Point and deletion mutations in the P/C, HN and L genes that attenuate HPIV1 for replication in the respiratory tract of hamsters or AGMs are indicated in Table 113141516. The C^R84G mutation is a single nucleotide substitution mutation that affects both the P and C proteins and that results in amino acid substitutions of R84 to G in C, and E87 to G in P (Table 1) 15. The C^R84G mutation is attenuating in the upper respiratory tract (URT) of AGMs, but only in the presence of the HN^T553A point mutation indicated in Table 115. The C^R84G and HN^T553A mutations are each based on single nucleotide substitutions (Table 1), and thus the att phenotype would be lost by reversion at either position. The C^Δ170 deletion mutation in HPIV1 involves a six-nucleotide deletion, a length that was chosen to comply with the "rule of six" 20. This deletion results in a loss of two amino acids and substitution of a third at codon positions 168–170 in C (RDF to S), and a deletion of amino acids GF in P at codon positions 172–173 (Table 1) 16. The changes in the C protein also would be present in the nested C', Y1, and Y2 proteins (not shown) 16. The Y942A mutation in L has three nucleotide changes in codon 942 and specifies a genetically and phenotypically stabilized ts att phenotype 13.

In the present study, the L^Δ1710–11 deletion mutation in HPIV1 was created at a site that corresponds by sequence alignment to a ts att point mutation originally identified in BPIV3 19. Importation of this BPIV3 point mutation has previously been shown to attenuate HPIV2 21. Here, the L^Δ1710–11 mutation contains a six-nucleotide deletion that results in a deletion of amino acids AE at codon positions 1710–11 of the L gene of HPIV1 (Table 1).

The mutations in Table 1 were introduced into the HPIV1 antigenomic cDNA individually or in combinations to yield the panel of rHPIV1 viruses listed in Table 2. These viruses were recovered following transfection of cDNAs into HEp-2, BHK-T7 or Vero cells and biologically cloned in LLC-MK2 cells, and each was sequenced in its entirety to confirm the presence of the engineered mutation(s) and the absence of adventitious mutations. Unexpectedly, we were unable to isolate rHPIV1 containing the L^Δ1710–11 mutation by itself and without adventitious mutations despite four attempts to do this using multiple replicates each time. However, we were able to recover virus bearing L^Δ1710–11 in the presence of C^R84G without adventitious mutations. Thus, our analysis of the phenotype of the L^Δ1710–11 mutation was performed in the presence of the C^R84G mutation, which is neither ts nor att 15.

We first sought to characterize the rHPIV1 mutants bearing the four single att mutations (the C^R84GHN^T553A set, C^Δ170, L^Y942A, and L^Δ1710–11) to define the contributions of the individual mutations to the phenotypes of the rHPIV1 mutants (Groups 3, 4, 5, 7 in Tables 2 and 3). We previously generated and evaluated the rHPIV1-C^R84GHN^T553A and rHPIV1-C^Δ170 viruses (each containing a single non-ts att mutation) in vitro and in vivo 131516. These previously evaluated single-mutation viruses were included here for the purpose of comparison with viruses containing the other individual mutations as well as combinations of mutations. An rHPIV1 mutant, rHPIV1-L^Y942A, bearing the Y942A mutation in L was generated for the present study. We had previously generated and characterized a virus, rHPIV1-C^R84GHN^T553AL^Y942A, containing the L^Y942A mutation in combination with the C^R84GHN^T553A pair of mutations 13. The newly generated rHPIV1-L^Y942A virus would permit evaluation of its specific contribution to the level of temperature sensitivity in vitro and attenuation in vivo. The rHPIV1 mutant bearing the individual att mutation L^Δ1710–11 (rHPIV1-C^R84GL^Δ1710–11) also contained the C^R84G mutation, although this latter mutation is phenotypically silent on its own, as already noted.

