against Human Respiratory Syncytial Virus Infection

JOURNAL OF VIROLOGY, Mar. 199, p. 1-11 22-8X/9/1-8$2./ Copyright 199, American Society for Microbiology Vol. 67, No. Bovine Respiratory Syncytial Virus Protects Cotton Rats against Human Respiratory Syncytial Virus Infection FRANCO M. PIAZZA,"* SUSAN A. JOHNSON,' MIRIAM E. R. DARNELL,2 DAVID D. PORTER, VAL G. HEMMING,4 AND GREGORY A. PRINCE2 Children's National Medical Center and Department of Pediatrics, The George Washington University School of Medicine and Health Sciences, Washington, D.C. 211; Virion Systems, Inc., Rockville, Maryland 282; Department of Pathology and Laboratory Medicine, University of California School of Medicine, Los Angeles, California 924; and Department of Pediatrics, Unifonned Services University of the Health Sciences, Bethesda, Maryland 28144 Received 24 August 1992/Accepted November 1992 Human respiratory syncytial virus (HRSV) is the most frequent cause of severe respiratory infections in infancy. 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 HRSV infection. Cotton rats were primed intranasally with graded doses of BRSV/7 or HRSV/Long or were left unprimed. Three weeks later, they were challenged intranasally with either BRSV/7, HRSV/Long (subgroup A), or HRSV/187 (subgroup B). At intervals postchallenge, animals were sacrificed for virus titration and histologic evaluation. Serum neutralizing antibody titers were determined at the time of viral challenge. BRSV/7 replicated to low titers in nasal tissues and lungs. Priming with 1i PFU of BRSV/7 effected a - to 1,-fold reduction in peak nasal HRSV titer and a greater than 1,-fold reduction in peak pulmonary HRSV titer upon challenge with HRSV/Long or HRSV/187. In contrast to priming with HRSV, priming with BRSV did not induce substantial levels of neutralizing antibody against HRSV and was associated with a delayed onset of clearance of HRSV upon challenge. Priming with BRSV/7 caused mild nasal and pulmonary pathology and did not cause exacerbation of disease upon challenge with HRSV/Long. Our findings suggest that BRSV may be a potential vaccine against HRSV and a useful tool for studying the mechanisms of immunity to HRSV. Human respiratory syncytial virus (HRSV) was discovered in 19 during an outbreak of respiratory disease in a chimpanzee colony (22) and was later shown to be a ubiquitous human respiratory pathogen (7). HRSV is the single most important respiratory pathogen of infancy and early childhood worldwide (). No vaccine to prevent HRSV infection has yet been protective. Antiviral drugs have been of limited utility in the treatment of HRSV disease. In 1967, a virus related to HRSV was isolated from cattle in Europe (28). This agent, known as bovine respiratory syncytial virus (BRSV), causes severe lower respiratory tract disease in calves similar to the illness caused by HRSV in infants. BRSV occurs and causes serious disease in bovine 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 of the first vaccine. Jenner used cowpox virus, a natural bovine pathogen, to immunize humans against smallpox. The Jennerian approach is currently being used in the development of live-virus vaccine strains to immunize against parainfluenza type virus (PIV), rotavirus, and influenza virus infections (6). In the present study, we have used the cotton rat, a well-established model of HRSV infection (2) and vaccine-induced potentiation of HRSV lung disease (1), to evaluate the feasibility of using BRSV as a vaccine against HRSV. * Corresponding author. 1 MATERIALS AND METHODS Animals. Young adult inbred cotton rats (Sigmodon hispidus) were obtained from a colony maintained by Virion Systems, Inc., Rockville, Md., and were housed and fed as previously described (2). Animals were shown to be free of serum neutralizing antibody against HRSV prior to inclusion in the study. Viruses. The BRSV strain used in these studies, BRSV/ 7, was obtained from Howard Lehmkuhl, U.S. Department of Agriculture, Ames, Iowa (19). Virus stocks were grown in bovine nasal turbinate cell monolayers (obtained from the same source) to a titer of 16 PFU/ml. BRSV/7 was chosen for evaluation as an immunogen against HRSV because previous studies showed that among six different strains of BRSV tested in cotton rats, BRSV/7 was isolated from the highest percentage of inoculated animals and replicated to the highest titer (29a). Two prototype strains of HRSV were used: the Long strain (subgroup A) and the 187 strain (subgroup B). Stocks of these viruses were prepared in HEp-2 cells and contained 16 PFU/ml. PIV (lot 691) was obtained from Program Resources, Inc., Rockville, Md., and contained 16 PFU/ml. Experimental protocol. Cotton rats were anesthetized by methoxyflurane inhalation and inoculated with either BRSV/ 7, HRSV/Long, HRSV/187, or PIV. All inoculations (priming and challenge) were performed intranasally, using.1 ml of virus suspension. Animals were sacrificed by carbon dioxide intoxication at intervals following inoculation. For virus titration, nasal tissues and lungs were homogenized in 1 parts (wt/vol) of Hanks balanced salt solution Downloaded from http://jvi.asm.org/ on December 2, 218 by guest

