WO2009030676A2

WO2009030676A2 - Pharmaceutical composition comprising a japanese encephalitis virus for vaccination of patients of at least 40 years of age

Info

Publication number: WO2009030676A2
Application number: PCT/EP2008/061527
Authority: WO
Inventors: Erich Tauber; Shailesh Dewasthaly; Christoph Klade; Astrid KALTENBÖCK; Elisabeth Schuller
Original assignee: Intercell Ag
Priority date: 2007-09-03
Filing date: 2008-09-02
Publication date: 2009-03-12
Also published as: WO2009030676A3

Abstract

The present invention relates to a pharmaceutical composition comprising a cell culture derived, inactivated JEV for use in the treatment or prevention of an infection with JEV in a human subject with at least 40 years of age.

Description

Pharmaceutical composition comprising a Japanese Encephalitis Virus for administration to a specific patient population

Japanese encephalitis (JE), a disease affecting the central nervous system, is caused by the Japanese encephalitis virus (JEV) transmitted by mosquitos. It is endemic in South East Asia. In the last three decades its geographical range has extended to previously unaffected parts of Asia-Pacific region and northern Australia. Thirty-five percent of the 50,000 individuals with JE die annually from their disease and 50% of survivors are left with permanent neurologic or psychiatric sequelae. JE will continue to be a significant public health problem to both persons who live in endemic countries, as well as civilian travelers and military personnel who travel or are deployed to endemic countries.

JEV belongs to the family Flaviviridae, genus Flavivirus. Flaviviruses were classified serologically into several antigenic complexes, such as the Japanese encephalitis (JE) serocomplex or the tick- borne encephalitis (TBE) serocomplex. Amino acid sequence homologies between viruses in different serocomplexes range from 40-53%, with significantly higher homologies ranging from -62-80% to as high as 96-98% within the serocomplexes.

The mature flavivirus particle is composed of three structural proteins: the capsid protein C; the membrane protein prM/M and the envelope protein E. Multiple copies of the capsid protein C enclose the viral RNA and form the nucleocapsid, which is further surrounded by a lipid bilayer derived from the host cell. The membrane protein prM/M and the envelope protein E are anchored inside this lipid bilayer by their C-terminal domains and form an outside layer. The envelope glycoprotein E is responsible for the attachment of the virus to the host cells and thus is associated with infectivity.

The beneficial role of humoral immunity against JEV infection has been well characterized and is used to measure the efficacy of JEV immunization. Primary JEV infection triggers a rapid and potent IgM response in serum and cerebrospinal fluid. The failure to mount an IgM response is associated with a fatal outcome. In surviving JE patients a class- switching to IgG occurs. Antibodies against JEV are thought to protect the host by restricting viral replication before the virus crosses the blood-brain barrier. They may also limit damage during established encephalitis by neutralizing extracellular virus and facilitating lysis of infected cells by antibody-dependent cellular cytotoxicity. The E protein is the primary target for virus-specific neutralizing antibodies and seems to be responsible for the induction of a protective immune response. Antibodies against prM and NSl have also been reported to be capable of inducing protective immunity [I]. It is generally accepted that a neutralizing antibody titer of 1 :10 or greater is protective. The most accepted assay to demonstrate functional antibodies able to inactivate virus is the plaque-reduction neutralization test (PRNT).

As there are no effective antiviral drugs available to treat JEV infections, vaccination to prevent disease remains the most effective measure. Three kinds of JEV vaccines are currently in use. The first JEV vaccine was developed in 1954. This formalin- inactivated whole virus vaccine was based on the virulent Nakayama strain and prepared in infant mouse brains [2, 3]. The purity of such a vaccine was low, so that it might induce an allergic neurological disorder in the central nervous system. Thereafter, an improved high-purity vaccine was put into practical use in 1965. Currently commercially available vaccine JE-V AX^® is produced by the Research Foundation for Microbial Diseases of Osaka University (BIKEN^®) in Japan and is purified, whole virus mouse brain-derived vaccine formulated with stabilizers and Thimerosal. Since the middle of the 1960s this vaccine has been widely used and dramatically reduced the burden of the disease. So far it is the only vaccine against JEV licensed in the U.S., Canada and Australia, but not in Europe.

The protective efficacy of the JE -VAX^® is only achieved if three doses are administered followed by a booster after 12 to 18 months. It is commonly recommended that a booster should be administered after 2 or 3 years for persons who remain at risk for JEV [4]. Although effective, the use of JE -VAX^® has been troubled by safety concerns. Serious side effects, such as anaphylaxis occurring typically 1-3 days (up to 17 days) after vaccination, have been noted with an incidence of about 15-62 per 10,000 among U.S. citizens. While the exact cause of these reactions is unknown, most experts blame the porcine gelatin stabilizers included in the JE-VAX^® formulation to be responsible for these severe side effects. In addition, the neural tissue content of the vaccine has raised concerns about safety and the possibility of vaccine-related neurological side effects. The problems of strain variation and the protection offered by the vaccine based on Nakayama strain have been noted as well.

