Fusion with extracellular domain of cytotoxic T-lymphocyte-associated-antigen 4 leads to enhancement of immunogenicity of Hantaan virus DNA vaccines in C57BL/6 mice


Hantaan virus (HTNV) is the causative agent of the most severe form of a rodent-borne disease known as hemorrhagic fever with renal syndrome (HFRS). A safe and effective HTNV vaccine is needed. Vaccination with DNA constructs expressing fused antigen with bioactive factors, has shown promising improvement of immunogenicity for viral agents in animal models, but the effect of fusion strategy on HTNV DNA vaccine has not been investigated.


DNA plasmids encoding the HTNV nucleocapsid protein (N) and glycoprotein (Gn and Gc) in fusion to the extracellular domain of cytotoxic T-lymphocyte-associated-antigen 4 (eCTLA-4) targeting to antigen presenting cells (APCs) were constructed. Intramuscular immunization of mice with plasmids expressing eCTLA-4-HTNV-N/GP fusion proteins leads to a significant enhancement of the specific antibody response as well as cytotoxic T-lymphocyte (CTL) response in C57BL/6 mice. Moreover, this effect could be further augmented when co-administered with CpG motifs.


Modification of viral antigen in fusion to bioactive factor will be promising to confer efficient antigen presentation and improve the potency of DNA vaccine in mice.


Hantaan virus (HTNV) (Bunyaviridae family, Hantavirus genus) is the causative agent of the most severe form of a rodent-borne disease known as hemorrhagic fever with renal syndrome (HFRS). Other hantaviruses that are known to cause HFRS include Seoul virus (SEOV), Dobrava virus (DOBV) and Puumala virus (PUUV), which cause disease in Asia, Europe, Scandinavia, and western Russia respectively. In addition, a few hantaviruses have been identified to associate with outbreaks of a highly lethal disease, hantavirus pulmonary syndrome (HPS), in the Americas. Since hantaviruses can cause epidemics with high morbidity, and currently there is no proven therapy for hantaviral disease, a safe and effective vaccine(s) against hantaviruses infection is necessary. HTNV causes the most severe form of HFRS and around 150,000 cases of HFRS are reported worldwide annually, with the majority of HFRS occurring in Asia.

Hantaviruses are enveloped, negative strands RNA viruses consisting of three single RNA segments designated S (small), M (medium), and L (large), which encode the nucleocapsid (N) protein, envelope glycoproteins (Gn and Gc), and the RNA polymerase respectively. As a key surface antigen, glycoproteins (Gn and Gc) bear the epitopes which could elicit neutralizing antibodies against hantavirus infection. N-specific antibodies are neither neutralizing nor protective, but may play a role through cellular immune response.

Immunization with DNA vaccines encoding antigen has been used to induce both humoral and cellular immune responses and holds potential for developing vaccines to a variety of viral antigens. Application of DNA vaccine to hantavirus was also promising and previously explored. DNA vaccination with a plasmid containing the SEOV M segment elicited neutralizing antibody responses in mice and hamsters as well as a certain level of cross-protection against HTNV. A HTNV M gene-based DNA vaccine conferred good protection against infection in hamster model and elicited high levels of neutralizing antibodies in Rhesus monkeys. However, there are still concerns about the potency of DNA vaccines, like a low level of protein expression after DNA immunization.

One of interesting approaches, to improve the potency of DNA vaccine, is to fuse a bioactive domain, like cytotoxic-T-lymphocyte-associated protein 4 (CTLA-4), to viral antigens. CTLA-4 consists of extracellular domain, transmembrane domain and cytoplasmic domain. As an inhibitory costimulatory molecule, CTLA-4 normally plays a key role to downmodulate T-cell activation by interaction with its ligand, B7 on antigen presenting cells (APCs). However, the affinity of CTLA-4 to the shared ligands, B7 is 10-20 times higher than that of its counterpart, CD28 which provides a costimulatory signal to APCs. Recently, Axel et al demonstrated that without the cytoplasmic domain of CTLA-4, the extracellular domain of CTLA-4 (eCTLA-4) alone can enhance TCR activation instead of inhibitory function in the full-length form. Lu et al has observed an enhancement of specific immune response in mice and woodchuck models conferred by eCTLA4 fused with woodchuck hepatitis virus nucleoprotein. In addition, adjuvant effects of CpG motifs have been shown to enhance antigen-specific immune responses to protein vaccine in mice and human. While the effects of CpG motifs co-delivery on immune responses to DNA vaccination in mice are diverse.

In this study, we first report to generate recombinant HTNV DNA vaccine plasmids encoding HTNV N or GP fused to eCTLA4, and evaluated their immunogenicity in C57BL/6 mice as well as the strategy of co-delivery with CpG motifs. Our results indicated that eCTLA4 fusion strategy could enhance specific antibody response and cellular immune response in mice generated by HTNV DNA vaccine. This adjuvant effect could be further augmented when co-delivery with CpG motifs.