Development of multivalent vaccine targeting M segment of Crimean Congo Hemorrhagic Fever Virus (CCHFV) using immunoinformatic approaches

Crimean-Congo Hemorrhagic Fever (CCHF) is a tick-borne viral infection with no licensed vaccine or therapeutics available for its treatment. In the present study we have developed the first multi-epitope subunit vaccine effective against all the seven genotypes of CCHF virus (CCHFV). The vaccine contains five B-cell, two MHC-II (HTL), and three MHC-I (CTL) epitopes screened from two structural glycoproteins (Gc and Gn in M segment) of CCHFV with an N-terminus human β-defensin as an adjuvant, as well as an N-terminus EAAAK sequence. The epitopes were rigorously investigated for their antigenicity, allergenicity, IFN gamma induction, anti-inflammatory responses, stability, and toxicity. The three-dimensional structure of the vaccine was predicted and docked with TLR-3, TLR-8, and TLR-9 receptors to find the strength of the binding complexes via molecular dynamics simulation. After codon adaptation, the subunit vaccine construct was developed in a pDual-GC plasmid and has population coverage of 98.47% of the world's population (HLA-I & II combined). The immune simulation studies were carried out on the C-ImmSim in-silico interface showing a marked increase in the production of cellular and humoral response (B-cell and T-cell) as well as TGFβ, IL-2, IL-10, and IL-12 indicating that the proposed vaccine would be able to sufficiently provoke both humoral and cell-mediated immune responses. Thus, making it a new and promising vaccine candidate against CCHFV.     

Influenza A virus nucleoprotein derived from Escherichia coli or recombinant vaccinia (Tiantan) virus elicits robust cross-protection in mice

Immunity to conserved viral antigens is an attractive approach to develop a universal vaccine against epidemic and pandemic influenza. A nucleoprotein (NP)-based vaccine has been explored and preliminary studies have shown promise. However, no study has explored the immunity and cross-protective efficacy of recombinant NP derived from Escherichia coli compared with recombinant vaccinia virus (Tiantan). Recombinant NP protein (rNP) from influenza virus A/Jingke/30/95(H3N2) was obtained from E. coli and recombinant vaccinia virus (Tiantan) RVJ1175NP. Purified rNP without adjuvant and RVJ1175NP were used to immunize BALB/c mice intramuscularly. Humoral immune responses were detected by ELISA, while cell-mediated immune responses were measured by ex vivo IFN-γ ELISPOT and in vivo cytotoxicity assays. The cross-protective efficacy was assessed by a challenge with a heterosubtype of influenza virus A/PR/8/34(H1N1). Our results demonstrate that a high dose (90 μg) of rNP induced NP-specific antibodies and T cell responses that were comparable with those of RVJ1175NP in mice. Importantly, the survival ratio (36, 73, and 78%) of the vaccinated mice after the influenza virus A/PR/8/34(H1N1) challenge was rNP vaccine dose-dependent (10, 30, and 90 μg, respectively), and no significant differences were observed between the rNP- and RVJ1175NP-immunized (91%) mice. Influenza A virus NP derived from E. coli or recombinant vaccinia (Tiantan) virus elicited cross-protection against influenza virus in mice, and the immune response and protective efficacy of rNP were comparable to RVJ1175NP. These data provide a basis for the use of prokaryotically expressed NP as a candidate universal influenza vaccine.     

Evaluation of the cell culture based and the mouse brain derived inactivated vaccines against Crimean-Congo hemorrhagic fever virus in transiently immune-suppressed (IS) mouse model

Crimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne virus in the Nairoviridae family within the Bunyavirales order of viruses. Crimean-Congo hemorrhagic fever (CCHF) is the most widespread among tick-borne human viral diseases. It is endemic in many areas of Africa, Asia, the Middle East, in the Balkans, Russia and countries of the former Soviet Union. The confirmed CCHF cases were seen in Spain in 2016 to signify expansion of the virus into new geographical areas. CCHFV causes a viral human disease characterized by sudden onset of fever, headache, abdominal pain, nausea, hypotension, hemorrhage, and hepatic dysfunction with fatality rates up to 30%. Currently, there are no spesific treatments or licensed vaccines available for CCHFV. The absence of a susceptible animal model for CCHFV infection was severely hindered work on the development of vaccines. However, several animal models of CCHFV infection have been recently developed and used to assess vaccine efficacy. In this study, we have used the transiently immune-suppressed (IS) mouse model that MAb-5A3 was used to block IFN-I signaling in immune intact, wild-type mice at the time of CCHFV infection to evaluate the immune response and efficacy of the cell culture based and the mouse brain derived inactivated vaccines against CCHFV. Both vaccine preparations have provided complete protection but the cell culture based vaccine more effectively induced to CCFHV spesific antibodies and T cell responses. This is the first comparison of the cell culture based and the mouse brain derived vaccines for assessing the protective efficacy and the immunogenicity in the IS mouse CCHFV model.     

Serologic and molecular evidence for circulation of Crimean-Congo hemorrhagic fever virus in ticks and cattle in Zambia

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne zoonosis with a high case fatality rate in humans. Although the disease is widely found in Africa, Europe, and Asia, the distribution and genetic diversity of CCHF virus (CCHFV) are poorly understood in African countries. To assess the risks of CCHF in Zambia, where CCHF has never been reported, epidemiologic studies in cattle and ticks were conducted. Through an indirect immunofluorescence assay, CCHFV nucleoprotein-specific serum IgG was detected in 8.4% (88/1,047) of cattle. Among 290 Hyalomma ticks, the principal vector of CCHFV, the viral genome was detected in 11 ticks. Phylogenetic analyses of the CCHFV S and M genome segments revealed that one of the detected viruses was a genetic reassortant between African and Asian strains. This study provides compelling evidence for the presence of CCHFV in Zambia and its transmission to vertebrate hosts.     

Fine Epitope Mapping of the Central Immunodominant Region of Nucleoprotein from Crimean-Congo Hemorrhagic Fever Virus (CCHFV)

Crimean-Congo hemorrhagic fever (CCHF), a severe viral disease known to have occurred in over 30 countries and distinct regions, is caused by the tick-borne CCHF virus (CCHFV). Nucleocapsid protein (NP), which is encoded by the S gene, is the primary antigen detectable in infected cells. The goal of the present study was to map the minimal motifs of B-cell epitopes (BCEs) on NP. Five precise BCEs (E1, ²⁴⁷FDEAKK²⁵²; E2a, ²⁵⁴VEAL²⁵⁷; E2b, ²⁵⁸NGYLNKH²⁶⁴; E3, ²⁶⁷EVDKA²⁷¹; and E4, ²⁷⁴DSMITN²⁷⁹) identified through the use of rabbit antiserum, and one BCE (E5, ²⁵⁸NGYL²⁶¹) recognized using a mouse monoclonal antibody, were confirmed to be within the central region of NP and were partially represented among the predicted epitopes. Notably, the five BCEs identified using the rabbit sera were able to react with positive serum mixtures from five sheep which had been infected naturally with CCHFV. The multiple sequence alignment (MSA) revealed high conservation of the identified BCEs among ten CCHFV strains from different areas. Interestingly, the identified BCEs with only one residue variation can apparently be recognized by the positive sera of sheep naturally infected with CCHFV. Computer-generated three-dimensional structural models indicated that all the antigenic motifs are located on the surface of the NP stalk domain. This report represents the first identification and mapping of the minimal BCEs of CCHFV-NP along with an analysis of their primary and structural properties. Our identification of the minimal linear BCEs of CCHFV-NP may provide fundamental data for developing rapid diagnostic reagents and illuminating the pathogenic mechanism of CCHFV.     

