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Original=20 research

Volume 363, Number 9427 =20 26 June 2004


	Articles

Mucosal = immunisation of=20 African green monkeys (Cercopithecus aethiops) = with an=20 attenuated parainfluenza virus expressing the SARS = coronavirus=20 spike protein for the prevention of SARS=20

Alexander Bukreyev, Elaine W Lamirande, Ursula J = Buchholz,=20 Leatrice N Vogel, William R Elkins, Marisa St Claire, Brian = R=20 Murphy, Kanta Subbarao, Peter L Collins

Laboratory of Infectious Diseases, National Institute = of=20 Allergy and Infectious Diseases, National Institutes of = Health,=20 Bethesda, MD, 20892, USA (A Bukreyev PhD, E W Lamirande = BS, U J=20 Buchholz PhD, L N Vogel MS, W R Elkins DVM, B R Murphy MD, K = Subbarao MBBS, P L Collins PhD); and Bioqual, Rockville, = MD,=20 20850 (M St Claire DVM)

Correspondence to: Alexander Bukreyev, Building = 50, Room=20 6505, NIAID, NIH, 50 South Drive, Bethesda, MD 20892 = (e-mail:AB176v@nih.gov)

Summary=20
Introduction=20
Methods=20
Results=20
Discussion=20
References=20

Summary =

Background The outbreak of severe acute = respiratory=20 syndrome (SARS) in 2002 was caused by a previously unknown=20 coronavirus--SARS coronavirus (SARS-CoV). We have developed = an=20 experimental SARS vaccine for direct immunisation of the = respiratory=20 tract, the major site of SARS- coronavirus transmission and = disease.=20

Methods We expressed the complete SARS = coronavirus=20 envelope spike (S) protein from a recombinant attenuated=20 parainfluenza virus (BHPIV3) that is being developed as a = live=20 attenuated, intranasal paediatric vaccine against human=20 parainfluenza virus type 3 (HPIV3). We immunised eight = African green=20 monkeys, four with a single dose of BHPIV3/ SARS-S and four = with a=20 control, BHPIV3/Ctrl, administered via the respiratory = tract. A=20 SARS-coronavirus challenge was given to all monkeys 28 days = after=20 immunisation.=20

Findings Immunisation of animals with = BHPIV3/SARS-S=20 induced the production of SARS-coronavirus-neutralising = serum=20 antibodies, indicating that a systemic immune response = resulted from=20 mucosal immunisation. After challenge with SARS coronavirus, = all=20 monkeys in the control group shed SARS coronavirus, with = shedding=20 lasting 5-8 days. No viral shedding occurred in the group = immunised=20 with BHPIV3/SARS-S.=20

Interpretation A vectored mucosal vaccine = expressing=20 the SARS-coronavirus S protein alone may be highly effective = in a=20 single-dose format for the prevention of SARS.=20

Lancet 2004; 363: 2122-27=20

See Commentary=20

Introduction =

Severe acute respiratory syndrome (SARS) emerged in = southeast=20 Asia in late 2002 and subsequently spread internationally. = To date,=20 it has resulted in more than 8000 cases and 774 = deaths.¹=20 The causative agent was quickly identified as a previously = unknown=20 member of the Coronaviridae family.^2-8 = SARS=20 infection of human beings has since been contained through=20 infection-control measures. However, resurgence is still a = threat=20 because the causative agent remains in animal reservoirs = that are=20 not fully understood and sporadic cases continue to be = reported. We=20 have an incomplete understanding of the genesis of SARS = coronavirus=20 (SARS-CoV), and there may be the potential for the emergence = of=20 variants capable of greater transmissibility. Coronaviruses = are=20 enveloped viruses with a genome that is a single strand of = positive=20 sense RNA of 30 kilobases or more in length. Sequence = analysis of=20 the RNA genome of the SARS coronavirus identified 11 open = reading=20 frames encoding proteins typical of coronaviruses, including = the=20 envelope spike (S) protein of the virus = particle.^5-7=20