14 PIAZZA ET AL. supplemented with.218 M sucrose-4.4 mm glutamate-.8 mm KH2PO4-.2 mm K2HPO4, and the resulting suspension was stored at -7 C until assayed. For histologic preparation of nasal tissues, the skin was removed from the head and the skull was fixed in 1% neutral buffered formalin. For preparation of lungs, these were removed from the thorax and inflated through the trachea with 1% formalin. Animals were bled from the retro-orbital venous plexus, and serum was stored at -2 C until assayed for neutralizing antibody titers. Virus assays. Virus titers for HRSV and PIV were determined by plaque assay on HEp-2 cell monolayers as previously described (2). Titration for BRSV was performed on bovine nasal turbinate cell monolayers, which were incubated with tissue homogenates for 6 to 7 (instead of 4) days. Virus titers were expressed as the geometric mean of individual titers (PFU per gram of tissue plus or minus the standard error) for each animal group. Antibody assays. Serum neutralizing antibody against HRSV was measured by a plaque reduction neutralization assay on HEp-2 cell monolayers, using a 6% plaque reduction end point, as previously described (2). Neutralizing antibody against BRSV was determined by a similar assay using bovine nasal turbinate cells and BRSV instead of HEp-2 cells and HRSV, respectively. Antibody titers were expressed as the geometric mean of individual titers for all animals at a given time. Pathologic study. Formalin-fixed nasal tissues (decalcified) and lungs were embedded in paraffin, cut into coronal sections, and stained with hematoxylin-eosin (2). Three sections of nasal tissues and one of lungs from each animal were examined by light microscopy. Pathologic changes were scored blindly by using a qualitative scoring system as previously described (29). Statistical analysis. Geometric means of virus titers of experimental groups were compared with those of control groups by using the two-tailed Student t test of summary data. Antigenic relatedness between BRSV/7 and HRSV/ Long was determined by using Archetti and Horsfall's formula (1), r = Vrj x r2, where r, is heterologous titer of virus 2/homologous titer of virus 1 and r2 is heterologous titer of virus 1/homologous titer of virus 2. This formula stipulates that values of r of 2 or more indicate significant antigenic dissimilarity. RESULTS Growth of BRSV. Cotton rats were inoculated with 14 PFU of BRSV/7 and sacrificed at min, 12, 18, and 24 h, and 2,, 4,, 6, and 8 days for virus titration (Fig. 1A). After an eclipse phase at 12 h, BRSV/7 reached a peak titer at 24 h in the lungs and at 2 days in nasal tissues. BRSV/7 replicated to low titers but was isolated from all the animals studied on days 1 through. For comparison, cotton rats were inoculated with 14 PFU of HRSV/Long and sacrificed at 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 to much higher titers than did BRSV/7. Effect of priming with BRSV on replication of subgroup A HRSV. The effect of priming with various doses of BRSV upon subsequent challenge with subgroup A HRSV was assessed. Cotton rats inoculated intranasally with graded doses of BRSV/7 (11 to 1 PFU in a volume of.1 ml) or left unprimed were challenged intranasally weeks later with 1 PFU of HRSV/Long and sacrificed 4 days post- ) ō) ọ I- F cn M r- 4-. 2-6 A. BRSV/7 J. VIROL. -U-- * Lungs --- - Nose 2 4 6 8 Lungs Nose 4 6 8 DAYS POST-INOCULATION FIG. 1. Replication of BRSV/7 and HRSV/Long. Cotton rats were inoculated with 14 PFU of BRSV/7 and sacrificed at min, 12, 18, and 24 h, and 2,, 4,, 6, and 8 days (A) or HRSV/Long and sacrificed at 12 h and at 2, 4, 6, and 8 days (B). Each point represents the geometric mean virus titer (log,o) of four to eight animals; bars indicate standard errors. challenge (at the time of peak viral replication in unprimed animals) (Fig. 2A). The effectiveness of priming with BRSV in the induction of protection against challenge with HRSV was compared with that of priming with HRSV. Cotton rats inoculated with graded doses of HRSV/Long (11 to 1 PFU in a volume of.1 ml) or left unprimed were challenged weeks later with 1 PFU of the same virus and sacrificed 4 days postchallenge (Fig. 2B). The animals primed with the highest dose of BRSV/7 (1 PFU) and subsequently challenged with HRSV/Long had HRSV titers 4 days postchallenge of 1.2 x 12 PFU/g in nasal tissues and <12 PFU/g (undetectable) in lungs (Fig. 2A). Unprimed control animals had HRSV titers in nasal tissues and lungs of 1.6 x 1 and.7 x 14 PFU/g, respectively. These differences in virus titers were highly significant (P <.1 for both comparisons). In the animals primed with lower doses of BRSV/7, the postchallenge HRSV titers increased as the dose of BRSV/7 decreased. A significant reduction in nasal titers was demonstrated with a priming BRSV dose as low as 1 PFU. A significant reduction in lung titers occurred with a priming BRSV dose as low as 12 PFU. The titer of serum neutralizing antibody against HRSV/ Long at the time of challenge with this virus was very low Downloaded from http://jvi.asm.org/ on December 2, 218 by guest