Other two JEV vaccines are currently in use in China. The first one is prepared by formalin- inactivating the cell culture supernatant obtained from primary hamster kidney cells infected with the P3 strain of JEV. The second one, which gradually replaces the inactivated vaccine since 1988, is a live-attenuated vaccine based on the strain SAi 4- 14-2. It is produced by serial cell culture passage of a virus in hamster kidney-derived cells. The immunization schedule consists of two doses at 1 and 2 years of age, with an efficacy of greater than 95% after the first dose. The efficacy and safety of the live-attenuated vaccine has been proven in large-scale human studies [5]. Recently, this vaccine has been licensed for use in South Korea [2, 4]. However, as a live attenuated vaccine contains agents capable of causing infection, concerns about reversion to virulence are always an issue.

A new JEV vaccine IC51 is developed by Intercell AG (Vienna, Austria) and manufactured by Intercell Biomedical Ltd (Livingston, U.K.). The Intercell vaccine is a purified inactivated vaccine, prepared in a cell culture substrate in lieu of mouse brain tissue. It is based on the attenuated JEV strain SA₁₄-14-2, adapted to growth in Vera cells. Pre-clinical and human phase I, II and III clinical studies have shown that IC51 induces higher SCR, is safer, and more convenient to administer than the licensed vaccine JE -V AX^®.

Another flavivirus-related disease of the central nervous system is tick-borne encephalitis (TBE). TBE is caused by tick-borne encephalitis virus (TBEV) which, like JEV, is a member of the family Flaviviridae, genus Flavivirus. TBE is a large public health problem in many parts of Europe, Siberia, and the Far East of Russia. Annually approximately 10,000 cases of TBE are reported worldwide. Central European TBE appears to be less severe (the case-fatality rate is 1-2%) compared to Russian spring-summer encephalitis (case- fatality rate -20%). Neurological sequelae occur in 30-60% of survivors. Tick bites are the primary and most effective way to transmit TBEV. Another route of transmission is via ingestion of raw milk.

Active immunization appears to be the most effective means of preventing TBE. In Europe, two TBE vaccines are currently available. A purified formalin- inactivated vaccine, based on an Austrian Central European encephalitis virus isolate (strain Neudoerfl), became commercially available in 1976 (FSME-IMMUN^®; Baxter BioScience, formerly ImmunoAG, Orth/Donau) [2]. The virus is grown on chick embryo cells, purified, formalin-inactivated and adsorbed onto aluminum hydroxide A1(OH)3 as adjuvant. The second European tick-borne encephalitis vaccine, Encepur^® (Novartis Vaccines, Germany), was first licensed in Germany in 1991. It is similar to the Austrian vaccine, but based on the virus strain K23 [2]. Both inactivated vaccines provide safe and reliable protection. The antigenic components of the two available vaccines are highly homologous, with only a few base exchanges within the genes encoding the E protein, and can be assumed to elicit the same immune response. Cross-reactivity against various European and Asian isolates has been documented for both vaccines [6]. The TBE vaccines are administered in at least two doses for primary immunization, followed by a booster at 1 year. The protection rate is 96-99% after completing the series of three doses [7]. Until recently, subsequent periodic boosters every 3 years were recommended for people living in, or traveling to, TBE-endemic countries. However, the results of recently published serologic follow-up studies led to the recommendation that later regular boosters should be administered every 5 years for individuals younger than 60 years of age [8]. Prophylactic TBE vaccines have been used for more than 30 years; they have proven to be the most effective means of prevention of TBE. For instance, Austria had a very high recorded morbidity of TBE in the pre-vaccination era. In high risk areas, the average annual incidence in the population exposed to ticks in their working environment was 0.9/1000. A mass vaccination campaign was started in 1982. The vaccination coverage of the total Austrian population reached 86% in 2001 [7].

The flaviviruses JEV and TBEV possess a sequence identity of around 40% between their structural proteins. Therefore, a potential cross-reactivity between JEV and TBEV vaccines could occur. Such a heterologous immune response may affect the outcome of JE vaccination.

Thus, the problem underlying the present invention was to determine such potential interference and to provide safe and effective JEV vaccines for TBEV vaccinated subjects and to provide safe and effective vaccination schedules to administer a JEV vaccine to subjects which have previously been vaccinated with a TBEV vaccine. More particularly, the problem was to provide a pharmaceutical composition comprising a JEV for use in the treatment or prevention of an infection with JEV in a subject which has previously been vaccinated with a TBEV vaccine. Another problem was to provide a method for treating or preventing an infection with JEV, comprising the steps of administering a therapeutically effective amount of a TBEV vaccine to a subject, and subsequently administering a therapeutically effective amount of a pharmaceutical composition comprising a JEV to said subject.

A further general problem related to vaccinations is the waning immunity with age. The major phenomenon is the depressed T cell response as a part of the aging process. Immune responses to JEV vaccination is required by each individual, because a herd immunity effect does not prevent JE disease in an individual.

Thus, another problem underlying the present invention was to provide a JEV vaccine which is effective especially in the elderly subject group. More particularly, the problem was to provide a pharmaceutical composition comprising a cell culture derived, inactivated JEV for use in the treatment or prevention of an infection with JEV in a human subject with at least 40 years of age. Furthermore, the problem was to provide a method for treating or preventing an infection with JEV, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a cell culture derived, inactivated JEV to a human subject with at least 40 years of age.

In one aspect, the problem underlying the present invention is solved by a pharmaceutical composition comprising a cell culture derived, inactivated JEV for use in the treatment or prevention of an infection with JEV in a human subject with at least 40 years of age.