SARS illness in humans is chiefly a pneumonia,=20 notwithstanding the occurrence of systemic disease signs and = detection of virus or viral RNA in other = organs.^4,9,10=20 The primary mode of transmission of SARS seems to be through = mucosal=20 membranes of the eyes, nose, or mouth;^1,9 = faecal-oral=20 transmission has also been suggested but its occurrence and = relative=20 importance have not yet been documented. The prominent role = of the=20 respiratory tract in SARS transmission and disease suggest = that=20 direct immunisation of the respiratory mucosa would be an = effective=20 strategy for immunoprophylaxis against SARS. Furthermore, = mucosal=20 immunisation of the respiratory tract with live attenuated=20 respiratory virus vaccines efficiently induces systemic as = well as=20 local immunity.^11-16

As an approach for the development of a vaccine = against SARS=20 coronavirus, we took advantage of an existing live = attenuated=20 vaccine virus, BHPIV3, that is being developed for = intranasal=20 paediatric immunisation against HPIV3 infection and=20 disease.^11,14 BHPIV3 was derived from bovine=20 (B)PIV3,^14,16 a closely-related bovine = counterpart of=20 HPIV3 that is attenuated in primates because of a natural = host=20 range-restriction. It has also been shown to be attenuated = and=20 immunogenic in humans, and is a candidate vaccine against=20 HPIV3.^14,16 BPIV3 was modified previously with=20 recombinant DNA methods to replace its F and HN protective = surface=20 antigen genes with their HPIV3 counterparts, yielding=20 BHPIV3.¹¹ BHPIV3 was an improved HPIV3 vaccine, = since it=20 bears protective antigens that exactly match HPIV3.^{11 =}

In our study, BHPIV3 was further modified by the = insertion of=20 a transcriptional cassette containing the coding sequence of = the=20 full-length S protein of SARS coronavirus. The S protein was = chosen=20 because studies with other coronaviruses showed that it is a = major=20 viral surface protein important in initiating infection.=20 Additionally, immunisation of experimental animals with the = S=20 protein of other coronaviruses induced virus-specific = immunity and,=20 in some cases, conferred protection against subsequent=20 challenge.^17-19 We investigated the replication,=20 immunogenicity, and protective effects of this experimental=20 BHPIV3/SARS-S vaccine in African green monkeys = (Cercopithecus=20 aethiops).

Methods =

Construction of BHPIV3/SARS-S and BHPIV3/Ctrl = viruses=20

The Urbani strain of SARS coronavirus was provided by = L J=20 Anderson and T G Ksiazek of the Centers for Disease Control = and=20 Prevention, Atlanta, GA, USA, and propagated in Vero=20 cells.²⁰ All experiments involving infectious = SARS=20 coronavirus were done under approved biosafety level 3=20 conditions.²⁰ Viral genomic RNA was isolated and = used in=20 RT-PCR to synthesise and amplify a 3768 bp cDNA containing = the=20 complete SARS-coronavirus S coding sequence. The cDNA was = designed=20 so that the SARS coding sequence was flanked by short = HPIV3-specific=20 transcription signals that are necessary for the foreign = gene to be=20 expressed by the transcriptional program of the BHPIV3 = vector=20 (sequences of the oligonucleotide primers and details of = RT-PCR and=20 DNA construction are available from A Bukreyev).^{11,12 =}

A PCR product of the expected length was purified and = inserted into a NotI restriction endonuclease site = that had=20 previously been introduced into a complete cloned cDNA of=20 BHPIV3,¹¹ and the sequence of the insert and = flanking=20 regions was confirmed. In this configuration, the = SARS-coronavirus S=20 insert is present as an added gene located between the = BHPIV3 P and=20 M genes (figure 1) and would be expressed as a separate mRNA = by the=20 BHPIV3 polymerase. Certain idiosyncratic features of HPIV3 = molecular=20 genetics were also accommodated: the insert was designed so = that the=20 BHPIV3 genome length remained an even multiple of six, which = is=20 required for efficient HPIV3 replication and is thought to = reflect a=20 nucleocapsid spacing requirement.²¹ In addition, = the=20 insert was designed to maintain the spacing of the = transcriptional=20 units within this hexamer organisation.²¹ The = recombinant=20 BHPIV3/SARS-S virus was recovered and propagated in cell = culture as=20 described elsewhere.^11,12

=20

Figure 1: RNA genome maps for=20 BHPIV3/SARS-S and control BHPIV3/Ctrl viruses =

N=3Dnucleocapsid protein. P=3Dphosphoprotein. = F=3Dfusion=20 glycoprotein. HN=3Dhaemagglutinin-neuraminidase = glycoprotein.=20 L=3Dpolymerase protein. Black bars at beginning and = end of each=20 gene represent PIV3-specific transcription signals, = and gap=20 between rectangles represents the PIV3 intergenic=20 trinucleotide. Leader (Le) and trailer (Tr) sequences = at 3'=20 and 5' ends of the PIV3 genome are short extragenic = regions=20 containing promoter sequences. =

