitial pneumonia were induced (Qin etl., 2006).
Interestingly, no side effects were observed even in the pres-
nce of low titer neutralizing antibodies, indicating that theurified SARS-CoV vaccine is safe in monkeys.
In other approaches, SARS-CoV partially purified in sucroseushions was completely inactivated with formaldehyde. This
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irus efficiently competed the binding of infectious virus toells, indicating that the inactivated virus kept a functional RBSQu et al., 2005). Polyethyleneglycol precipitated virus aloner in the presence of cholera toxin B (CTB, Sigma) or CpG,dministered to mice by the intranasal route elicited serumARS-CoV-specific neutralizing antibodies, and IgA specificntibodies in the trachea and lungs (Qu et al., 2005).
In another approach, formaldehyde inactivated whole virus,repared in Vero cells, was used in intramuscular immuniza-ion of 2–5 year-old rhesus monkeys (Zhou et al., 2005). Afterweeks, monkeys were challenged with SARS-CoV. Doses of
.5 or 5 �g/monkey protected most of the monkeys against chal-enge with 108 pfu of SARS-CoV, and higher doses (50 �g)onferred complete protection. In contrast, the control animalseveloped a typical SARS-CoV infection after challenge. Themmunization preferentially induced Th1 responses, but alsonhanced other cellular immune responses, including the pro-uction of IFN-� that can increase the activity of natural killerells and inhibit virus replication. No systemic side effects werebserved in vaccinated animals post-immunization, even at theigh dose (5000 �g/monkey) and after two injections.
One vaccine manufactured to large scale using fermenter cul-ures of Vero cells in serum free medium has been based in aouble-inactivated, whole virus vaccine (Spruth et al., 2006).irus infection at a moi of 0.001 resulted in generation of highiral titers (around 108 TCID50/ml). Culture supernatants werearvested and inactivated by formalin treatment followed by UVnactivation. This two step inactivation procedure was utilizedn order to ensure a high safety margin with respect to resid-al infectivity. Mice immunized twice with l �g of SARS-CoVaccine using adjuvant (0.2% aluminium hydroxide) developedigh antibody titers against SARS-CoV spike protein, as deter-ined by an ELISA test. The use of the adjuvant Al(OH)3 had
nly a minor effect on the immunogenicity of the vaccine. Inddition, cell mediated immunity, as measured by the produc-ion of INF-� and IL-4 stimulation, was elicited. The vaccineonfers 100% protection that was correlated to antibody titergainst the SARS-CoV S protein and to neutralizing antibodyiter. Induction of neutralizing antibodies with titers ≥114 andn S-specific ELISA titer ≥25,600 resulted in 100% protectiongainst intranasal challenge with 105 TCID50 of infectious virus.
.2. Subunit vaccines
A polypeptide containing amino acids 14–762 of the SARS-oV spike protein has been expressed using the baculovirus
ystem (Bisht et al., 2005). The affinity purified protein wasdministered to mice with either saponin or Ribi as adjuvants.oth regimens induced binding and neutralizing monoclonalntibodies, although the best results were obtained with saponinnd polypeptide, which provided the highest antibody response.fter challenge, protective immunity was shown by the reduc-
ion of SARS-CoV titers in the upper and lower respiratory
ract. This subunit vaccine induced higher neutralizing antibodynd more complete protection against an intranasal challengehan that achieved by inoculation of mice with live SARS-CoVSubbarao et al., 2004), vaccinia virus Ankara (MVA) expressing
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he full length S protein (Bisht et al., 2004), or DNA expressinghe full-length S or S protein lacking the transmembrane andytoplasmic domains (Yang et al., 2004).
N protein by itself does not provide protection against SARS-oV infections. Still, a large number of reports using N proteins an antigen have been published. The immune response ofice vaccinated with a purified N protein fused to glutathione S-
ransferase (GST) was analyzed and compared with the responsef two DNA-based vaccination approaches (Gupta et al., 2006).he immunization with N-GST elicited a strong T-cell IL-4, andntibody responses but minimal IFN-� response. This responseiffered markedly with the immune response shown by micemmunized with both DNA encoding unmodified cytoplasmic
protein, and DNA encoding N as a LAMP-1 chimera targetedo the lysosomal MHC II compartment. DNA immunizationslicited a strong T-cell IFN-� and CTL responses. Neverthe-ess, the T-cell responses to the three immunogens were elicitedy the same N peptides as shown by the ELISPOT analysis ofntigen-activated T cells. In vivo protection experiments wereot performed with these vaccines.
The N protein of SARS-CoV was expressed in E. colind purified (Liu et al., 2006). Balb/c mice were vaccinatedith N protein emulsified in Montanide ISA-51 containing theligodeoxynucleotide CpG, or in PBS. In the first case, anti-
antibodies were found to be mainly IgG2a, suggesting arevalence of Th1 immune response. In contrast, anti-N pro-ein antibodies of mice immunized with N protein in PBS wereound to be mainly IgG1. Reactivity of antisera raised against
protein formulated in ISA-51/CpG in mice and monkeys andhat of sera from patients were tested with a panel of overlap-ing peptides. The region around residues 156–175 of N proteins immunogenic in the three models. In addition, peptides cor-esponding to residues 1–30, 86–100, 306–320 and 351–365ontained murine immunodominant T-cell epitopes. Using andFN-� secretion cell assay, peptides containing residues 81–95ere capable of stimulating CD4+ and CD8+ cell proliferation
n vitro. Peptides corresponding to residues 336–350 were capa-le of stimulating INF-� production in T-cell cultures derivedrom peripheral blood mononuclear cells (PBMCs) of macaquesmmunized with the N protein emulsified in ISA-51/CpG. Norotection experiments were performed with this immunogen.
