MedImmune Vaccines Inc., 297 North Bernardo Avenue, Mountain View, CA 94043, USA
Correspondence
Aurelia Haller
hallera{at}medimmune.com
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ABSTRACT |
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Present address: VaxGen Inc., Brisbane, CA 94005, USA.
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INTRODUCTION |
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Recently, a live, attenuated, cold-passaged (cp), temperature-sensitive (ts), intranasally administered RSV candidate vaccine (cpts-248/404) was evaluated in adults and children for safety, infectivity and immunogenicity (Crowe et al., 1994a, b
; Karron et al., 1997
). cpts-248/404 did not cause fever or lower respiratory tract illness and was not transmitted from infant to infant. However, nasal congestion, which occasionally led to difficulty in eating and irritability, was judged sufficiently disruptive to the infant and family to make this an undesirable vaccine (Karron et al., 1997
). Based on these experiences with live attenuated vaccines, new strategies are needed to produce a RSV vaccine that will effectively protect infants against RSV infection, yet will not result in upper respiratory tract congestion.
In this study, we wanted to determine whether bovine parainfluenza virus type 3 (bPIV3) would tolerate the insertion of two translationally active transcription units in its genome as well as generate a novel bPIV3-vectored RSV vaccine candidate. bPIV3 constitutes a promising virus vector, which was shown in human clinical trials to be attenuated, immunogenic, non-transmissible in a day care centre setting and genetically stable in children as young as 2 to 6 months of age (Karron et al., 1996). The capacity of the bPIV3 vaccine to replicate in the nasal cavity without causing respiratory illness demonstrated its attenuation in young seronegative children (Karron et al., 1996
). Recombinant bPIV3 (r-bPIV3) has been used successfully as a virus vector to express the highly conserved human PIV3 (hPIV3) fusion (F) and haemagglutininneuraminidase (HN) genes (Haller et al., 2000
; Schmidt et al., 2000
). To determine whether bPIV3 could be used to express heterologous antigens in addition to bPIV3 antigens, the G and F proteins of RSV were selected as they represent the major antigens responsible for eliciting neutralizing antibodies. The safety and attenuation profile of bPIV3 in infants support its use as a vector to deliver RSV antigens to this population. The r-bPIV3 expressing RSV surface glycoproteins is expected to elicit production of RSV serum antibodies and cell-mediated immune responses, yet not cause disease in infants.
The chimeric bPIV3 virus characterized in this study contained the RSV G and F genes inserted downstream of the HN gene of bPIV3. The recombinant bPIV3 harbouring the RSV surface glycoproteins, bPIV3/RSV(I), was shown by biochemical and immunological assays to express the RSV G and F proteins. Studies in small animals showed that bPIV3/RSV(I) was restricted for replication in hamsters, yet bPIV3/RSV(I)-immunized animals were protected from challenge with either hPIV3 or RSV. The bPIV3/RSV(I) construct validated the use of bPIV3 as a virus vector. The insertion of two foreign transcription units was tolerated and both genes were expressed. The recombinant bPIV3/RSV(I) described here will be evaluated further for safety and efficacy in primates as a RSV vaccine candidate.
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METHODS |
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Construction of full-length bPIV3/RSV(R) and bPIV3/RSV(I) cDNAs.
Oligonucleotides used to amplify by PCR the RSV G and F genes from RSV-infected Vero cells were complementary to the 5' end of the RSV G gene and encoded an NheI restriction enzyme site or complementary to the 3' end of the RSV F gene and encoded a SalI site. The 2·8 kb PCR fragment was digested with NheI/SalI and cloned into pGEM3 (Promega). The RSV G and F genes were sequenced to confirm the presence of open reading frames. A bPIV3 gene end/gene start sequence was introduced into the intergenic sequence between the RSV G and F genes using QuickChange mutagenesis (Stratagene). The plasmid bPIV3/N/S, which contains the bPIV3 genome, described previously (Haller et al., 2001), was cleaved with NheI (nt 5042)/SalI (nt 8530) to remove the bPIV3 F and HN genes, and the RSV G and F genes were inserted to yield the full-length cDNA of bPIV3/RSV(R). The MG and FL intergenic junctions of this plasmid were sequenced.
