Vol. 67, No. 3JOURNAL OF VIROLOGY, Mar. 1993, p. 1503-15100022-538X/93/031503-08$02.00/0Copyright © 1993, American Society for Microbiology

Bovine Respiratory Syncytial Virus Protects Cotton Ratsagainst Human Respiratory Syncytial Virus Infection

FRANCO M. PIAZZA,"* SUSAN A. JOHNSON,' MIRIAM E. R. DARNELL,2 DAVID D. PORTER,3VAL G. HEMMING,4 AND GREGORY A. PRINCE2

Children's National Medical Center and Department of Pediatrics, The George Washington University Schoolof Medicine and Health Sciences, Washington, D.C. 200101; Virion Systems, Inc., Rockville, Maryland208502; Department of Pathology and Laboratory Medicine, University of California School of Medicine,Los Angeles, California 900243; and Department of Pediatrics, Unifonned Services University of the

Health Sciences, Bethesda, Maryland 208144

Received 24 August 1992/Accepted 30 November 1992

Human respiratory syncytial virus (HRSV) is the most frequent cause of severe respiratory infections ininfancy. No vaccine against this virus has yet been protective, and antiviral drugs have been of limited utility.Using the cotton rat model of HRSV infection, we examined bovine respiratory syncytial virus (BRSV), a cause

of acute respiratory disease in young cattle, as a possible vaccine candidate to protect children against HRSVinfection. Cotton rats were primed intranasally with graded doses of BRSV/375 or HRSV/Long or were leftunprimed. Three weeks later, they were challenged intranasally with either BRSV/375, HRSV/Long (subgroupA), or HRSV/18537 (subgroup B). At intervals postchallenge, animals were sacrificed for virus titration andhistologic evaluation. Serum neutralizing antibody titers were determined at the time of viral challenge.BRSV/375 replicated to low titers in nasal tissues and lungs. Priming with 10i PFU of BRSV/375 effected a 500-to 1,000-fold reduction in peak nasal HRSV titer and a greater than 1,000-fold reduction in peak pulmonaryHRSV titer upon challenge with HRSV/Long or HRSV/18537. In contrast to priming with HRSV, priming withBRSV did not induce substantial levels of neutralizing antibody against HRSV and was associated with a delayedonset of clearance ofHRSV upon challenge. Priming with BRSV/375 caused mild nasal and pulmonary pathologyand did not cause exacerbation of disease upon challenge with HRSV/Long. Our findings suggest that BRSV maybe a potential vaccine against HRSV and a useful tool for studying the mechanisms of immunity to HRSV.

Human respiratory syncytial virus (HRSV) was discov-ered in 1955 during an outbreak of respiratory disease in achimpanzee colony (22) and was later shown to be a ubiqui-tous human respiratory pathogen (7). HRSV is the singlemost important respiratory pathogen of infancy and earlychildhood worldwide (5). No vaccine to prevent HRSVinfection has yet been protective. Antiviral drugs have beenof limited utility in the treatment of HRSV disease.

In 1967, a virus related to HRSV was isolated from cattlein Europe (28). This agent, known as bovine respiratorysyncytial virus (BRSV), causes severe lower respiratorytract disease in calves similar to the illness caused by HRSVin infants. BRSV occurs and causes serious disease inbovine herds throughout the world (18).The idea of using an agent of animal origin to protect

humans against a related human pathogen was the basis ofthe first vaccine. Jenner used cowpox virus, a natural bovinepathogen, to immunize humans against smallpox. The Jen-nerian approach is currently being used in the developmentof live-virus vaccine strains to immunize against parainflu-enza type 3 virus (PIV3), rotavirus, and influenza virusinfections (6). In the present study, we have used the cottonrat, a well-established model of HRSV infection (32) andvaccine-induced potentiation of HRSV lung disease (31), toevaluate the feasibility of using BRSV as a vaccine againstHRSV.

* Corresponding author.

