This study focused on the antigenic cross-reactivity between tick-borne encephalitis virus (TBEV) and Omsk hemorrhagic fever virus (OHFV) to assess the efficacy of the commercial TBE vaccine against OHFV infection. Neutralization tests performed on sera from OHFV- and TBEV-infected mice showed that neutralizing antibodies are cross-protective. The geometric mean titers of antibodies against TBEV and OHFV from TBEV-infected mice were similar. However, the titers of anti-TBEV antibodies in OHFV-infected mice were significantly lower than those of anti-OHFV antibodies in the same animals. In mouse vaccination and challenge tests, the TBE vaccine provided 100% protection against OHFV infection. Eighty-six percent of vaccinees seroconverted against OHFV following complete vaccination, and the geometric mean titers of neutralizing antibodies against OHFV were comparable to those against TBEV. These data suggest that the TBE vaccine can prevent OHFV infection.
baby hamster kidney
focus forming unit
geometric mean titer
lethal dose at the 50th percentile
minimum essential media
Omsk hemorrhagic fever
tick-borne encephalitis virus
Tick-borne encephalitis virus and (OHFV) are tick-borne flaviviruses belonging to the TBE serocomplex [1, 2]. This serocomplex consists of tick-borne flaviviruses that are cross-reactive in serological tests . Other members of this group include Langat virus, Powassan virus, Louping Ill virus, Alkhurma virus and Kyasanur Forest disease virus [1, 4, 5]. The majority of the viruses in this serocomplex cause encephalitis; however OHFV, Alkhurma virus and Kyasanur Forest disease virus are known to cause hemorrhagic fever syndrome [1, 4]. TBEV is an important human pathogen, causing over 10,000 cases of encephalitis annually . There are three subtypes of the TBEV: European, far-eastern, and Siberian [7-12]. Mortality rates vary depending on the TBEV subtype. The far-eastern subtype has the highest mortality rate, namely 5–20% . TBEV has a wide geographic range that extends over Europe and Asia [14-17]. The virus is transmitted by Ixodes ticks, Ixodes ricinus and Ixodes persulcatus being the principle vectors in Europe and Asia, respectively . In contrast, OHFV is endemic in the Omsk and Novosibirsk regions of Russia. The Siberian subtype of TBEV is also endemic in these regions . In these regions, Demercentor ticks are thought to be the principle vectors of OHFV [2, 18]. The incidence of OHF is unclear, but most outbreaks are seasonal and coincide with an increase in tick populations or muskrat (Ondatra zibethicus) hunting [2, 18]. Muskrats, which are highly susceptible to the virus, are thought to be the amplifying host.
Tick-borne flaviviruses are zoonotic arthropod-borne viruses belonging to the family Flaviviridae, genus Flavivirus. The flavivirus genome contains a single open reading frame that encodes 10 proteins. Three of these, the C, prM/M and E proteins, are structural proteins. The remaining seven are non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) [19, 20]. Despite the differences in disease manifestations, phylogenetic analysis has revealed that OHFV is more closely related to TBEV than to the other two hemorrhagic fever viruses . A comparison of the E proteins of OHFV and of two Siberian/far-eastern TBE viruses showed that only 28 amino acids are specific to OHFV .
Vaccination is the most important means of reducing the risk of flavivirus infection. Four vaccines against TBE are currently available on the global market. Two of these, Encepur (Chiron Behring GmbH, Marburg, Germany) and FSME-IMMUN (Baxter AG, Vienna, Austria), are based on European subtypes of TBEV. The other two, EnceVir (Virion, Tomsk, Russia) and TBE-Moscow vaccine (Chumakov Institute of Poliomyelitis and Viral Encephalitides, Moscow, Russia), are based on the far-eastern subtypes [14, 21]. All of these vaccines are reportedly highly immunogenic against each of the viral subtypes [14, 21-25]. The cross-protection amongst the subtypes is thought to be because the E protein has an amino acid homology in excess of 95% [1, 26]. Protection against flaviviruses is mediated primarily by neutralizing antibodies against the E protein, and to a lesser extent the prM and NS1 proteins [27-31]. The E protein forms three structural domains, which are designated I–III [4, 32]. Epitopes recognized by neutralizing antibodies have been identified in all three domains [33, 34]. Domain III is thought to be highly immunogenic—most serotype- and virus-type-specific neutralizing antibodies target this region [33-35]. In addition, many cross-reactive antibodies recognize the fusion loop of domain II [36, 37].