We constructed and recovered the control virus using = the same=20 methods as those described for BHPIV3/SARS-S, except that = the S open=20 reading frame of the insert was replaced by a foreign = sequence of=20 identical length (derived from a complementary copy of the = genome of=20 human respiratory syncytial virus) that did not have any = significant=20 open reading frame. Thus, this control gene would be = expressed as a=20 separate mRNA, but would not encode a significant foreign = protein=20 (details of this construction available from A Bukreyev).=20

Virus growth and virological and serological = assays

BHPIV3/SARS-S and BHPIV3/Ctrl were propagated on = LLC-MK2=20 monkey kidney cells, and viral titres were determined by = limiting=20 dilution on the same cells using haemadsorption with guinea = pig=20 erythrocytes (a property of the HPIV3 HN protein) to detect = virus=20 infection.^11,12 Titres are expressed in units of = tissue=20 culture 50% infectious dose (TCID₅₀), which are = similar=20 in magnitude to a plaque forming unit.=20

Serum antibodies specific to HPIV3 were quantified by = an=20 haemagglutination inhibition (HAI) assay in which dilutions = of serum=20 were tested for the ability to block agglutination of guinea = pig=20 erythrocytes in vitro by HPIV3, in parallel with known = positive and=20 negative control standards.^11,12 SARS coronavirus = was=20 propagated in Vero monkey kidney cells, and viral titres = were=20 measured by limiting dilution in the same cells scored by a = 50% end=20 point of visible cytopathology.²⁰

Serum antibodies specific to SARS coronavirus were = quantified=20 in Vero cells by testing dilutions for the ability to = neutralise 100=20 TCID₅₀ units of SARS coronavirus per well, in = parallel=20 with known positive and negative control = standards.²⁰=20 Each sample involved four wells per dilution in a 96 well = plate, and=20 viral cytopathic effect was read on day 3 and day 4 and = expressed as=20 a 50% endpoint.=20

Detection of the S protein=20

LLC-MK2 cells were infected with BHPIV3/SARS-S or the = control=20 virus at multiples of 5 TCID₅₀ units of infection = per=20 cell, and cells were harvested 18 h = post-infection.¹² In=20 addition, the medium overlying other infected cells was = harvested at=20 48 h post-infection, and virus was concentrated by = centrifugation at=20 8000 g at 4=BAC for 18 h. The viral pellets were resuspended = and=20 subjected to centrifugation on a 30-60% w/v discontinuous = sucrose=20 gradient at 130 000 g at 4=BAC for 90 min, after which the = banded=20 virus was harvested. Western blot analysis was done with = NuPage=20 protein electrophoresis system and WesternBreeze = immunodetection kit=20 (Invitrogen, Carlsbad, CA, USA). We detected S protein with = serum=20 samples from mice infected with SARS = coronavirus²⁰ and a=20 second antibody of alkaline-phosphatase-conjugated goat = anti-mouse=20 IgG (Invitrogen).=20

Infection and challenge of African green = monkeys=20

We used eight young adult African green monkeys of = either sex=20 (bodyweight 3=B76-5=B79 kg) with a confirmed absence of = detectable serum=20 antibodies against HPIV3 or SARS coronavirus. We immunised = four=20 animals with BHPIV3/SARS-S and four with BHPIV3/Ctrl. We = used a=20 single combined intranasal and intratracheal inoculation = with=20 10⁶ TCID₅₀ units of virus in a 1 mL = inoculum=20 per site (combined dose of 10^6.3 = TCID₅₀=20 units).=20