.3. Virus like particles (VLPs)
The requirements for SARS-CoV VLP formation differssing different expression systems and cell types. Productionf viral proteins in insect cells using baculovirus has shownntracellular SARS-CoV VLPs assembly by expressing M and
proteins (Ho et al., 2004). Secretion of these VLPs to the extra-ellular media required the co-expression of S protein (Mortoland Roy, 2004). In these experiments secretion of VLPs was rela-ively efficient (200 �g per 1 × 109 infected cells). These resultsre at variance with those obtained by expressing SARS-CoV
roteins in human 293 renal epithelial cells under the control ofytomegalovirus promoter, using DNA plasmids (Huang et al.,004). In this system, any combination of genes that expressed
and N proteins, with or without S or E proteins, gener-
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ted intracellular VLPs, and the pseudoparticles did not formn the absence of the M and N proteins. No single viral geneas able to support the formation of viral capsids within these
ells. The additional expression of the S protein allowed the for-ation of budding particles with morphology typical of SARS
nd related coronaviruses. Different types of VLPs could beormed depending of the protein composition of these SARS-oV VLPs. Protection by the administration of VLPs has noteen reported.
. Vaccines produced in plants
The development of plant-based vaccines against differentoronaviruses [TGEV, infectious bronchitis virus (IBV), andorcine epidemic diarrhea virus (PEDV)] using the oral deliv-ry of recombinant S protein that elicit protective immunity haseen reported (Bae et al., 2003; Lamphear et al., 2004; Tubolyt al., 2000; Zhou et al., 2003). One of these studies include aprotein plant-based vaccine candidate against TGEV that has
dvanced into early phase farming trials (Tuboly et al., 2000).ore recently, to develop a safe, effective, and an inexpen-
ive vaccine candidate, the S1 domain of SARS-CoV S proteinas been expressed in tomato and low-nicotine tobacco plantsPogrebnyak et al., 2005). High expression levels of recombi-ant S1 protein (>0.1% total soluble protein) were observed ineveral transgenic lines by Western blot analysis using S pro-ein specific antibodies. Plant-derived antigen induced systemicnd mucosal immune responses in mice, which showed signif-cantly increased levels of SARS-CoV specific IgA after oralngestion of tomato fruits expressing S1 protein (Pogrebnyakt al., 2005). Sera of mice parenterally primed with tobacco-erived S1 protein revealed the presence of SARS-CoV-specificgG.
. DNA vaccines
Several approaches based on DNA vaccination have beenescribed in order to elicit protection against SARS. Two of themsed prime-boost strategies and showed that the combination ofhe DNA vaccine and the whole chemically inactivated vaccinean be used to enhance the magnitude of the immune response,nd also to change the balance of humoral to cellular immuneesponse (Zakhartchouk et al., 2005a). A combination of theNA and inactivated virus induces Th1 immune responses while
he whole killed virus vaccine induces Th2 immune responses.Mice immunized intramuscularly with a DNA vaccine
xpressing S protein and intraperitoneally boosted with E.oli expressing S peptides showed high neutralization titers>1:1280). This vaccine might have a practical value to immu-ize in farms growing civet cats due to its low cost (Woo et al.,005).
Other DNA vaccines express N protein alone or linked to cal-eticulin. The first ones preferentially induce IgG responses of
he IgG2a isotype, IFN-� and IL-2, and CD8+ CTL responseso N protein, but produce strong delayed-type hypersensitivityDTH) that could have undesired side effects (Zhao et al., 2005).he expression of N protein linked to calreticulin increases
isiT
arch 133 (2008) 45–62 51
ajor histocompatibility complex (MHC) class I presentation toD8+ T cells, in the absence of reported adverse effects in mice
Kim et al., 2004). These vaccines led to the generation of strong-specific humoral and T-cell-mediated immune responses inice, but no protection experiments were shown. N protein has
lso been expressed linked to hLAMP (N-hLAMP), that tar-et antigen to the MHC class II, leading to a stronger and higheremory cellular immune response associated to high IFN-� pro-
uction than immunization with N protein alone (Gupta et al.,006).
Studies on DNA immunization to protect against SARS,sing three forms of the spike protein: full-length S, and Sroteins with the cytoplasmic or the transmembrane domainseleted have been reported (Yang et al., 2004). These vaccinesnduced neutralizing antibodies and T-cell responses, resultingn protective immunity in mice. Viral replication was reduced
ore than six orders of magnitude in the lungs of mice vacci-ated with these S plasmid DNA expression vectors. Protectionas mediated by a humoral but not a T-cell dependent immuneechanism, as shown by adoptive T-cell transfer in which donor
-cells were unable to reduce pulmonary viral replication inecipient animals. By contrast, passive transfer of purified IgGrom immunized mice, but not control IgG provided immunerotection against SARS-CoV (Yang et al., 2004). The vectorxpressing the S protein with the cytoplasmic domain partiallyeleted induced the most potent neutralizing antibody response.
