A variety of approaches have been used in the past to immunize against disease caused by human RSV infection. To date, only inactivated, subunit, and live attenuated RSV vaccines have been evaluated in human clinical trials (22
). Formalin-inactivated RSV did not protect vaccinees against RSV infection, and vaccinated individuals were more likely to develop severe RSV-associated disease than naïve individuals during subsequent RSV infection (22
Following the failure of inactivated RSV vaccine, vaccine development focused on immunization with live attenuated RSV. The first live attenuated RSV vaccines tested in human trials were cold-passaged and/or chemically mutagenized viruses displaying temperature sensitivity. These vaccine candidates failed because they were either over- or underattenuated, and wild-type revertants were often isolated from vaccinees (23
). Cold-passaged, temperature-sensitive viruses 248/955 and 530/1009, a more current series of RSV strains, were evaluated in RSV-seronegative children as young as 6 months. However, both of these vaccine candidates were insufficiently attenuated for further evaluation in infants (19
). Cold-passaged, temperature-sensitive virus 248/404, the most attenuated live RSV tested in humans to date, caused mild to moderate congestion in the upper respiratory tract of infants 1 to 2 months old and therefore was still underattenuated as a vaccine for early infancy (40
While there are currently no suitably attenuated live RSV vaccines for use in young infants, the clinical trials showed that immunization with a live RSV (i) did not result in enhanced disease during RSV reinfection, (ii) could elicit protective immunity against RSV infection in infants 1 to 2 months old, (iii) could be achieved in the presence of maternal antibodies, and (iv) might require two or more doses to achieve satisfactory infection rates and antibody responses.
Subunit vaccines consisting of purified RSV F were also evaluated as potential vaccines for immunization of the elderly (9
) and high-risk children (12
) and for maternal immunization (F. M. Munoz, P. A. Piedra, M. Maccato, C. Kozinetz, and W. P. Glezen, RSV after 45 Years, abstr., p. 45). In the elderly, purified RSV F was moderately immunogenic (6
); 25 to 48% of the elderly vaccinees showed a rise equal to or greater than fourfold in RSV-neutralizing antibody titers. A phase 3 trial of 298 children with cystic fibrosis immunized with purified RSV F showed no statistically significant differences in the frequency of LRT infections between the vaccinated and those receiving placebo (9
Other RSV subunit vaccines that have been evaluated in clinical trials include BBG2Na, a fusion protein consisting of highly conserved residues 130 to 230 of the G protein from RSV conjugated to the albumin binding domain of streptococcal protein G (32
). BBG2Na was well tolerated in healthy adults and moderately immunogenic; 33 to 71% of those immunized had a rise equal to or greater than twofold in neutralizing antibody titer. RSV subunit vaccines had minimum reactogenicity and did not cause enhanced disease; however, they were only moderately immunogenic (34
The approach presented here utilizes a virus vector to deliver RSV F with the aim of inducing both humoral and cell-mediated immunity against RSV infection. Other virus vectors have been used in the past for delivery of RSV F and RSV G proteins. Vaccinia viruses F and G were separately able to induce long-term protection against wild-type RSV challenge in BALB/c mice (4
). However, vaccinia viruses F and G failed to induce adequate levels of neutralizing antibody in seronegative chimpanzees. No protection was detected in the URT and incomplete protection was found in the LRT when the chimpanzees were challenged with RSV (3
). Adenoviruses expressing RSV F, RSV G, and RSV F and G have also been tested in RSV-seronegative chimpanzees and found to be poorly immunogenic (16
Our vector delivery system did not elicit the level of neutralizing antibodies seen with wild-type RSV infection, presumably because only one RSV antigen was expressed. However, both the upper and lower respiratory tracts of AGMs immunized with both vaccine candidates were protected against RSV challenge 1 month postimmunization. It is not clear how long the immune response to RSV and/or hPIV3 will persist in AGMs. In hamsters, we were unable to detect any decay in RSV-neutralizing antibody and HAI antibody titer 53 days after immunization with 105
PFU of b/h PIV3/RSV F2 (R. Tang, unpublished data). Tao et al. showed that PIV3 immunity can last up to 4 months in hamsters (37
The chimeric b/h PIV3/RSV F vaccines produced RSV-neutralizing antibodies specific for both RSV subgroups A and B. The high degree of conservation of the amino acid sequences between the RSV F proteins of subgroup A and B resulted in shared neutralizing epitopes. Not surprisingly, the levels of RSV-neutralizing antibody titers were lower by 5 log2 for b/h PIV3/RSV F than those observed for primate sera obtained from AGMs infected with wild-type RSV A2.
