• DNA vaccine;
  • needle-free injection;
  • subviral particles;
  • tick-borne encephalitis


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Tick-borne encephalitis virus (TBEV) causes severe encephalitis in humans. It is endemic in one area of Japan; however no commercial vaccine is available in that country. In this Japan-based study, the efficacy of subviral particles (SPs) of TBEV administered by needle-free injector was evaluated as a vaccine candidate. Inoculation with SP-encoding DNA by needle-free injector induced neutralizing antibodies more efficiently than when administered by needle and syringe, and mice vaccinated with one dose by needle-free injector survived challenge with a lethal dose of TBEV. These results suggest that SP vaccines delivered by needle-free injector can protect against TBEV infection.

List of Abbreviations: 

biosafety level






focus forming units




subviral particle


tick-borne encephalitis


tick-borne encephalitis virus

Tick-borne encephalitis virus belongs to the family Flaviviridae, genus Flavivirus, and causes severe encephalitis in humans. TBEV is distributed widely in Europe, Russia, and far-eastern Asia including Japan; thousands of human cases are reported annually (1). TBEV can be divided into three subtypes; European, Siberian, and far-eastern (2). TBEV has a significant impact on public health in these endemic regions.

Tick-borne encephalitis was first diagnosed in Japan in 1993 in a patient with severe encephalitis in Hokkaido and TBEV was subsequently isolated from dogs, ticks, and rodents in this region. The virus was identified as a far-eastern subtype of TBEV by nucleotide sequence analysis and believed to possess a higher pathogenic potential for mice (3–5). Our research has demonstrated that the virus has persisted for a number of years in one particular area of Hokkaido. Therefore, it is important to be prepared for a future TBE outbreak.

Because no anti-viral therapy for TBE infection is available, control of TBE depends on prophylaxis, such as active immunization by vaccination. Formalin-inactivated vaccines based on the European subtype have been used in European countries and shown to induce highly protective immunity. In our previous studies, this vaccine was shown to prevent infection with the Siberian and far-eastern subtypes of TBEV (6, 7). However, because these vaccines are not licensed in Japan, there is an urgent need to establish preventive measures for residents of TBEV-endemic regions and travelers to Europe and Russia. Because TBEV is classified as a BSL 3 or 4 virus, the production and inactivation of live TBEV is restricted by safety considerations; furthermore, formalin inactivation affects several epitopes of the virion structural proteins (8, 9). Thus, a new TBEV vaccine candidate is required to replace the current formalin-inactivated formulation.

The flavivirus genome consists of a positive-sense, single-stranded RNA strand of approximately 11 kb that encodes three structural proteins: the C, prM, and E proteins, and seven non-structural proteins within a single long open reading frame (10). The flavivirus virion has both an envelope and nucleocapsid (11). The nucleocapsid consists of genomic RNA and C protein, and the envelope consists of a regular lattice formed by E and M proteins. The E protein mediates virus entry via receptor-mediated endocytosis and carries the major epitopes, leading to a protective immune response (12, 13).

In our previous study, a SP system of Japanese TBEV was developed by the co-expression of viral prM and E proteins(14). SPs are smaller than authentic virions but possess E proteins that have similar characteristics to those of authentic virions (15). They have been shown to induce host responses (9, 15). SPs do not produce progeny virus because they lack a nucleocapsid. Therefore, SPs could be potent vaccine candidates that do not require formalin inactivation. Additionally, SP-encoding DNA may be a DNA vaccine candidate.

In this Japan-based study, we investigated vaccination with both SPs of the far-eastern subtype of TBEV and SP-encoding DNA. We also attempted to improve the induction of neutralizing antibodies by use of a needle-free injector instead of needle injection.

The pCAG-TBEME plasmid, which is a pCAGGS-based plasmid encoding the signal sequence of the prM, prM, and E genes of TBE virus (Oshima 510 strain), was constructed as described previously (14). This plasmid was used both to prepare the SP antigens of TBEV and as a DNA vaccine. It was amplified in Escherichia coli JM109 (Takara, Shiga, Japan) and purified using a Qiagen Plasmid kit (Qiagen, Tokyo, Japan) according to the manufacturer's instructions. For preparation of SPs, 293T cells grown in a 150-cm2 culture flask were transfected with 40 μg of pCAG-TBEME combined with 120 μL TransIT-LT1 transfection reagent (Mirus, Madison, WI, USA) in Opti-MEM (Gibco BRL, Gaithersburg, MD, USA). At 36 hr post-transfection, the supernatants were harvested and cleared by centrifugation at 12,000 g for 20 min at 4°C. SPs in the cleared supernatant were incubated with 10% polyethylene glycol (MW = 8000) and 1.9% NaCl for 2 hr at 4°C and then centrifuged at 12,000 g for 30 min at 4°C. Pelleted SPs were purified by equilibrium density centrifugation in a 10–50% sucrose gradient at 140,000 g for 16 hr at 4°C (P50AT2 rotor, Hitachi, Japan). Fractions (0.5 mL) were collected from the tops of tubes, and amounts of E protein were quantified by ELISA, as described previously (14).