. Development of SARS-CoV vaccines based on viralxpression vectors
.1. Poxvirus expression vector based vaccines
Using the highly attenuated modified MVA, SARS-CoV Srotein has been expressed by several groups (Bisht et al., 2004;hen et al., 2005). The first one has shown that intranasal or
ntramuscular immunization of Balb/c mice elicited protectivemmunity as shown by the reduction of SARS-CoV titers in thepper and lower respiratory tract after challenge. Furthermore,assive transfer of serum from mice immunized with the recom-inant MVA expressing S protein to naıve mice also reducedhe replication of SARS-CoV in the respiratory tract after chal-enge, demonstrating a role for S protein specific antibodies inrotection. The second group (Chen et al., 2005) showed thenduction of neutralizing antibodies in mice, ferrets, and mon-eys, although protection experiments were not performed. In anntibody absorption assay, the majority of the antibodies raisedy the MVA recombinant expressing the full-length S proteinere absorbed by an S protein fragment including aa 400–600,
hat includes the RBD, indicating that the major SARS-CoVeutralization mechanism likely occurs through blocking thenteraction between the virus and the cellular receptor ACE2.
In contrast, other authors immunized ferrets using MVAxpressing the SARS-CoV S protein, inducing a vigorous
mmune response that did not prevent virus infection andpreading (Czub et al., 2005; Weingartl et al., 2004). Livernflammation was found in all MVA-spike vaccinated ferrets.hese authors suggested that their results indicate the induction
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f antibody dependent enhancement (ADE) of disease similaro that caused by feline infectious peritonitis virus (FIPV).
.2. Adenovirus vector based vaccines
SARS vaccines based on the use of adenovirus vectors havehown that expression of S protein alone or in combination with
protein led to the protection of mice against the challenge withARS-CoV. The efficacy of immunization with adenovirus vec-
ors was compared with that of chemically inactivated partiallyurified virus. Whole-killed virus vaccine was more effective inonferring protective immunity against live SARS-CoV (Seet al., 2006). Other adenovirus vaccines tested in mice havexpressed either the S or the N protein (Zakhartchouk et al.,005b) and shown that the S2 domain and the N protein containtrong T-cell epitopes, but reported no challenge experiments.n the monkey model, adenovirus-based vaccines induce strongARS-CoV-specific immune responses, indicating that theseectors are promising vaccine candidates but, again, no infor-ation on protection have been provided (Gao et al., 2003). The
denovirus Ad5 vector with a deletion in the E1 and E3 regions,ave also been used to express the S1 domain of the SARS-CoVprotein (490 aa) (Liu et al., 2005). Wistar rats immunized
hree times throughout consecutive weeks produce antiserumapable of protecting from SARS-CoV infection in cell culture.istopathological examination found no evident side effects in
he immunized animals. Nevertheless, in vivo protection exper-ments were not performed. Therefore, additional experimentsre required with the adenovirus based SARS vaccines.
Severe disease and high death rates were noted in senescentuman populations infected with SARS-CoV, while childrennder 12 years of age did not develop the severe disease thatas seen in adults (Baric et al., 2006; Deming et al., 2006; Ng et
l., 2004). These data suggest that the quality of the immuneesponse may play a role in the outcome of virus infection.he ability of vaccines to induce robust immune responses inenescent populations has been evaluated to determine if pro-ection can be elicited in elderly populations with senescentmmune systems. To evaluate vaccine efficacy against homol-gous and heterologous strains, the Urbani S glycoprotein anducleocapsid genes were inserted in VEE virus replicon par-icles (VRP-S or VRP-N) (Baric et al., 2006; Deming et al.,006). In addition, expression of the influenza A HA glycopro-ein (VRP-HA) was used as a control. Using reverse genetics,ynthetically resurrected recombinant viruses bearing the GD03glycoprotein that replicated to high titers in Vero and human
irway epithelial cells have been obtained (Baric et al., 2006;eming et al., 2006). Importantly, human convalescent seraad plaque reduction neutralization titre of 50% (PRNT50) val-
es of about 1:1600 against late phase isolates like Urbani,et were reduced about 10–15 fold against the heterologouscGD03 virus (PRNT50 1:150) (Fig. 3A). Young and senescentalb/c mice with ages exceeding 1 year at the time of challenge,
8
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arch 133 (2008) 45–62
ere vaccinated with VRP-HA, VRP-S, VRP-N, or a combina-ion of VRP-S and VRP-N, and challenged with recombinantARS-CoV expressing the Urbani S protein or the antigeni-ally different GD03 S protein (Fig. 3B). In vaccinated animals,RP-S vaccines provided complete short- and long-term protec-
ion against homologous challenge, protecting both young andenescent mice from the Urbani strain replication. After chal-enge, VRP-S and VRP-S + VRP-N vaccinated mice displayedittle if any pathologic lesions in the lung, whereas VRP-HA vac-inated aged mice demonstrate pathological lesions in the lungimilar to that reported in the literature (Roberts et al., 2005a).RP-S vaccines also provided short-term protection in youngice challenged with the heterologous GD03 S strain, despite