Whenever it is referred to age herein, the age of the human subject at the time of the first administration of the pharmaceutical composition according to the present invention is meant.

In an embodiment, the human subject is from 40 to 50 years of age.

In another embodiment, the human subject is at least 65 years of age.

In still another embodiment, the human subject is from 65 to 80 years of age.

In another embodiment of the pharmaceutical composition as described above, the cell culture derived, inactivated JEV comprises the JEV strain SAi 4- 14-2.

In yet another embodiment of said pharmaceutical composition, the cell culture derived, inactivated JEV is produced in Vera cells.

In still another embodiment of the pharmaceutical composition, the cell culture derived, inactivated JEV is formalin inactivated.

In a further embodiment, the cell culture derived, inactivated JEV is adsorbed to 0.1% aluminum hydroxide.

According to a preferred embodiment, the pharmaceutical composition is a liquid formulation.

In another aspect, the problem is solved by the use of a cell culture derived, inactivated JEV for the preparation of a pharmaceutical composition for the treatment or prevention of an infection with JEV, wherein the pharmaceutical composition is administered to a human subject with at least 40 years of age.

In a preferred embodiment of the use as described above, the pharmaceutical composition is administered to a human subject from 40 to 50 years of age.

In another preferred embodiment of said use, the pharmaceutical composition is administered to a human subject with at least 65 years of age.

In an especially preferred embodiment of the use as defined above, the pharmaceutical composition is administered to a human subject from 65 to 80 years of age.

In another embodiment of the use as described above, the cell culture derived, inactivated JEV comprises the JEV strain SAi ₄- 14-2.

In another embodiment of the use, the cell culture derived, inactivated JEV is produced in Vero cells.

In still another embodiment, the cell culture derived, inactivated JEV is formalin inactivated.

In yet another embodiment, the cell culture derived, inactivated JEV is adsorbed to 0.1% aluminum hydroxide.

According to a preferred embodiment of said use, the pharmaceutical composition is a liquid formulation.

A further aspect of the present invention relates to a method for treating or preventing an infection with JEV, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a cell culture derived, inactivated JEV to a human subject, wherein the human subject is at least 40 years of age.

In an embodiment of said method, the human subject is from 40 to 50 years of age.

In another embodiment of the method, the human subject is at least 65 years of age.

In still another embodiment of the method, the human subject is from 65 to 80 years of age.

In an embodiment of the method as described above, the cell culture derived, inactivated JEV comprises the JEV strain SAi ₄- 14-2.

In another embodiment, the cell culture derived, inactivated JEV is produced in Vero cells.

According to a preferred embodiment of the method, the pharmaceutical composition is a liquid formulation.

In an embodiment of the pharmaceutical composition as described above, or of the use as described above, or of the method as described above, the pharmaceutical composition comprising a cell culture derived, inactivated JEV is IC51. IC51 is a purified, formalin inactivated vaccine, containing the JEV strain SAi ₄- 14-2 and manufactured by Intercell Biomedical Ltd, Livingston, UK. This attenuated SAi 4- 14-2 vaccine strain was adapted to growth in Vero cells. The vaccine is prepared by using a purification and inactivation process consistent with current good manufacturing practices (cGMP). The finished product does not contain Thimerosal or gelatins, is adjuvanted with aluminum hydroxide and filled as a single dose in syringes. One dose of IC51 contains 6 μg of purified and inactivated virus adsorbed to 0.1% aluminum hydroxide in 0.5 mL of a liquid formulation.

Suitable JEV vaccines including the IC51 vaccine and their production are described in Still another aspect of the invention relates to a pharmaceutical composition comprising a JEV for the treatment or prevention of an infection with JEV in a subject which has previously been vaccinated with a TBEV vaccine.

In an embodiment of said pharmaceutical composition, the subject which has previously been vaccinated with a TBEV vaccine is a human subject younger than 50 years of age.

In another embodiment of said pharmaceutical composition, the subject which has previously been vaccinated with a TBEV vaccine is a human subject which is at least 18 years of age and younger than 50 years of age.

In still another embodiment, the pharmaceutical composition as defined above is administered once.

In an especially preferred embodiment of the pharmaceutical composition, said pharmaceutical composition is administered once to a human subject which has previously been vaccinated with a TBEV vaccine and which is younger than 50 years of age.

In another embodiment of the pharmaceutical composition, the JEV is a cell culture derived, inactivated JEV.

In still another embodiment of the pharmaceutical composition as described above, the JEV comprises the JEV strain SAi ₄- 14-2.

In yet another embodiment of said pharmaceutical composition, the JEV is produced in Vero cells.

In still another embodiment of the pharmaceutical composition, the JEV is formalin inactivated.

In a further embodiment, the JEV is adsorbed to 0.1% aluminum hydroxide.

A further aspect of the present invention relates to the use of a JEV for the preparation of a pharmaceutical composition for the treatment or prevention of an infection with JEV, wherein the pharmaceutical preparation is administered to a subject which has previously been vaccinated with a TBEV vaccine.

In an embodiment of said use, the subject which has previously been vaccinated with a TBEV vaccine is a human subject younger than 50 years of age.

In still another embodiment, the subject which has previously been vaccinated with a TBEV vaccine is a human subject which is at least 18 years of age and younger than 50 years of age.