A novel role for non-neutralizing antibodies against nucleoprotein in facilitating resistance to influenza virus

Current influenza vaccines elicit Abs to the hemagglutinin and neuraminidase envelope proteins. Due to antigenic drift, these vaccines must be reformulated annually to include the envelope proteins predicted to dominate in the following season. By contrast, vaccination with the conserved nucleoprotein (NP) elicits immunity against multiple serotypes (heterosubtypic immunity). NP vaccination is generally thought to convey protection primarily via CD8 effector mechanisms. However, significant titers of anti-NP Abs are also induced, yet the involvement of Abs in protection has largely been disregarded. To investigate how Ab responses might contribute to heterosubtypic immunity, we vaccinated C57BL/6 mice with soluble rNP. This approach induced high titers of NP-specific serum Ab, but only poorly detectable NP-specific T cell responses. Nevertheless, rNP immunization significantly reduced morbidity and viral titers after influenza challenge. Importantly, Ab-deficient mice were not protected by this vaccination strategy. Furthermore, rNP-immune serum could transfer protection to naive hosts in an Ab-dependent manner. Therefore, Ab to conserved, internal viral proteins, such as NP, provides an unexpected, yet important mechanism of protection against influenza. These results suggest that vaccines designed to elicit optimal heterosubtypic immunity to influenza should promote both Ab and T cell responses to conserved internal proteins.     

Induction of caspase activation and cleavage of the viral nucleocapsid protein in different cell types during Crimean-Congo hemorrhagic fever virus infection

Regulation of apoptosis during infection has been observed for several viral pathogens. Programmed cell death and regulation of apoptosis in response to a viral infection are important factors for host or virus survival. It is not known whether Crimean-Congo hemorrhagic fever virus (CCHFV) infection regulates the apoptosis process in vitro. This study for the first time suggests that CCHFV induces apoptosis, which may be dependent on caspase-3 activation. This study also shows that the coding sequence of the S segment of CCHFV contains a proteolytic cleavage site, DEVD, which is conserved in all CCHFV strains. By using different recombinant expression systems and site-directed mutagenesis, we demonstrated that this motif is subject to caspase cleavage. We also demonstrate that CCHFV nucleocapsid protein (NP) is cleaved into a 30-kDa fragment at the same time as caspase activity is induced during infection. Using caspase inhibitors and cells lacking caspase-3, we clearly demonstrate that the cleavage of NP is caspase-3-dependent. We also show that the inhibition of apoptosis induced progeny viral titers of ～80-90%. Thus, caspase-3-dependent cleavage of NP may represent a host defense mechanism against lytic CCHFV infection. Taken together, these data suggest that the most abundant protein of CCHFV, which has several essential functions such as protection of viral RNA and participation in various processes in the replication cycle, can be subjected to cleavage by host cell caspases.     

Improved immune response to recombinant influenza nucleoprotein formulated with ISCOMATRIX

Current influenza vaccines elicit antibodies effective against homologous strains, but new strategies are urgently needed for protection against emerging epidemic or pandemic strains. Although influenza vaccine candidates based on the viral nucleoprotein (NP) or matrix protein do not elicit sterilizing immunity, they have the advantage of inducing immunity that may cover a larger number of viral strains. In this study, recombinant NP produced in Escherichia coli was purified and formulated in combination with the adjuvant ISCOMATRIX. This formulation increased a NP-specific immunity in mice, with a Th1 profile, and may constitute a promising low-cost influenza vaccine candidate, with ability to stimulate humoral and cellular immune responses..     

Crimean-Congo hemorrhagic fever virus activates endothelial cells

Crimean-Congo hemorrhagic fever virus (CCHFV) causes viral hemorrhagic fever with high case-fatality rates and is geographically widely distributed. Due to the requirement for a biosafety level 4 (BSL-4) laboratory and the lack of an animal model, knowledge of the viral pathogenesis is limited. Crimean-Congo hemorrhagic fever (CCHF) is characterized by hemorrhage and vascular permeability, indicating the involvement of endothelial cells (ECs). The interplay between ECs and CCHFV is therefore important for understanding the pathogenesis of CCHF. In a previous study, we found that CCHFV-infected monocyte-derived dendritic cells (moDCs) activated ECs; however, the direct effect of CCHFV on ECs was not investigated. Here, we report that ECs are activated upon infection, as demonstrated by upregulation of mRNA levels for E-selectin, vascular cell adhesion molecule 1 (VCAM1), and intercellular adhesion molecule 1 (ICAM1). Protein levels and cell surface expression of ICAM1 responded in a dose-dependent manner to increasing CCHFV titers with concomitant increase in leukocyte adhesion. Furthermore, we examined vascular endothelial (VE) cadherin in CCHFV-infected ECs by different approaches. Infected ECs released higher levels of interleukin 6 (IL-6) and IL-8; however, stimulation of resting ECs with supernatants derived from infected ECs did not result in increased ICAM1 expression. Interestingly, the moDC-mediated activation of ECs was abrogated by addition of neutralizing tumor necrosis factor alpha (TNF-α) antibody to moDC supernatants, thereby identifying this soluble mediator as the key cytokine causing EC activation. We conclude that CCHFV can exert both direct and indirect effects on ECs.     

