The goal of this study was to examine the suitability of recombinant chimeric bovine/human parainfluenza viruses (rB/HPIV3) expressing RSV glycoproteins for use as bivalent, live attenuated mucosal vaccine against RSV and PIV3. rB/HPIV3 was shown previously to be attenuated and immunogenic in rhesus monkeys (46
), and its biological parent virus, BPIV3, was safe and immunogenic in infants 2 to 6 months of age (33
). The attenuation phenotype reflects a host range restriction of replication in primates that is specified by the BPIV3 backbone. These properties suggested that rB/HPIV3 would be an appropriate vector for constructing multivalent pediatric vaccines. Here, rB/HPIV3 was evaluated as a vector for the major protective antigens of RSV, the most important cause of pediatric viral respiratory tract disease. This approach offers the possibility of obviating the problems that have been encountered in developing a live attenuated RSV vaccine that is adequately attenuated yet retains sufficient immunogenicity. The live attenuated RSV vaccine candidates that have been produced during 3 decades of research have been either overattenuated and lacking immunogenicity (32
) or underattenuated and pathogenic to some degree (34
). More recently, reverse genetics has provided a new set of vaccine candidates whose suitability will be evaluated clinically (reference 59
and unpublished data).
The use of rB/HPIV3 that expresses RSV protective antigens as a vaccine against RSV has a number of specific advantages over the previously tested RSV vaccines. First, the expression of RSV glycoproteins as additional, heterologous antigens from rB/HPIV3 makes it possible to vaccinate simultaneously against HPIV3 and RSV. This offers the advantage of simplified vaccine preparation, testing, and administration. In this regard, it is important to note that the F and HN genes of the rB/HPIV3 vector are derived from HPIV3 and thus provide homologous immunity. Multivalent vaccines would reduce the number of necessary separate vaccinations and help simplify the crowded pediatric vaccination schedule.
Second, rB/HPIV3 grows efficiently in vitro and reaches final titers that exceed those of RSV by a factor of 10 to 100. In this respect, vaccine production and evaluation would be more efficient using PIV3-based chimeric viruses. In addition, RSV has the property of unstable infectivity, and a PIV3-based RSV vaccine would facilitate vaccine production, transport, storage, and administration due to increased stability. Thus, use of PIV3 as a vector eliminates the difficulty inherent in attenuating, propagating, handling, and administering RSV.
A third advantage is that the host range restriction phenotype of rB/HPIV3 would be expected to be phenotypically stable, as has been the case with all Jennerian vaccines studied to date (7
). Although this will need to be confirmed for BPIV3, clinical studies to date suggest that the attenuation phenotype is indeed stable. This contrasts, for example, with studies of several RSV vaccines in which partial reversion was observed (21
). Furthermore, swaps between HPIV3 and BPIV3 involving the N gene, or the HN and F genes together, indicate that the host range restriction of BPIV3 is specified by multiple genes and likely is specified by many of the nucleotide and amino acid differences between BPIV3 and HPIV3 (2
). This should contribute to phenotypic stability.
Similar to attenuated RSV vaccine candidates, rB/HPIV3 can infect the respiratory tract efficiently and stimulate local and systemic immunity (33
). This route of administration reduces the virus-neutralizing and immunosuppressive effects of maternally derived serum antibodies present in young infants and children (40
). These two separate phenomena, neutralization of vaccine virus and antibody-mediated suppression of the immune response, can greatly reduce vaccine efficacy in young infants and children (41
). The ability to directly immunize the respiratory tract is critical for the use of rB/HPIV3 as a vector to express antigens for respiratory tract pathogens such as RSV (61
In the present study, we expressed RSV G and F individually from rB/HPIV3. This was done to assess the effect of each glycoprotein alone on replication of the chimeric virus in vitro and in vivo. Since both RSV glycoproteins are independent neutralization antigens, it would be preferable to express both from a single rB/HPIV3 virus, and this work is in progress. In the present paper, insertion of the 1.8-kb F gene into rB/HPIV3 resulted in an eightfold reduction in growth in vitro. However, the observation that this virus induced extensive syncytium formation suggests that the reduced growth might be due to increased cytopathology resulting from the expression of a second fusion protein, rather than to a limitation of the vector's ability to accommodate large genes. In other work, HPIV3 was shown to accommodate a gene insert of 3.9 kb with little effect on replication in vitro and only a modest attenuation effect in vivo (49
). In a separate study, the 15,462-nt HPIV3 genome was increased by nearly 50% with little effect on replication in vitro (M. Skiadopoulos, unpublished data). Thus, the addition of the RSV G gene to rB/HPIV3-F, which would increase the total insert size from 1.8 to 2.7 kb, will likely be tolerated.