MATERIALS AND METHODSAnimals. Young adult inbred cotton rats (Sigmodon hispi-

dus) were obtained from a colony maintained by VirionSystems, Inc., Rockville, Md., and were housed and fed aspreviously described (32). Animals were shown to be free ofserum neutralizing antibody against HRSV prior to inclusionin the study.

Viruses. The BRSV strain used in these studies, BRSV/375, was obtained from Howard Lehmkuhl, U.S. Depart-ment of Agriculture, Ames, Iowa (19). Virus stocks weregrown in bovine nasal turbinate cell monolayers (obtainedfrom the same source) to a titer of 106 PFU/ml. BRSV/375was chosen for evaluation as an immunogen against HRSVbecause previous studies showed that among six differentstrains of BRSV tested in cotton rats, BRSV/375 was iso-lated from the highest percentage of inoculated animals andreplicated to the highest titer (29a).Two prototype strains of HRSV were used: the Long

strain (subgroup A) and the 18537 strain (subgroup B).Stocks of these viruses were prepared in HEp-2 cells andcontained 106 PFU/ml. PIV3 (lot 0691) was obtained fromProgram Resources, Inc., Rockville, Md., and contained106 5PFU/ml.Experimental protocol. Cotton rats were anesthetized by

methoxyflurane inhalation and inoculated with either BRSV/375, HRSV/Long, HRSV/18537, or PIV3. All inoculations(priming and challenge) were performed intranasally, using0.1 ml of virus suspension. Animals were sacrificed bycarbon dioxide intoxication at intervals following inocula-tion.For virus titration, nasal tissues and lungs were homoge-

nized in 10 parts (wt/vol) of Hanks balanced salt solution

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supplemented with 0.218 M sucrose-4.4 mM glutamate-3.8mM KH2PO4-3.2 mM K2HPO4, and the resulting suspensionwas stored at -70°C until assayed.For histologic preparation of nasal tissues, the skin was

removed from the head and the skull was fixed in 10%neutral buffered formalin. For preparation of lungs, thesewere removed from the thorax and inflated through thetrachea with 10% formalin.Animals were bled from the retro-orbital venous plexus,

and serum was stored at -20°C until assayed for neutralizingantibody titers.Virus assays. Virus titers for HRSV and PIV3 were deter-

mined by plaque assay on HEp-2 cell monolayers as previ-ously described (32). Titration for BRSV was performed onbovine nasal turbinate cell monolayers, which were incu-bated with tissue homogenates for 6 to 7 (instead of 4) days.Virus titers were expressed as the geometric mean of indi-vidual titers (PFU per gram of tissue plus or minus thestandard error) for each animal group.Antibody assays. Serum neutralizing antibody against

HRSV was measured by a plaque reduction neutralizationassay on HEp-2 cell monolayers, using a 60% plaque reduc-tion end point, as previously described (32). Neutralizingantibody against BRSV was determined by a similar assayusing bovine nasal turbinate cells and BRSV instead ofHEp-2 cells and HRSV, respectively. Antibody titers wereexpressed as the geometric mean of individual titers for allanimals at a given time.

Pathologic study. Formalin-fixed nasal tissues (decalcified)and lungs were embedded in paraffin, cut into coronalsections, and stained with hematoxylin-eosin (32). Threesections of nasal tissues and one of lungs from each animalwere examined by light microscopy. Pathologic changeswere scored blindly by using a qualitative scoring system aspreviously described (29).

Statistical analysis. Geometric means of virus titers ofexperimental groups were compared with those of controlgroups by using the two-tailed Student t test of summarydata. Antigenic relatedness between BRSV/375 and HRSV/Long was determined by using Archetti and Horsfall'sformula (1), r = Vrj x r2, where r, is heterologous titer ofvirus 2/homologous titer of virus 1 and r2 is heterologoustiter of virus 1/homologous titer of virus 2. This formulastipulates that values of r of 2 or more indicate significantantigenic dissimilarity.