To generate a cDNA for bPIV3/RSV(I), a DNA fragment harbouring the RSV G and F genes flanked by SalI restriction enzyme sites was generated by PCR and cloned into pGEM3. bPIV3 gene end/gene start sequences were introduced upstream of the RSV G and the RSV F genes by PCR. This plasmid was digested with SalI, resulting in a 2·8 kb DNA fragment. The cDNA containing bPIV3/N/S was linearized with SalI (nt 8530) and the RSV G and F gene fragment was inserted to construct the plasmid bPIV3/RSV(I). The HNG and the RSV FbPIV3 L gene junctions were sequenced. Both cDNA constructs were designed to obey the rule of six.
Transfection of full-length viral cDNA-containing plasmids.
Transfections of viral cDNAs were carried out as described previously (Haller et al., 2000). The presence of bPIV3/RSV(R) or bPIV3/RSV(I) was confirmed by immunostaining of virus-infected monolayers using RSV antiserum (Biogenesis). Following three cycles of plaque purification at 33 °C, virus stocks were prepared in Vero cells.
Growth curves.
Vero or BHK-21 cells were grown to 90 % confluency and infected at an m.o.i. of 0·1 with r-bPIV3, bPIV3/RSV(I) or RSV. Infected monolayers were incubated at 33, 37, 39 and 40 °C. At 0, 24, 48, 72, 96 or 120 h post-infection (p.i.), the cells and media were harvested. Virus titres for each time-point were determined by plaque assay on Vero cells that were immunostained using RSV antiserum.
Immunoblot and immunoprecipitation analyses.
For Western blot analysis, Vero cells were infected with r-bPIV3, bPIV3/RSV(I) or RSV at an m.o.i. of 0·1. At 48 h p.i., proteins were extracted with lysis buffer. The cell lysate was fractionated on a 10 % protein gel, transferred onto a nylon membrane and probed with RSV G68 mAb (Martinez et al., 1997). Proteinantibody complexes were visualized by chemiluminescence (Amersham).
For immunoprecipitation, Vero cells were infected with r-bPIV3, bPIV3/RSV(I) or RSV at an m.o.i. of 1·0. At 24 h p.i., the cells were washed once with cysteine- and methionine-free DME (ICN) and incubated in the same media for 30 min. The medium was then removed and 0·5 ml DME lacking cysteine and methionine but containing 100 µCi [35S]Pro-Mix (Amersham) was added to the cells. Infected cells were incubated in the presence of 35S-labelled isotopes for 5 h at 37 °C. Medium was removed and infected cells were lysed in 0·3 M RIPA buffer containing protease inhibitors. The cell lysate was incubated with RSV F 1200 mAb (Beeler & van Wyke Coelingh, 1989) and bound to anti-mouse IgGagarose (Sigma). After washing three times with 0·5 M RIPA buffer, samples were fractionated on a 12 % protein gel. The gel was dried and exposed to MR-X film.
Flow cytometric analysis.
Vero cells were infected with r-bPIV3, RSV or bPIV3/RSV(I) at an m.o.i. of 0·1 or were mock infected. Infected monolayers were incubated for 24 h, trypsinized using Versene (Gibco-BRL), washed with PBS containing 0·1 % BSA and incubated with the primary bPIV3 (VMRD), RSV G68 or RSV F 1200 antisera. After 30 min of incubation, cells were washed and incubated with the secondary sheep anti-mouse FITC-conjugated (Biodesign) or rabbit anti-goat PE-conjugated (Sigma) antisera for 30 min. Cells were then washed twice and analysed using a FACSCalibur flow cytometer (BD Immunocytometry Systems).
Small animal studies.
Hamsters (5-week-old, 812 animals per group) were infected intranasally with 1x106 p.f.u. r-bPIV3, bPIV3/RSV(I), RSV or placebo (Opti-MEM) in a 0·1 ml volume. The four different groups were maintained separately in microisolator cages. At 4 days p.i., the nasal turbinates and lungs of the animals were harvested and homogenized. The titre of virus present in the tissues was determined by plaque assay on Vero cells that were immunostained with RSV or bPIV3 polyclonal antiserum. For challenge studies, animals were inoculated on day 21 intranasally with 1x106 p.f.u. of hPIV3 or RSV. At 4 days post-challenge, the nasal turbinates and lungs of the animals were assayed by plaque assay on Vero cells for quantification.
Neutralization assay.