VOL. 67, 199 BOVINE RSV IN COTTON RATS 1 6. 4, E cm IL a.c I- 6-4' * * Lungs E Nose * P <.1 C] T- o) Lu a- F z -J Ir 6 1 4 <2 Control 1 (9) (8) Control 11 12 1 (1) (8) (6) (7) 12 1 14 1 (14) (1) (1) (1) protection was comparable to that observed in animals primed with the same dose of BRSV/7 and challenged with HRSV/Long (Fig. 2A). ) x 12 PFU/g in Antigenic cross-reactivity between BRSV and HRSV. Sera Inprimed control obtained from convalescent cotton rats infected weeks Id lungs of 1.9 x earlier with 1 PFU of BRSV/7 were tested for neutral- izing antibodies against BRSV/7 and HRSV/Long. Also,.1 for both sera obtained from animals infected weeks earlier with 1 with BRSV, the PFU of HRSV/Long were tested for antibodies against ;priming dose of HRSV/Long and BRSV/7 (Table 1). The degree of anti- (1:21) or undetectable (<1:2) for all grn primed animals (Fig. 2A). The animals primed with the highest dose of HRSV/Long (1 PFU) and subsequently challenged witlh the same virus had HRSV titers 4 days postchallenge of 1.C nasal tissues and <12 in lungs (Fig. 2B). U animals had HRSV titers in nasal tissues an 1 and 2. x 1 PFU/g, respectively. These differences in virus titers were highly significant (P < comparisons). As seen in animals primed' level of protection was proportionate to the <2 Control (17) 4 1 (1) * * DOSE OF PRIMING VIRUS (PFU/animal) FIG.. Effect of priming with BRSV/7 on virus titers following HRSV/LONG HRSV/187 challenge. Cotton rats were inoculated with 14 or 1 PFU of BRSV/7 or left unprimed, challenged weeks later with 1 PFU of HRSV/187, and sacrificed 4 days postchallenge. The geometric mean virus titers (log1o) and standard errors are shown. * Lungs Virus titers significantly lower than control values are marked with Nose asterisks. The number of animals studied is in parentheses. P <.1 HRSV. A significant reduction in nasal and in lung titers was seen with a priming HRSV dose as low as 12 PFU. The titers of serum neutralizing antibody against HRSV/ Long at the time of challenge with this virus were 1: and * * * * 1:418 for the groups primed with 14 and 1 PFU of HRSV, 418 418 respectively (Fig. 2B). Those groups primed with lower doses had titers of 1:7 or lower. 4 In comparison with BRSV/7, a lower priming dose of 1 1 HRSV/Long effected a comparable degree of protection (7) (1) against challenge with HRSV/Long (Fig. 2). Effect of priming with BRSV on replication of subgroup B DOSE OF PRIMING VIRUS (F'FU/animal) HRSV. The effect of priming with BRSV upon subsequent FIG. 2. Effect of the priming dose of BRSV/7 or HRSV/Long challenge with subgroup B HRSV was assessed. Cotton rats on virus titers following HRSV/Long challenge. Cotton rats were inoculated with 14 or 1 PFU of BRSV/7 or left inoculated with graded doses (11 to 1 PFU) off BRSV/7 (A) or unprimed were challenged weeks later with 1 PFU of HRSV/Long (B) or left unprimed, challenged w( eeks later with 1 HRSV/187 and sacrificed 4 days postchallenge (Fig. ). PFU of HRSV/Long, and sacrificed 4 days postchallenge. The The animals primed with the highest dose of BRSV/7 geometric mean virus titers (log1) and standard errors are Virus titers significantly lower than control value asterisks. The serum neutralizing antibody tite showni and challenged with HRSV/187 had HRSV titers 4 days rs against HRSV postchallenge of 1.1 x 12 PFU/g in nasal tissues and <12 (reciprocal of the geometric mean) at the time of challenge are PFU/g in lungs (Fig. ). The respective titers in unprimed shown. The number of animals studied is in parentheses. control animals were.1 x 1 and.7 x 14 PFU/g. These differences in virus titers were again highly significant (P <.1 for both comparisons). The animals primed with 14 PFU of BRSV/7 had HRSV titers that were significantly oups of BRSV- lower than those of unprimed controls (Fig. ). The level of D cn IL 1 (1) Downloaded from http://jvi.asm.org/ on December 2, 218 by guest

16 PIAZZA ET AL. TABLE 1. Seruma Antigenic cross-reactivity between BRSV and HRSV in cotton rats Reciprocal of geometric mean neutralizing antibody titer (no. of animals tested) BRSV/7 HRSV/Long BRSV/7 86 () 22 () HRSV/Long 1 (1) 418 (11) a Sera obtained from cotton rats infected weeks earlier with 1 PFU of BRSV/7 or HRSV/Long were assayed individually for neutralizing antibody titers against each of the viruses by plaque reduction assay. genic relatedness between BRSV and HRSV in vitro was determined by using Archetti and Horsfall's formula (1). This formula yielded an r value of 4., which indicates highly significant antigenic divergence between BRSV/7 and HRSV/Long. Specificity of BRSV in the induction of immunity to HRSV. The specificity of priming with BRSV in the induction of immunity to HRSV was examined. Cotton rats inoculated with 1 PFU of BRSV/7 or left unprimed were challenged weeks later with either 1- PFU of PIV or 1 PFU of HRSV/Long and sacrificed 4 days postchallenge for virus titration (Fig. 4). The animals primed with BRSV/7 and subsequently challenged with PIV had PIV titers of.8 x 14 PFU/g in nasal tissues and 2.6 x 1 PFU/g in lungs (Fig. 4A). The respective titers in unprimed control animals were 2.4 x 1 and 2.7 x 1 PFU/g. Nasal PIV titers were lower in the BRSV-primed group than in