Animals were first anaesthetised with ketamine = hydrochloride=20 given intramuscularly at a dose of 10 mg/kg, and then placed = in=20 dorsal recumbency. Through each nostril, we administered = 0=B75 mL of=20 inoculum with a sterile Luer syringe introduced about 3-5 mm = into=20 each nostril. For intratracheal inoculation, a sterile = stainless=20 steel laryngoscope was used to observe the epiglottis, and = the 1 mL=20 inoculum was delivered through a sterile flexible catheter = that had=20 been inserted about 3 cm past the epiglottal opening. We = used=20 separate sterilised instruments for every animal. = Nasopharyngeal=20 swabs were taken on days 0-10, 12, and 14, and tracheal = lavages were=20 done on days 2, 4, 6, 8, 10, and 14,²² and = titration of=20 BHPIV3/SARS-S and BHPIV3/Ctrl was done as described = previously. For=20 tracheal lavage, we used a laryngoscope and catheter placed = as=20 described above, with a 2 mL wash volume of = phosphate-buffered=20 saline that was instilled and aspirated back. For the=20 SARS-coronavirus challenge, the animals were given a similar = combined intranasal and intratracheal inoculation with=20 10⁶ TCID₅₀ units of virus in a 1 mL = inoculum=20 at each site. Nasopharyngeal swabs and tracheal lavages were = obtained on the same schedule as that used after the initial = immunisation. These experiments were done in an approved = animal=20 biosafety level 3 facility. Virus titration was done as = described=20 earlier. We took serum samples 1 day before immunisation = (day -1), 1=20 day before the challenge (day 27), and 28 days post = challenge (day=20 56), which were analysed for virus-specific antibodies as = described=20 above.=20

The primate study was approved by the National = Institutes of=20 Health (USA) Animal Care and Use Committee and was done in a = laboratory approved by the Association for Assessment and=20 Accreditation of Laboratory Animal Care International.=20

Role of the funding source

The sponsor of the study had no role in study design, = data=20 collection, data analysis, data interpretation, or in the = writing of=20 the report.

Results =

BHPIV3 was modified to express the envelope spike S = protein=20 of SARS coronavirus (figure 1). The recombinant = BHPIV3/SARS-S virus=20 was recovered and propagated in cell culture in parallel = with the=20 control virus, BHPIV3/Ctrl. The presence of foreign inserts = did not=20 affect the efficiency of replication of the vector in vitro. = The=20 BHPIV3 genome is a single strand of negative sense RNA of = 15=B75=20 kilobases that contains six non-overlapping genes (figure = 1). The=20 virally-encoded polymerase initiates at the left end of the = genome=20 and transcribes each gene in turn, including any inserted = foreign=20 gene, into a separate mRNA. The correct expression of the S = sequence=20 as a separate mRNA was confirmed by northern blot analysis = of=20 intracellular RNA isolated from LLC-MK2 cells infected with=20 BHPIV3/SARS-S (data not shown).=20

Expression of the SARS-coronavirus S protein was = confirmed by=20 western blot analysis of lysates of BHPIV3/SARS-S-infected = cells=20 with serum of mice that had been infected with SARS=20 coronavirus²⁰ (figure 2A, lane 3). This showed = that the S=20 protein migrated as two major, diffuse bands of about 170 = and 200=20 kDa. The two forms of the S protein probably differ by the = extent of=20 glycosylation; based on the nucleotide sequence, the = predicted full=20 length unmodified S protein is 138 kDa and has 23 potential=20 glycosylation sites.^5-7 There was no evidence of=20 proteolytic cleavage of the SARS S protein into two = subunits, as=20 occurs with some coronaviruses. Since the SARS-coronavirus S = protein=20 is a transmembrane virion component and BHPIV3 also is an = enveloped=20 virus, we wanted to determine whether the S protein was = incorporated=20 into BHPIV3 particles. Western blot analysis did not detect = the S=20 protein in purified, concentrated BHPIV3/SARS-S virus = particles=20 (figure 2A, lane 1) under conditions where direct Coomassie = staining=20 (figure 2B, lane 2) showed that viral protein was present in = quantities greater than that required for detection by = western blot.=20 The absence of significant incorporation of S protein into = virions=20 suggests that its expression would be unlikely to affect the = tropism=20 of the vector and is a safety factor.=20

=20

Figure 2: Expression of = SARS-coronavirus S=20 protein by BHPIV3/SARS-S in cell culture and its = absence in=20 the BHPIV3 virus particle

A: western blot analysis shows presence of the=20 SARS-coronavirus S protein in lysates of cells = infected with=20 BHPIV3/SARS-S (lane 3) and its absence in lysates from = cells=20 infected with BHPIV3/Ctrl (lane 4). S protein was not=20 detectable in purified, concentrated BHPIV3/SARS-S = virus=20 particles (lane 1) or in the negative control = BHPIV3/Ctrl=20 (lane 2). B: direct Coomassie staining of a gel loaded = with=20 replicate samples of purified, concentrated = BHPIV3/Ctrl (lane=20 1) and BHPIV3/SARS-S (lane 2) shows viral protein was = present=20 in excess of what should be necessary for detection by = western=20 blot analysis.