Microneutralization assays were performed for bPIV3, bPIV3/RSV(I) or RSV using Vero cells. Antibodies [RSV (Biogenesis), bPIV3 (VMRD) polyclonal antisera or RSV F 1153, RSV F 1243 (Beeler & van Wyke Coelingh, 1989) or Synagis (MedImmune) monoclonal antisera] were used at serial twofold dilutions; the starting dilution was 1 : 4. Samples were then incubated with 100 p.f.u. bPIV3, bPIV3/RSV(I) or RSV at 4 °C for 60 min. Following incubation, virus/serum mixtures were transferred to 96-well plates of Vero cell monolayers, overlaid with media and incubated at 35 °C for 6 days. Neutralization titres were expressed as the reciprocal of the highest serum dilution that inhibited CPE.
Plaque-reduction neutralization assays were carried out for serum obtained on day 21 p.i. from hamsters immunized with r-bPIV3, bPIV3/RSV(I) or RSV. Hamster sera were serially twofold diluted and incubated with 100 p.f.u. RSV in the presence of guinea pig complement for 1 h at 4 °C. Then the virus/serum mixtures were transferred to Vero cell monolayers and overlaid with methylcellulose. After 5 days of incubation at 35 °C, monolayers were immunostained using RSV polyclonal antiserum for quantification. Neutralization titres were expressed as the reciprocal log2 of the highest serum dilution that inhibited 50 % of virus titres.
Haemagglutination inhibition assay.
Haemagglutination-inhibition (HAI) assays were performed by incubating serial twofold dilutions of hamster serum with hPIV3 at 25 °C for 30 min. Guinea pig erythrocytes were added to each well and incubation was continued for 90 min to allow haemagglutination to occur. HAI titres were expressed as the reciprocal log2 of the highest serum dilution that resulted in inhibition of red blood cell agglutination.
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RESULTS |
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Since the RSV F and G proteins are not essential for replication of bPIV3/RSV(I), it was necessary to determine whether a mixed virus population was present in these virus stocks, i.e. bPIV3 expressing only RSV G or bPIV3 expressing only RSV F, as well as bPIV3 expressing both RSV G and F. The virus stocks characterized in this study were passaged five times in Vero cells. Flow cytometry was used to assess the presence of both RSV and PIV3 antigens in bPIV3/RSV(I)-infected cells (Fig. 4). As a control, r-bPIV3-infected cells were incubated with bPIV3-specific primary antiserum or non-specific goat isotype antiserum and both were labelled with PE. Only the peak representing cells labelled with bPIV3 antiserum shifted to the right (Fig. 4A
). Similarly, Vero cells infected with bPIV3/RSV(I) or mock-infected cells were incubated with a bPIV3 polyclonal antiserum linked to PE and a shift was only observed for the bPIV3/RSV(I)-infected cells expressing the PIV3 surface glycoproteins (Fig. 4A
). RSV-infected cells incubated with RSV G- or F-specific mAbs linked to FITC shifted to the right, unlike RSV-infected cells labelled with non-specific mouse isotype antiserum labelled with FITC (Fig. 4B
). bPIV3/RSV(I)-infected cell populations expressing RSV F and G proteins or mock-infected cells were also probed with RSV mAbs labelled with FITC. bPIV3/RSV(I)-infected cells displaying RSV proteins exclusively displayed a distinct peak apart from the mock-infected cell population (Fig. 4B
). A double-labelling experiment showed that bPIV3/RSV(I)-infected cells incubated with both bPIV3 PE-conjugated and either RSV F or G FITC-conjugated antisera sorted to the upper right quadrant (Fig. 4C
). In contrast, mock-infected cells incubated with bPIV3- and RSV-specific antisera were observed solely in the lower left quadrant (Fig. 4C
). These results showed that bPIV3/RSV(I) expressed the surface glycoproteins of both bPIV3 and RSV (G and F). A spurious deletion of the RSV G or F genes would have resulted in cell populations expressing solely bPIV3 proteins, which should be observed in the upper left quadrant. If cells were infected with viruses that only expressed RSV proteins, a separate cell population in the lower right quadrant would be expected, but this was not seen in these studies (Fig. 4C
). The results obtained from the flow cytometric analysis confirmed that bPIV3/RSV(I)-infected cells expressed both bPIV3 and RSV surface glycoproteins.