he significantly reduced ability of anti-Urbani S antibody toeutralize virus expressing GD03 S. In contrast, vaccination ofenescent mice with VRP-S provided limited protection (∼38%)nd the combination of VRP-S + VRP-N vaccines provided lit-le long-term protection against infection by the antigenicallyifferent SARS-CoV GD03, although virus titers were reducedbout 10-fold compared with VRP-HA controls. The SARS-oV GD03 challenge also produced pathological lesions in both
he VRP-HA and SARS-vaccinated animals that were virtu-lly indistinguishable from those produced by infection with theARS-CoV-Urbani strain. Therefore, it is likely that declining
mmunity of senescent animals in combination with the reducedbility of antibody to neutralize heterologous challenge virusesesulted in vaccine failure in aged animals. It seems that vaccinepproaches that induce less robust neutralization responses likeNA and killed vaccines, might completely fail in protecting
enescent populations against SARS-CoV GD03 challenge.SARS-CoV disease severity was linked to age and other
o-morbidities, with mortality rates increasing with age andxceeding 50% in individuals over 65. It is also known thatlderly respond poorly to new antigens as compared to youngeropulations, but overall this phenomena is poorly studied. Con-equently, vaccine efficacy in the elderly is a key property offficacious SARS-CoV vaccines. Immunosenescence is com-on in animal models and in clinical studies that occur during
ging, and vaccine efficacy is often attenuated in the elderlyFrasca et al., 2005; Song et al., 1997; Zheng et al., 1997).mmune complications include a generalized decrease in theunction of B and T cell and innate immune function, diminishedacrophage and granulocyte function, diminished cellular traf-c, cell growth and differentiation and decreased natural killerell numbers and activity. New and different vaccine regimenshould be developed and tested with the ultimate goal of elicitingomplete protection against antigenically heterologous formsf SARS-CoV, especially in the most vulnerable elderly popu-ations, and that there is a need for further testing developingaccines that induce an anti-N response in more animal mod-ls, similarly to what has been described in other viral systemsFrech et al., 2005; McElhaney, 2005).
.4. Parainfluenza based vectors
A vector based in an existing live attenuated parainfluenzairus, that is being developed for intranasal pediatric immu-
L. Enjuanes et al. / Virus Research 133 (2008) 45–62 53
Fig. 3. Neutralization of SARS-CoV pseudotypes and heterologous challenge studies. (A) Cross neutralization responses between wt Urbani (�) and icGD03 (�).About 100 PFU of each virus was incubated for 30 min with varying concentrations of human antisera from a convalescent SARS patient or control serum andtitered by plaque assay. (B) Compilation of vaccination results in mice inoculated with VRP-vectored vaccines and challenged with icSARS or icGDO3-S (Deminget al., 2006). The percent of mice without detectable replicating virus are shown as bars while the average titers of detectable virus are shown as red circles. Errorbars represent the standard deviation of the measured samples. Mice were vaccinated with VRP-S, VRP-N, a cocktail of VRP-S and VRP-N (VRP-S+N), or mockvaccinated with either VRP-HA (VRP expressing influenza A HA protein) or PBS. Mice were intranasally challenged with either Urbani derived from the infectiousclone (icSARS) or a chimeric virus expressing the GD03 spike glycoprotein (GD03-S). Mice challenged with icSARS were vaccinated when young (4–5 weeks),boosted 4 weeks later, and challenged either 8 weeks post boost (young) or 54 weeks post boost when old (Senescent). VRP-S and VRP-S+N provided protectioni cinatea or 32
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n both groups against icSARS. Mice challenged with GD03-S were either vacpproximately 4 weeks later, and challenged either 7 weeks post boost (young)
ization against human parainfluenza virus type 3 (HPIV3) wassed to express SARS-CoV S protein (Bukreyev et al., 2004).ector administration to monkeys resulted in the productionf systemic immune response by mucosal immunization. Afterhallenge with SARS-CoV, all monkeys in the control grouphed SARS-CoV. In contrast, no viral shedding occurred in theroup immunized with the parainfluenza vector expressing the Srotein. Recombinant viruses expressing SARS-CoV structural, M, and N proteins, individually or in combination, have beenvaluated for immunogenicity and protection in hamsters thatupport the replication of both SARS-CoV and parainfluenzaector (Buchholz et al., 2004). A single intranasal adminis-ration of the vector expressing the S glycoprotein induced aigh titer of SARS-CoV neutralizing antibodies, only two fold
ower than that induced by SARS-CoV infection. This responserovided complete protection against SARS-CoV challenge inhe lower respiratory tract, and partial protection in the upperespiratory tract. In contrast, expression of M, N, or E pro-
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eins did not induce detectable serum SARS-CoV neutralizingntibodies.