Table 1 shows the monkeys' responses to immunisation. = Analysis of the nasal swab samples taken after immunisation = showed=20 that the peak titre (3=B79 vs 4=B73 log₁₀=20 TCID₅₀/mL) and duration of shedding (5=B78 = vs 7=B78=20 days) from the upper respiratory tract did not differ = significantly=20 between BHPIV3/SARS-S and BHPIV3/ Ctrl. Analysis of the = tracheal=20 lavage samples, a measure of shedding from the lower = respiratory=20 tract, showed that two of the four animals infected with=20 BHPIV3/SARS-S did not have detectable virus shedding, = whereas the=20 other two showed some shedding at 2 days but this was less = than for=20 the BHPIV3/Ctrl-immunised animals (table 1). Thus, = expression of the=20 SARS S protein had an attenuating effect on the replication = of the=20 vector, particularly in the lower respiratory tract. = Nevertheless,=20 BHPIV3/SARS-S and BHPIV3/Ctrl each induced a moderate titre = of=20 HPIV3-specific antibodies as measured by an HAI assay (table = 1). In=20 addition, BHPIV3/SARS-S induced a detectable level of serum=20 antibodies that neutralised SARS-S coronavirus in vitro = (mean=20 reciprocal titre of 3=B79 log₂).=20

	BHPIV3 shedding				Serum antibodies
	Nasal swab		Tracheal lavage		HPIV3 HAI titre (reciprocal log₂)	SARS-coronavirus neutral-ising titre (reciprocal=20 log₂)
	Duration (days)	Peak titre (log₁₀ = TCID₅₀/mL)	Duration (days)	Peak titre (log₁₀ = TCID₅₀/mL)
BHPIV3/SARS-S
Monkey
V101 (F)	3	3=B77	0	0=B75*	9	4=B71
V104 (F)	5	3=B75	0	0=B75	8	3=B72
V117 (F)	7	3=B72	1	2=B70	8	4=B72
V191 (M)	8	5=B70	3	2=B75	9	4=B72
BHPIV3/Ctrl
Monkey
V099 (F)	9	4=B75	9	3=B75	8	2=86
V103 (F)	6	5=B70	5	6=B75	9	2
V107 (F)	6	3=B75	3	3=B75	9	2
122 (F)	10	4=B72	5	2=B77	10	2
F=3Dfemale. M=3Dmale. Serum samples = taken 1 day=20 before immunisation did not have detectable HAI = antibodies=20 (detection limit 1=B70 reciprocal log₂ = dilution=20 units) or detectable SARS-coronavirus-neutralising = antibodies=20 (lower limit of detection was 2 reciprocal log₂). *Lower limit = of=20 detection of virus. =86Lower limit of detection of = serum=20 neutralising antibodies.
Table 1: Responses to mucosal=20 immunisation with BHPIV3/SARS-S and=20 BHPIV3/Ctrl

Table 2 shows responses to challenges with SARS = coronavirus.=20 All four monkeys immunised with BHPIV3/Ctrl shed challenge = SARS=20 coronavirus from the upper and lower respiratory tract. By = contrast,=20 none of the animals immunised with BHPIV3/SARS-S shed SARS=20 coronavirus on any day from either the upper or lower = respiratory=20 tract. SARS-coronavirus-neutralising serum antibody titres = 28 days=20 after the challenge are shown in table 2. The increase in = titre in=20 the BHPIV3/SARS-S group after the challenge could be the = result of=20 an immune response to the antigen present in the virus = inoculum or=20 to a low level of replication of SARS coronavirus.=20