Synonymous codon usage pattern among the S,M, and L segments in Crimean-congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) virus is one among the major zoonosis viral diseases that use the Hyalomma ticks as their transmission vector to cause viral infection to the human and mammalian community. The fatality of infectious is high across the world especially in Africa, Asia, Middle East, and Europe. This study regarding codon usage bias of S, M, and L segments of the CCHF virus pertaining to the host Homo sapiens, reveals in-depth information about the evolutionary characteristics of CCHFV. Relative Synonymous Codon Usage (RSCU), Effective number of codons (ENC) were calculated, to determine the codon usage pattern in each segment. Correlation analysis between Codon adaptation index (CAI), GRAVY (Hydrophobicity), AROMO (Aromaticity), and nucleotide composition revealed bias in the codon usage pattern. There was no strong codon bias found among any segments of the CCHF virus, indicating both the factors i.e., natural selection and mutational pressure shapes the codon usage bias.     

Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine

In this study, we compared the immunogenicity and tumor-protective activity of anti-idiotypic antibodies mimicking a single tumor-associated epitope and tumor-associated antigen expressing multiple potentially immunogenic epitopes. We focused our study on the colorectal-carcinoma(CRC)-associated antigen GA733 (also known as CO17-1A/KS1-4/KSA/EpCAM). Monoclonal anti-idiotypic antibody (Ab2) BR3E4 was produced against murine anti-CRC mAb CO17-1A (Ab1) in rats. Full-length native GA733 protein was isolated from human tumor cells, and the extracellular domain protein (GA733-2E) was isolated from supernatants of recombinant baculovirus-infected insect cells by immunoafffinity chromatography. The immunomodulatory activity of the Ab2 was compared with that of the antigen, both in rabbits and in mice. Mice, like humans but not rabbits, express a GA733 antigen homologue on some of their normal tissues. Thus, these in vivo models allow the comparison of the immunogenicity of Ab2 and antigen in the presence (mice) and absence (rabbits) of normal tissue expression and immunological tolerance of the GA733 antigen homologue. In rabbits, aluminum-hydroxide(alum)-precipitated native GA733 antigen was superior to alum-precipitated Ab2 in inducing specific humoral immunity. In mice, alum-precipitated recombinant GA733-2E antigen, but not alum-precipitated Ab2, induced specific humoral immunity. However, when the Ab2 was administered to mice in Freund's complete adjuvant, specific humoral immune responses were elicited. Ab2 in complete Freund's adjuvant and GA733-2E in alum were compared for their capacity to induce antigen-specific cellular immunity in mice. Whereas lymphoproliferative responses were obtained with the recombinant antigen only, delayed-type hypersensitivity responses were obtained with both recombinant antigen and Ab2, although these responses were lower than after antigen immunization. The recombinant antigen in alum did not protect mice against challenge with antigen-positive syngeneic murine CRC cells. Similar studies with Ab2 BR3E4 mimicking the CO17-1A epitope were not possible because the tumor cells do not express this epitope after transfection with the human GA733-2 cDNA. However, similar studies with Ab2 mimicking the epitope defined by mAb GA733, which is expressed by the transfected tumor cells, indicated a lack of tumor-protective activity of this Ab2. In contrast, the full-length antigen expressed by recombinant adenovirus inhibited the growth of established tumors in mice. In conclusion, soluble antigen is a more potent modulator of humoral and cellular immune responses than Ab2, both administered in adjuvant. However, for induction of protective immunity, the immunogenicity of the antigen must be further enhanced, e.g., by expression of the antigen in a viral vector. Received: 27 December 1999 / Accepted: 27 January 2000     