rB/HPIV3-G1 and rB/HPIV3-F1 expressed RSV G and F glycoproteins in most or all of the infected cells, as indicated by the similarity in titers determined by RSV or HPIV3 staining of viral plaques. Although we did not formally determine the genome stability of B/HPIV3-G1 and B/HPIV3-F1, we did not detect any loss of activity in biological clones of HPIV3 bearing foreign inserts after eight passages in cell culture (unpublished data). This observation agrees with previously published reports on the genetic stability of modified recombinant parainfluenza viruses (24
rB/HPIV3-G1 and rB/HPIV-F1 were comparable to RSV in their ability to confer protection against challenge of hamsters with wt RSV. The hamster model was selected over the more common mouse model for RSV infection because PIV3 is severely restricted in mice whereas hamsters are semipermissive for both PIV3 and RSV. The hamster model does not allow evaluation of the attenuation phenotype of BPIV3, since that is specific to primates, but this phenotype was confirmed in earlier work for rB/HPIV3 (46
). The level of replication of rB/HPIV3-G1 in the respiratory tract was similar to, and statistically indistinguishable from, that of the control viruses, BPIV3 and HPIV3. rB/HPIV-F1 replicated to a slightly lower titer than did the other viruses on days 4 and 5. This may be an effect of having this large gene in a promoter-proximal position, an effect of the expression of a second fusogenic protein, or both. In any case, this difference was not statistically significant in comparison with the biological BPIV3 virus, which in previous primate and clinical studies replicated sufficiently well to induce a protective immune response (33
The titer of G-specific or F-specific antibodies induced by the rB/HPIV3-G1 or rB/HPIV3-F1 virus, respectively, was two- to fourfold higher than that induced by wt RSV, and RSV-neutralizing antibody titers induced by B/HPIV3-G1 and -F1 also tended to be higher than those induced by wt RSV. However, since RSV replicated less efficiently in hamsters than did the rB/HPIV3 chimeric viruses, the difference in immunogenicity should be interpreted with caution. It is important to note that the antibody response to rB/HPIV3-G1 or rB/HPIV3-F1 is similar to that induced by RSV in that high RSV glycoprotein-specific ELISA titers are matched by high RSV neutralizing titers, indicating that conformationally correct glycoproteins are expressed. This is fundamentally different from the picture seen following immunization with formalin-inactivated or subunit RSV vaccines, where antibodies with high ELISA titers but low RSV neutralizing activity are induced (16
). Each of the rB/HPIV3 viruses induced a titer of PIV3-specific antibody that was indistinguishable from that of their parent virus, rB/HPIV3, indicating that the presence and expression of the insert antigen did not compromise the immunogenicity of the vector glycoproteins. Consistent with the high RSV- and HPIV3-specific antibody titers, rB/HPIV3-G1 and rB/HPIV3-F1, alone or in combination, induced a level of resistance to challenge with RSV that was indistinguishable from that induced by previous infection with wt RSV. Both viruses also induced a level of resistance to challenge with HPIV3 that was similar to that induced by previous infection with rB/HPIV3.
We anticipate that the expression of RSV G and F from rB/HPIV3 will not be associated with immune-mediated enhanced pathogenicity upon RSV challenge in primates, since this was our experience with vaccinia virus recombinants expressing G or F (21
). In certain inbred strains of mice, dermal immunization with RSV G alone, expressed from a vaccinia virus vector, primed for enhanced disease upon RSV challenge (1
). It is not clear whether this phenomenon is specific to these particular conditions in the mouse model or whether it has wider significance. Since BPIV3 grows poorly in mice, we have not evaluated possible immunopotentiation in that model. However, the possibility of vaccine-specific effects will be carefully evaluated in future studies in primates.
In summary, the in vitro and in vivo characteristics of rB/HPIV3-G1 and rB/HPIV3-F1 indicate that these viruses are candidates for a bivalent mucosal vaccine against both RSV and HPIV3. Both viruses, as well as a planned virus expressing both G and F, need further characterization with regard to their level of attenuation and their immunogenicity in primates before clinical studies can be initiated. The lack of internal RSV proteins in rB/HPIV3-G1 and rB/HPIV3-F1, and therefore the loss of several cytotoxic T-lymphocyte epitopes, did not seem to impact the immunogenicity and protective efficacy of these viruses (17
). In consideration of the complex immune response induced by RSV, a vectored approach to developing an RSV vaccine may help to achieve the fine balance between immunogenicity and attenuation that is needed.