	SARS-coronavirus shedding			Serum antibodies
	Nasal swab		Tracheal lavage		SARS-coronavirus neutralising titre (reciprocal=20 log₂)
	Duration (days)	Peak titre (log₁₀ = TCID₅₀/mL)	Duration (days)	Peak titre (log₁₀ = TCID₅₀/mL)
BHPIV3/SARS-S
Monkey
V101 (F)	0	0=B75*	0	0=B75*	7=B72
V104 (F)	0	0=B75	0	0=B75	8=B72
V117 (F)	0	0=B75	0	0=B75	7=B72
V191 (M)	0	0=B75	0	0=B75	7=B72
BHPIV3/Ctrl
Monkey
V099 (F)	7	1=B75	3	3=B75	7=B71
V103 (F)	5	1=B77	0	0=B75	6=B72
V107 (F)	7	3=B70	3	2=B75	7=B70
V122 (F)	7	1=B75	1	3=B70	7=B72
F=3DFemale. M=3Dmale. *Lower limit of = detection of=20 virus.
Table 2: Responses to challenge = with=20 SARS coronavirus in monkeys immunised with = BHPIV3/SARS-S or=20 BHPIV3/Ctrl

Discussion =

We have developed an experimental SARS vaccine using = an=20 existing live attenuated HPIV3 vaccine candidate, BHPIV3, as = a=20 vector to express the SARS-coronavirus S protein. The use of = this=20 respiratory virus as a vector provides for direct = immunisation of=20 the respiratory tract, the main site of SARS coronavirus=20 transmission and disease. The monkeys that were vaccinated = with=20 BHPIV3/SARS-S were highly protected against the challenge = infection.=20 This finding identified the S protein as a major protective = antigen=20 of SARS coronavirus and indicates that vaccines against SARS = should=20 include this protein.=20

Topical immunisation of the respiratory tract also = induced=20 detectable SARS-coronavirus-neutralising serum antibodies, = evidence=20 of a systemic immune response. However, the = post-immunisation titre=20 induced by BHPIV3/SARS-S was almost eight-fold lower than = that=20 achieved by SARS-coronavirus infection of the = BHPIV3/Ctrl-immunised=20 animals. This difference in immunogenicity might reflect the = natural=20 restriction of HPIV3 to the respiratory tract, whereas SARS=20 coronavirus disseminates systemically, at least in non-human = primates.^8,9 There was no evidence of = immune-mediated=20 enhancement of infection or disease, which occurs for one=20 coronavirus, feline infectious peritonitis = virus.²³ Thus,=20 our results show that one mucosal immunisation with vectored = SARS S=20 protein was sufficient to protect against shedding after a = large=20 challenge dose of SARS coronavirus.=20

Some cynomolgus monkeys (Macaca fascicularis) = infected=20 with SARS coronavirus have been reported to develop clinical = disease=20 signs including a transient rash, respiratory distress, and=20 lethargy.^24,8 We have investigated three species = of=20 monkeys, namely cynomolgus, rhesus (M mulatta), and = African=20 green monkeys, for permissiveness for SARS-coronavirus = replication=20 and possible disease (unpublished data). Of the three, = African green=20 monkeys supported the highest levels of replication as = measured by=20 virus shedding and hence were used in this study. None of = the 12=20 animals in any of the three species developed overt disease = signs,=20 and we did not note disease signs in this study. Mice, = ferrets, and=20 cats have been shown to support high levels of pulmonary=20 SARS-coronavirus replication, and some of the infected = ferrets=20 became lethargic.^{25, 20} We did not assess = BHPIV3/SARS-S=20 in mice because the HPIV3 vaccine vector is severely = restricted for=20 replication in this animal, and the cat and ferret models = were only=20 recently reported. Thus, although our study showed complete=20 protection against shedding of challenge virus, we could not = assess=20 whether protection would be afforded against clinical = disease signs.=20 This study limitation is not unusual. With respect to human = viruses,=20 experimental animals rarely provide faithful models of the = infection=20 and disease that is observed in humans, and disease signs = often are=20 minimal, altered, or absent. The measurement of infectious = challenge=20 virus in secretions or other samples from experimental = animals is a=20 general standard for measuring the efficacy of viral = vaccines. The=20 assessment of an experimental vaccine in a non-human primate = is=20 especially important in view of the phylogenetic and = anatomical=20 similarity to humans, and is an appropriate last step before = clinical trials.=20