.5. Rhabdovirus based vector
A recombinant rabies virus vector has been used to expresshe S protein of SARS-CoV (Faber et al., 2005). Immunogenicitytudies in mice showed the induction of SARS-CoV neutral-zing antibodies after a single dose, but no protection studiesave been shown. Similarly, an attenuated vesicular stomati-is virus (VSV) vector was used to express the S protein ofARS-CoV (Kapadia et al., 2005). Mice vaccinated with VSV-developed SARS-CoV neutralizing antibodies that controlled
hallenge with SARS-CoV performed at 1 or 4 months after a
ingle vaccination.
In summary, immunization to prevent SARS using differentive vector systems has shown that protection is mainly mediatedy humoral immune responses to the S protein. A warning signal
5 s Research 133 (2008) 45–62
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Fig. 4. Propagation of SARS-CoV with a deleted E gene in different cell lines.Vero E6 (A), Huh-7 (B), and CaCo-2 (C) cells were infected at a moi of 0.5 witheither the rSARS-CoV-�E or the recombinant wild-type virus. At different timespost-infection, virus titers were determined by plaque assay on Vero E6 cells.E
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as that, at least with some vectors such as VEE virus, thexpression of N protein may lead to side effects.
. SARS-CoV vaccine candidates engineered by reverseenetics
.1. Vaccines based on the deletion of single or severalccessory combinations of genes
The effect of the deletion of group-specific genes in differentoronaviruses has been studied. Reports using mouse hepati-is virus (MHV) as a model have shown that deletion mutantsemoving ORFs 4, 5a, 7a, and HE are attenuating in the naturalost (de Haan et al., 2002a). Similarly, studies deleting ORFof TGEV (Ortego et al., 2003) and ORFs 3abc and 7ab of
IPV (Haijema et al., 2004) led to virus attenuation. However,ARS-CoV deletion mutants lacking ORFs 3a, 3b, 6, 7a, or 7bid not significantly influence in vitro and in vivo replicationfficiency in the mouse model (Yount et al., 2005). All recombi-ant viruses replicated to wild-type levels in the murine model,uggesting that either the group-specific ORFs play little rolen in vivo replication efficiency or that the mouse model is notf sufficient quality for discerning the role of the group-specificRFs in disease. In fact it has been surprising that ORFs likea, 7a, and 7b, that encode structural virus proteins, (Huang etl., 2006; Schaecher and Pekosz, 2006; Yount et al., 2005) haveittle influence on in vivo virus replication in the mouse model.nly deletion of ORF 3a has shown a minor decrease (aboutne log unit) in virus growth (Yount et al., 2005). Furthermore,eletion of more than one gene, such as deletion of ORFs 3a andb, and ORF6, showed a 1–1.5 log reduction in Vero cells butittle effect on growth in the murine model at day 2 postinfec-ion. Moreover, little difference in growth or pathogenesis wereoted in the mice model between wt and deletion mutants lack-ng ORF3a and ORF7. Therefore, the effect of SARS-CoV geneeletions needs to be tested in more relevant animal models.nterestingly, the simultaneous deletion of larger combinationsf group-specific genes such as 6, 7a, 7b, 8a, 8b, and 9b has leado the production of an infectious SARS-CoV deletion-mutanthat propagates in cell culture with a titer similar to that of thearental wild type virus. The potential of this deletion-mutant aspromising vaccine candidate is being studied (M.L. DeDiego,nd L. Enjuanes, unpublished results).
.2. Vaccines based on the deletion of structural proteins
A recombinant SARS-CoV (rSARS-CoV) that lacks the Eene generated from a bacterial artificial chromososme (BAC)as attenuated in vitro and in an animal model (DeDiego et al.,007). The E gene was previously shown to be a non-essentialene for the group 2 MHV coronavirus (Kuo and Masters, 2003),lthough elimination of this gene from MHV genome reducedirus growth in cell culture more than one thousand-fold. In con-
rast, for group 1 TGEV coronavirus, expression of the E generoduct was essential for virus release and spread. Propagationf E gene deleted TGEV (TGEV-�E) was restored by providingprotein in trans (Curtis et al., 2002; Ortego et al., 2002).
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rror bars represent standard deviations from the mean from three experiments.
The role of E protein in SARS-CoV propagation was studiedy constructing SARS-CoV with a deleted E gene (DeDiego etl., 2007). Interestingly, viable viruses were recovered in Vero6 cells with a relatively high titer (around 106 pfu/ml) andlso from Huh-7 and CaCo-2 cells with reduced titers (Fig. 4),ndicating that SARS-CoV E protein is not essential for viruseplication in cell culture. Electron microscopy observation ofero E6 cells infected with the SARS-CoV wt or the �E dele-
ion mutant showed much higher assembly efficiency for the wtirus (Fig. 5). In this respect, SARS-CoV-�E behaves as MHV,he other group 2 coronavirus studied, although SARS-CoV-�Erows to a considerably higher titer. The differential behavior of
E mutant viruses from different coronavirus groups may indi-
ate basic differences in virion assembly or life cycles amonghe different groups.