Crimean-Congo hemorrhagic fever

Crimean-Congo hemorrhagic fever (CCHF) is an acute infectious disease caused by CCHF virus (CCHFV), a member of the family Bunyaviridae, genus Nairovirus. The case fatality rate of CCHF ranges from 10-40%. Because CCHF is not present in Japan, many Japanese virologists and clinicians are not very familiar with this disease. However, there remains the possibility of an introduction of CCHFV or other hemorrhagic fever viruses into Japan from surrounding endemic areas. Development of diagnostic laboratory capacity for viral hemorrhagic fevers is necessary even in countries without these diseases. At the National Institute of Infectious Diseases, Tokyo, Japan, laboratory-based systems such as recombinant protein-based antibody detection, antigen-capture and pathological examination have been developed. In this review article, epidemiologic and clinical data on CCHF in the Xinjiang Uygur Autonomous Region, compiled through field investigations and diagnostic testing utilizing the aforementioned laboratory systems, are presented. CCHFV infections are closely associated with the environmental conditions, life styles, religion, occupation, and human economic activities. Based on these data, preventive measures for CCHFV infections are also discussed.     

Identification of potential microRNA markers related to Crimean-Congo hemorrhagic fever disease

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease caused by the arbovirus Crimean-Congo hemorrhagic fever virus (CCHFV). The CCHFV has a single-stranded RNA genome of negative sense. MicroRNAs (miRNAs) are key players in virus-host interactions and viral pathogenesis. We investigated the miRNA gene expression profiles in patients with CCHF using microarray for the first time in the world. Microarray analysis was performed using mirBase Ver 21 (Agilent Technologies, Santa Clara, CA). All statistical analyses were performed across the case-control, fatal-control, and fatal-nonfatal case groups using Genespring (Ver 3.0). Fifteen miRNAs were statistical significant in patients with CCHF compared with the controls (5 were upregulated, 10 were downregulated). Seventy-five and sixty-six miRNAs are in fatal compared with control and nonfatal case, respectively (fold change ([FC] ≥50) were statistically significant. In this study, the target genes of important miRNAs were identified and Gene Ontology analyses were performed across all groups. As a result of this study, we propose that the detection of miRNAs in patients with CCHF will allow the determination of therapeutic targets in diseases. CCHF is an important public health problem that can often be fatal. In this study, we investigated miRNA expression in case-control, fatal-control, and fatal-nonfatal case groups. Significant miRNAs associated with fatality were detected in CCHF. This study will serve as a source of data for the development of an antagomir-based therapy against CCHF using miRNAs in the future.     

Risk factors associated with exposure to Crimean-Congo haemorrhagic fever virus in animal workers and cattle,and molecular detection in ticks,South Africa

Crimean-Congo haemorrhagic fever (CCHF) is a severe tick-borne viral zoonosis endemic to parts of Africa, Europe, the Middle East and Central Asia. Human cases are reported annually in South Africa, with a 25% case fatality rate since the first case was recognized in 1981. We investigated CCHF virus (CCHFV) seroprevalence and risk factors associated with infection in cattle and humans, and the presence of CCHFV in Hyalomma spp. ticks in central South Africa in 2017–18. CCHFV IgG seroprevalence was 74.2% (95%CI: 64.2–82.1%) in 700 cattle and 3.9% (95%CI: 2.6–5.8%) in 541 farm and wildlife workers. No veterinary personnel (117) or abattoir workers (382) were seropositive. The prevalence of CCHFV RNA was significantly higher in Hyalomma truncatum (1.6%) than in H. rufipes (0.2%) (P = 0.002). Seroprevalence in cattle increased with age and was greater in animals on which ticks were found. Seroprevalence in cattle also showed significant geographic variation. Seroprevalence in humans increased with age and was greater in workers who handled livestock for injection and collection of samples. Our findings support previous evidence of widespread high CCHFV seroprevalence in cattle and show significant occupational exposure amongst farm and wildlife workers. Our seroprevalence estimate suggests that CCHFV infections are five times more frequent than the 215 confirmed CCHF cases diagnosed in South Africa in the last four decades (1981–2019). With many cases undiagnosed, the potential seriousness of CCHF in people, and the lack of an effective vaccine or treatment, there is a need to improve public health awareness, prevention and disease control.     