Attenuated versions of HPIV3 are under active = development as=20 vaccines for intranasal immunisation of infants and young = children,=20 as are HPIV3-based vectored vaccines that also express = protective=20 antigens of respiratory pathogens such as measles virus, = respiratory=20 syncytial virus, and metapneumovirus.^12,13,15 = HPIV3=20 efficiently infects the respiratory tract but does not = spread far=20 beyond it, which is an important safety factor. HPIV3-based = vectors=20 have proven effective in inducing local and systemic = immunity=20 against a number of foreign antigens.^12,13,15=20 Furthermore, safe intranasal administration of attenuated = HPIV3 and=20 related viruses has been shown possible.^16,26,27 = An=20 additional safety feature is that RNA recombination is = almost=20 non-existent in nature for the family of viruses represented = by=20 HPIV3, whereas recombination is extremely frequent in = coronaviruses,=20 and the potential for recombination with circulating human=20 coronavirus would be a concern for a live-attenuated=20 SARS-coronavirus vaccine virus.=20

As currently constructed, the BHPIV3/SARS-S vector is = an=20 excellent candidate for clinical testing as a vaccine that = is likely=20 to be highly attenuated, safe, and effective against both = HPIV3 and=20 SARS for infants and young children, in whom the vector = would=20 replicate efficiently. This vaccine would be especially = useful if a=20 more transmissible version of SARS coronavirus emerges and=20 immunisation of infants and children is needed. However, any = replicating viral vector bearing the protective antigens of = a common=20 human pathogen, such as adenovirus or HPIV3, is unlikely to=20 replicate sufficiently well in adults to be immunogenic = because of a=20 prevalence of neutralising antibodies to such=20 pathogens.^26,27 Fortunately, parainfluenza = viruses have=20 been amenable to swapping of the surface proteins without = loss of=20 infectivity in vitro or in vivo.^28,29 Therefore, = it=20 should be possible to replace the HPIV3 HN and F surface = proteins=20 with those of an antigenically-distinct parainfluenza virus = for=20 which the general population lacks immunity--in particular = one of=20 the many avian parainfluenza viruses such as an attenuated = strain of=20 Newcastle disease virus. The resulting vector would be a = useful SARS=20 vaccine for mucosal immunisation of the entire human = population.=20

Contributors=20

A Bukreyev participated in the design of the = constructs,=20 construction and in-vitro characterisation of the = recombinant=20 viruses, the design of the monkey experiment, = SARS-coronavirus=20 titrations, and interpretation of the results. E Lamirande=20 participated in the construction and in-vitro = characterisation of=20 the recombinant viruses, SARS-coronavirus titrations, and = serum=20 neutralisation assays. U Buchholz gave useful = recommendations on=20 BHPIV3 recovery, did northern blot analysis, and = participated in=20 SARS-coronavirus titrations. L Vogel prepared challenge = virus and=20 participated in serum neutralisation assays. W R Elkins and = M St=20 Claire supervised the animal experiments. B Murphy = participated in=20 planning of the experiments and interpretation of the = results. K=20 Subbarao prepared SARS-coronavirus RNA and mouse=20 SARS-coronavirus-immune serum, established the African green = monkey=20 model for SARS coronavirus, and participated in the planning = of the=20 experiments and interpretation of the results. P Collins=20 participated in design of the constructs, planning of the=20 experiments and interpretation of the results, and providing = general=20 guidance for the project.

Conflict of = interest=20 statement

None declared.

Acknowledgments=20

We thank Kim-Chi Tran for technical assistance and = Kathryn=20 Hanley for assistance with statistical analysis. This = project was=20 funded as part of the NIAID Intramural Program.

References =

1 WHO. Communicable disease surveillence and = response: severe=20 acute respiratory syndrome (SARS) http://www.who.int/csr/sars/en.=20 (accessed Dec 31, 2003).=20

2 Ksiazek TG, Erdman D, Goldsmith CS, et al. A novel=20 coronavirus associated with severe acute respiratory = syndrome.=20 N Engl J=20 Med 2003; 348: 1953-66. [PubMed]=20

3 Poutanen SM, Low DE, Henry B, et al. Identification = of=20 severe acute respiratory syndrome in Canada. N Engl = J=20 Med 2003; 348: 1995-2005. [PubMed]=20

4 Drosten C, Gunther S, Preiser W, et al. = Identification of a=20 novel coronavirus in patients with severe acute respiratory=20 syndrome. N Engl J=20 Med 2003; 348: 1967-76. [PubMed]=20

5 Rota PA, Oberste MS, Monroe SS, et al. = Characterization of=20 a novel coronavirus associated with severe acute respiratory = syndrome.=20 = Science 2003; 300: 1394-99. [PubMed]=20

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7 The complete genome of the SARS associated = Coronavirus.=20 Gene Bank sequence AY274119 2003.=20

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