The level of temperature sensitivity of replication of the four viruses with single att mutations was first studied (Table 2, groups 3, 4, 5, and 7) and compared to that of rHPIV1 wt and rHPIV1-C^R84G. Viruses containing only P/C gene mutations with or without the HN mutation were non-ts, whereas each of the L gene mutations specified a ts phenotype in vitro. The single L^Y942A mutation specified a shut-off temperature of 37°C, a level of temperature sensitivity that was equivalent to that previously observed for rHPIV1-C^R84GHN^T553AL^Y942A (Table 2, compare Groups 5 and 6). These data indicate that the L^Y942A mutation is responsible for the observed ts phenotype of rHPIV1-C^R84GHN^T553AL^Y942A (Table 2). The L^Δ1710–11 mutation specified an even stronger ts phenotype than the L^Y942A mutation (Table 2). The L^Δ1710–11 mutation clearly contributes significantly to the ts property of rHPIV1-C^R84GL^Δ1710–11 since rHPIV1-C^R84G was confirmed to be non-ts (Table 2, compare Groups 2 and 7). Therefore, both L^Y942A and L^Δ1710–11 are ts mutations in HPIV1. In a multiple cycle growth curve, the two newly generated rHPIV1 mutants with single att mutations, rHPIV1-L^Y942A and rHPIV1-C^R84GL^Δ1710–11, reached a titer equivalent to that of rHPIV1 wt in both LLC-MK2 and Vero cells (Figure 1). Thus, these individual mutations do not significantly restrict replication in vitro at the permissive temperature of 32°C and therefore could be useful mutations in vaccine candidates.

The level of replication of rHPIV1-L^Y942A and rHPIV1-C^R84GL^Δ1710–11 in AGMs was next evaluated and compared to that of rHPIV1 wt and the other two single att mutants (Table 3, Groups 1, 3, 4, 5, 7). A rHPIV1 mutant was considered attenuated if it exhibited a significant (P < 0.05) reduction in replication in either the mean peak virus titer or the mean sum of the daily virus titers (a measure of the total amount of virus shed over the duration of the infection) in either the nasopharyngeal (NP) swab (representative of the upper respiratory tract, URT) or tracheal lavage (TL) samples (representative of the lower respiratory tract, LRT) compared to the HPIV1 wt group. We have previously demonstrated that rHPIV1-C^R84G replicates to levels equivalent to HPIV1 wt in AGMs, whereas rHPIV1-C^R84GHN^T553A and rHPIV1-C^R84GHN^T553AL^Y942A were attenuated in AGMs 1516. Here, both rHPIV1-L^Y942A and rHPIV1-C^R84GL^Δ1710–11 were significantly attenuated in the URT and LRT of AGMs in comparison to HPIV1 wt. The levels of attenuation of rHPIV1-L^Y942A and rHPIV1-C^R84GHN^T553AL^Y942A were comparable, indicating that the L^Y942A mutation is an attenuating mutation by itself and that the attenuation specified by the L^Y942A mutation is not additive to that specified by the C^R84GHN^T553A att mutation. The rHPIV1-C^R84GL^Δ1710–11 mutant also was significantly attenuated in AGMs, reducing virus titer in comparison to HPIV1 wt by 2.7 and 3.0 log₁₀ 50%-tissue-culture-infectious-doses (TCID₅₀)/ml in the URT and LRT, respectively (Table 3). Since rHPIV1-C^R84G was confirmed not to be attenuated in AGMs (Table 3, Group 2) 16, this suggests that the L^Δ1710–11 mutation contributes significantly to the observed attenuation phenotype.

The immunogenicity and protective efficacy resulting from immunization with rHPIV1s containing single att mutations were evaluated in AGMs by measuring post-immunization HPIV1 hemagglutination inhibiting (HAI) serum antibody titers and by challenging immunized and control animals with HPIV1 wt 28 days following immunization and determining challenge virus titers in the URT and LRT (Table 4). AGMs immunized with rHPIV1s containing single att mutations (Groups 3, 4, 5, and 7) developed post-immunization HAI serum antibodies and manifested resistance to replication of the challenge virus. The rHPIV1-C^R84GL^Δ1710–11 mutant, which showed a strong level of attenuation following immunization of AGMs, was protective only at a low level in the URT.

Having identified the in vitro and in vivo properties of the four single att mutations, we used this information to generate two live attenuated HPIV1 vaccine candidates containing both non-ts and ts attenuating mutations. These vaccine candidates were designed to incorporate a backbone containing one stabilized non-ts attenuating mutation, C^Δ170, as well as the C^R84GHN^T553A att mutation. The addition of this second mutation (the C^R84GHN^T553A att mutation) would be expected to increase the overall stability of the virus by increasing the total number of attenuating mutations present in the vaccine candidate. To generate the two live attenuated HPIV1 vaccine candidates, either the stabilized ts att L^Y942A mutation or the L^Δ1710–11 deletion mutation was added to the rHPIV1-C^R84G/Δ170HN^T553A backbone. We then evaluated the resulting combination mutants, rHPIV1-C^R84G/Δ170HN^T553AL^Y942A and rHPIV1-C^R84G/Δ170HN^T553AL^Δ1710–11, as potential vaccine candidates.