Anti-nucleocapsid protein immune responses counteract pathogenic effects of Rift Valley fever virus infection in mice

The known virulence factor of Rift Valley fever virus (RVFV), the NSs protein, counteracts the antiviral effects of the type I interferon response. In this study we evaluated the expression of several genes in the liver and spleen involved in innate and adaptive immunity of mice immunized with a RVFV recombinant nucleocapsid protein (recNP) combined with Alhydrogel adjuvant and control animals after challenge with wild type RVFV. Mice immunized with recNP elicited an earlier IFNβ response after challenge compared to non-immunized controls. In the acute phase of liver infection in non-immunized mice there was a massive upregulation of type I and II interferon, accompanied by high viral titers, and the up- and downregulation of several genes involved in the activation of B- and T-cells, indicating that both humoral and cellular immunity is modulated during RVFV infection. Various genes involved in pro-inflammatory responses and with pro-apoptotic effects were strongly upregulated and anti-apoptotic genes were downregulated in liver of non-immunized mice. Expression of many genes involved in B- and T-cell immunity were downregulated in spleen of non-immunized mice but normal in immunized mice. A strong bias towards apoptosis and inflammation in non-immunized mice at an acute stage of liver infection associated with suppression of several genes involved in activation of humoral and cellular immunity in spleen, suggests that RVFV evades the host immune response in more ways than only by inhibition of type I interferon, and that immunopathology of the liver plays a crucial role in RVF disease progression.     

First international external quality assessment of molecular detection of Crimean-Congo hemorrhagic fever virus

Crimean-Congo hemorrhagic fever (CCHF) is a zoonosis caused by a Nairovirus of the family Bunyaviridae. Infection is transmitted to humans mostly by Hyalomma ticks and also by direct contact with the blood or tissues of infected humans or viremic livestock. Clinical features usually include a rapid progression characterized by hemorrhage, myalgia and fever, with a lethality rate up to 30%. CCHF is one of the most widely distributed viral hemorrhagic fevers and has been reported in Africa, the Middle East and Asia, as well as parts of Europe. There is no approved vaccine or specific treatment against CCHF virus (CCHFV) infections. In this context, an accurate diagnosis as well as a reliable surveillance of CCHFV infections is essential. Diagnostic techniques include virus culture, serology and molecular methods, which are now increasingly used. The European Network for the Diagnostics of "Imported" Viral Diseases organized the first international external quality assessment of CCHVF molecular diagnostics in 2011 to assess the efficiency and accurateness of CCHFV molecular methods applied by expert laboratories. A proficiency test panel of 15 samples was distributed to the participants including 10 different CCHFV preparations generated from infected cell cultures, a preparation of plasmid cloned with the nucleoprotein of CCHFV, two CCHFV RNA preparations and two negative controls. Forty-four laboratories worldwide participated in the EQA study and 53 data sets were received. Twenty data sets (38%) met all criteria with optimal performance, 10 (19%) with acceptable performance, while 23 (43%) reported results showing a need for improvement. Differences in performance depended on the method used, the type of strain tested, the concentration of the sample tested and the laboratory performing the test. These results indicate that there is still a need for improving testing conditions and standardizing protocols for the molecular detection of Crimean-Congo hemorrhagic fever virus.     

Analysis of immune responses against nucleocapsid protein of the Hantaan virus elicited by virus infection or DNA vaccination

Even though neutralizing antibodies against the Hantaan virus (HTNV) has been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. In this report, we have examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination. To examine the cellular immune response against HTNV NP, we used H-2K(b) restricted T-cell epitopes of NP. The NP-specific CD8(+) T cell response was analyzed using a (51)Cr-release assay, intracellular cytokine staining assay, enzyme-linked immunospot assay and tetramer binding assay in C57BL/6 mice infected with HTNV. Using these methods, we found that HTNV infection elicited a strong NP-specific CD8(+) T cell response at eight days after infection. We also found that several different methods to check the NP-specific CD8(+) T cell response showed a very high correlation among analysis. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, the NP-specific antibody response was elicited 2 approximately 4 weeks after immunization and maximized at 6 approximately 8 weeks. NP-specific CD8(+) T cell response reached its peak 3 weeks after immunization. In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100-fold compared to the negative control mice.