In still another embodiment of said use, the pharmaceutical composition as defined above is administered once.

In an especially preferred embodiment of said use, the pharmaceutical composition according to the present invention is administered once to a human subject which has previously been vaccinated with a TBEV vaccine and which is younger than 50 years of age.

In an embodiment of the use as described above, the JEV is a cell culture derived, inactivated JEV.

In another embodiment of said use, the JEV comprises the JEV strain SAi ₄- 14-2.

In another embodiment of the use, the JEV is produced in Vero cells.

In yet another embodiment, the JEV is adsorbed to 0.1% aluminum hydroxide.

In still another aspect, the invention relates to a method for treating or preventing an infection with JEV, comprising the steps of administering at least once a therapeutically effective amount of a TBEV vaccine to a subject, and subsequently administering at least once a therapeutically effective amount of a pharmaceutical composition comprising a JEV to said subject. In an embodiment of said method, the therapeutically effective amount of a TBEV vaccine is administered three times, before a therapeutically effective amount of a pharmaceutical composition comprising a JEV is administered to said subject.

In an embodiment of the method, the subject is a human subject younger than 50 years of age at the time of administration of the first dose of a pharmaceutical composition comprising a JEV.

In another embodiment, the therapeutically effective amount of a pharmaceutical composition comprising a JEV is administered once.

In an especially preferred embodiment of said method, the therapeutically effective amount of the pharmaceutical composition according to the present invention is administered once to a human subject which has previously been vaccinated with a TBEV vaccine and which is younger than 50 years of age.

In an embodiment of the method as described above, the JEV is a cell culture derived, inactivated JEV.

According to a preferred embodiment of said method, the JEV comprises the JEV strain SAi4-14-2.

In another embodiment, the JEV is produced in Vera cells.

In still another embodiment, the JEV is formalin inactivated.

In yet another embodiment of the method, the JEV is adsorbed to 0.1% aluminum hydroxide.

In an embodiment of the pharmaceutical composition as described above, or of the use as described above, or of the method as described above, the pharmaceutical composition comprising a cell culture derived, inactivated JEV is IC51, which has been further described above. According to another embodiment of the pharmaceutical composition as described above, or of the use as described above, or of the method as described above, the interval between the first TBEV vaccination and the first administration of the pharmaceutical composition according to the invention is at most 50 years, preferably at most 45, 40, 35, 30, 25, 20, 15, 10, or 5 years. The first TBEV vaccination is the first time of an administration of a TBEV vaccine to the subject.

According to another embodiment of the pharmaceutical composition as described above, or of the use as described above, or of the method as described above, the interval between the latest TBEV vaccination and the first administration of the pharmaceutical composition according to the invention is at most 10 years, preferably at most 7, 5, 4, 3, 2, or 1 year. The latest TBEV vaccination is the last time of an administration of a TBEV vaccine to the subject and may be the first, second or third time of an administration of TBEV vaccine, or any booster vaccination, e.g. as described below.

In an embodiment, the subject which has previously been vaccinated with a TBEV vaccine has been vaccinated with a commercially available TBEV vaccine according to the prescription of said vaccine. The TBEV vaccine may be e.g. FSME-Immun^® (Baxter BioScience, formerly ImmunoAG, Orth/Donau) or Encepur^® (Novartis Vaccines, Germany). The TBEV vaccine may have been administered at least once, preferably at least twice, more preferably at least three times before the pharmaceutical composition according to the present invention is being administered. Preferred intervals between the first and second administration of the TBEV vaccine are 7 days to 1 year, more preferably 2 to 4 weeks. Preferred intervals between the second and third vaccination are 2 weeks to 10 years, more preferably 9 to 12 months. Preferably, at least one booster dose of the TBEV vaccine may have been administered 3 to 10 years after the first TBEV vaccination, preferably 3 to 5 years after the first TBEV vaccination. For example, the TBEV vaccine may have been administered on days 0, 7 and 21, and a booster dose may have been administered 12 to 18 months after the first vaccination with a TBEV vaccine. In another example, the TBEV vaccine may have been administered on days 0 and 14, and a booster dose may have been administered 3 years after the first vaccination with a TBEV vaccine.

In an embodiment of the present invention, the subject which has previously been vaccinated with a TBEV vaccine has established an immunity against TBEV, preferably a long-lasting immunity, before a pharmaceutical composition according to the present invention is administered to said subject. Such pre-existing TBEV immunity may be determined e.g. with a TBEV-ELISA test as described herein. For example, the ELISA test as described in Holzmann et al. [33] may be used to identify baseline (pre-IC51 vaccination) anti-flavi virus immune status by determining the presence of pre-existing anti-flavi virus antibodies (mainly due to TBE vaccination, but potentially also due to Yellow fever vaccination or exposure to Dengue virus or West Nile virus). Said ELISA does not discriminate between neutralizing and non-neutralizing antibodies and cross-reactive anti-flavi virus antibodies. A subject may be considered to be anti-TBEV immune positive at baseline, if the concentration of anti-flavivirus antibodies in serum is > 155 Vienna Units (VIEU) [33]. Accordingly, sera with an anti-flavivirus antibody concentration <155 VIEU may be considered negative. The term "at baseline" as used herein refers to the time before the first administration of a pharmaceutical composition according to the present invention to the subject.