L. Enjuanes et al. / Virus Research 133 (2008) 45–62 55
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Fig. 6. Growth of rSARS-CoV in the respiratory tract of hamsters. Hamsterswere inoculated with 103TCID50 of rSARS-CoV or rSARS-CoV-�E. Animalswere sacrified and tissues were harvested at different times post-infection. Viraltiters in lung (A) and nasal turbinates (B) were determined in Vero E6 cellsmonolayers. The non-parametric Mann–Whitney U-statistical method was usedfwd
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ig. 5. Assembly of SARS-CoV-�E deletion mutant in the ERGIG compart-ent. Electron micrographs of Vero E6 cells infected (moi 1.0) with SARS-CoV
A) and SARS-CoV-�E (B) at 24 h post-infection.
The hamster model has been used to study SARS-CoV-�Eirus pathogenicity, because it demonstrates elements presentn human cases of SARS-CoV infections including interstitialneumonitis and consolidation (DeDiego et al., 2007). An idealnimal model that completely reproduces human clinical diseasend pathological findings has not been identified. Nevertheless,he hamster model reproducibly supports SARS-CoV replica-ion in the respiratory tract to a higher titer and for a longeruration than in mice or non-human primates. Virus replica-ion in this model is accompanied by histological evidence ofneumonitis, and the animals develop viremia and extrapul-onary spread of virus (Roberts et al., 2005b). Although overt
linical disease is absent, the hamster model is a useful modelor the evaluation of SARS-CoV infection. Titers of recombi-ant SARS-CoV (rSARS-CoV) achieved in the respiratory tractf hamsters (Fig. 6) were similar to those previously reportedRoberts et al., 2005b) and were 100–1000-fold higher than titersf the rSARS-CoV-�E virus, suggesting that this mutant virus isttenuated. Histopathology examination of lungs from infectedamsters was performed at two and five days post-infection,
ecause it has been shown that pulmonary disease was mostotable at these time points. Detection of viral antigen waseduced in lungs from rSARS-CoV-�E infected hamsters andulmonary inflammation was less prominent in these animals
sfnc
or ascertaining the significance of observed differences. Statistical significanceas indicated by (*p-value < 0.05). The dotted line indicates the lower limit ofetection.
han in rSARS-CoV-infected animals, indicating that rSARS-oV-�E is attenuated in vivo (DeDiego et al., 2007). In fact,
eduction of SARS-CoV titers in patients has been associatedith a considerable reduction in pathogenicity and survival rates
Chu et al., 2004; Hung et al., 2004). In contrast, challenge ofamsters with recombinant viruses lacking single group spe-ific ORFs3a and ORF7 did not result in significant reductionsn virus titier or pathology (Baric et al., unpublished). Therefore,ARS-CoV-�E attenuated virus is a promising vaccine candi-ate that is being evaluated in different animal models (mice,errets and macaques).
.3. Future trends on SARS-CoV reverse genetic vaccines
Live attenuated virus vaccines face a series of potential con-erns including reversion to wt and recombination repair withirculating heterogeneous human coronaviruses or zoonoticARS strains. Consequently, live virus vaccine formulations
hould include rational approaches for minimizing the potentialor reversion to wt phenotype and simultaneously resist recombi-ation repair. It is clear that modifications of SARS-CoV genomeould lead to viruses with an attenuated phenotype that could be
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onsidered safe and effective vaccine candidates. The replicases a target for attenuating coronaviruses is undiscovered ter-itory, likely ripe with alleles that might influence replicationfficiency and virulence. The SARS-CoV replicase represents aajor target of future research endeavors.Coronaviruses have a characteristic, strictly conserved
enome organization with structural genes occurring in the order′-polymerase (pol)-S-E-M-N-3′. MHV virus mutants with theenes encoding the structural proteins located in different orderere constructed (de Haan et al., 2002b). These recombinantiruses were tested for the ability to replicate in the natural host,he mouse. The results indicate that the canonical coronavirusenome organization is not essential for in vivo replication.ome of the mutants showed an attenuated phenotype, similarly
o what has been observed for the VSV (Ball et al., 1999). There-ore, deliberate rearrangement of the viral genes may be usefuln the generation of attenuated coronaviruses, which due to theireduced risk of generating viable viruses by recombination withirculating field viruses would make safer vaccines.
Vaccines based on modifications of the replicase gene couldn principle be generated by mutagenesis, as modifications intro-uced in the MHV nsp-1coding regions have identified residuesmportant for protein processing and viral RNA replication that
ay affect virus virulence and could be introduced in vac-ine candidates (Brockway and Denison, 2005). SARS nsp1locks host macromolecular synthesis and abrogates IFN sig-aling (Kamitani et al., 2006), providing further evidence thatsp1 coding regions represent potential virulence determinants.lternatively, Tyr6398His substitution in open reading frame
ORF) 1b-nsp14 has been demonstrated that attenuate MHVeplication in mice (Sperry et al., 2005). Similarly, deletion of
he nsp2 gene in MHV and SARS-CoV has been shown to yieldiable attenuated mutant viruses that replicate about one logess efficiently than wt virus in cell culture and in animals, anday also provide a foundation for the design of live vaccines
Srmc
ig. 7. (A) Genome organization of SARS-CoV recombinant viruses to generate safeere changed to CCGGAT (red circles). Since the wild-type and mutant TRS signalsrecombination event resulting in a viral genome with mixed TRS signals is not viaboronaviruses. TRS sequences for select group 1, 2 and 3 coronaviruses are summari
arch 133 (2008) 45–62
Graham et al., 2005). As the nsp14 Tyr residue and nsp 2 areompletely conserved, it may be possible to engineer commonoronaviridae attenuating alleles via recombinant DNA tech-iques. Alternatively, changes in gene order within the replicaser even relocation to the 3′-end of the genome, if tolerated, mayed to attenuated virus phenotypes.