Four-week-old male C57BL/6J mice (Charles River, Yokohama, Japan) were used. Animal work was approved by the Committee on Animal Experimentation of Hokkaido University and performed following that institution's guidelines for animal experimentation. Animals used for vaccinations and infections were handled under BSL-3 containment conditions. To evaluate the immunogenicity of SPs, groups of 8–10 mice were inoculated once or twice at 10-day intervals with 0.1 μg or 1.0 μg of SPs via a needle-free jet injector (ShimaJET; Shimadzu, Kyoto, Japan) intramuscularly or via a needle and a syringe subcutaneously. SPs were diluted in PBS to 50 μL per mouse.

The inoculum was inoculated from the lower base of the thigh toward the upper back of the leg so as not to leak any liquid. No physical damage was observable in the inoculated area. At 10 days post-vaccination, the mice were killed and blood samples collected. Neutralizing antibodies against TBEV were assayed by neutralization tests of sera as described previously (6).

No neutralizing antibodies were induced by a single inoculation of SPs; however the use of two doses induced an antibody response in 20–60% of mice (Table 1). The neutralizing titer was not very high (NT50 = 40–80), and there was no significant difference in the neutralizing titers between needle-free and needle injections or between 0.1 and 1 μg of inoculum of SPs.

Table 1.  Neutralization antibody titers against TBEV after SPs inoculation
Injection Frequency of injection Amount (μg)Needle-free injectorNeedle syringe + adjuvanta
 0.1   1 0.1   1 0.1   1 0.1   1
  1. a0.2-0.3 mg/mice of 0.2 % aluminum hydroxide was used as adjuvant.

  2. bThe serum samples were collected 10 days after the last SP inoculation. Each titer was determined as the reciprocal of highest dilution that reduced the virus focus counts by 50% in neutralization test.

  3. cThe average titers were calculated by the average exponent of each neutralizing titers (10 × 2x).

Neutralizing titer of each mouseb<40<40  40  40<40<40  80  80
 <40<40  40  40<40<40  80  80
 <40<40  40<40<40<40  40  40
 <40<40  40<40<40<40  40  40
 <40<40  40<40<40<40  40<40
 <40<40  40<40<40<40  40<40
Average titer of seropositive micec  40  40  49  57
Number of seropositive animals with neutralizing0/80/86/102/100/100/106/104/10
 antibodies/number in group(0%)(0%)(60%)(20%)(0%)(0%)(60%)(40%)

Next, we examined the efficacy of pCAG-TBEME as a DNA vaccine. Groups of 8–10 mice were inoculated with 1.0 or 5.0 μg of pCAG-TBEME once or twice at 10-day intervals using a needle-free jet injector intramuscularly or with 5.0 or 50 μg of plasmid twice at 10 day intervals using a needle and syringe subcutaneously. At 10 days post-vaccination, blood samples were collected. As a negative control, four mice were inoculated with the vector plasmid, pCAGGS. DNA was diluted in PBS to 50 μL per mouse. As shown in Table 2, both the ratios and average neutralizing titers of seropositive mice were significantly higher in mice that had received needle-free injections than in those that had received needle injections. A single inoculation of 5 μg of DNA by needle-free injector induced neutralizing antibodies in all mice; in contrast, 50 μg of DNA inoculated using a needle and syringe induced antibodies in only 40% of the mice. Vector plasmids did not induce neutralizing antibodies. Although two of eight mice inoculated with one dose of 1 μg DNA by needle-free injector did not produce neutralizing antibodies, a positive response was more efficiently induced by both one and two doses of DNA by needle-free injection. Compared with the results of SP vaccination, inoculation with pCAG-TBEME DNA induced both higher neutralizing antibodies titers and higher seropositivity rates. These data indicate that pCAG-TBEME DNA is an effective inducer of anti-TBEV neutralizing antibody and that needle-free injection may be a useful substitute for a needle and syringe.