These two viruses were first evaluated for their level of temperature sensitivity of replication in vitro (Table 2). The level of temperature sensitivity of rHPIV1-C^R84G/Δ170HN^T553AL^Y942A and rHPIV1-C^R84G/Δ170HN^T553AL^Δ1710–11 (Groups 8 and 9 in Table 2) was equivalent to that of the corresponding L gene single-mutation viruses from which they were derived (namely rHPIV1-L^Y942A and rHPIV1-C^R84GL^Δ1710–1, Groups 5 and 7 in Table 2). This indicates that combining the non-ts and ts mutations in rHPIV1-C^R84G/Δ170HN^T553AL^Y942A and rHPIV1-C^R84G/Δ170HN^T553AL^Δ1710–11 did not significantly alter their overall level of temperature sensitivity of replication in vitro. A multiple cycle growth curve at 32°C demonstrated that each virus achieved titers in Vero cells that will allow efficient manufacture. Specifically, the rHPIV1-C^R84G/Δ170HN^T553AL^Y942A and rHPIV1-C^R84G/Δ170HN^T553AL^Δ1710–11 vaccine candidates reached peak titers of 7.9 and 7.2 log₁₀ TCID₅₀/ml, respectively, in Vero cells (Figure 1).

The level of replication of rHPIV1-C^R84G/Δ170HN^T553AL^Y942A and rHPIV1-C^R84G/Δ170HN^T553AL^Δ1710–11 in AGMs were next evaluated and compared to that of rHPIV1 wt and the other two single att mutants (Table 3, Groups 1, 3, 4, 5, 7, 8, and 9). The rHPIV1-C^R84G/Δ170HN^T553AL^Y942A virus was strongly attenuated compared to rHPIV1 mutants bearing the corresponding single att mutations only in C/P, C/P/HN or L. The mean peak titer of rHPIV1-C^R84G/Δ170HN^T553AL^Y942A in the URT and LRT was reduced by 3.0 and 3.3 log₁₀ TCID₅₀/ml, respectively, in comparison to HPIV1 wt (Table 3). Similarly, the addition of the HN^T553A and C^Δ170 mutations to rHPIV1-C^R84GL^Δ1710–11 to generate the rHPIV1-C^R84G/Δ170HN^T553AL^Δ1710–11 further attenuated the virus in AGMs, restricting virus replication in comparison to HPIV1 wt by 3.1 and 3.4 log₁₀ TCID₅₀/ml in the URT and LRT, respectively (Table 3). Therefore these two HPIV1 vaccine candidates demonstrate strong attenuation phenotypes in vivo. Considering the 9 viruses in Table 3 together, a relationship was found to exist between level of temperature sensitivity of replication in vitro and the attenuation manifested in vivo, i.e., the lower the shut off temperature, the higher the level of in vivo attenuation (Figure 2). Evaluation of these data using the Spearman rank test gives correlation coefficients of 0.47 and 0.67 for the URT and LRT, respectively, based on the mean daily sum of virus titers for individual AGMs. This indicates a moderate positive correlation with a stronger association between the level of temperature sensitivity and virus replication in the LRT. However, as might be expected, viruses bearing only the non-ts attenuating P/C gene mutations, including the C^Δ170 and the C^R84GHN^T553A set of mutations, did not follow this pattern (Figure 2), and we would expect a higher correlation coefficient if these non-ts viruses were not included in the analysis.

The levels of immunogenicity and protective efficacy against HPIV1 wt challenge following immunization with rHPIV1-C^R84G/Δ170HN^T553AL^Y942A and rHPIV1-C^R84G/Δ170HN^T553AL^Δ1710–11 were also determined (Groups 8 and 9 in Table 4). The two vaccine candidates failed to induce detectable HAI antibodies. However, immunization with the rHPIV1-C^R84G/Δ170HN^T553AL^Y942A was protective against HPIV1 wt challenge in both the URT and LRT (Table 4). In contrast, immunization with rHPIV1-C^R84G/Δ170HN^T553AL^Δ1710–11 did not offer significant protection against HPIV1 wt challenge in the AGMs (Table 4), i.e., it appeared overattenuated in this animal model. A relationship was found between the level of replication of the immunizing virus and its ability to induce resistance to replication of the challenge virus (Tables 3 and 4), and this is graphically displayed in Figure 3.