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To determine whether the restricted replication phenotype of bPIV3/RSV(I) in hamsters can be mimicked in tissue culture, the replication kinetics of bPIV3/RSV(I), r-bPIV3 and RSV were compared in Vero and BHK-21 cells (Fig. 5). All three viruses, bPIV3/RSV(I), r-bPIV3 and RSV, replicated to peak titres of 8·5, 9·0 and 7·1 log10 p.f.u. ml-1 in Vero cells, respectively (Fig. 5A
). Replication of r-bPIV3 was reduced by
1·0 log10 in BHK-21 cells and a peak titre of 8·0 log10 p.f.u. ml-1 was achieved (Fig. 5B
). The peak of r-bPIV3 replication in Vero cells occurred at 48 h p.i. (Fig. 5A
). In BHK-21 cells, the peak titre of r-bPIV3 was observed at 96 h p.i. (Fig. 5B
). RSV replicated to a low titre of 5·9 log10 p.f.u. ml-1 in BHK-21 cells and displayed unstable virus titres after 48 h p.i. (Fig. 5B
). A 1·0 log10 reduction in RSV peak titre in BHK-21 cells compared to titres achieved in Vero cells was observed in this study. bPIV3/RSV(I) replication peaked at 48 h p.i. in Vero cells, while a delayed onset of virus replication in BHK-21 cells was observed (Fig. 5A, B
). Peak titres of bPIV3/RSV(I) in BHK-21 were lower by
1·5 log10 compared to the titres obtained in Vero cells. The data from this study showed that all three viruses, r-bPIV3, bPIV3/RSV(I) and RSV, replicated less efficiently in BHK-21 cells. However, the delayed replication of bPIV3/RSV(I) in BHK-21 cells cannot be correlated directly to the impaired in vivo phenotype observed in hamsters.
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bPIV3/RSV(I)-immunized hamsters that were given a RSV challenge dose intranasally also showed protection from RSV strain A2. bPIV3/RSV(I)-immunized animals displayed titres of 1·7 and 1·4 log10 p.f.u. g-1 tissue in the nasal turbinates and lungs, respectively (Table 2). Hamsters that were inoculated with RSV initially and then challenged with RSV displayed 1·2 and 1·5 log10 p.f.u. g-1 tissue in the nasal turbinates and lungs (Table 2
). Animals that had been immunized with placebo medium or r-bPIV3 and were challenged with RSV showed titres of 3·8 and 3·0 log10 p.f.u. g-1 tissue or 3·3 and 2·9 log10 p.f.u. g-1 tissue, respectively, in the nasal turbinates and lungs (Table 2
). bPIV3/RSV(I) immunization reduced RSV virus loads in the upper and lower respiratory tracts of hamsters by 2·1 and 1·6 log10 compared to the RSV-challenged animals that had received placebo medium (Table 2
). Immunization with RSV reduced virus titres by 2·6 and 1·5 log10 p.f.u. g-1 tissue in the nasal turbinates and lungs, respectively (Table 2
). Therefore, bPIV3/RSV(I) protected the lower respiratory tract of hamsters as well as wt RSV and showed slightly less protection of the upper respiratory tract than RSV-immunized animals. bPIV3-RSV(I) animals were completely protected from challenge with hPIV3.
Hamsters vaccinated with bPIV3/RSV(I) elicited RSV neutralizing and PIV3 HAI serum antibody responses
Hamster sera obtained 21 days post-immunization were analysed for the presence of RSV neutralizing and PIV3 HAI serum antibodies. Despite low levels of replication observed for bPIV3/RSV(I), RSV neutralizing antibodies were detected in the day 21 hamster sera (Table 3). RSV antibody titres of 5·4 log2 were observed for bPIV3/RSV(I) hamster sera, while wt RSV sera contained slightly higher antibody titres of 6·6 log2. HAI antibody titres were also determined for the day 21 hamster sera. Sera obtained from animals vaccinated with bPIV3/RSV(I) had slightly lower HAI titres of 2·2 compared to HAI titres present in bPIV3 hamster sera, which displayed titres of 3·3. In general, HAI titres produced by bPIV3-vaccinated hamsters were low; this has been observed previously (Haller et al., 2000
). Thus, bPIV3 and bPIV3/RSV(I) may mediate immune protection by cellular immune pathways.