In the b/h PIV3/RSV vaccines, RSV neutralizing antibodies were produced only in response to the RSV F protein rather than to the whole RSV virus particle. The levels of RSV B cross-neutralizing antibody for sera obtained from AGMs infected with RSV A2 were reduced by 5 log2 compared to the antibody levels observed when the homologous RSV A2 antigen was tested. In contrast, a decrease in RSV B specific-neutralizing antibody titers produced by b/h PIV3/RSV F2 and b/h PIV3/sol RSV F2 was not observed. These results suggested that the serum neutralizing antibody levels induced by the RSV F protein were sufficient to protect primates from RSV challenge 1 month postvaccination.
Although the RSV-neutralizing antibody titers were lower for b/h PIV3/RSV F primate sera, the neutralizing activity for subgroup A and B RSV strains was essentially identical. Primate sera derived from wild-type RSV infection, displayed high RSV-neutralizing titers for the homologous RSV A antigen and lower levels for the RSV B antigen, which were similar in titer to those observed for the vectored PIV3/RSV F vaccines. A rise (>6 log2) in RSV F IgG antibody titers was observed for primates infected with RSV A2 or immunized with the b/h PIV3/RSV F vaccines. A further increase in either RSV-neutralizing or IgG antibody titers was not observed for animals vaccinated with b/h PIV3/RSV F or b/h PIV3/sol RSV F in response to the RSV challenge. Since the RSV neutralizing antibody titers measured for PIV3/RSV F vaccines were lower than those observed for sera obtained from primates infected with wild-type RSV, cellular immune responses may have played a role in generating such effective protection from RSV challenge. Future studies will address the contribution of the cellular immune system and of secretory IgA antibodies to the efficacy of the live attenuated PIV3/RSV vaccines.
The b/h PIV3 vector is expected to be attenuated in humans because the majority of the viral genome is derived from bPIV3, which was demonstrated to be safe in children (20
). Skiadopoulos et al. clearly showed, using a rhesus monkey attenuation model, that the bPIV3 attenuation phenotype was polygenic in nature (35
). While the bPIV3 F and HN genes contain some genetic determinants specifying attenuation, the greatest contribution to the attenuation phenotype was ascribed to the bPIV3 N and P proteins. Schmidt et al. evaluated a number of b/h PIV3-expressing RSV antigens from different PIV3 genome positions for replication in the respiratory tract of rhesus monkeys (33
). All of the chimeric b/h PIV3-expressing RSV proteins replicated less efficiently than b/h PIV3 in the URT. Slightly higher titers (~0.5 log10
/ml) were observed in the LRT of rhesus monkeys compared to the vector b/h PIV3. Taken together, these data further validate the expectation that b/h PIV3/RSV will be attenuated in humans.
Infants do not possess a well-developed immune system, and therefore multiple vaccine administrations may be necessary to develop long-lasting and protective immunity to RSV. Vaccination at 2, 4, and 6 months of age may be conceivable, ideally to be scheduled concurrently with other routine childhood vaccinations. PIV3 is highly immunogenic, and the first PIV/RSV vaccination induces high levels of PIV3 antibodies. This may result in vector immunity, such that subsequent immunizations with PIV/RSV may not produce a further rise in antibody titer. While we have not directly addressed this issue experimentally, a recent study by Karron et al. presented data showing that multiple doses of PIV3 will not result in vector immunity provided the dose administrations are spaced far enough apart (18
The administration of a single dose of cp-45 PIV3 vaccine, a cold-passaged, temperature- sensitive virus, restricted the magnitude of vaccine replication after the second dose. However, the frequency of infection with a second dose of vaccine was clearly influenced by the dosing interval. Only 24% of infants shed virus when a second dose of vaccine was administered 1 month later. In contrast, 62% of infants shed virus when the second dose was administered 3 months after the first dose. These results suggested that to minimize PIV3 vector immunity effects, the interval between vaccinations should be >1 month but <3 months.
While the main goal of this study was to evaluate b/h PIV3/RSV F2 and b/h PIV3/sol RSV F2 as potential RSV vaccines, we also wanted to determine whether hPIV3 serum HAI and neutralizing antibody titers were produced in response to vaccination. The levels of hPIV3 HAI and neutralizing antibodies observed for the primate sera obtained from animals immunized with both kinds of b/h PIV3/RSV F vaccines were similar to the titers displayed by rhesus monkeys vaccinated with b/h PIV3 (30
). Rhesus monkeys immunized with b/h PIV3 were effectively protected from challenge with wild-type hPIV3. These results suggested that b/h PIV3 vectored RSV vaccines may be developed in the future as bivalent vaccines to protect infants from both RSV and hPIV3 infections and disease.