RESULTS

Growth of BRSV. Cotton rats were inoculated with 104PFU of BRSV/375 and sacrificed at 5 min, 12, 18, and 24 h,and 2, 3, 4, 5, 6, and 8 days for virus titration (Fig. 1A). Afteran eclipse phase at 12 h, BRSV/375 reached a peak titer at 24h in the lungs and at 2 days in nasal tissues. BRSV/375replicated to low titers but was isolated from all the animalsstudied on days 1 through 5. For comparison, cotton ratswere inoculated with 104 PFU of HRSV/Long and sacrificedat 12 h and at 2, 4, 6, and 8 days for virus titration (Fig. 1B).After an eclipse phase at 12 h, HRSV/Long replicated tomuch higher titers than did BRSV/375.

Effect of priming with BRSV on replication of subgroup AHRSV. The effect of priming with various doses of BRSVupon subsequent challenge with subgroup A HRSV wasassessed. Cotton rats inoculated intranasally with gradeddoses of BRSV/375 (101 to 105 PFU in a volume of 0.1 ml) orleft unprimed were challenged intranasally 3 weeks laterwith 105 PFU of HRSV/Long and sacrificed 4 days post-

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DAYS POST-INOCULATIONFIG. 1. Replication of BRSV/375 and HRSV/Long. Cotton rats

were inoculated with 104 PFU of BRSV/375 and sacrificed at 5 min,12, 18, and 24 h, and 2, 3, 4, 5, 6, and 8 days (A) or HRSV/Long andsacrificed at 12 h and at 2, 4, 6, and 8 days (B). Each point representsthe geometric mean virus titer (log,o) of four to eight animals; barsindicate standard errors.

challenge (at the time of peak viral replication in unprimedanimals) (Fig. 2A).The effectiveness of priming with BRSV in the induction

of protection against challenge with HRSV was comparedwith that of priming with HRSV. Cotton rats inoculated withgraded doses of HRSV/Long (101 to 105 PFU in a volume of0.1 ml) or left unprimed were challenged 3 weeks later with105 PFU of the same virus and sacrificed 4 days postchal-lenge (Fig. 2B).The animals primed with the highest dose of BRSV/375

(105 PFU) and subsequently challenged with HRSV/Longhad HRSV titers 4 days postchallenge of 1.2 x 102 PFU/g innasal tissues and <102 PFU/g (undetectable) in lungs (Fig.2A). Unprimed control animals had HRSV titers in nasaltissues and lungs of 1.6 x 105 and 3.7 x 104 PFU/g,respectively. These differences in virus titers were highlysignificant (P < 0.001 for both comparisons). In the animalsprimed with lower doses of BRSV/375, the postchallengeHRSV titers increased as the dose of BRSV/375 decreased.A significant reduction in nasal titers was demonstrated witha priming BRSV dose as low as 103 PFU. A significantreduction in lung titers occurred with a priming BRSV doseas low as 102 PFU.The titer of serum neutralizing antibody against HRSV/

Long at the time of challenge with this virus was very low

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FIG. 3. Effect of priming with BRSV/375 on virus titers followingHRSV/LONG HRSV/18537 challenge. Cotton rats were inoculated with 104 or 10

PFU of BRSV/375 or left unprimed, challenged 3 weeks later with105 PFU of HRSV/18537, and sacrificed 4 days postchallenge. Thegeometric mean virus titers (log1o) and standard errors are shown.

