The advent of a reverse genetics system for the generation of infectious paramyxoviruses from full-length cDNA plasmids has greatly facilitated the development of live attenuated HPIV1 vaccine candidates [13–16]. The reverse genetics system for HPIV1 has allowed site-directed manipulation of the viral genome via cDNA intermediates, permitting the introduction of attenuating mutations in desired combinations into vaccine candidates. It has also been possible to genetically modify some of the attenuating mutations to optimize genetic and phenotypic stability of viruses bearing the mutations, both by the use of gene deletions and by using codons chosen for a low probability of reversion. This process enables us to optimize the safety profile of the live attenuated HPIV1 vaccine candidates before these viruses are tested in humans.
We are focusing our efforts on the development of live attenuated rHPIV1 vaccines since they have a number of advantages over inactivated or subunit vaccines, including the ability to: (i) induce the full spectrum of protective immune responses including serum and local antibodies as well as CD4+ and CD8+ T cells ; (ii) infect and replicate in the presence of maternal antibody permitting immunization of young infants [22, 23]; (iii) cause an acute, self-limited infection that is readily eliminated from the respiratory tract; and (iv) replicate to high titers in cell substrates acceptable for products for human use, including qualified Vero cells, making manufacture of these vaccines commercially feasible. In the present study, two new rHPIV1 viruses containing single att mutations in L, LΔ1710–11 and LY942A, were generated and characterized, and these ts att mutations were used in combination with previously described non-ts att mutations in the P/C gene and HN gene to generate two new live attenuated HPIV1 vaccine candidates.
A major result of the present study was the creation of the LΔ1710–11 mutation that was found to specify a strong ts att phenotype. The LΔ1710–11 mutation was originally identified as an attenuating point mutation, LT1711I, in BPIV3 . It was evaluated as a deletion mutation in the present study since a deletion mutation offers a higher level of genetic stability than a point mutation, a property that is desirable for mutations in a vaccine candidate. Indeed, since this deletion occurs in an ORF (in which the triplet nature of the codons must be maintained) and in a virus that conforms to the rule of six (in which the hexamer organization must be maintained), same-site reversion would require the precise restoration of six nucleotides. We unfortunately were not able isolate a rHPIV1 mutant with only the LΔ1710–11 mutation since each rHPIV1-LΔ1710–11 mutant that was isolated also possessed one or more adventitious mutations. The LΔ1710–11 mutation could only be recovered free of adventitious mutations when it was in combination with the CR84G mutation, and thus had to be studied in that context. We acknowledge that it is possible that the phenotypes that we observed for the rHPIV1-CR84GLΔ1710–11 are the result of an interaction between the CR84G and LΔ1710–11 mutations. However, we believe that this possibility is unlikely since the CR84G mutation does not contribute to the ts or att phenotype of HPIV1 as an independent mutation. Furthermore, the high level of temperature sensitivity and attenuation of rHPIV1-CR84GLΔ1710–11 versus that of rHPIV1-CR84G suggests a major independent role of the LΔ1710–11 mutation in these two phenotypes. rHPIV1-CR84GLΔ1710–11 manifested a shut-off temperature of 37°C in vitro and was restricted in replication in the URT and LRT of AGMs by 2.5 log10 or 3.0 log10, respectively. Therefore, we suggest that the LΔ1710–11 deletion mutation specifies a ts att phenotype for HPIV1, and, as such, it is a suitable mutation to include in a HPIV1 vaccine candidate.
