Growth restriction of an experimental live attenuated human parainfluenza virus type 2 vaccine in human ciliated airway epithelium in vitro parallels attenuation in African green monkeys - PubMed (original) (raw)

Growth restriction of an experimental live attenuated human parainfluenza virus type 2 vaccine in human ciliated airway epithelium in vitro parallels attenuation in African green monkeys

Anne Schaap-Nutt et al. Vaccine. 2010.

Abstract

Human parainfluenza viruses (HPIVs) are common causes of severe pediatric respiratory viral disease. We characterized wild-type HPIV2 infection in an in vitro model of human airway epithelium (HAE) and found that the virus replicates to high titer, sheds apically, targets ciliated cells, and induces minimal cytopathology. Replication of an experimental, live attenuated HPIV2 vaccine strain, containing both temperature sensitive (ts) and non-ts attenuating mutations, was restricted >30-fold compared to rHPIV2-WT in HAE at 32 degrees C and exhibited little productive replication at 37 degrees C. This restriction paralleled attenuation in the upper and lower respiratory tract of African green monkeys, supporting the HAE model as an appropriate and convenient system for characterizing HPIV2 vaccine candidates.

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Figures

Fig. 1

rHPIV2-WT infects HAE and progeny virus is shed from the apical surface. HAE were inoculated via the apical surface with rHPIV2-WT at high MOI (5.0 TCID50/cell). (A) Titers of virus shed into the apical (circles) and basolateral (squares) compartments were determined at the indicated times. Virus titers shown are the means of 6 cultures from 2 donors (3 cultures per donor) ± S.E. The limit of detection is 1.2 log10TCID50/ml. (B) Representative photomicrographs of HAE fixed at the indicated times post-inoculation (pi) and immunoprobed en face for the presence of HPIV2 antigen (green) after apical inoculation by HPIV2 or vehicle alone (Mock). (C) Quantification of the percentage of HPIV2-positive cells in HAE (FITC-positive surface area) per culture over time after apical inoculation with rHPIV2-WT. Mean across 10 fields ± S.E..

Fig. 2

rHPIV2-WT, but not rHPIV1-WT, infects HAE via the basolateral surface. (A) HAE was inoculated via the apical surface (top panels) or basolateral surface (bottom panels) with rHPIV2-WT at a MOI of 1.0 or 5.0 TCID50/cell or with rHPIV1-WT at a MOI of 5.0 TCID50/cell. Titers of virus shed into the apical and basolateral compartments were determined at the indicated times pi. The limit of detection is 1.2 log10TCID50/ml. (B) Representative photomicrographs of HAE fixed at day 6 pi and immunoprobed en face for the presence of viral antigen (green) after basolateral inoculation with either HPIV2 or HPIV1, or after apical inoculation with HPIV1.

Fig. 3

Infection of LLC-MK2 cells and HAE with rHPIV2-WT and rHPIV2-VAC at temperatures reflective of the upper and lower airways. (A) Comparison of replication of rHPIV2-WT (circles) and rHPIV2-VAC (squares) in LLC-MK2 cells at 32 and 37 °C. Triplicate cell monolayers were infected at a MOI of 5.0 TCID50/cell and aliquots were taken at 24-h intervals pi. The mean titer (log10) for each time point is indicated. (B) Comparison of single cycle growth curves in HAE inoculated with rHPIV2-WT (circles) or rHPIV2-VAC (squares). HAE cells were inoculated at the apical surface with rHPIV2-WT or rHPIV2-VAC at a MOI of 5.0 TCID50/cell. Cultures were incubated at either 32 or 37 °C, as indicated. Virus titers were determined in the apical compartments at the indicated times pi. Virus titers shown are the means of 6 cultures from 2 donors (3 cultures per donor) ± S.E., and the limit of detection is 1.2 log10TCID50/ml.

Fig. 4

HPIV2 infects ciliated cells in HAE cultures without causing obvious cytopathic effects or cell–cell fusion. (A) Photomicrographs of histological sections of HAE inoculated at a MOI of 5.0 TCID50/cell with rHPIV2-WT (at 37 °C) or rHPIV2-VAC (at 32 °C) or with control fluid (mock). At day 6 pi, cells were fixed and prepared as histological sections and then immunoprobed with antibodies to HPIV2 (green) and acetylated alpha tubulin (red) to detect virus antigen and ciliated cells, respectively. HPIV2 antigen was detected only in ciliated columnar epithelial cells and not in mock-inoculated HAE. (B) Representative histological cross-sections of infected HAE counterstained with hematoxylin and eosin.

Fig. 5

Cellular cytotoxicity induced by HPIV2 infection. Cytotoxicity was quantified by measuring adenylate kinase activity in apical wash samples (circles, scale on left _y_-axis). Adenylate kinase in the sample activated a bioluminescent detection reagent (ToxiLight BioAssay Kit), which was detected using a luminometer; activity is expressed as relative luminescence units (RLU). Virus titers in the apical samples are shown as dashed lines for reference and use the same scale as Fig. 2A (scale on right _y_-axis).

Fig. 6

Comparison of replication of rHPIV2-WT and rHPIV2-VAC in African green monkeys and HAE cultures. (A) Titers of rHPIV2-WT and rHPIV2-VAC in the URT (upper respiratory tract) of AGMs and in the apical wash of HAE incubated at 32 °C. (B) Virus titers in the LRT (lower respiratory tract) of AGMs and in the apical wash of HAE incubated at 37 °C. In both graphs, the peak virus titer, irrespective of sampling day, for each AGM is indicated by a circle (rHPIV2-WT infected AGMs) or square (rHPIV2-VAC infected AGMs). Virus titers from HAE were determined in high MOI growth curves (Fig. 3), and titers shown are from apical wash samples collected at day 3 pi. Lines indicate the mean peak titer for each group ± S.E. A single asterisk (*) indicates statistical significance with P value > 0.01, while (**) indicates a P value > 0.001 (two-way ANOVA with Bonferroni post-tests).

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Growth restriction of an experimental live attenuated human parainfluenza virus type 2 vaccine in human ciliated airway epithelium in vitro parallels attenuation in African green monkeys - PubMed (original) (raw)