the unprimed controls (P <.), but pulmonary titers were not different. The animals primed with BRSV/7 and challenged with HRSV/Long had HRSV titers of 1. x 12 PFU/g in nasal tissues and <12 PFU/g (undetectable) in lungs (Fig. 4B). The respective titers in unprimed control animals were 4.1 x 14 and 6. x 1 PFU/g. These differences in virus titers were highly significant (P <.1 for both comparisons). Effect of priming with BRSV on kinetics of HRSV clearance upon challenge. Cotton rats inoculated with 14 PFU of either BRSV/7 or HRSV/Long or left unprimed were challenged weeks later with 1 PFU of HRSV/Long. Groups of animals were sacrificed on days 2, 4, 6, and 8 postchallenge for virus titration (Fig. ). The unprimed animals had mean HRSV titers in nasal tissues and lungs that were high on day 2, peaked above 1 PFU/g on day 4, and decreased to near undetectable levels by day 8. The HRSV-primed animals had a mean HRSV titer in nasal tissues that was very low on day 2 and decreased to undetectable levels by day 4; HRSV was not detected in lungs on any day. The BRSV-primed animals had a mean HRSV titer in nasal tissues that was above 14 PFU/g on day 2 and decreased to undetectable levels by day 6; the HRSV titer in lungs was lower than 1 PFU/g on day 2 and below detectable levels by day 6. Effect of priming with BRSV on the pathology of HRSV infection. Cotton rats inoculated with 14 PFU of BRSV/7 or left unprimed were challenged weeks later with either 1 PFU of HRSV/Long or 1 PFU of BRSV/7 (Table 2). Subgroups of these animals were sacrificed on days 2, 4, 6, and 8 postchallenge for histologic evaluation and virus titration. Additional animals that were not inoculated with either virus were sacrificed for histologic evaluation. No pathological changes were identified in uninoculated control animals (Table 2, group D). Unprimed animals Re ) ) ~ If T- ML- a) V - ) IL ep z -J 6. - 4 <2. Unprimed Primed with (7) BRSV/7 6 (7) 4. T * Lungs Nose P <. B. HRSV/LONG CHALLENGE ** ** * Lungs Nose * P <. ** P <.1 J. VIROL. IL '2-m/ Unprimed Primed with (8) BRSV/7 (7) FIG. 4. Effect of priming with BRSV/7 on virus titers following PIV or HRSV/Long challenge. Cotton rats were inoculated with 1 PFU of BRSV/7 or left unprimed, challenged weeks later with either 1 PFU of PIV (A) or 1 PFU of HRSV/Long (B), and sacrificed 4 days postchallenge. The geometric mean virus titers (loglo) and standard errors are shown. Virus titers significantly lower than control values are marked with asterisks. The number of animals studied is in parentheses. challenged with BRSV/7 (Table 2, group C) experienced mild pathological changes. Epithelial clefting of the nasal mucosa was seen only on day 2. Bronchiolitis peaked on day 6, and interstitial pneumonia was minimal throughout. The mean BRSV titer in nasal tissues was 1.2 x 1 PFU/g on day 2 and dropped to undetectable levels by day 6. The BRSV titer in lungs remained very low (<2. x 12 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 day 6 (4+) and decreasing but still persisting by day 8. Bronchiolitis was clearly present on day 4, peaked on day 6 ( +/4+), and decreased by day 8. Interstitial pneumonia was more prominent than in any of the other groups, being easily discernible on day 4 and peaking on day 6. The mean HRSV titer in nasal tissues was above 1 PFU/g on day 2, peaked Downloaded from http://jvi.asm.org/ on December 2, 218 by guest

VOL. 67, 199 BOVINE RSV IN COTTON RATS 17 a- IL CL ij I C,, 6 2 A. NOSE * Unpnmed E BRSV Pnmed l9 HRSV Pnmed vl 4 6 8 4 6 B. LUNGS DAYS POST-CHALLENGE FIG.. Effect of priming with BRSV/7 or HRSV/Long on virus clearance following HRSV/Long challenge. Cotton rats were inoculated with 14 PFU of either BRSV/7 or HRSV/Long or left unprimed and challenged weeks later with 1 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 (log1) and standard errors are shown for nose (A) and lungs (B). at 2. x 1 PFU/g on day 4, and dropped progressively to 4.7 x 12 PFU/g by day 8. The HRSV titer in lungs followed a similar curve, peaking at 1.2 x 1 PFU/g on day 4 and decreasing to nearly undetectable levels by day 8. The animals primed with BRSV/7 and challenged with HRSV/Long (Table 2, group A) experienced milder pathological changes than did the unprimed, HRSV-challenged animals (group B). Clefting of the nasal mucosa was seen on day 4 but decreased subsequently and was not detected by day 8. Bronchiolitis was less marked than in group B, peaking on day 6 (2+) and decreasing subsequently. Interstitial pneumonia was very mild ( to 1+) throughout. There was no evidence of potentiation of pathology in any of the BRSV-primed, HRSV-challenged animals. The mean HRSV titer in nasal tissues was 1. x 14 PFU/g on day 2 but decreased rapidly to undetectable levels by day 6. The HRSV titer in lungs was 4.6 x 12 PFU/g on day 2 and decreased progressively to undetectable levels by day 6. TABLE 2. Determination Effect of priming with BRSV on the pathology of HRSV infectiona Group Days postchallenge 2 4 6 8 Pathology scoreb Bronchiolitis A 1+ 1+/2+ 2+ 1+/2+ B 1+ 2+1+ +14+ 1+ C 1+ 1+ + 1+ D Interstitial A 1+ /1+ pneumonia B 2+ + C 1+ 1+/2+ D Nasal pathology A 1+/2+ 1+ B 1+ 2+/+ 4+ 2+ C /1+ D Virus titerc Lung A 2.67 2.29 <2. <2. B 4.78.8.18 2.19 C 2.1 2.19 2.12 <2. Nose A 4.17. <2. <2. B.26.1.19 2.67 C.8 2.61 <2. 