Despite the availability of several TBE vaccines, no vaccine for OHFV exists. It is therefore necessary to determine if the available TBE vaccines can be used to prevent OHFV infection. Although OHFV and TBEV are cross-reactive in serological tests, limited data regarding whether this cross-reactivity is protective are available. Nucleotide analysis of TBEV Neudoerfl (the prototype of the European subtype of TBEV used in the production of FSME-IMMUN vaccine) revealed an 81.8% similarity with OHFV  in the gene encoding the E protein. Because of the similarities between the E proteins of TBEV and OHFV, the available TBE vaccines could provide effective protection against OHFV. Therefore, the aim of this study was to evaluate the antigenic cross-reactivity of these two viruses and determine if the commercially available TBE vaccines, which are based on the European subtype of the virus, provide sufficient cross-protection against OHFV infection.
MATERIALS AND METHODS
Viruses and cell lines
The TBEV-Sofin-HO strain (accession No. 062064) and OHF-infectious clone were used for neutralization tests and mouse inoculation. The OHF- infectious clone strain was derived from the Guriev strain of OHFV (accession No. AB507860) . The Sofin-HO strain was first isolated from the brain of a TBE patient in Khabarovsk in 1937. This virus (of unknown passage history) was provided by Dr. Ohya (National Institute of Infectious Disease, Tokyo, Japan) in 1967, and further passaged seven times in suckling mouse brain and twice in BHK cells. The BHK cell line was grown at 37°C in Eagles MEM (Gibco, Gaithersburg, MD, USA) and supplemented with FCS.
Four- to 5-week-old female C57Bl/6J mice (Charles River Laboratories Japan, Yokohama, Japan) were used throughout the experiment. Water and food pellets were supplied ad libitum. The animals were handled under Biosafety Level 3 containment conditions. All experiments were approved following review by the Animal Care and Use Committee and President of Hokkaido University.
For cross-neutralization tests, mice were inoculated subcutaneously with 1000 FFU of either TBEV or OHFV. Sera were collected at the onset of clinical signs, which occurred around Days 8 and 9 for OHFV and TBEV, respectively.
Virus neutralization assay
An equal volume of serially diluted serum was mixed with stock virus and incubated at 37°C for 1 hr. Monolayers of BHK cells grown in 12-well plates were then inoculated with the mixtures (100 µL/well) and incubated at 37°C for 1 hr. After the viral inocula had been removed, the cells were rinsed once with 2% FCS–MEM. The cells were then layered with 1 mL MEM containing 1.5% carboxyl methylcellulose and 2% FCS. These monolayers were next incubated in a CO2 incubator at 37°C for 4 days for TBEV and 5 days for OHFV. Following incubation, the mixed carboxyl methylcellulose-MEM medium was removed and the cells fixed and stained with formalin-crystal violet. Neutralizing antibody titers were determined as the reciprocal of the highest serum dilution that reduced the virus counts by at least 50%. Results were considered to be positive at a titer ≥20.
Tick-borne encephalitis vaccines Encepur Adult (Chiron Behring GmbH) and FSME-IMMUN inject (Baxter AG) were used in human studies. Only FSME-IMMUN inject was used in the mouse experiments.
Human volunteers received a total of either two or three i.m. injections on Days 0 and 28 or on Days 0 and 28 followed by a booster after 12 months. Sera were collected 4 weeks after the second or third vaccination.