In the context of the present invention, the seroconversion rate (SCR) among the subject population may be determined as a measure for the efficacy of the administration and/or the administration schedule of the pharmaceutical compositions according to the invention. The term "seroconversion" as used herein means the induction of a neutralizing immune response in a subject. For example, JEV specific neutralizing antibodies may be measured by the Plaque Reduction Neutralization Test (PRNT). Seroconversion may be defined as PRNT50 titer > 1 :10.

The administration and/or the administration schedule of the pharmaceutical compositions according to the present invention may also be considered effective, if the titer of JEV specific antibodies in a sample obtained from a subject after the administration of the pharmaceutical composition is at least four times the titer determined in the same subject before the administration of the pharmaceutical composition. If no antibodies could be detected in the sample obtained from the subject before administration, absolute antibody titers of at least superior or equal to 1 :10, preferably 1 :20, more preferably 1 :30, even more preferably 1 :40 may be considered positive for the presence of neutralizing antibodies, i.e. being indicative for an efficient immunization or treatment. The antibody titer may be expressed as reciprocal of dilution, i.e. the greatest dilution ratio that still gives a positive result as measured e.g. in an ELISA assay.

The neutralizing antibody titers of the patient population, for example determined by PRNT, may be expressed as geometric mean titer (GMT) value(s). Alternatively or in addition, the titer of JEV specific antibodies may be compared to the average concentration of JEV specific antibodies in a group of JEV naϊve subjects. JEV naϊve subjects are subjects with no documented and/or certified vaccination against JEV for at least 10 years. Alternatively or in addition, the ratio of the JEV specific antibodies to the general immunoglobulin content in the sample may be determined.

The sample obtained from the subject is preferably a serum sample.

The pharmaceutical compositions according to the present invention may further comprise at least one pharmaceutically acceptable carrier and/or excipient.

The pharmaceutically acceptable carriers and/or excipients useful for the pharmaceutical compositions according to the present invention are conventional and may include buffers, stabilizers, diluents, preservatives, and solubilizers. Compositions and formulations suitable for pharmaceutical delivery are well known in the art. In general, the nature of the carrier or excipients will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e. g. powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

The pharmaceutical compositions according to the present invention may be administered parenterally, including, for example, administration that is subcutaneous, intramuscular, intravenous, intradermal, intranasal or transdermal. Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain antioxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the body fluid, preferably the blood, of the individual; and aqueous and non-aqueous sterile suspensions which may include suspending agents or thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried condition requiring only the addition of the sterile liquid carrier immediately prior to use.

In a preferred embodiment, the pharmaceutical composition is a vaccine composition. Preferably, such vaccine composition is conveniently in injectable form. Conventional adjuvants may be employed to enhance the immune response.

In a preferred embodiment, the adjuvant is aluminum hydroxide, also called alum.

In another embodiment, the at least one immunostimulatory substance is selected from the group consisting of polycationic polymers, especially polycationic peptides, preferably polyarginine, more preferably peptides containing at least two LysLeuLys motifs, especially KLKLLLLLKLK. Such substances are described e.g. in WO 97/030721 [10], WO 99/038528 [11], WO 01/093903 [12], WO 02/032451 [13], and in Ganz, T., 1999 [14].

In another embodiment, the at least one immunostimulatory substance is selected from the group consisting of immunostimulatory oligo-deoxynucleotides (ODNs), especially Oligo(dIdC)13, neuroactive compounds, especially human growth hormone, alum, Freund's complete or incomplete adjuvants, or combinations thereof. Such substances are described e.g. in WO 96/002555 [15], WO 01/054720 [16], WO 01/093903 [12], WO 01/093905 [17], WO 02/095027 [18], and WO 03/047602 [19].

It is also within the present invention that any of the aforementioned polycationic compounds is combined with any of the immunostimulatory nucleic acids as aforementioned. Preferably, such combinations are according to the ones as described in WO 01/054720 [16], WO 01/093903 [12], WO 01/093905 [17], WO 02/013857 [20], WO 02/032451 [13], WO 02/095027 [18], and WO 03/047602 [19].

A suitable unit dose for vaccination with the pharmaceutical composition according to the present invention is e.g. from 0.06 μg to 0.1 μg of purified JEV per kg of body weight. A preferred dose is 6 μg of purified JEV per unit dose, especially for adults. A more preferred dose, especially for children, is 3 μg of purified JEV per unit dose. In an especially preferred embodiment, the purified virus is adsorbed to 0.1% aluminum hydroxide.

Such dose is preferably administered one, two or three times. If the vaccine is administered three times, it may be administered e.g. on days 0, 7-14 and 28-30. If the vaccine is administered twice, it may preferably be administered within an interval of 20 to 35 days, more preferably within an interval of 25 to 30 days, most preferably within an interval of 28 to 30 days. In an especially preferred embodiment, the dose is administered on days 0 and 28. Optionally, at least one booster dose may be administered from 6 months to 10 years after administration of the first dose, e.g. 6 months, 1 year, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years after administration of the first dose. The booster vaccine may be different from or advantageously identical to the vaccine previously administered.

Treatment in the context of the present invention refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) the targeted pathologic condition or disorder. Those in need of treatment include those already with the disorder as well as those prone to have the disorder or those in whom the disorder is to be prevented.