Other options to include safeguards into the genetically engi-eered vaccines, particularly those that can prevent the recoveryf the original virulent phenotype by recombination betweenhe vaccine strain and viruses circulating in the field (such asCoV-229E, -OC43, or -NL63) have been developed. One of
hem is the construction of replication-competent, propagation-efective viruses (pseudovirions) that are defective in one geneonferring an attenuated phenotype or even the ability for virusropagation (Enjuanes et al., 2005). These viruses could berown in packaging cell lines providing in trans the missingrotein. In the case of SARS-CoV, vaccine candidates withouthe E gene have been constructed. In order to prevent the rescuef the virulent phenotype by recombination with a circulatinguman coronavirus, the deletion of an essential gene, located inposition distant from gene E, and the relocation of the deletedene to the position previously occupied by gene E has beenroposed. A potential recombination leading to the rescue ofene E would lead to the loss of the essential gene (Enjuanes,005).
An alternative approach for developing safer, recombina-ion resistant live coronavirus vaccines has been developed by
odifying the transcriptional regulatory sequences (TRS) ofvaccine strain to a sequence incompatible with the TRS of
ny known circulating coronavirus. The idea being that recom-inant events between wt coronaviruses and TRS remodeled
ARS-CoV would result in genomes containing lethal mixedegulatory sequences that block expression of subgenomicRNAs (Fig. 7A) (Yount et al., 2006). TRS sequences among
oronaviruses are highly conserved and direct the expression
attenuated viruses. The wild-type SARS-CoV TRS, ACGAAC (blue circles),are not compatible in regulating subgenomic transcription (Yount et al., 2006),le. (B) The icSARS-CoV TRS sequence is unique from that of other describedzed. The TRS selected for the remodeled virus is shown at the bottom.
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f subgenomic mRNAs. Using a molecular clone, the SARS-oV TRS network was remodeled from ACGAAC to CCGGAT
Fig. 7B). This rewiring of the genomic transcription networkllows for efficient replication of the mutant virus, icSARS-RG. The icSARS-CRG recombinant virus replicated to titersquivalent to wt virus and expressed the typical ratios of subge-omic mRNAs and proteins. Interestingly, some new transcriptsere noted initiating from the replicase gene, most of which
ould encode N-terminal truncated ORF1a polyproteins. It is notlear if these novel transcripts might influence pathogenic out-omes, although in some instances nsp3 is truncated potentiallyllowing for the establishment of dominant negative phenotypesn replication in cell culture and animals. An attractive SARS-oV vaccine could further be modified by building attenuatingutations on the genetic template of the recombination-resistantRS rewired virus either for use as a safe, high titer seed stock foraking killed vaccines or as a live virus vaccine. One interesting
efinement of this approach would be to include secondary trapshat are activated in recombinant genomes. In this instance, wtRS sequences can be designed into intragenic sites in essen-
ial ORFs like the S and M glycoprotein genes. In recombinantiruses encoding wt leader TRS sequences, subgenomic tran-cription might initiate from within the essential structural genesnd the resulting N-terminal deletions would likely be lethal oreverely attenuating.
0. Potential side effects of SARS-CoV vaccines
Both humoral and T-cell-mediated responses to animal coro-aviruses may exacerbate disease or cause new health problemsZhong et al., 2005). T-cell responses have been implicated inhe demyelinization of the brain and spinal cord following infec-ion with neurotropic MHV (Castro and Perlman, 1995; Wut al., 2001), a group 2 coronavirus like SARS-CoV. Adverseumoral responses to another group 2 coronavirus, bovine coro-avirus (BCoV), have also been linked to the development ofshipping fever” in cattle (O’Connor et al., 2001). Moreover,revious exposure to FIPV, or passive or active immunizationgainst this virus, a group 1 coronavirus, was found to cause theearly death syndrome” instead of providing immune protectionPedersen et al., 1981; Weiss et al., 1980). This disease exacer-ation was due to the virus-specific antibodies that facilitatednd enhanced uptake and spread of the virus, causing ADE ofnfectivity (Porterfield, 1986; Vennema et al., 1990; Weiss andcott, 1981), that is caused by spike protein specific antibodiesCorapi et al., 1995; Corapi et al., 1992; Olsen et al., 1993; Olsent al., 1992).