Mice were immunized subcutaneously on Days 0 and 10 with 0.2 mL of a fourfold dilution of the TBE vaccine containing 0.2% Al (OH)3. Mice in the control group were immunized with an equal volume of 0.2% Al (OH)3 in PBS. Blood samples were collected 10 days after the second vaccination. Sera were heat-inactivated at 56°C for 30 min.
Vaccination and virus challenge
For vaccine protection, mice were immunized with the TBE vaccine or PBS as described above. Groups of 10 mice were challenged with 100 LD50 of TBEV (68 FFU/mouse) or 100 LD50 OHFV (209 FFU/mouse) 10 days after the second vaccination. The mice were observed for 24 days after the virus challenge.
Statistical analyses were performed using Student's t-test (Microsoft Office Excel). A value of P ≤ 0.05 was considered to indicate statistical significance.
Antigenic cross-reactivity between TBEV and OHFV
To determine if there are antigenic differences between TBEV and OHFV, cross-neutralization tests were performed using sera from mice infected with the TBEV-Sofin and OHFV-Gruiev strains. Figure 1a,b shows neutralizing antibody titers of mouse sera. All animals had detectable antibodies to both TBEV and OHFV. Sera from those infected with TBEV had consistently high antibody titers to both TBEV and OHFV, with no significant difference in GMTs. However, there was a significant difference (P = 0.002) in antibody titers of sera from OHFV-infected mice; these had high titers of antibody to OHFV but only moderate titers to TBEV. High anti-OHFV antibody titers were observed in all OHFV infected mice.
Efficacy of TBE vaccine against OHFV infection in mice
To determine if the commercial TBE vaccine could protect mice against OHFV infection, mice were vaccinated s.c. with FSME-IMMUN and re-vaccinated 10 days later. Seventy-one percent of vaccinated mice had detectable titers of TBEV antibodies and 57% had antibodies to OHFV (Table 1). There was no significant difference in the GMTs of neutralizing antibody to either of the two viruses. To further evaluate the efficacy of the commercial TBE vaccine in protecting against OHFV, vaccinated mice were challenged with a lethal dose (100 LD50) of OHFV. Their survival rates are shown in Figure 2: the TBE vaccine provided 100% protection against lethal doses of OHFV in these animals. In addition, the vaccinated mice developed no clinical signs during the observation period. Control mice, which received an equal dosage of PBS) had a 100% mortality rate. Therefore, the TBE vaccine induced a protective immune response against OHFV infection.
|Neutralizing antibodies||Seroconversion rate (%)||Geometric mean titer†|
Efficacy of the TBE vaccine against OHFV infection in humans
To evaluate the efficacy of the commercial TBE vaccines against human OHFV infection, antibody responses to the vaccines were assessed. The resulting neutralizing antibody titers against TBEV and OHFV are shown in Table 2. Seroconversion rates to TBEV and OHFV after the second vaccination were 87% and 79%, respectively, with no significant differences in neutralizing antibody GMTs. After the third vaccination, seroconversion rates increased to 100% for TBEV and 86% for OHFV. Statistical analysis showed no significant difference between the two viruses in GMTs. Therefore the commercial TBE vaccine is able to induce efficient antibody responses against OHFV in humans.
|Number of vaccinationsa||Neutralizing antibodies||Seroconversion rate (%)b||Geometric mean titerc|
The antigenic cross-reactivity of TBEV and OHFV was investigated and assessed to determine the efficacy of the European-subtype-based commercial TBE vaccine. Neutralizing antibodies from mice infected with the virus showed that TBEV and OHFV are cross-protective. However, significant differences in antibody titers were found in these animals. Sera from TBEV-infected mice had equally high titers of neutralizing antibodies against both TBEV and OHFV. In contrast, sera from animals infected with OHFV had high neutralizing antibodies against OHFV, but only moderate neutralizing antibodies against TBEV. This difference in neutralizing activity may be due to differences in amino acid residues in the E protein that could have an effect on epitope accessibility and tertiary structure [1, 4, 37, 39-41]. Domain III of the E protein differs from other TBE complex viruses at 18 distinct residues . Domain II has one amino acid residue that is specific to the three hemorrhagic viruses but is absent in TBEV . It is thought that these amino acid variations play a role in protein conformation and antigenic variation [1, 4]. Other factors, such as differences in virus cell tropism and induction of immune responses, may contribute to the difference in cross-neutralization activity. Studies in mice have demonstrated that TBEV primarily targets the brain whereas OHFV targets visceral organs . The NS1 protein of flaviviruses interacts with various components of the host immune system; some of these interactions are thought to be virus specific . Furthermore, the NS 1 protein may also contribute to the pathogenesis of hemorrhagic fever flaviviruses [44, 45].