The terms "vaccinated" or "vaccination" as used herein comprises the administration of an antigenic pharmaceutical composition to a subject to induce a protective immune response.

The subject as described herein may be a human or any animal. In a preferred embodiment, the subject is a human. In another preferred embodiment, the subject is an animal, more preferably a swine, a bird, or a hamster.

A "therapeutically effective amount" of a pharmaceutical composition may be calculated as that amount capable of exhibiting an in vivo effect, e.g. preventing or ameliorating a sign or symptom of infection with e.g. JEV or TBEV. Such amounts may be determined by one of skill in the art.

The present invention is further illustrated by the following Examples, Tables and Figures, from which further features, embodiments and advantages may be taken. It is to be understood that the present examples are given by way of illustration only and not by way of limitation of the disclosure.

Figure 1 shows the seroconversion rates (SCR) stratified by age group on day 56 (28 days after the 2^nd vaccination with IC51 and 28 days after the 3^rd vaccination with JE -V AX^®).

Figure 2 shows the geometric mean titers (GMT) stratified by age group on day 56 (28 days after the 2^nd vaccination with IC51 and 28 days after the 3^rd vaccination with JE -VAX^®). Table 1 shows the seroconversion rates (SCR) 28 days after each IC51 vaccination, stratified by anti-Tick Borne Encephalitis (TBE) immune status, in the ITT Population.

Table 2 shows the geometric mean titers (GMT) 28 days after each IC51 vaccination, stratified by anti-Tick Borne Encephalitis (TBE) immune status, in the ITT Population.

Table 3 shows the seroconversion rate (SCR) 28 days after each IC51 vaccination, stratified by anti- Tick Borne Encephalitis (TBE) immune status and age group, in the ITT population.

Table 4 shows the geometric mean titer (GMT) 28 days after each IC51 vaccination, stratified by anti- Tick Borne Encephalitis (TBE) immune status and age group, in the ITT population.

Table 5 shows the sensitivity analysis in the worst case: Seroconversion rate (SCR) 28 days after each IC51 vaccination, stratified by anti- Tick Borne Encephalitis (TBE) immune status and age group, in the ITT population.

Table 6 shows the sensitivity analysis in the best case: Seroconversion rate (SCR) 28 days after each IC51 vaccination, stratified by anti- Tick Borne Encephalitis (TBE) immune status and age group, in the ITT population.

Table 7 shows the sensitivity analysis in the worst case: Geometric mean titer (GMT) 28 days after each IC51 vaccination, stratified by anti- Tick Borne Encephalitis (TBE) immune status and age group, in the ITT population.

Table 8 shows the sensitivity analysis in the best case: Geometric mean titer (GMT) 28 days after each IC51 vaccination, stratified by anti- Tick Borne Encephalitis (TBE) immune status and age group, in the ITT population.

Table 9 shows the immunogenicity results stratified by previous TBE vaccination in the ITT population. EXAMPLES Example 1

1. Methods

Subjects: The study population consisted of healthy male and female volunteers, aged at least 18 years. A total of 867 subjects were randomized at 11 sites between September 5, 2005 and March 17, 2006. Of this total, 664 subjects were recruited at 8 sites in the United States and 203 subjects were recruited at 3 sites in Austria and Germany.

Vaccines:

IC51 is a purified inactivated vaccine, containing the JE virus strain SAi 4- 14-2 and manufactured by Intercell Biomedical Ltd, Livingston, UK. This attenuated SA_H-14-2 vaccine strain was adapted to growth in Vera cells. The vaccine is prepared by using a purification and inactivation process consistent with current good manufacturing practices (cGMP). The finished product does not contain Thimerosal or gelatins, is adjuvanted with aluminum hydroxide and filled as a single dose in syringes. One dose of IC51 contains 6 μg of purified and inactivated virus adsorbed to 0.1% aluminum hydroxide in a liquid formulation. The vaccine is being administered by i.m. injection into the deltoid muscle on days 0 and 28.

JE -VAX^® is a mouse-brain derived vaccine manufactured in Japan [21].

Immunogenicity: JEV specific neutralizing antibodies as measured by the Plaque Reduction Neutralization Test (PRNT) provide a reasonable immune correlate of protection [22]. Seroconversion is commonly defined as PRNT50 titer > 1 :10, a cut-off established by animal experiments [23] and correlation with protective efficacy demonstrated in the Phase 3 study in Thailand that supported licensure of JE-VAX^® in the US [24].

Serum samples were collected from all subjects prior to any vaccination on day 0, day 28 and day 56. Samples were stored at -8O⁰C and shipped on dry-ice. All analyses were done under GLP using a validated PRNT [25] in a central lab at Intercell AG, Vienna, Austria. Briefly, serial dilutions of test serum (1 :10; 1 :40; 1 :160; 1 :640 or higher when needed) were incubated for one hour at 35°C with a defined number of JEV plaque forming units (400 pfu/mL) and plated in triplicate onto a monolayer of Vera cells with a methylcellulose overlay to restrict virus spread. After 5 days of incubation (35°C, 5% CO₂, saturated H₂O), the viral plaques were fixed, stained with crystal violet, and automatically counted (ProtoCOL HR Colony Counter, Synbiosis, Cambridge, UK).