The LY942A mutation was identified previously as an attenuating mutation for introduction into potential HPIV1 vaccine candidates and was stabilized by codon optimization studies . These studies demonstrated that only three amino acids were shown to specify a wild type phenotype at this codon position (the wild type tyrosine, cysteine and phenylalanine) all of which would require three nucleotide changes to convert the GCG alanine to a codon specifying the wild type phenotype codon in the vaccine virus . In addition, the LY942A mutation was shown to be highly stable under selective pressure during passage at permissive and restrictive temperatures . Previous studies have evaluated the LY942A mutation only in the presence of the CR84GHNT553A set of mutations that attenuates HPIV1 for AGMs [13, 15]. To determine the specific contribution of the LY942A mutation to the ts and att phenotypes associated with the rHPIV1-CR84GHNT553ALY942A virus, a rHPIV1 containing only the LY942A mutation was generated and was found to be as attenuated as rHPIV1-CR84GHNT553ALY942A for AGMs. This indicated that the LY942A mutation independently attenuated HPIV1 for AGMs and can be used in the absence of the CR84GHNT553A mutation to attenuate HPIV1 for AGMs. The attenuation specified by the CR84GHNT553A mutation was not additive with that of LY942A. This actually is a desirable property, since it permits the inclusion of a greater number of mutations while avoiding over-attenuation, and these additional mutations would become unmasked in the case of the loss of one or more other mutations and would thus maintain the att phenotype. Thus, LY942A is a stable mutation that specifies a ts att phenotype for HPIV1 and is suitable for introducing into a HPIV1 vaccine candidate as an independent attenuating mutation.
The LY942A and LΔ1710–11
ts att mutations were used in conjunction with two of the non-ts att mutations, the CR84GHNT553A and CΔ170 mutations , to develop two live attenuated vaccine candidates for HPIV1, namely, rHPIV1-CR84G/Δ170HNT553ALY942A and rHPIV1-CR84G/Δ170HNT553ALΔ1710–11. These vaccine candidates thus each contain three independent attenuating mutations (two non-ts att and one ts att mutation), two of which have been genetically stabilized. The combination of mutations present in these two vaccine candidates should enhance the genetic and phenotypic stability of the viruses, although this will require formal demonstration in a clinical trial using clinical grade virus preparations.
Evaluation of the two vaccine candidates revealed that they are reasonable candidates for further study in clinical trials. Both candidates replicated well in Vero cells, a characteristic that is important for manufacturing purposes. Both viruses also demonstrated a strong ts phenotype in vitro (shut-off temperature of ≤38°C) that was similar to that of their ts parent virus, but the two viruses differ in their level of temperature sensitivity in vitro. Since the level of temperature sensitivity of respiratory viruses , including HPIV1 as demonstrated here, correlates with level of attenuation, it was anticipated that this difference in the ts phenotype would be reflected in a difference in the level of attenuation and immunogenicity in vivo, and this indeed was seen. The HPIV1 vaccine candidates were both strongly attenuated in the URT and LRT of AGMs, with rHPIV1-CR84G/Δ170HNT553ALY942A replicating to slightly higher levels than the more ts rHPIV1-CR84G/Δ170HNT553ALΔ1710–11. Both vaccines were weakly immunogenic and failed to induce a detectable level of serum HAI antibodies in AGMs. A low level of protective efficacy was observed in AGMs immunized with rHPIV1-CR84G/Δ170HNT553ALY942A, but the rHPIV1-CR84G/Δ170HNT553ALΔ1710–11 was not protective. This low level of immunogenicity and efficacy was not unexpected since each vaccine was highly restricted in replication and since there is a strong correlation between the level of replication of vaccine virus and its immunogenicity and ability to restrict replication of HPIV1 challenge virus. These results can be interpreted to indicate that the two vaccine candidates are over-attenuated, but we think that this conclusion would be premature. It is likely that these viruses will be more immunogenic, and therefore more efficacious, in humans compared to AGMs since they should replicate more efficiently in humans. The reasons for this are two-fold. First, HPIV1 is a human virus, and it should replicate more efficiently in its natural host in which it causes disease than in AGMs in which it causes only an asymptomatic infection. The actual level of replication of HPIV1 in seronegative humans is unknown, but it replicates efficiently even in adults with pre-existing immunity [25, 26]. Second, these vaccine candidates are highly ts and should replicate more efficiently in humans, which have a lower body core temperature (36.7°C), than in AGMs (approximately 39°C). Therefore, although these vaccine candidates appear to be over-attenuated in AGMs, it is expected that the viruses should replicate somewhat more efficiently in humans and would be more immunogenic than in AGMs. It also is fortunate that the two vaccine candidates appear to differ somewhat in their level of attenuation, since this provides two chances to achieve an optimal balance between safety and efficacy.