In the vaccine efficacy study, 57% of vaccinated mice had neutralizing antibodies against OHFV. No difference was found in the GMTs of anti-OHFV and anti-TBEV neutralizing antibodies in vaccinated animals. These findings correspond with those concerning cross neutralization that were seen in TBEV-infected mice. Efficient protection was observed in vaccinated animals challenged with a lethal dose of OHFV. The mice that survived the viral challenge had significantly lower titers of neutralizing antibodies against OHFV than did unvaccinated OHFV-infected mice (data not shown). This suggests that viral replication is inhibited in vaccinated mice. Although the vaccine resulted in moderate seroconversion rates, it still provided complete protection against a lethal dose of OHFV. This may be due to protection by non-neutralizing antibodies. Prior studies have demonstrated that poorly neutralizing or non-neutralizing antibodies are able to protect against flavivirus infection via Fc-receptor mechanisms and complement-mediated cell lysis .
The human sera of vaccinated subjects were collected from 1995 to 2013. During this period, Encepur Adult or FSME-IMMUN inject vaccines were used depending on vaccine availability. Both vaccines are produced from closely related European subtypes of TBEV and have been shown to be highly immunogenic with comparable efficacy [21, 47, 48]. Neutralization tests performed on human sera showed that complete vaccination (vaccine administered on Days 0, 28 and a booster after 12 months) induced production of anti-TBEV and anti-OHFV neutralizing antibodies in 100% and 86% of subjects, respectively. These results suggest that high titers of neutralizing antibodies against OHFV are achieved after complete vaccination. Furthermore, there was no significant difference in the GMTs of anti-TBEV and anti-OHFV neutralizing antibodies, similar to the neutralization test results in the vaccinated mice and unvaccinated TBEV-infected mice. The seroconversion rate for TBEV following complete vaccination is similar to that reported previously [26, 49]. In a study using West Nile virus chimera containing OHFV prME proteins, there was 98% seroconversion, suggesting that the TBE vaccine can protect against OHFV infection . Our study supports these findings, although the seroconversion rate against OHFV is lower than that against West Nile virus chimera. Several studies have reported on the “original antigenic sin phenomenon,” where pre-existing immunity to one or more flaviviruses can affect the immune response following vaccination [51-54]. The majority of the TBE vaccinated human subjects had previously been immunized against Japanese encephalitis. However, the findings indicate that the TBE vaccine is effective despite any prior immunization to Japanese encephalitis virus.
In 1991, the Russian government authorized the use of TBE vaccine as a preventative measure during an OHFV outbreak, despite lack of conclusive evidence on this vaccine's cross-protective potential . The present findings show that TBE vaccine can provide effective protection against OHFV infection. Moreover, it is likely that recovered TBEV patients also have cross-protective antibodies against OHFV.
In summary, complete vaccination with the TBE vaccine based on the European subtype of the virus provides effective protection against OHFV infection and therefore has potential for the prevention of Omsk hemorrhagic fever.
This work was supported by the Grants-in-Aid for Scientific Research (22780268 and 21405035) and the Global COE Program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and Health Sciences Grants for Research on Emerging and Re-emerging Infectious Disease from the Ministry of Health, Labour and Welfare of Japan.
The authors have no commercial or other associations that might pose a conflict of interest.