2. * Unpnmed E BRSV Pnmed a Cotton rats were divided into four groups. Animals in group A (2 El HRSV Pnmed animals) were inoculated with 14 PFU of BRSV/7 and challenged weeks later with 1 PFU of HRSV/Long. Animals in group B (2 animals) remained unprimed and were challenged with 1 PFU of HRSV/Long (at the time of group A challenge). Animals in group C (2 animals) remained unprimed and were challenged with 14 PFU of BRSV/7 (at the time of group A challenge). Animals in group D (eight animals) were not inoculated with either virus. On days 2, 4, 6, and 8 postchallenge, four animals from each of groups A, B, and C were sacrificed for histologic evaluation and four were sacrificed for virus titration. Two animals from group D were sacrificed on the same days for histologic evaluation. b Mean of individual scores for animals examined at each time period (, no _, pathology; 4+, maximal pathology). c Geometric mean titer for animals examined at each time period (PFU per 8 gram, log1o). DISCUSSION Shortly after HRSV was recognized as an important pathogen of infants and children, an intensive effort was initiated to develop a formalin-inactivated vaccine against this virus. Clinical trials showed that inoculation of infants with formalin-inactivated HRSV provided little protection and, moreover, enhanced the severity of naturally occurring HRSV disease among vaccinees (8, 12, 1, 17). Subsequent efforts to develop a vaccine against HRSV have included the use of temperature-sensitive mutant strains with restricted replication in the lower respiratory tract (16, 4), unmodified virus administered parenterally (2, 4, ), and recombinant vaccines that express HRSV glycoproteins (9, 11, 2, 26, 27, 44) and purified viral glycoproteins (, 1, 14, 24, 2, 7-4, 42, 4). To date, however, no candidate 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 crossreactivity with their HRSV counterparts, the BRSV G glycoprotein is antigenically different from that of HRSV and -is not cross-reactive (21). The deduced amino acid sequence of the BRSV G glycoprotein shows only 29 to % identity with the G glycoprotein of either the subgroup A or B HRSV (2). BRSV causes a severe lower respiratory tract disease in calves that is similar to the disease caused by HRSV in infants (18). It is not known whether BRSV can infect Downloaded from http://jvi.asm.org/ on December 2, 218 by guest

18 PIAZZA ET AL. humans (and cause disease) or whether BRSV can be used as a vaccine against HRSV. It is possible to experimentally infect cattle with HRSV (1, 6). Moreover, attempts have been made to use HRSV for immunization of cattle. A temperature-sensitive mutant of HRSV given intramuscularly has been found to provide partial protection against BRSV infection in cattle (4). This finding suggests that there is some level of cross-protection between bovine and human strains of respiratory syncytial virus in vivo. In this study, we have used the cotton rat to evaluate the feasibility of using BRSV as a vaccine against HRSV. Despite its limited replication in cotton rat tissues, BRSV/ 7 effected a high level of protection in nose and lungs against infection with HRSV of both subgroups (A and B). At a priming dose of 1 PFU, BRSV/7 effected a - to 1,-fold reduction in peak nasal HRSV titer and a greater than 1,-fold reduction in peak pulmonary HRSV titer upon challenge with HRSV of either subgroup. Priming with BRSV/7 not only decreased peak HRSV titers substantially upon challenge but also shortened shedding of HRSV. At lower priming doses, the level of pulmonary protection was always greater than that of nasal protection. This observation is consistent with previous work showing that after parenteral immunization of cotton rats with live HRSV () or recombinant vaccines that express HRSV glycoproteins (11) or purified viral glycoproteins (2), the lungs are more resistant than the nose to challenge with HRSV. To determine whether the protection against HRSV infection effected by BRSV priming is a specific phenomenon, we tested the effectiveness of priming with BRSV against infection with PIV, a related respiratory virus that replicates well in cotton rat tissues (29). BRSV/7 effected a marginal level of nasal protection and no pulmonary protection against PIV infection. This finding indicates that the in vivo protection against HRSV infection effected by BRSV priming is in fact specific and is likely to be a function of the antigenic similarity between the two viruses. The in vitro antigenic relatedness between BRSV and HRSV was examined by using Archetti and Horsfall's formula (1). Even though the calculated r value of 4. reveals significant in vitro antigenic divergence between BRSV and HRSV, the impressive degree of in vivo protection against HRSV infection conferred by BRSV priming suggests substantial antigenic similarity between these two viruses, which, while not reflected in neutralizing antibody titers, is indicative of other immunologic effectors. Previous studies using passive transfer of anti-hrsv antiserum