The biologically-derived HPIV3cp45 vaccine was previously shown to be highly infectious and well-tolerated in infants as young as 1 month of age and to induce serum HAI antibody responses in children over 6 months of age. [10
] While this virus has promising properties as an HPIV3 vaccine candidate, it has a long passage history in primary cells and cell lines. Here, a recombinant rcp45 virus was derived from cDNA to generate a new investigational vaccine with a short, well-defined passage history using qualified reagents and cells. Derivation of a live-attenuated virus from cDNA minimizes the risk of contamination with known or currently unidentified adventitious agents that could potentially be present in the original clinical isolate or could have been introduced during passage, arising either from the primary and continuous cells or from serum, trypsin, or other reagents. Several sequence differences exist between the biologically-derived and the cDNA-derived vaccine virus. While these did not include known attenuating mutations and appeared to be inconsequential for attenuation and immunogenicity in non-human primates, it was essential to confirm comparability of the biologically-derived and cDNA-derived cp45 vaccines in infants and young children.
In this study, we sought to determine whether rcp45 was as well tolerated, immunogenic and infectious as the biologically-derived vaccine virus. We initially evaluated the vaccine in an unscreened population of 6-to-12 month old children. However, serologic testing that was performed subsequent to inoculation showed that 5 of 23 children in this cohort were HPIV3 seropositive (HAI antibody titers >1:8), and all of these children received vaccine. This rate of seropositivity is consistent with epidemiologic studies that highlight the importance of HPIV3 as a respiratory pathogen in the first year of life. [4
] Replication of rcp45 was highly restricted in these seropositive infants, as we previously observed with the cp45 vaccine. [28
] This study confirms that in children over 6 months of age, serum HPIV3 HAI antibody is an appropriate marker for resistance to infection with a live-attenuated HPIV3 vaccine.
In contrast to the limited infectivity observed in HPIV3 seropositive children, rcp45 was highly infectious in HPIV3 seronegative children. Following the first dose of vaccine, 22 of 24 seronegative children (92%) were infected, and the titer and duration of vaccine virus shedding were comparable to what had been previously observed with the biological cp45 vaccine. [10
] Seroconversion following the first dose was observed in 83% of the seronegative vaccinees. The HAI titers achieved in vaccinees were lower than the prevaccination HAI titers in the seropositive subjects in this study and lower than has been observed following natural infection with wt HPIV3 [11
], indicating that the restricted replication of the vaccine virus induces a less robust antibody response than does infection with wt HPIV3. This finding suggests that two or more doses of vaccine will be necessary to achieve an immune response comparable to that induced by a single infection with wt HPIV3. However, we found that the first dose of rcp45 induced an immune response that was sufficient to prevent vaccine virus shedding in most children who received a second dose of vaccine 4 to 8 weeks after the first dose. This restriction of replication of the second dose of vaccine also prevented boosting of serum antibody titers, though we cannot exclude the possibility that subclinical infection may have occurred and that other components of immunity, such as as secretory or cellular immunity, were augmented by this second dose. This protection against replication of a second dose of rcp45 vaccine virus would likely translate to restriction of replication of a wt HPIV3 during this interval.
Of note, this high level of protection against ‘challenge’ with a second dose of vaccine in children 6 months of age and older is different from what we previously observed when biologically-derived cp45 vaccine was administered to 1 and 2 month old infants. [11
] In that study, replication of the second dose of vaccine virus was observed in 4 of 17 (24%) of infants who received a second dose at approximately 4 weeks and 8 of 13 (62%) of infants who received a second dose at 12 weeks after the first dose of vaccine. While the numbers of vaccinees are small and the intervals between doses are not identical in these two studies, these data suggest that rcp45 HPIV3 vaccine may induce a more potent and durable local immune response in older HPIV3-naïve infants and children than in the very youngest infants, which would be consistent with what has been previously observed following natural infection with respiratory syncytial virus (RSV) in young children. [29
] Additional studies would be needed to confirm this finding.
Studies are also underway to determine whether a longer interval between doses might be sufficient to allow reinfection and boosting of the antibody response. Currently, we are conducting studies to determine the infectivity and immunogenicity of a second dose of rcp45 vaccine administered 6 months after the first dose. This interval was chosen based upon previous studies that suggest that local immunity to other respiratory viruses or live-attenuated respiratory virus vaccines wanes within 3 to 6 months following the initial infection. [30
] Data provided from this ongoing study could be helpful in selecting dosing intervals for subsequent phase II studies of the rcp45 vaccine.
The cp45 vaccine virus has been shown to be biologically stable in vitro,
through extensive phenotypic analysis of viral isolates, [10
] and in vivo,
by administration of viral isolates to susceptible non-human primates. [18
] In this study, we extended this biological analysis by assessing the genetic stability of 9 attenuating mutations in rcp45 isolates obtained from the 11 children in cohort 1 who shed vaccine virus. We chose to evaluate specimens obtained from the last day of vaccine virus recovery to determine whether revertant viruses might have emerged that had a selective advantage over viruses containing the attenuating mutations. Because of the inherently high rate of nucleotide substitution in RNA viruses, we had designed rcp45 so that two of the major attenuating mutations (one in the F gene and one in the L gene) would each require two (rather than one) nucleotide substitutions to revert to the wild type codon. The presence of 9 rcp45 mutations, including all 6 amino acid substitutions that contribute to the attenuation phenotype of rcp45 in non-human primates, was confirmed for isolates from 10 children. For the 11th
child, the presence of four attenuating mutations was confirmed in the L, N, and C genes, but sequence for the remaining mutations could not be derived. While additional data from larger studies will be needed, the preliminary data generated from this clinical trial support previous findings regarding the biologic stability of the cp45 virus and suggest that the attenuating mutations present in the rcp45 vaccine virus are stable following replication in seronegative children.
In summary, rcp45 appears to be an appropriately attenuated vaccine that is infectious, immunogenic, and genetically stable in HPIV3 seronegative infants and children. The infectivity, tolerability, and immune response to rcp45 are comparable to what was previously observed following administration of the biologically-derived cp45 vaccine. However, rcp45 is preferred for future evaluation in phase 2 and 3 clinical trials because of its derivation from cDNA using qualified reagents and cells. Further clinical development of this promising investigational recombinant respiratory virus vaccine is warranted and in progress.