* Lungs Virus titers significantly lower than control values are marked with

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FIG. 2. Effect of the priming dose of BRSV/3on virus titers following HRSV/Long challenge.inoculated with graded doses (101 to 105 PFU) ofHRSV/Long (B) or left unprimed, challenged 3 w(

PFU of HRSV/Long, and sacrificed 4 days p

geometric mean virus titers (log10) and standardVirus titers significantly lower than control valueasterisks. The serum neutralizing antibody tite(reciprocal of the geometric mean) at the timeshown. The number of animals studied is in pare

(1:21) or undetectable (<1:20) for all grnprimed animals (Fig. 2A).The animals primed with the highest dose

(105 PFU) and subsequently challenged witlhad HRSV titers 4 days postchallenge of 1.Cnasal tissues and <102 in lungs (Fig. 2B). Uanimals had HRSV titers in nasal tissues an

105 and 2.0 x 105 PFU/g, respectively. Thevirus titers were highly significant (P <

comparisons). As seen in animals primed'level of protection was proportionate to the

P < 0.001

HRSV. A significant reduction in nasal and in lung titers wasseen with a priming HRSV dose as low as 102 PFU.The titers of serum neutralizing antibody against HRSV/

Long at the time of challenge with this virus were 1:305 and* * * * 1:418 for the groups primed with 104 and 105 PFU of HRSV,

305 305 418 418 respectively (Fig. 2B). Those groups primed with lower_ _ __ doses had titers of 1:73 or lower.

4 5 In comparison with BRSV/375, a lower priming dose of10 10 HRSV/Long effected a comparable degree of protection(7) (10) against challenge with HRSV/Long (Fig. 2).

Effect of priming with BRSV on replication of subgroup B'FU/animal) HRSV. The effect of priming with BRSV upon subsequent

75 or HRSV/Long challenge with subgroup B HRSV was assessed. Cotton ratsCotton rats were inoculated with 104 or 105 PFU of BRSV/375 or left

f BRSV/375 (A) or unprimed were challenged 3 weeks later with 105 PFU ofeeks later with 105 HRSV/18537 and sacrificed 4 days postchallenge (Fig. 3).ostchallenge. The The animals primed with the highest dose of BRSV/375

errors are showni and challenged with HRSV/18537 had HRSV titers 4 daysrs against HRSV postchallenge of 1.1 x 102 PFU/g in nasal tissues and <102of challenge are PFU/g in lungs (Fig. 3). The respective titers in unprimed

ntheses. control animals were 3.1 x 105 and 5.7 x 104 PFU/g. Thesedifferences in virus titers were again highly significant (P <

0.001 for both comparisons). The animals primed with 104PFU of BRSV/375 had HRSV titers that were significantly

oups of BRSV- lower than those of unprimed controls (Fig. 3). The level ofprotection was comparable to that observed in animals

of HRSV/Long primed with the same dose of BRSV/375 and challenged withh the same virus HRSV/Long (Fig. 2A).) x 102 PFU/g in Antigenic cross-reactivity between BRSV and HRSV. SeraInprimed control obtained from convalescent cotton rats infected 3 weeksId lungs of 1.9 x earlier with 105 PFU of BRSV/375 were tested for neutral-se differences in izing antibodies against BRSV/375 and HRSV/Long. Also,0.001 for both sera obtained from animals infected 3 weeks earlier with 105with BRSV, the PFU of HRSV/Long were tested for antibodies against;priming dose of HRSV/Long and BRSV/375 (Table 1). The degree of anti-

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TABLE 1. Antigenic cross-reactivity between BRSV and HRSVin cotton rats

Reciprocal of geometric mean neutralizingSeruma antibody titer (no. of animals tested)

BRSV/375 HRSV/Long

BRSV/375 586 (33) 22 (33)HRSV/Long 551 (10) 418 (11)

a Sera obtained from cotton rats infected 3 weeks earlier with 105 PFU ofBRSV/375 or HRSV/Long were assayed individually for neutralizing antibodytiters against each of the viruses by plaque reduction assay.

genic relatedness between BRSV and HRSV in vitro wasdetermined by using Archetti and Horsfall's formula (1).This formula yielded an r value of 4.5, which indicates highlysignificant antigenic divergence between BRSV/375 andHRSV/Long.