in cotton rats showed that circulating neutralizing antibody titers in excess of 1: were required for prevention of pulmonary infection with HRSV (). The present study has shown, however, that complete resistance to HRSV infection can be induced despite undetectable or very low levels of serum neutralizing antibody. The impressive protection afforded by BRSV/7 against HRSV infection, in the face of a weak neutralizing antibody response, serves to highlight the importance of understanding immunologic factors other than serum neutralizing antibody. Comparison of growth curves for HRSV upon challenge in animals primed with HRSV or BRSV showed that in HRSVprimed animals, complete restriction of viral replication occurred very early, whereas in BRSV-primed animals, it did not occur until 4 days postchallenge. In the first case, the presence of substantial levels of serum neutralizing antibodies at the time of challenge suggests that these antibodies would be responsible for the early onset of restriction of viral replication. In the second case, the delayed onset of viral J. VIROL. clearance in the absence of serum neutralizing antibodies is suggestive of cell-mediated immunity. However, since we did not measure antibody levels at the time of sacrifice, we cannot rule out a rapid rise in neutralizing antibody after challenge. Furthermore, it is possible that protection was due to nonneutralizing antibodies (, 7, 41) or to other means of protection such as antibody-dependent cell cytotoxicity. Even though HRSV does not cause overt clinical illness in cotton rats, it does cause distinct pathological changes in nasal mucosal epithelium, bronchioles, and lung interstitium. After experimental HRSV infection, these pathological changes peak on day 6, 2 days after peak viral titers are achieved (2). In the present study, infection with BRSV (which was associated with low levels of viral replication) caused bronchiolitis that was less marked than with HRSV as well as minimal nasal pathology and pulmonary interstitial inflammation. Priming with BRSV decreased the severity of nasal and pulmonary pathology caused by subsequent HRSV infection and shortened viral shedding. A major concern in the development of a vaccine against HRSV and other respiratory viruses has been the possibility of vaccine-induced potentiation of disease. This phenomenon, which occurred in children during the formalin-inactivated HRSV vaccine trials (8, 12, 1, 17) and was reproduced in the cotton rat model (1), has also been shown to occur with nonreplicating candidate vaccines containing purified HRSV glycoproteins (1, 24). Although the mechanism for this phenomenon has not been well characterized, alteration of critical viral epitopes and an abnormal immune response would appear to be necessary. By contrast, studies of replicating candidate HRSV vaccines have not, to date, provided evidence of disease potentiation (1). Therefore, the use of an unmodified, replicating immunogen such as BRSV would appear to be unlikely to induce an aberrant immune response and cause vaccine-induced enhancement of HRSV disease. In our study, priming with BRSV did not induce potentiation of disease upon challenge with HRSV weeks later. This lack of vaccine potentiation at an early time postimmunization, however, does not guarantee lack of potentiation at a later time. Studies are currently being conducted to assess the safety of BRSV vaccination in animals 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 causes less pathology than does HRSV, can be used effectively to immunize against HRSV infection without causing potentiation of disease weeks after immunization. The fact that BRSV produces less pathology than does HRSV in the cotton rat does not guarantee safety in humans. However, given that BRSV is a wild-type virus in its stable form, there is little reason to believe that it could revert to a more pathogenic form, as can happen with unstable mutant strains, such as some of the attenuated strains of HRSV, when subjected to selective pressure. If BRSV is capable of causing limited infection in humans, it is possible that it could be used as a vaccine against HRSV without requiring any attenuation. ACKNOWLEDGMENTS This work was supported in part by a research grant from the American Lung Association of Maryland and by a Scientific Award from the Research Advisory Council, Children's National Medical Center, Washington, D.C. We thank Victor Tineo for expert assistance in the care and maintenance of the cotton rat colony. Downloaded from http://jvi.asm.org/ on December 2, 218 by guest