With this scenario of side effects caused by some coro-avirus vaccines, a safety concern is that SARS-CoV couldnduce similar antibody- or cell-mediated immune pathologies.his concern was increased mainly by three reports. One studytilizing lentivirus pseudotyped with various SARS-CoV S pro-eins (Yang et al., 2005) indicated that within the S protein, the
ajor target for vaccine and immunotherapy, there are minorifferences among eight strains transmitted during human out-reaks in early 2003, whereas substantial functional changesere detected in S derived from a case in late 2003 from Guan-
snto
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ong province (isolate GD03) and from two palm civets (SZ16nd SZ3). The GD03 spike pseudotyped virus is markedly resis-ant to antibody neutralization. Alternatively, antibodies thateutralized most human S glycoproteins, enhanced virus entryediated by two civet cat virus S glycoproteins related to theD03 isolate (Yang et al., 2005). In another report, it has been
hown that the administration into ferrets of MVA-based SARS-oV S vaccine, but not MVA alone, followed by live SARS-CoVhallenge, resulted in enhanced hepatitis (Weingartl et al., 2004).hese side effects have not been reported in other studies withARS-CoV in ferrets, in which it has been shown that ferrets areuseful model for SARS-CoV (Martina et al., 2003). Similarly,
hese data were not reproduced by other groups (He et al., 2006).urthermore, ADE of disease has not been observed with anyuman SARS-CoV strain, therefore it will be important to assessaccines in relevant animal models as they become available.ntibodies directed against SARS-CoV were found to be protec-
ive and not to enhance viral infectivity in the mouse or hamsterodels (Bisht et al., 2004; Roberts et al., 2005b; Subbarao et
l., 2004; Yang et al., 2004), using inactivated SARS-CoV ormmunization with recombinant adenovirus vectors expressinghe S and N proteins of SARS-CoV (See et al., 2006), althoughheir effect in humans remains unknown (Zhong et al., 2005).ide effects have not been observed in other animal modelsuch as African green monkeys that, 2 months after adminis-ration of SARS-CoV into the respiratory tract, were challengedith SARS-CoV and no evidence of enhanced disease upon
e-challenge was shown (McAuliffe et al., 2004).Consistent with these results, cynomolgus macaques immu-
ized with different amounts of purified virus, in the absencer the presence of adjuvant, challenged by the nasal route 30ays post-immunization showed no side effects even in the pres-nce of low titer neutralizing antibodies. Temperature, breathing,ppetite, mental state, and all biochemical indexes were normalor immunized monkeys, and no abnormalities were observedn major organs such as lung, liver, kidney, etc. All control non-accinated animals showed interstitial pneumonia. These resultsndicate that the purified SARS-CoV vaccine is safe in monkeysQin et al., 2006). In summary, immunization of mice usingither S protein or whole inactivated virus (Spruth et al., 2006), orf monkeys with whole inactivated SARS-CoV (Qin et al., 2006;hou et al., 2005), most frequently resulted in the absence of sideffects after providing different types of SARS-CoV vaccines.
In contrast, VEE virus expressing N protein failed to inducerotection in either young or senescent animals, and resulted innhanced immunopathology following viral challenge betweenays 4 and 14 post-infection. Therefore, caution has to be takenefore including N in vaccine formulations by expressing N pro-ein using DNA immunization, or VEE vectors (Deming et al.,006), as no protection was elicited in mice against homologoushallenge, and no benefit to vaccination with a cocktail of both Snd N proteins was observed. Rather, the co-expression of N pro-ein in vaccine regimens which failed to simultaneously induce a
trong neutralizing anti-S antibody response led to an increasedumber of lymphocytic and eosinophilic inflammatory infil-rates, which are also characteristic of the immune pathologybserved with respiratory syncytial virus (RSV) infection fol-
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owing vaccination with formalin inactivated RSV (De Swartt al., 2002; Hancock et al., 1996). Therefore, the concern haseen raised that expression of N protein may result in vaccine-nhanced pulmonary disease, as previously described for virusesike RSV (Kim et al., 1969). The data suggest that the presencef N protein in vaccines should be evaluated in each vaccineormulation (Deming et al., 2006). Although thus far, no humanARS-CoV S vaccine has been shown to be involved in ADE ofisease, possible immunopathological complications of SARSaccine candidates require rigorous clinical and immunologicalvaluation.
1. Concluding remarks
Whereas the production of effective and safe vaccines fornimal coronavirus previously reported has not been satisfac-ory (Cavanagh, 2003; Enjuanes et al., 1995; Saif, 2004), theroduction of inactivated, subunit, or vaccines based on DNA,ecombinant vectors, or by reverse genetics using SARS-CoVenomes seem more promising. An optimum animal model forARS-CoV vaccine evaluation is still required. After preclinical
rials in animal models efficacy and safety evaluation of the mostromising vaccine candidates described has to be performed inumans.
2. Acknowledgements
This work was supported by grants from the Comision Inter-inisterial de Ciencia y Tecnologıa (CICYT), the Consejerıa deducacion y Cultura de la Comunidad de Madrid, Fort Dodgeeterinaria, and the European Communities (Frame VI, DIS-ECT project SP22-CT-2004-511060 and RIVIGENE projectSPE-CT-2005-022639) to LE. Marta L. DeDiego received aellowship from the Department Education and Science of Spain.nrique Alvarez received a contract from the National Institutef Health (ISCIII) of Spain. The work is also supported by theational Institutes of Health AI059136 and AI059443 to RSB.
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