The anti-tumor effect of Newcastle disease virus HN protein is influenced by differential subcellular targeting

Abstract

Background: Immunotherapy is emerging as a major player in the current standard of care for aggressive cancers such as non-small cell lung cancer (NSCLC). The Newcastle disease virus with its tumor-specific replicative and oncolytic abilities is a promising immunotherapeutic candidate. A DNA vaccine expressing the major immunogenic hemagglutinin-neuraminidase (HN) protein of this virus has shown promising results as an immunotherapeutic agent.

Methods: In the present study, three different DNA vaccine constructs encoding differentially targeted HN proteins (cytoplasmic or Cy-HN, secreted or Sc-HN and membrane-anchored or M-HN) were generated to evaluate their anti-tumor effect in vitro and in vivo.

Results: Although all three DNA constructs elicited an immune response, tumor-bearing mice intratumorally injected with M-HN demonstrated a significantly better anti-tumor effect than those injected with Cy-HN or Sc-HN. We also showed that this anti-tumor effect was mediated by higher lymphocyte proliferative response and CTL activity in mice intratumorally injected with M-HN.

Conclusion: The membrane-anchored form of the HN protein appears to be an ideal candidate to develop as an immunotherapeutic agent for NSCLC.

Fig. 1

Plasmids used for expression analysis in vitro and for DNA treatment. pcDNA3.1(+) was used to express three different versions of HN encoding cytoplasmic (Cy-HN), secreted (Sc-HN) and membrane-anchored (M-HN) HN protein, respectively. CMV cytomegalovirus immediate-early promoter/enhancer, TPA tissue plasminogen activator signal sequence, polyA polyadenylation signal, TM the transmembrane anchor domains (TM) of influenza virus HA, HN HN gene of new castle disease virus strain D90. The boxed arrows show the position of the CMV promoter and direction of transcription

Fig. 2

Expression and intracellular localization analyses of modified HN protein in transfected A549 cells. Approximately 80–90% confluent A549 cells were transfected with 4 μg of plasmid DNA. At 48-h post-transfection, the cells were fixed with absolute ethanol and processed for an indirect immunofluorescence assay. Representative fluorescence images of cells transfected with Cy-HN (a), Sc-HN (b), M-HN (c) or the empty vector pcDNA3.1(+) (d) are shown. For SDS-PAGE (e), cell lysates (lane 3) or supernatants (lane 2) from cells transfected with Sc-HN were prepared at 48-h post-transfection. Arrow indicates the expressed HN protein

Fig. 3

Cell death rate in A549 cells transfected with Cy-HN, Sc-HN, M-HN or the empty vector pcDNA3.1(+) for 120 h (n = 3 or 4 per group). One-way ANOVA followed by LSD or Dunnett’s T3 test was performed to test for differences among the four subcellular localizations at a specific time-point and the difference between five time-points for a given subcellular localization (significantly different from Cy-HN, *P < 0.05; significantly different from Sc-HN, ^† P < 0.05; significantly different from M-HN, ^‡ P < 0.05; a indicates significantly different from 24 h; b indicates significantly different from 48 h; c indicates significantly different from 72 h). Data are presented as mean ± standard deviation (SD)

Fig. 4

Detection of apoptosis of A549 by flow cytometry. Approximately 80–90% confluent A549 cells were transfected with 4 μg of plasmid DNA in six-well tissue culture plates. At 24-h (1) or 72-h (2) post-transfection, the cells were collected and stained with a TACS annexin V-FITC kit as described

Fig. 5

Representative images of apoptotic bodies. 1 Demonstration of apoptotic bodies in A549 cells induced by Cy-HN (a), Sc-HN (b) and M-HN (c) by electron microscopy. 2 AO/PI double staining of A549 cell after transfection with Cy-HN (a), Sc-HN (b), M-HN (c) and the empty vector pcDNA3.1(+) (d) by CLSM

Fig. 6

Stimulation index in A549 cells transfected with PC3.1(+), Cy-HN, Sc-HN and M-HN (n = 4 per group). One-way ANOVA was used; LSD was utilized for post hoc tests. Significantly different from PC3.1(+), ^¶ P < 0.05. Data are presented as mean ± standard deviation (SD)

Fig. 7

The CTL activity in tumor-bearing mice treated with PC3.1(+), Cy-HN, Sc-HN and M-HN (n = 4 per group). One-way ANOVA followed by LSD or Dunnett’s T3 test was performed to test the difference between the four subcellular localizations at a specific E/T ratio and the difference between the three E/T ratios for each subcellular localization. Significantly different from PC3.1(+), ^¶ P < 0.05. Significantly different from M-HN, ^‡ P < 0.05. a indicates significantly different from E/T ratio of 25:1, P < 0.05. Data are presented as mean ± standard deviation (SD)