Table 2.  Neutralization antibody titers against TBEV after DNA vaccine inoculation
Injection Frequency of injection DNA Amount (μg)Needle-free injectorNeedle syringea 2
  1  5  1   5  5 50
  1. aThe DNA vaccine was inoculated intramuscularly with 20 μl/mouse of transfection detergent (DOTAP)

  2. bThe serum samples were collected 10 days after the last SP inoculation. Each titer was determined as the reciprocal of highest dilution that reduced the virus focus counts by 50% in neutralization test.

  3. cThe average titers were calculated by the average exponent of each neutralizing titers (10 × 2x)

Neutralizing titer of each mouseb640640<406401280<40320160
 640320<40640 640<40160  80
 320160<40320 160<40  80  80
   80160<40320 160<40  40  40
   80160 320  80  40<40
   80160 160  80 <40<40
 <40  80   80  40 <40<40
 <40  80   80  40 <40<40
       40 <40<40
Average titer of seropositive micec201190247127  92  80
Number of seropositive animals with neutralizing antibodies6/88/80/48/89/90/45/104/10

To evaluate the protective effect of the DNA vaccine against virus challenge, vaccinated mice were challenged with a lethal dose of TBEV. Groups of 10 male 6-week-old C57BL/6J mice were inoculated with 5.0 or 1.0 μg of pCAG-TBEME. As a control, five mice were inoculated with PBS once or twice using a needle-free jet injector. Ten days after inoculation, the mice were challenged intraperitoneally with 10 plaque forming units (>100 LD50) of the far-eastern subtype of the TBEV Sofjin-HO strain that had been isolated from the brain of a TBE patient in Khabarovsk in 1937 (16). The mice were monitored for 28 days post-infection to obtain survival curves and body weight changes. As shown in Fig. 1a, all mice vaccinated with pCAG-TBEME survived for 28 days, regardless of the amount or frequency of vaccination, whereas all mock-vaccinated mice died. The average body weights of pCAG-TBEME-vaccinated groups increased without their showing any clinical symptoms, whereas mock-vaccinated mice lost weight and evidenced neurological signs of illness (Fig. 1b). These data indicate that DNA vaccination using a needle-free jet injector is able to effectively prevent TBEV infection.


Figure 1. (a) Survival curves and (b) weight changes of mice inoculated with TBEV. C57BL/6J mice were inoculated with either one (left panel) or two (right panel) doses of 5 μg pCAG-TBEME DNA (diamond, = 10), 1 μg pCAG-TBEME DNA (open square, = 10), or PBS (cross, = 5) using a needle-free injector at 10-day intervals. At 10 days post-vaccination, each group of mice was challenged intraperitoneally with the Sofjin-HO strain of TBE virus, 10 ffu/mouse (LD50 < 0.1 ffu).

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In this study, we examined the efficacy of vaccination with SPs and SP-encoding DNA based on a Japanese TBEV isolate using a needle-free injector. SPs induced neutralizing antibodies to some extent; the DNA vaccine induced them very efficiently. Our results also showed that a single dose of a small quantity of DNA protected mice from challenge with a lethal dose of TBEV. These data suggest that recombinant vaccines administered with a needle-free injector are able to protect against TBEV infection.

The needle-free injector was shown to be a more effective method of inoculation of the DNA vaccine than a needle and syringe. Small quantities of DNA administered by needle-free injection efficiently induced neutralizing antibodies without use of a transfection reagent. Because needle-free injectors diffuse the solution widely in the subcutaneous tissue from a narrow opening using high pressure, the injected DNA is taken up by more cells. It has also been reported that a higher degree of protein expression is detected in the inoculated area when needle-free injectors have been used (17). The wide diffusion and strong expression of antigen likely lead to efficient induction of neutralizing antibodies. These data indicate that needle-free injection may be a safe and painless substitute for the traditional needle and syringe injection method.

A mixture of DNA and recombinant vaccines has been reported to enhance immunogenicity. In a Japanese encephalitis study, vaccination with a combination of SPs and SP-encoding DNA was shown to induce protective immunity (18, 19). Although induction of neutralizing antibodies was low when SPs alone were used, a more effective TBE vaccine may result from use of a combination of SPs and DNA.

In summary, we have demonstrated that needle-free injection of SP-encoding DNA efficiently induces protective immunity against TBEV infection. These data will be useful in establishing an effective preventative strategy in Japan.


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We thank Dr. Konishi (Kobe University Graduate school of Medicine, Kobe, Japan) for helpful information about needle-free jet injectors. This work was supported by Grants-in-Aid for Scientific Research (22780268) and the Global CEO Program from the Ministry of Education, Culture, Sports, Sciences and Technology of Japan, and Health Sciences Grants for Research on Emerging and Re-emerging Infectious Disease from the Ministry of Health, Labor and Welfare of Japan.


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