LLC-MK2 cells (ATCCCCL7.1) and HEp-2 cells (ATCCCCL23) were maintained in Opti-MEM I (Gibco-Invitrogen, Inc. Grand Island, NY) supplemented with 5% FBS and gentamicin sulfate (50 μg/ml). Vero cells (ATCC CCL-81) were maintained in Opti-PRO SFM (Gibco-Invitrogen, Inc.) in the absence of FBS and supplemented with gentamicin sulfate (50 μg/ml) and L-glutamine (4 mM). BHK-T7 cells, which constitutively express T7 RNA polymerase 27, were kindly provided by Dr. Ulla Buchholz, NIAID, and were maintained in GMEM (Gibco-Invitrogen, Inc.) supplemented with 10% FBS, geneticin (1 mg/ml), MEM amino acids, and L-glutamine (2 mM). Biologically-derived wt HPIV1 Washington/20993/1964, the parent for the recombinant virus system, was isolated previously from a clinical sample in primary African green monkey kidney (AGMK) cells and passaged 2 additional times in primary AGMK cells 25 and once in LLC-MK2 cells 15. This preparation has a wild type phenotype in AGMs, and will be referred to here as HPIV1 wt. It was previously described as HPIV1_LLC1 15. HPIV1 wt and rHPIV1 mutants were grown in LLC-MK2 cells in the presence of 1.2% Tryple select, a recombinant trypsin (Gibco-Invitrogen, Inc.), as described previously 8.

P/C, HN and L gene mutations (Table 1) were introduced into the appropriate rHPIV1 subgenomic clones 14 using the Advantage-HF PCR Kit (Clontech Laboratories, Palo Alto, CA) with a modified PCR mutagenesis protocol described elsewhere 28. The entire PCR amplified subgenomic clone was sequenced using a Perkin-Elmer ABI 3100 sequencer with the Big Dye sequencing kit (Perkin-Elmer Applied Biosystems, Warrington, UK) to confirm that the subclone contained the introduced mutation and to confirm the absence of adventitious mutations introduced during PCR amplification. Full-length antigenomic cDNA clones (FLCs) of HPIV1 containing the desired mutations were assembled using standard molecular cloning techniques 8, and the region containing the introduced mutation in each FLC was sequenced as described above to confirm the presence of the introduced mutation and absence of adventitious changes. Each virus was designed to conform to the rule of six, which is a requirement by HPIV1 and numerous other paramyxoviruses that the nucleotide length of their genome be an even multiple of six for efficient replication 20.

Three different recovery methods were used to generate rHPIV1 mutants that differed in the source of the T7 polymerase needed to synthesize RNA from the transfected virus-specific plasmids and, in one case, a different transfection method was used. First, using previously described procedures 8, rHPIV1 virus was recovered from HEp-2 cells that were transfected with plasmids encoding the antigenome and N, P, and L support proteins and infected with an MVA-T7 vaccinia virus recombinant as a source of T7 polymerase. Second, Vero cells were grown to 80% confluency and transfection experiments were performed using the AMAXA Cell Line Nucleofector Kit V, according to manufacturer's directions (AMAXA, Koeln, Germany), as previously described 29. Briefly, the cells were transfected with 5 μg each of the FLC and the pCL-Neo-BCI-T7 plasmid (expressing T7 polymerase under the control of a eukaryotic promoter) 30, 0.2 μg each of the N and P, and 0.1 μg of the L support plasmids. The transfection mixture was removed after 24 h at 37°C, and cells were washed and overlaid with Opti-PRO with L-glutamine (4 mM) supplemented with 1.2% Tryple select. The cells and supernatant were transferred to LLC-MK2 cells in T25 cm² flasks (Corning, NY) 7 days following transfection. Third, BHK-T7 cells constitutively expressing T7 polymerase 27 were grown to 90 to 95% confluence in six-well plates. The cells were transfected with 5 μg of the FLC, 0.8 μg each of the N and P, and 0.1 μg of the L support plasmids in a volume of 100 μl of Opti-MEM per well. Transfection was carried out with Lipofectamine 2000 (Invitrogen, Inc., Carlsbad, CA), according to the manufacturer's directions. The transfection mixture was removed after a 24 h incubation period at 37°C, and the cells were washed and maintained in GMEM. On day 2 following transfection, the media was supplemented with 1.2% trypsin, and the recovered virus was harvested on days 2–4. All viruses were amplified by passage on LLC-MK2 cells, and each was cloned by two successive rounds of terminal dilution using LLC-MK2 monolayers in 96-well plates (Costar, Corning Inc., Acton, MA). To confirm that the recovered rHPIV1 mutants contained the appropriate mutations and lacked adventitious mutations, viral RNA (vRNA) was isolated from infected cell supernatants using the Qiaquick vRNA kit (Qiagen Inc., Valencia, CA), reverse transcribed using the SuperScript First-Strand Synthesis System (Invitrogen, Inc., Carlsbad, CA) and amplified using the Advantage cDNA PCR Kit (Clontech Laboratories). Each viral genome was sequenced in its entirety.

