Expression and identification of eCTLA4-N/GP fusion proteins

The expressing of eCTLA4-N fusion protein was firstly determined in prokaryotic system. Supernatant and cell lysates of E.coli DH5α transformed with pET30a/eCTLA-S or pET30a/S plasmid were analyzed by SDS-PAGE (Figure 1A). Neither eCTLA4-N fusion protein nor HTNV N was expressed into supernatant (Figure 1A, lane 1 and lane 3). Instead, eCTLA4-N fusion protein was shown in cell lysate with about 66KD of molecular weight (MW). HTNV N was consistently seen in 50KD of MW which matches previous result [23]. The expression of eCTLA4-N fusion protein was further examined by western-blot from 293T cells transiently transfected with pcDNA3/eCTLA4-S. eCTLA4-N fusion protein was detected by either N-specific monoclonal antibody of L13F3 [28,29] (eCTLA4-N/anti-N) or anti-mouse eCTLA-4 (eBioscience, USA) (eCTLA4-N/anti-eCTLA4). As a control, the HTNV N protein without fusion with eCTLA-4 was also detected by monoclonal antibody L13F3 (NP/anti-N).

IFA was also used to verify the expression of eCTLA4-N and eCTLA4-GP fusion proteins as described in Methods. BHK cells were transiently transfected with pcDNA3/eCTLA4-M or pcDNA3/eCTLA4-S construct. The expression of eCTLA4-GP or eCTLA4-N fusion protein was detected with Gc- specific antibody (Y22) or N-specific antibody (L13F3) [28,29] respectively as demonstrated in (Figure 1C, a and 1c). Furthermore, eCTLA4-GP and eCTLA4-N fusion proteins could also be captured by monoclonal antibody of anti-mouse eCTLA-4.

Antibody responses to HTNV N and GP induced in mice following immunization with plasmids expressing eCTLA4-N/GP fusion protein

To evaluate whether eCTLA4 fusion strategy could enhance immunogenicity on HTNV DNA vaccine, C57 mice were immunized with DNA plasmids expressing HTNV N and GP, or eCTLA4-N and GP fusion proteins with or without 10 μg of CpG1826 motifs. The antibody immune response to HTNV N or GP was determined by N-specific ELISA or IFA assays. (Figure 2) The levels of N protein-specific IgG were found to be substantially induced one week after first immunization in mice that received pcDNA3/eCTLA4-S+M DNA plasmids alone or with CpG1826 (Figure 2A), and significantly higher than that of mice receiving pcDNA3/S+M DNA plasmids alone or with CpG1826. One week after second injection, mice immunized with pcDNA3/eCTLA4-S+M plasmids plus CpG1826 showed significantly higher N protein-specific IgG antibody titers compared to groups of mice that received pcDNA3/eCTLA4-S+M or pcDNA3/S+M DNA plasmids alone (p < 0.05), and about 3.5-fold higher than that of mice receiving pcDNA3/S+M DNA plasmids plus CpG1826 though not achieved statistic significance. After two boosts, all mice that received HTNV DNA vaccine plasmids had substantial increase of N protein-specific IgG antibody titers. DNA plasmids expressing eCTLA4-N and GP fusion proteins, combined with CpG1826, elicited the highest N protein-specific IgG antibody titers one week after third immunization compared to all the other groups (p < 0.05). In addition, we also observed that the magnitude of glycoprotein specific IgG antibody was significantly improved by vaccination with DNA plasmids expressing eCTLA4-N and GP fusion proteins, especially when combined with CpG1826. No eCTLA-4-specific antibodies were detected in sera of mice receiving DNA plasmids expressing fusion proteins (data not shown), which is consistent with the results of Lu et al. These results indicate that eCTLA4 fusion strategy and CpG motif could improve the immunogenicity of HTNV DNA vaccine.