Specificity of BRSV in the induction of immunity to HRSV.The specificity of priming with BRSV in the induction ofimmunity to HRSV was examined. Cotton rats inoculatedwith 105 PFU of BRSV/375 or left unprimed were challenged3 weeks later with either 105-5 PFU of PIV3 or 105 PFU ofHRSV/Long and sacrificed 4 days postchallenge for virustitration (Fig. 4).The animals primed with BRSV/375 and subsequently

challenged with PIV3 had PIV3 titers of 5.8 x 104 PFU/g innasal tissues and 2.6 x 105 PFU/g in lungs (Fig. 4A). Therespective titers in unprimed control animals were 2.4 x 105and 2.7 x 105 PFU/g. Nasal PIV3 titers were lower in theBRSV-primed group than in the unprimed controls (P <0.005), but pulmonary titers were not different.The animals primed with BRSV/375 and challenged with

HRSV/Long had HRSV titers of 1.0 x 102 PFU/g in nasaltissues and <102 PFU/g (undetectable) in lungs (Fig. 4B).The respective titers in unprimed control animals were 4.1 x104 and 6.5 x 103 PFU/g. These differences in virus titerswere highly significant (P < 0.001 for both comparisons).

Effect of priming with BRSV on kinetics of HRSV clearanceupon challenge. Cotton rats inoculated with 104 PFU ofeither BRSV/375 or HRSV/Long or left unprimed werechallenged 3 weeks later with 105 PFU of HRSV/Long.Groups of animals were sacrificed on days 2, 4, 6, and 8postchallenge for virus titration (Fig. 5).The unprimed animals had mean HRSV titers in nasal

tissues and lungs that were high on day 2, peaked above 105PFU/g on day 4, and decreased to near undetectable levelsby day 8. The HRSV-primed animals had a mean HRSV titerin nasal tissues that was very low on day 2 and decreased toundetectable levels by day 4; HRSV was not detected inlungs on any day. The BRSV-primed animals had a meanHRSV titer in nasal tissues that was above 104 PFU/g on day2 and decreased to undetectable levels by day 6; the HRSVtiter in lungs was lower than 103 PFU/g on day 2 and belowdetectable levels by day 6.

Effect of priming with BRSV on the pathology of HRSVinfection. Cotton rats inoculated with 104 PFU of BRSV/375or left unprimed were challenged 3 weeks later with either105 PFU of HRSV/Long or 10 PFU of BRSV/375 (Table 2).Subgroups of these animals were sacrificed on days 2, 4, 6,and 8 postchallenge for histologic evaluation and virustitration. Additional animals that were not inoculated witheither virus were sacrificed for histologic evaluation.No pathological changes were identified in uninoculated

control animals (Table 2, group D). Unprimed animals

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FIG. 4. Effect of priming with BRSV/375 on virus titers followingPIV3 or HRSV/Long challenge. Cotton rats were inoculated with105 PFU of BRSV/375 or left unprimed, challenged 3 weeks laterwith either 105 5 PFU of PIV3 (A) or 105 PFU of HRSV/Long (B),and sacrificed 4 days postchallenge. The geometric mean virus titers(loglo) and standard errors are shown. Virus titers significantly lowerthan control values are marked with asterisks. The number ofanimals studied is in parentheses.

challenged with BRSV/375 (Table 2, group C) experiencedmild pathological changes. Epithelial clefting of the nasalmucosa was seen only on day 2. Bronchiolitis peaked on day6, and interstitial pneumonia was minimal throughout. Themean BRSV titer in nasal tissues was 1.2 x 103 PFU/g onday 2 and dropped to undetectable levels by day 6. TheBRSV titer in lungs remained very low (<2.0 x 102 PFU/g)throughout.Unprimed animals challenged with HRSV/Long (Table 2,

group B) had much more impressive pathological changes.Clefting of the nasal mucosa was prominent, peaking on day6 (4+) and decreasing but still persisting by day 8. Bronchi-olitis was clearly present on day 4, peaked on day 6 (3 +/4+),and decreased by day 8. Interstitial pneumonia was moreprominent than in any of the other groups, being easilydiscernible on day 4 and peaking on day 6. The mean HRSVtiter in nasal tissues was above 105 PFU/g on day 2, peaked

