Comparative analysis of macrophage associated vectors for use in genetic vaccine

Abstract

Background: Antigen presentation by non professional antigen presenting cells (APC) can lead to anergy. In genetic vaccines, targeting the macrophages and APC for efficient antigen presentation might lead to balanced immune response. One such approach is to incorporate APC specific promoter in the vector to be used.

Methods: Three promoters known to be active in macrophage were selected and cloned in mammalian expressing vector (pAcGFP1-N1) to reconstruct (pAcGFP-MS), (pAcGFP-EMR) and (pAcGFP-B5I) with macrosialin, EmrI and Beta-5 Integrin promoters respectively. As a positive control (pAcGFP-CMV) was used with CMV promoter and promoterless vector (pAcGFP-NIX) which served as a negative control. GFP gene was used as readout under the control of each of the promoter. The expression of GFP was analyzed on macrophage and non-macrophage cell lines using Flow cytometry and qRT-PCR with TaqMan probe chemistries.

Results: All the promoters in question were dominant to macrophage lineage cell lines as observed by fluorescence, Western blot and quantitative RT-PCR. The activity of macrosialin was significantly higher than other macrophage promoters. CMV promoter showed 1.83 times higher activity in macrophage cell lines. The expression of GFP driven by macrosialin promoter after 24 hours was 4.40 times higher in macrophage derived cell lines in comparison with non macrophage cell lines.

Conclusions: Based on this study, macrosialin promoter can be utilized for targeting macrophage dominant expression. In vivo study needs to be carried out for its utility as a vaccine candidate.

Figure 1

Schematic representation of reconstructed promoters constructs with GFP as a reporter gene.

Figure 2

Sequence alignment of GFP variants in GenBank showing the location of primers and probe. GFP sequences were selected from data bank and aligned using MEGA4 software. The references of sequences are mentioned with the Accession number of GenBank. The sequence used for the primer and probe design was: Accession number-AY233272, GI-34421677.

Figure 3

PCR analysis of amplified promoters. M: 1 Kb+ Ladder (Invitrogen); 2: Macrosialin; 3: Beta-5 Integrin; 4: EMR1 are the respective amplicons of promoters documented in 1% Agarose gel in TAE buffer.

Figure 4

Fluorescent Microscopy pictures of cells transfected with respective plasmid. Expression of GFP in transfected RAW 264.7 (R) and L929 (L) cells at different time points as: A-6 hrs, B-12 hrs, C-24 hrs, D-36 hrs and E-48 hrs. The constructs for the transfected cells are mentioned at the top which follows throughout the respective column followed by (pAcGFP-). CC represents cell control.

Figure 5

PAGE/Western blot Analysis. (A) 12% SDS-PAGE gel (B) Western blot analysis of the total cell lysates of the RAW 264.7 cells. M: PageRuler™ (Fermentas); 1: pAcGFP-CMV; 2: pAcGFP-MS; 3: pAcGFP-EMR; 4: pAcGFP-B5I; 5: pAcGFP-NIX. The blot shows expressed GFP protein from different constructs after 24 hours of transfection.

Figure 6

Flow cytometry analysis. Mean fluorescence of cells of different constructs transfected in (A) RAW264.7, (B) L929 cells. (C) Ratio of (RAW264.7/L929) were determined as an expression of macrophage specificity. The activity was measured at various time points. The average and SEM shown are from three independent assays. For ststistical analysis, General Linear Model (GLM), Tukey's comparison test was performed to compare the significance difference on fluorescence level amongst transfected plasmid.

Figure 7

Standard curve plot of log10 diluted in vitro transcribed RNA for GFP.

Figure 8

Transcript profiling of GFP. Transcript profiling of RAW264.7 (A) and L929 (B) cells transfected with different promoter constructs at the given time interval.