PRNT50 titers (the serum dilution giving a 50% plaque reduction compared to plaque formation in virus-only controls) were calculated using a linear regression (probit) analysis program. In a dose- response analysis, the probit model, transforms a sigmoid-shaped observed response proportion into a linear shaped response variable (probit).

Study design: Anti-JEV antibody titers of subjects after administration of IC51 have been compared to anti-JEV-antibody titers of subjects after administration of JE -V AX^® in a multicenter, observer blinded, randomized, controlled phase 3 clinical study. Randomization was stratified by study center and age. The vaccines were prepared and injected by an unblinded staff member at the site, whereas the study subjects and all other investigators and staff members remained blinded throughout the trial.

The primary aim of the study was to investigate the immunogenicity of IC51 (0.5 mL given i.m. on days 0 and 28) compared to JE -VAX^® (1.0 mL given s.c. on days 0, 7 and 28) in terms of seroconversion rates (SCRs) and geometric mean titers (GMTs) of JEV neutralizing antibody at 4 weeks after the last vaccine dose (day 56). The secondary aim was to evaluate and compare the safety of the two study vaccines.

2. Results

Primary Endpoint: On day 56 (28 days after the 2^nd vaccination with IC51 and 28 days after the 3^rd vaccination with JE-V AX^®), 96% of the subjects who received the Intercell JEV vaccine seroconverted versus 94% for JE-VAX^®. GMTs were 244 for IC51 versus 102 for JE-VAX^®, a GMT ratio of 2.3.

Age aspects: Interestingly, IC51 exhibited an especially appealing immunogenicity profile in the old and elderly population: GMT in the group older than 65 years of age, IC51 had a Seroconversion Rate of 96%, compared with 90% in the JE-VAX (Figure 1). Moreover, the GMT was 260, compared with 100 (Figure 2). In general, IC51 had equally appealing immunogenicity results in the elderly population, whereas in other vaccines, immune responses are generally lower in the older population. Example 2

1. Methods

Subjects: The study population consisted of healthy male and female subjects, aged at least 18 years. The Intent-to-Treat (ITT) population comprised of 430 subjects who received the JEV vaccine IC51. The population with available baseline anti-TBE immune status consisted of 420 IC51 vaccinees. The reasons for exclusion from randomization were inclusion/exclusion criteria not met, informed consent not signed and other reasons. Exclusion criteria comprised previous flavivirus infection, previous JE or Yellow Fever vaccine immunization, use of any investigational or non-registered drug or vaccine other than the study vaccine during the study period or within 30 days preceding the first dose of study vaccine, administration of immunosuppressants or other immune-modifying drugs within six months of vaccination, administration of another vaccine during the study, and prior history of severe hypersensitivity reactions. A written informed consent obtained from the subject; female subject had to be of non-childbearing potential, or, if of childbearing potential, a negative urine pregnancy test prior to each vaccination and avoidance of pregnancy until 30 days after the last dose of vaccine. The study protocol was approved by Independent Review Boards and the FDA in U.S., and by the Ethics Committees and the national health authorities in Austria and in Germany.

Vaccine: IC51 and JE -VAX^® have been administered as described above in Example 1.

Immunogenicity: JEV specific neutralizing antibodies were measured by the Plaque Reduction Neutralization Test (PRNT) as described above in Example 1.

Study design: The treatment with IC51 was part of a multicenter, observer blinded, controlled, randomized phase 3 study, carried out in the US, Germany and Austria. After a two-week screening period, during which inclusion and exclusion criteria were checked, subjects were randomized in equal proportions stratified by age (< 50 versus >50 years) to receive either: two injections of IC51 (6 μg in 0.5 mL) intramuscularly (i.m.) on days 0 and 28 and one 0.5 mL injection with placebo (phosphate-buffered saline [PBS] solution containing 0.1% aluminum hydroxide as an adjuvant) on day 7 or three injections of JE -V AX^® (1.0 mL dose) subcutaneously (s.c.) on days 0, 7 and 28. A final evaluation took place four weeks after last vaccination on day 56 or in the event of early termination. The primary objective was to demonstrate the non-inferiority of IC51 (Japanese encephalitis purified inactive vaccine; 2 x 6 μg) compared to JE -VAX^® (Japanese encephalitis vaccine; 3 x 1.0 mL) in terms of the seroconversion rate (SCR) and geometric mean titer (GMT) at day 56; four weeks after the last vaccination. Secondary objectives included the comparison of the SCR and GMT between baseline anti-TBE immune positive and negative subjects receiving IC51. To assess the immunogenicity of IC51 in subjects with pre-existing TBE immunity, anti flavivirus immune status was assessed at baseline with a TBEV-ELISA test in the ITT population. All subjects were immunologically naϊve to JEV. JEV specific immunity after one and two IC51 vaccinations was analyzed with the Plaque Reduction Neutralization Test (PRNT50).

