Attenuation and efficacy of human parainfluenza virus type 1 (HPIV1) vaccine candidates containing stabilized mutations in the P/C and L genes

Abstract

Background: Two recombinant, live attenuated human parainfluenza virus type 1 (rHPIV1) mutant viruses have been developed, using a reverse genetics system, for evaluation as potential intranasal vaccine candidates. These rHPIV1 vaccine candidates have two non-temperature sensitive (non-ts) attenuating (att) mutations primarily in the P/C gene, namely CR84GHNT553A (two point mutations used together as a set) and CDelta170 (a short deletion mutation), and two ts att mutations in the L gene, namely LY942A (a point mutation), and LDelta1710-11 (a short deletion), the last of which has not been previously described. The latter three mutations were specifically designed for increased genetic and phenotypic stability. These mutations were evaluated on the HPIV1 backbone, both individually and in combination, for attenuation, immunogenicity, and protective efficacy in African green monkeys (AGMs).

Results: The rHPIV1 mutant bearing the novel LDelta1710-11 mutation was highly ts and attenuated in AGMs and was immunogenic and efficacious against HPIV1 wt challenge. The rHPIV1-CR84G/Delta170HNT553ALY942A and rHPIV1-CR84G/Delta170HNT553ALDelta1710-11 vaccine candidates were highly ts, with shut-off temperatures of 38 degrees C and 35 degrees C, respectively, and were highly attenuated in AGMs. Immunization with rHPIV1-CR84G/Delta170HNT553ALY942A protected against HPIV1 wt challenge in both the upper and lower respiratory tracts. In contrast, rHPIV1-CR84G/Delta170HNT553ALDelta1710-11 was not protective in AGMs due to over-attenuation, but it is expected to replicate more efficiently and be more immunogenic in the natural human host.

Conclusion: The rHPIV1-CR84G/Delta170HNT553ALY942A and rHPIV1-CR84G/Delta170HNT553ALDelta1710-11 vaccine candidates are clearly highly attenuated in AGMs and clinical trials are planned to address safety and immunogenicity in humans.

Figures

Figure 1

Comparison of the replication of HPIV1 wt and rHPIV1 mutant viruses containing the indicated mutations in the P/C, HN and L genes in a multiple cycle growth curve. Monolayer cultures of LLC-MK2 cells and Vero cells were infected at a multiplicity of infection of 0.01 TCID50/cell and incubated at 32°C. The medium was removed on days 0 (residual inoculum), 2 and 4–11 post-infection, frozen for later determination of virus titers, and replaced by fresh medium containing trypsin. The virus titers shown are the means of 3 replicate cultures.

Figure 2

Representation of the association between the in vitro shut-off temperature and the attenuation phenotype in AGMs for HPIV1 wt (W) and rHPIV1 mutant viruses. For each virus (number designations correspond to the virus group numbers assigned in tables 2-4), the shut-off temperature (°C), as determined by an in vitro temperature sensitivity assay (Table 2), was plotted against the mean sum of daily virus titers (log10 TCID50/ml; Table 3) in the URT (A) and LRT (B) of AGMs. rHPIV1 wt and non-ts rHPIV1 mutants were assigned a shut-off temperature of 40°C for the purposes of this schematic. The limit of detection for the mean sum of daily virus titers is shown by a dashed line and viruses containing a single or set of non-ts attenuating mutation (**) or a single ts attenuating mutation (*) are highlighted, as shown. A linear trend line fit using the individual daily data is shown (solid line). The Spearman rank-correlation coefficient was determined to be 0.47 for the URT and 0.67 for the LRT, indicating a moderate positive correlation between shut-off temperature and mean daily sum of virus titer in the URT and a stronger association for the LRT.

Figure 3

Representation of the relationship between the level of replication of HPIV1 wt and rHPIV1 mutants in AGMs and the subsequent level of replication of HPIV1 wt challenge virus in the immunized animals. The mean peak virus titer (log10 TCID50/ml) in the URT following immunization (y-axis) was plotted for viruses 1–9 (Table 3) against the mean peak challenge virus titers (log10 TCID50/ml; x-axis) in the same groups (Table 4). A curve of best fit has been inserted (solid line) to demonstrate the association between these two data sets.