The recombinant HPIV1 mutants were compared to HPIV1 wt on LLC-MK2 and Vero cells at 32°C in a multiple cycle growth curve. Confluent monolayer cultures in 6-well plates were infected in triplicate at a multiplicity of infection (MOI) of 0.01 50%-tissue-culture-infectious-doses (TCID₅₀) per cell in media containing trypsin. The residual inoculum was withdrawn 2 h post infection as the day 0 sample and was replaced by medium with trypsin. On days 2, and 4–11 post-infection, the total medium supernatant was removed for virus quantitation and was replaced with fresh medium with trypsin. Supernatants containing virus were frozen at -70°C, and all samples were tested together for virus titer with endpoints identified by hemadsorption.

The ts phenotype for each mutant rHPIV1 virus was determined by comparing its level of replication to that of HPIV1 wt at 32°C and at 1°C increments from 35°C to 40°C, as described previously 31. Briefly, each virus was serially diluted 10-fold in 96-well LLC-MK2 monolayer cultures in L-15 media (Gibco-Invitrogen, Inc.) containing trypsin with four replicate wells per plate. Replicate plates were incubated at the temperatures indicated above for seven days, and virus infected wells were detected by hemadsorption with guinea pig erythrocytes. The virus titer at each temperature was determined in three to sixteen separate experiments and is expressed as the mean log₁₀ TCID₅₀/ml. The mean titer at an elevated temperature was compared to the mean titer at 32°C, and the reduction in mean titer was determined. The shut-off temperature of an rHPIV1 mutant is defined as the lowest temperature at which the reduction in virus titer compared to its titer at 32°C was 100-fold greater than the reduction in HPIV1 wt titer between the same two temperatures. A mutant is defined as having a ts phenotype if its shut-off temperature is ≤40°C.

AGMs in groups of two to four animals at a time were inoculated intranasally (i.n.) and intratracheally (i.t.) with 10⁶ TCID₅₀ of either HPIV1 wt or mutant rHPIV1 in a 1 ml inoculum at each site. NP swab samples were collected daily from days 1 to 10 post-inoculation, and TL fluid samples were collected on days 2, 4, 6, 8 and 10 post-inoculation. The specimens were flash frozen and stored at -80°C and were subsequently assayed in parallel. Virus present in the samples was titered in dilutions on LLC-MK2 cell monolayers in 96-well plates and an undiluted 100 μl aliquot was also tested in 24-well plates. These were incubated at 32°C for 7 days. Virus was detected by hemadsorption, and the mean log₁₀ TCID₅₀/ml was calculated for each sample day. The limit of detection was 0.5 log₁₀TCID₅₀/ml. The mean peak titer for each group was calculated using the peak titer for each animal, irrespective of the day of sampling. The mean sum of the virus titers for each group was calculated from the sum, calculated for each animal individually, of the virus titers on each day of sampling, up to day 10. The sum of the lower limit of detectability was 5.0 log₁₀ TCID₅₀/ml for NP swabs and 2.5 log₁₀ TCID₅₀/ml for TL samples.

On day 28 post-inoculation, the AGMs were challenged i.n. and i.t. with 10⁶ TCID₅₀ of HPIV1 wt in 1 ml at each site. NP swab and TL samples were collected for virus quantitation on days 2, 4, 6 and 8 post-challenge.

All animal studies were performed under protocol LID22, as approved by the National Institute of Allergy and Infectious Disease (NIAID) Animal Care and Use Committee (ACUC).

Serum was collected from each monkey on days 0 and 28 post-immunization and on day 28 post-challenge (day 56 post-immunization). HPIV1 HAI antibody titers were determined at 21°C, as described previously 32, using 0.5% v/v guinea pig erythrocytes and HPIV1 wt as the antigen. The HAI antibody titer was defined as the end-point serum dilution that inhibited hemagglutination and is expressed as the mean reciprocal log₂ ± standard error (SE).

The Prism 4 (GraphPad Software Inc., San Diego, CA) one-way ANOVA test, (Student-Newman-Keuls multiple comparison test) was used to assess statistically significant differences between data groups (P < 0.05). The R software programme 33 was used to perform a Spearman rank test to determine correlation between data sets.