VOL. 67, 199 REFERENCES 1. Archetti, I., and F. L. Horsfall. 19. Persistent antigenic variation of influenza A viruses after incomplete neutralization in ovo with heterologous immune serum. J. Exp. Med. 92:441-462. 2. Belshe, R. B., L. P. van Voris, and M. A. Mufson. 1982. Parenteral administration of live respiratory syncytial virus vaccine: results of a field trial. J. Infect. Dis. 14:11-19.. Brideau, R. J., R. R. Walters, M. A. Stier, and M. W. Wathen. 1989. Protection of cotton rats against human respiratory syncytial virus by vaccination with a novel chimeric FG glycoprotein. J. Gen. Virol. 7:267-2644. 4. Buynak, E. B., R. E. Weibel, A. A. McClean, and M. R. Hilleman. 1978. Live respiratory syncytial virus vaccine administered parenterally. Proc. Soc. Exp. Biol. Med. 17:66-642.. Centers for Disease Control. 199. Respiratory syncytial virus and parainfluenza virus surveillance-united States, 1989-9. Morbid. Mortal. Weekly Rep. 9:82. 6. Chanock, R. M., B. R. Murphy, P. L. Collins, K. V. W. Coelingh, R. A. Olmsted, M. H. Snyder, M. K. Spriggs, G. A. Prince, B. Moss, J. Flores, M. Gorziglia, and A. Z. Kapikian. 1988. Live viral vaccines for respiratory and enteric diseases. Vaccine 6:129-1. 7. Chanock, R. M., B. Roizman, and R. Myers. 197. Recovery from infants with respiratory illness of a virus related to chimpanzee coryza agent. I. Isolation, properties and characterization. Am. J. Hyg. 66:281-29. 8. Chin, J., R. L. Magoffin, L. A. Shearer, J. H. Schieble, and E. H. Lennette. 1969. Field evaluation of a respiratory syncytial virus vaccine and a trivalent parainfluenza virus vaccine in a pediatric population. Am. J. Epidemiol. 89:449-46. 9. Collins, P. L., R. H. Purcell, W. T. London, L. A. Lawrence, R. M. Chanock, and B. R. Murphy. 199. Evaluation in chimpanzees of vaccinia virus recombinants that express the surface glycoproteins of human respiratory syncytial virus. Vaccine 8:16-169. 1. Connors, M., P. L. Collins, C.-Y. Firestone, A. V. Sotnikov, A. Waitze, A. R. Davis, P. P. Hung, R. M. Chanock, and B. R. Murphy. 1992. Cotton rats previously immunized with a chimeric RSV FG glycoprotein develop enhanced pulmonary pathology when infected with RSV, a phenomenon not encountered following immunization with vaccinia-rsv recombinants or RSV. Vaccine 1:47-484. 11. Elango, N., G. A. Prince, B. R. Murphy, S. Venkatesan, R. M. Chanock, and B. Moss. 1986. Resistance to human respiratory syncytial virus (RSV) infection induced by immunization of cotton rats with a recombinant vaccinia virus expressing the RSV G glycoprotein. Proc. Natl. Acad. Sci. USA 8:196-191. 12. Fulginiti, V. A., J. J. Eller,. F. Sieber, J. W. Joyner, M. Minamitani, and G. Meiklejohn. 1969. Respiratory virus immunization. I. A field trial of two inactivated respiratory virus vaccines: an aqueous trivalent parainfluenza virus vaccine and an alum-precipitated respiratory syncytial virus vaccine. Am. J. Epidemiol. 89:4-448. 1. Jacobs, J. W., and N. Edington. 197. Experimental infection of calves with respiratory syncytial virus. Res. Vet. Sci. 18:299-6. 14. Johnson, P. R., R. A. Olmsted, G. A. Prince, B. R. Murphy, D. W. Alling, E. E. Walsh, and P. L. Collins. 1987. Antigenic relatedness between glycoproteins of human respiratory syncytial virus subgroups A and B: evaluation of the contributions of F and G glycoproteins to immunity. J. Virol. 61:16-166. 1. Kapikian, A. Z., R. H. Mitchell, R. M. Chanock, R. A. Schvedoff, and C. E. Stewart. 1969. An epidemiologic study of altered clinical reactivity to respiratory syncytial (RS) virus infection in children previously vaccinated with an inactivated RS virus vaccine. Am. J. Epidemiol. 89:4-421. 16. Kim, H. W., J.. Arrobio, C. D. Brandt, P. Wright, D. Hodes, R. M. Chanock, and R. H. Parrott. 197. Safety and antigenicity of temperature-sensitive (ts) mutants of respiratory syncytial virus (RSV) in infants and children. Pediatrics 2:6-6. 17. Kim, H. W., J. G. Canchola, C. D. Brandt, G. Pyles, R. M. Chanock, K. Jensen, and R. H. Parrott. 1969. Respiratory BOVINE RSV IN COTTON RATS 19 syncytial virus disease in infants despite prior administration of antigenic inactivated vaccine. Am. J. Epidemiol. 89:422-44. 18. Kimman, T. G., and F. Westenbrink 199. Immunity to human and bovine respiratory syncytial virus. Arch. Virol. 112:1-2. 19. Lehmkuhl, H. D., P. M. Gough, and D. E. Reed. 1979. Characterization and identification of a bovine respiratory virus isolated from young calves. Am. J. Vet. Res. 4:124-126. 2. Lerch, R. A., K. Anderson, and G. W. Wertz. 199. Nucleotide sequence analysis and expression from recombinant vectors demonstrate that the attachment protein G of bovine respiratory syncytial virus is distinct from that of human respiratory syncytial virus. J. Virol. 64:9-69. 21. Lerch, R. A., E. J. Stott, and G. W. Wertz. 1989. Characterization of bovine respiratory syncytial virus proteins and mrnas and generation of cdna clones to the viral mrnas. J. Virol. 6:8-84. 22. Morris, J. A., R. E. Blount, and R. E. Savage. 196. Recovery of a cytopathogenic agent from chimpanzees with coryza. Proc. Soc. Exp. Biol. Med. 92:44-49. 2. Murphy, B. R., R. A. Olmsted, P. L. Collins, R. M. Chanock, and G. A. Prince. 1988. Passive transfer of respiratory syncytial virus (RSV) antiserum suppresses the immune response of the RSV fusion (F) and large (G) glycoproteins expressed by recombinant vaccinia viruses. J. Virol. 62:97-91. 24. Murphy, B. R., A. V. Sotnikov, L. A. Lawrence, S. M. Banks, and G. A. Prince. 199. Enhanced pulmonary histopathology is observed in cotton rats immunized with formalin-inactivated respiratory syncytial virus (RSV) or purified F glycoprotein and challenged with RSV -6 months after immunization. Vaccine 8:497-2. 