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TABLE 2. Effect of priming with BRSV on the pathology ofHRSV infectiona

Days postchallengeDetermination Group

2 4 6 8

Pathology scorebBronchiolitis A 1+ 1+/2+ 2+ 1+/2+

B 1+ 2+13+ 3+14+ 1+C 1+ 1+ 3+ 1+D 0 0 0 0

Interstitial A 0 1+ 0/1+ 0pneumonia B 0 2+ 3+ 0

C 0 1+ 1+/2+ 0D 0 0 0 0

Nasal pathology A 0 1+/2+ 1+ 0B 1+ 2+/3+ 4+ 2+C 0/1+ 0 0 0D 0 0 0 0

Virus titercLung A 2.67 2.29 <2.0 <2.0

B 4.78 5.08 3.18 2.19C 2.15 2.19 2.12 <2.0

Nose A 4.17 3.53 <2.0 <2.0B 5.26 5.31 3.19 2.67C 3.08 2.61 <2.0 2.0

Pnmed a Cotton rats were divided into four groups. Animals in group A (32Pnmed animals) were inoculated with 104 PFU of BRSV/375 and challenged 3 weeks

later with 105 PFU of HRSV/Long. Animals in group B (32 animals) remainedunprimed and were challenged with 105 PFU of HRSV/Long (at the time ofgroup A challenge). Animals in group C (32 animals) remained unprimed andwere challenged with 104 PFU of BRSV/375 (at the time of group A challenge).Animals in group D (eight animals) were not inoculated with either virus. Ondays 2, 4, 6, and 8 postchallenge, four animals from each of groups A, B, andC were sacrificed for histologic evaluation and four were sacrificed for virustitration. Two animals from group D were sacrificed on the same days forhistologic evaluation.

b Mean of individual scores for animals examined at each time period (0, no_, pathology; 4+, maximal pathology).

c Geometric mean titer for animals examined at each time period (PFU per8 gram, log1o).

DAYS POST-CHALLENGEFIG. 5. Effect of priming with BRSV/375 or HRSV/Long on

virus clearance following HRSV/Long challenge. Cotton rats were

inoculated with 104 PFU of either BRSV/375 or HRSV/Long or leftunprimed and challenged 3 weeks later with 105 PFU of HRSV/Long. Four to eleven animals from each group were sacrificed on

days 2, 4, 6, and 8 postchallenge. The geometric mean virus titers(log10) and standard errors are shown for nose (A) and lungs (B).

at 2.0 x 105 PFU/g on day 4, and dropped progressively to4.7 x 102 PFU/g by day 8. The HRSV titer in lungs followeda similar curve, peaking at 1.2 x 105 PFU/g on day 4 anddecreasing to nearly undetectable levels by day 8.The animals primed with BRSV/375 and challenged with

HRSV/Long (Table 2, group A) experienced milder patho-logical changes than did the unprimed, HRSV-challengedanimals (group B). Clefting of the nasal mucosa was seen on

day 4 but decreased subsequently and was not detected byday 8. Bronchiolitis was less marked than in group B,peaking on day 6 (2+) and decreasing subsequently. Inter-stitial pneumonia was very mild (0 to 1+) throughout. Therewas no evidence of potentiation of pathology in any of theBRSV-primed, HRSV-challenged animals. The mean HRSVtiter in nasal tissues was 1.5 x 104 PFU/g on day 2 butdecreased rapidly to undetectable levels by day 6. TheHRSV titer in lungs was 4.6 x 102 PFU/g on day 2 anddecreased progressively to undetectable levels by day 6.

