DNA vaccine has been demonstrated as a promising vaccination strategy for various viral infections. Previous studies have shown good immunogenicity and protection efficacy of hantavirus DNA vaccine. Hooper et al. demonstrated that DNA vaccination with a plasmid containing a cDNA representing the Seoul virus (SEOV) M segment elicited neutralizing antibody responses in mice and hamsters. Gene gun vaccination with this DNA construct protected hamsters against infection with SEOV and HTNV. They also reported a HTNV M gene-based DNA vaccine conferred sterile protection against infection in hamster model and elicited high levels of neutralizing antibodies in nonhuman primates. Kamrud et al. also demonstrated a good immunogenicity of SEOV S gene-based DNA vaccine in hamster model. Virus-neutralizing antibodies could be induced slightly in BALB/c mice following vaccination with DNA constructs encoding overlapped peptide fragments of Sin Nombre hantavirus (SNV) Gn and Gc protein. However, these authors failed to reproduce the neutralizing antibody findings in a subsequent study with deer mouse model. DNA vaccination with Puumala virus (PUUV) S segment also induced specific antibody response in mice. Consistently, in our study, an N or GP-specific antibody response was detected respectively in mice after immunized with equal mixture of HTNV S gene and M gene-based DNA plasmids. A substantial level of neutralizing antibody was elicited by HTNV DNA vaccine. As cellular immune response also plays an important role in limiting virus infection and replication, we further evaluated the HTNV N-specific cellular immune response in vitro, and did see a high frequency of CD8+/IFN+ T-cells in mice receiving HTNV DNA vaccine.
It’s generally accepted that modification of a viral antigen by fusion to a cellular protein, like eCTLA-4, could improve the efficacy of DNA vaccine. Here we constructed DNA plasmids encoding HTNV N or GP fused to eCTLA-4 protein (pcDNA3/eCTLA4-S or M). Compared to DNA vaccine encoding HTNV N or GP alone, pcDNA3/eCTLA4-S (M) greatly improved the speed and magnitude of HTNV specific humoral immune response in mice. Lu et al. reported similar modulation effect of eCTLA4 on woodchuck hepatitis virus nucleoprotein in mice and woodchuck models. Nicholas and his colleagues also observed the enhancement of immune responses to pro cathepsin B antigen in sheep model by fusion to eCTLA4 [40] In addition, there is a higher frequency of CD8+/IFN+ T-cells in mice immunized with pcDNA3/eCTLA4-S (M) DNA plasmids than that of pcDNA3/S (M). As the high affinity of eCTLA4 to its B7 ligand of APCs, our results indicated that eCTLA4 targeting may facilitate the antigen intake and processing by APCs, which will possibly improve the efficacy of DNA vaccine.
Another interesting finding of our study is that the efficacy of HTNV DNA vaccine was augmented by CpG motifs. When co-administration with CpG motifs, HTNV DNA vaccine induced better immune responses in mice compared with immunization with HTVN DNA vaccine alone. Vaccination with pcDNA3/eCTLA4-S (M) DNA plasmids plus CpG motifs elicited the highest antibody and cellular immune responses compared to all the other groups. Mice receiving pcDNA3/S (M) plus CpG motifs, though showed lower antibody titer one week after first immunization than that of mice vaccinated with pcDNA3/eCTLA4-S (M) DNA plasmids alone, exhibited comparable antibody response after the second injection. The recognition of CpG motifs is through toll-like receptor 9 (TLR-9) and then induces a broad range of immunological effects on APCs. Adjuvant effect of CpG motifs have been demonstrated in mice, humans as well as other species. Thus, after co-delivery of CpG motifs with HTNV DNA vaccine, it’s conceivable that APCs may be activated firstly by CpG motifs, then display enriched costimulatory molecules on the surface. This early event may provide a more efficient intake of antigen mediated by eCTLA4 later on through binding with B7 ligand. This may, if any, at least partially explain the observed augmentation of humoral and cellular immune responses induced by HTNV DNA vaccine in combination with CpG motifs.