2. Murphy, B. R., A. Sotnikov, P. R. Paradiso, S. W. Hildreth, A. B. Jenson, R. B. Baggs, L. Lawrence, J. J. Zubak, R. M. Chanock, J. A. Beeler, and G. A. Prince. 1989. Immunization of cotton rats with the fusion (F) and large (G) glycoproteins of respiratory syncytial virus (RSV) protects against RSV challenge without potentiating RSV disease. Vaccine 7:-4. 26. Olmsted, R. A., R. M. L. Buller, P. L. Collins, W. T. London, J. A. Beeler, G. A. Prince, R. M. Chanock, and B. R. Murphy. 1988. Evaluation in nonhuman primates of the safety, immunogenicity, and efficacy of recombinant vaccinia viruses expressing the F or G glycoprotein of respiratory syncytial virus. Vaccine 6:19-24. 27. Olmsted, R. A., N. Elango, G. A. Prince, B. R. Murphy, P. R. Johnson, B. Moss, R. M. Chanock, and P. L. Collins. 1986. Expression of the F glycoprotein of respiratory syncytial virus by a recombinant vaccinia virus: comparison of the individual contributions of the F and G glycoproteins to host immunity. Proc. Natl. Acad. Sci. USA 8:7462-7466. 28. Paccaud, M. F., and C. Jacquier. 197. A respiratory syncytial virus of bovine origin. Arch. Gesamte Virusforsch. :27-42. 29. Porter, D. D., G. A. Prince, V. G. Hemming, and H. G. Porter. 1991. Pathogenesis of human parainfluenza virus infection in two species of cotton rats: Sigmodon hispidus develops bronchiolitis, while Sigmodon fulviventer develops interstitial pneumonia. J. Virol. 6:1-111. 29a.Prince, G. A. Unpublished data.. Prince, G. A., R. L. Horswood, and R. M. Chanock. 198. Quantitative aspects of passive immunity to respiratory syncytial virus infection in infant cotton rats. J. Virol. :17-2. 1. Prince, G. A., A. B. Jenson, V. G. Hemming, B. R. Murphy, E. E. Walsh, R. L. Horswood, and R. M. Chanock. 1986. Enhancement of respiratory syncytial virus pulmonary pathology in cotton rats by prior intramuscular inoculation of formalin-inactivated virus. J. Virol. 7:721-728. 2. Prince, G. A., A. B. Jenson, R. L. Horswood, E. Camargo, and R. M. Chanock. 1978. The pathogenesis of respiratory syncytial virus infection in cotton rats. Am. J. Pathol. 9:771-792.. Prince, G. A., L. Potash, R. L. Horswood, E. Camargo, S. C. Suffin, R. A. Johnson, and R. M. Chanock. 1979. Intramuscular inoculation of live respiratory syncytial virus induces immunity in cotton rats. Infect. Immun. 2:72-728. 4. Stott, E. J., L. H. Thomas, G. Taylor, A. P. Collins, J. Jebbett, and S. Crouch. 1984. A comparison of three vaccines against Downloaded from http://jvi.asm.org/ on December 2, 218 by guest

11 PIAZZA ET AL. respiratory syncytial virus in calves. J. Hyg. 9:21-261.. Taylor, G., E. J. Stott, B. F. Fernie, P. J. Cote, A. P. Collins, M. Hughes, and J. Jebbett. 1984. Monoclonal antibodies that protect against respiratory syncytial virus infection in mice. Immunology 2:17-142. 6. Thomas, L. H., E. J. Stott, A. P. Collins, S. Crouch, and J. Jebbett. 1984. Infection of gnotobiotic calves with a bovine and human isolate of respiratory syncytial virus. Modification of the response by dexamethasone. Arch. Virol. 79:67-77. 7. Trudel, M., F. Nadon, C. Seguin, and H. Binz. 1991. Protection of Balb/C mice from respiratory syncytial virus infection by immunization with a synthetic peptide derived from the G glycoprotein. Virology 18:749-77. 8. Trudel, M., F. Nadon, C. Seguin, S. Brault, Y. Lusignan, and S. Lemieux. 1992. Initiation of cytotoxic T-cell response and protection of Balb/c mice by vaccination with an experimental ISCOMs respiratory syncytial virus subunit vaccine. Vaccine 1:17-112. 9. Trudel, M., F. Nadon, C. Seguin, C. Simard, and G. Lussier. 1989. Experimental polyvalent ISCOMs subunit vaccine induces antibodies that neutralize human and bovine respiratory syncytial virus. Vaccine 7:12-16. 4. Walsh, E. E., C. B. Hall, M. Briselli, M. W. Brandiss, and J. J. Schlesinger. 1987. Immunization with glycoprotein subunits of J. VIROL. respiratory syncytial virus to protect cotton rats against viral infection. J. Infect. Dis. 1:1198-124. 41. Walsh, E. E., C. B. Hall, J. J. Schlesinger, M. W. Brandiss, S. Hildreth, and P. Paradiso. 1989. Comparison of antigenic sites of subtype-specific respiratory syncytial virus attachment proteins. J. Gen. Virol. 7:29-2961. 42. Wathen, M. W., T. J. Kakuk, R. J. Brideau, E. C. Hausknecht, S. L. Cole, and R. M. Zaya. 1991. Vaccination of cotton rats with a chimeric FG glycoprotein of human respiratory syncytial virus induces minimal pulmonary pathology on challenge. J. Infect. Dis. 16:477-482. 4. Wathen, M. W., R. J. Brideau, and D. R. Thomsen. 1989. Immunization of cotton rats with the human respiratory syncytial virus F glycoprotein produced using a baculovirus vector. J. Infect. Dis. 19:2-26. 44. Wertz, G. W., E. J. Stott, K. K.-Y. Young, K. Anderson, and L. A. Ball. 1987. Expression of the fusion protein of human respiratory syncytial virus from recombinant vaccinia virus vectors and protection of vaccinated mice. J. Virol. 61:29-1. 4. Wright, P. F., R. B. Belshe, H. W. Kim, L. P. van Voris, and R. M. Chanock. 1982. Administration of a highly attenuated, live respiratory syncytial virus vaccine to adults and children. Infect. Immun. 7:97-4. Downloaded from http://jvi.asm.org/ on December 2, 218 by guest