DISCUSSION

Shortly after HRSV was recognized as an importantpathogen of infants and children, an intensive effort wasinitiated to develop a formalin-inactivated vaccine againstthis virus. Clinical trials showed that inoculation of infantswith formalin-inactivated HRSV provided little protectionand, moreover, enhanced the severity of naturally occurringHRSV disease among vaccinees (8, 12, 15, 17).Subsequent efforts to develop a vaccine against HRSV

have included the use of temperature-sensitive mutantstrains with restricted replication in the lower respiratorytract (16, 45), unmodified virus administered parenterally (2,4, 33), and recombinant vaccines that express HRSV glyco-proteins (9, 11, 23, 26, 27, 44) and purified viral glycoproteins(3, 10, 14, 24, 25, 37-40, 42, 43). To date, however, nocandidate vaccine has proven both safe and effective.BRSV is structurally similar to HRSV. However, while

the BRSV F, N, M, and P proteins show antigenic cross-reactivity with their HRSV counterparts, the BRSV Gglycoprotein is antigenically different from that of HRSV and-is not cross-reactive (21). The deduced amino acid sequenceof the BRSV G glycoprotein shows only 29 to 30% identitywith the G glycoprotein of either the subgroup A or B HRSV(20).BRSV causes a severe lower respiratory tract disease in

calves that is similar to the disease caused by HRSV ininfants (18). It is not known whether BRSV can infect

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humans (and cause disease) or whether BRSV can be used asa vaccine against HRSV. It is possible to experimentallyinfect cattle with HRSV (13, 36). Moreover, attempts havebeen made to use HRSV for immunization of cattle. Atemperature-sensitive mutant of HRSV given intramuscu-larly has been found to provide partial protection againstBRSV infection in cattle (34). This finding suggests that thereis some level of cross-protection between bovine and humanstrains of respiratory syncytial virus in vivo. In this study,we have used the cotton rat to evaluate the feasibility ofusing BRSV as a vaccine against HRSV.

Despite its limited replication in cotton rat tissues, BRSV/375 effected a high level of protection in nose and lungsagainst infection with HRSV of both subgroups (A and B).At a priming dose of 105 PFU, BRSV/375 effected a 500- to1,000-fold reduction in peak nasal HRSV titer and a greaterthan 1,000-fold reduction in peak pulmonary HRSV titerupon challenge with HRSV of either subgroup. Priming withBRSV/375 not only decreased peak HRSV titers substan-tially upon challenge but also shortened shedding of HRSV.At lower priming doses, the level of pulmonary protectionwas always greater than that of nasal protection. Thisobservation is consistent with previous work showing thatafter parenteral immunization of cotton rats with live HRSV(33) or recombinant vaccines that express HRSV glycopro-teins (11) or purified viral glycoproteins (25), the lungs aremore resistant than the nose to challenge with HRSV.To determine whether the protection against HRSV infec-

tion effected by BRSV priming is a specific phenomenon, wetested the effectiveness of priming with BRSV against infec-tion with PIV3, a related respiratory virus that replicateswell in cotton rat tissues (29). BRSV/375 effected a marginallevel of nasal protection and no pulmonary protectionagainst PIV3 infection. This finding indicates that the in vivoprotection against HRSV infection effected by BRSV prim-ing is in fact specific and is likely to be a function of theantigenic similarity between the two viruses.The in vitro antigenic relatedness between BRSV and

HRSV was examined by using Archetti and Horsfall's for-mula (1). Even though the calculated r value of 4.5 revealssignificant in vitro antigenic divergence between BRSV andHRSV, the impressive degree of in vivo protection againstHRSV infection conferred by BRSV priming suggests sub-stantial antigenic similarity between these two viruses,which, while not reflected in neutralizing antibody titers, isindicative of other immunologic effectors.