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DISCUSSION |
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The recombinant bPIV3 characterized in this study expressed both RSV G and F genes. bPIV3/RSV(I) was temperature sensitive for growth in Vero cells at 40 °C; however, it replicated to high virus titres at 33 °C. In vivo, bPIV3/RSV(I) was restricted for replication in the respiratory tract of hamsters. Despite lower levels of replication observed for bPIV3/RSV(I) in hamsters, immunized animals were protected from challenge with hPIV3 or RSV A2. The restricted replication of bPIV3/RSV(I) in hamsters could not be mimicked in BHK-21 cells. bPIV3/RSV(I) did not display a host cell-specific ts phenotype in BHK-21 cells at the body temperature of hamsters (37 °C), which could have explained its poor replication in hamsters. The impaired replication of bPIV3/RSV(I) in vivo may be due to inserting two transcription units between the HN and L genes of bPIV3, thereby adding 2900 nt to the viral genome. Skiadopoulos et al. (2000)
studied the effect of increasing the PIV3 genome on virus replication. Non-coding or coding sequences up to
3900 nt were inserted into a recombinant hPIV3 between the HN and L genes. Viruses harbouring the largest inserts replicated efficiently in vitro. However, viruses containing a genome that was increased by greater than 3000 nt exhibited restricted replication in hamsters. The genes inserted into bPIV3/RSV(I) were not only transcriptionally active but also translationally competent, the latter of which differed from the inserts used by Skiadopoulos and co-workers, which may be the reason for the increased in vivo attenuation observed in this study.
We demonstrated protective immunity of bPIV3/RSV(I)-vaccinated animals not only for RSV but also for hPIV3, thereby expanding the use of bPIV3 for the generation of bivalent paediatric vaccines. bPIV3/RSV(I)-immunized animals produced RSV neutralizing and PIV3 HAI serum antibodies at levels slightly lower than the parental virus strains. There are several advantages of using live, attenuated, intranasally administered vaccines: (1) live virus vaccines stimulate immune responses similar to wt virus infections, inducing both systemic and local immunity (Murphy et al., 1994); (2) maternal antibodies present in infants do not appear to interfere with vaccine virus replication in the nasopharynx and its ability to induce protective immunity (Murphy et al., 1994
); (3) live attenuated RSV vaccines are not likely to cause RSV disease potentiation during subsequent, naturally occurring, wt RSV infections in immunized infants (Chanock & Murphy, 1991
).
Results obtained by other investigators underscore further the high likelihood of success for bPIV3 as a virus vaccine vector. Recently, Schmidt et al. (2001, 2002)
generated recombinant chimeric bovine/human PIV3 expressing RSV G and/or F proteins. Hamsters immunized with these recombinant viruses were protected in challenge studies from RSV as well as hPIV3. The position of the RSV genes introduced into the PIV3 genome may specify a host range phenotype. The recombinant bovine/human PIV3 harboured the RSV genes at the 3' end of the viral genome, while bPIV3/RSV(I) contained the RSV G and F genes in the centre of the genome, between the HN and L genes of bPIV3. Schmidt et al. (2001
, 2002)
employed a chimeric bovine/human PIV3 as a vector backbone to enhance PIV3 antigenicity. However, the humanization of the bPIV3 surface glycoproteins may result in the loss of the critical attenuation phenotype observed in humans. The attenuation phenotype associated with bPIV3 is most likely the result of contributions from multiple viral gene products. Therefore, it is important to pursue development of bPIV3 as a vaccine vector since it retains all of the attenuation determinants.
bPIV3/RSV(I) represents a novel, rationally designed, vaccine candidate intended to protect children, the elderly and immunocompromised individuals from disease caused by RSV and hPIV3. While other virus vectors may be developed in the future to deliver RSV antigens, bPIV3 has a number of advantages, especially regarding safety and genetic stability, that warrant its development as a vaccine vector: (1) the greatest advantage of using bPIV3 is its prior experience in human clinical trials, where bPIV3 was shown to be attenuated in infants (Karron et al., 1996); (2) recombinant bPIV3 expressing both RSV G and F proteins has the advantage that only a single vaccine virus needs to be administered to afford immunity; (3) recombinant bPIV3/RSV(I) contains the entire bPIV3 RNA genome and, thus, is expected to retain all of the attenuation determinants; (4) RSV antigens are delivered within the context of a live virus and, thus, immunopotentiation upon infection with wt RSV is not expected to occur; (5) live bPIV3/RSV(I) vaccine should stimulate both systemic and mucosal immunity. The information gained from studying bPIV3/RSV(I) lends further support to developing bPIV3 as a vaccine vector for expression of viral and bacterial antigens.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 3 January 2003;
accepted 2 May 2003.