Previous studies using passive transfer of anti-HRSVantiserum in cotton rats showed that circulating neutralizingantibody titers in excess of 1:350 were required for preven-tion of pulmonary infection with HRSV (30). The presentstudy has shown, however, that complete resistance toHRSV infection can be induced despite undetectable or verylow levels of serum neutralizing antibody. The impressiveprotection afforded by BRSV/375 against HRSV infection, inthe face of a weak neutralizing antibody response, serves tohighlight the importance of understanding immunologic fac-tors other than serum neutralizing antibody.Comparison of growth curves for HRSV upon challenge in

animals primed with HRSV or BRSV showed that in HRSV-primed animals, complete restriction of viral replicationoccurred very early, whereas in BRSV-primed animals, itdid not occur until 4 days postchallenge. In the first case, thepresence of substantial levels of serum neutralizing antibod-ies at the time of challenge suggests that these antibodieswould be responsible for the early onset of restriction of viralreplication. In the second case, the delayed onset of viral

clearance in the absence of serum neutralizing antibodies issuggestive of cell-mediated immunity. However, since wedid not measure antibody levels at the time of sacrifice, wecannot rule out a rapid rise in neutralizing antibody afterchallenge. Furthermore, it is possible that protection wasdue to nonneutralizing antibodies (35, 37, 41) or to othermeans of protection such as antibody-dependent cell cyto-toxicity.Even though HRSV does not cause overt clinical illness in

cotton rats, it does cause distinct pathological changes innasal mucosal epithelium, bronchioles, and lung intersti-tium. After experimental HRSV infection, these pathologicalchanges peak on day 6, 2 days after peak viral titers areachieved (32). In the present study, infection with BRSV(which was associated with low levels of viral replication)caused bronchiolitis that was less marked than with HRSVas well as minimal nasal pathology and pulmonary interstitialinflammation. Priming with BRSV decreased the severity ofnasal and pulmonary pathology caused by subsequentHRSV infection and shortened viral shedding.A major concern in the development of a vaccine against

HRSV and other respiratory viruses has been the possibilityof vaccine-induced potentiation of disease. This phenome-non, which occurred in children during the formalin-inacti-vated HRSV vaccine trials (8, 12, 15, 17) and was repro-duced in the cotton rat model (31), has also been shown tooccur with nonreplicating candidate vaccines containingpurified HRSV glycoproteins (10, 24). Although the mecha-nism for this phenomenon has not been well characterized,alteration of critical viral epitopes and an abnormal immuneresponse would appear to be necessary. By contrast, studiesof replicating candidate HRSV vaccines have not, to date,provided evidence of disease potentiation (10). Therefore,the use of an unmodified, replicating immunogen such asBRSV would appear to be unlikely to induce an aberrantimmune response and cause vaccine-induced enhancementof HRSV disease. In our study, priming with BRSV did notinduce potentiation of disease upon challenge with HRSV 3weeks later. This lack of vaccine potentiation at an earlytime postimmunization, however, does not guarantee lack ofpotentiation at a later time. Studies are currently beingconducted to assess the safety of BRSV vaccination inanimals challenged at longer intervals after vaccination.The results of this study demonstrate that in the cotton rat

model, BRSV, which replicates to lower titers and causesless pathology than does HRSV, can be used effectively toimmunize against HRSV infection without causing potenti-ation of disease 3 weeks after immunization. The fact thatBRSV produces less pathology than does HRSV in thecotton rat does not guarantee safety in humans. However,given that BRSV is a wild-type virus in its stable form, thereis little reason to believe that it could revert to a morepathogenic form, as can happen with unstable mutantstrains, such as some of the attenuated strains of HRSV,when subjected to selective pressure. If BRSV is capable ofcausing limited infection in humans, it is possible that itcould be used as a vaccine against HRSV without requiringany attenuation.

ACKNOWLEDGMENTSThis work was supported in part by a research grant from the

American Lung Association of Maryland and by a Scientific Awardfrom the Research Advisory Council, Children's National MedicalCenter, Washington, D.C.We thank Victor Tineo for expert assistance in the care and

maintenance of the cotton rat colony.

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