Analyses: All tests were performed as two-tailed tests with a significance level of α = 0.05. Missing PRNT50 values in the ITT population originating from missing blood samples were imputed according to the last-observation-carried- forward approach. Missing values of other kinds were not replaced. All analyses and summaries were based on a by- visit basis. No reassignment of dates was done. Early termination visits were not assigned to any visit windows. Sensitivity analyses were performed by imputation of early termination results for missing values once per subject, further missings were replaced by not seroconverted for worst case and by seroconverted for best case. For SCR and GMT analyses stratified by age and anti-TBE immune status, sensitivity analyses were performed. Early termination results were imputed for missing values once per subject, further missings were replaced by not seroconverted for worst case and by seroconverted for best case. Sensitivity analyses were performed by imputation of early termination results for missing values once per subject, further missings were replaced by a PRNT50 of 5 for worst case and by the geometric mean at the respective visit for best case.

2. Results:

Demographics: When stratified by continent, the proportion of Caucasian subjects was higher in Europe than in the US: 97/99 (98.0%) vs. 241/330 (73%), respectively and the proportion of Black or African Americans was higher in North America (17% vs. 2%). The proportion of male and female subjects was similar. The median age was higher for North American subjects: 43 years vs. 33 years for European subjects. Median weight (82 kg), and BMI (29 kg/m²) were higher for subjects in the US than European subjects (73 kg and 27 kg/m², respectively).

The average age in the entire IC51 population was 42 years (SD 14.5), the average weight 80 kg (SD 20.8 kg) and height 170 cm (SD 10).

Seroconversion rates and geometric mean titers against JEV after one and two vaccinations:

81 of 420 persons (19.1 %) who received primary immunization with IC51 were anti-TBE positive at baseline as assessed by a TBEV-ELISA test. After the first IC51 vaccination, the SCR was significantly higher in subjects with positive baseline anti-TBE immune status than in those with negative immune status (76.5% vs. 49.0%, p<0.0001). After two IC51 vaccinations there was no significant difference in SCR between baseline anti-TBE positive and negative persons (Table 1).

Table 1: Seroconversion rates (SCR) 28 days after each IC51 vaccination, stratified by anti- Tick Borne Encephalitis (TBE) immune status. ITT Population

^ P-value of Fisher's exact test for difference in seroconversion rate between anti- tick borne encephalitis (TBE) positive and negative subjects.

Twenty-eight days after a single vaccination with IC51 the SCR was 76.5% (62/81) in TBE positives, whereas in TBE negatives the SCR was 49.0 % (166/339) (p < 0.0001 Fisher exact test). After two IC51 vaccinations the SCR was not significantly different in TBE positives 96.3% (78/81) compared to TBE negatives 91.4% (310/339). GMTs were also significantly higher in subjects with positive anti- TBE immune status after a single IC51 vaccination (28.4 vs. 16.0 p=0.0006). After the complete primary immunization of two IC51 vaccinations GMTs were 206.5 and 186.5 respectively (Table 2).

This advantageous effect of prior immunization against TBE was even more obvious in vaccinees younger than 50 years of age 28 days after a single IC51 vaccination. SCR was higher in subjects with positive anti-TBE immune status at baseline who were <50 years compared to those aged >50 years (81.5% vs. 56.3%) (Table 3). GMTs were also significantly higher in subjects with positive anti-TBE immune status after a single IC51 vaccination in persons who were <50 years (28.0 vs. 17.6 p=0.0006) (Table 4).

Table 2: Geometric mean titers (GMT) 28 days after each IC51 vaccination, stratified by anti- Tick Borne Encephalitis (TBE) immune status. ITT Population

^ P-value of ANOVA for difference in PRNT50 between anti- flavi virus immune status positive and negative subjects (factor anti- flavi virus immune status)

Table 3: Seroconversion rate (SCR) 28 days after each IC51 vaccination, stratified by anti- Tick Borne Encephalitis (TBE) immune status and age group. ITT population

Table 4: Geometric mean titer (GMT) 28 days after each IC51 vaccination, stratified by anti- Tick Borne Encephalitis (TBE) immune status and age group. ITT Population

There was no significant difference between TBE positives and TBE negative subjects after two IC51 vaccinations for SCR or GMT in both age groups. After two IC51 vaccinations SCR was similar for subjects with positive immune status who were aged <50 years or >50 years (95.4% vs. 100.0%). For subjects with negative TBE immune status, SCR was also similar for both age groups (93.9% vs. 86.2%, respectively). Results of the sensitivity analyses were very similar and supported the results presented (see Tables 5 to 7).

Table 5: Sensitivity analysis- Worst Case: Seroconversion rate (SCR) 28 days after each IC51 vaccination, stratified by anti- Tick Borne Encephalitis (TBE) immune status and age group. ITT population

Table 6: Sensitivity analysis- Best Case: Seroconversion rate (SCR) 28 days after each IC51 vaccination, stratified by anti- Tick Borne Encephalitis (TBE) immune status and age group. ITT population

Table 7: Sensitivity analysis- Worst Case: Geometric mean titer (GMT) 28 days after each IC51 vaccination, stratified by anti- Tick Borne Encephalitis (TBE) immune status and age group. ITT Population

Table 8: Sensitivity Analysis- Best Case: Geometric mean titer (GMT) 28 days after each IC51 vaccination, stratified by anti- Tick Borne Encephalitis (TBE) immune status and age group. ITT Population

Anti TBE Immune Status at Baseline

A within-treatment group comparison of SCRs between North America and Europe showed that SCRs were significantly higher in European subjects in the IC51 group after the first vaccination (70.0% vs. 48.8%, p=0.0002) but there was no difference after the second vaccination (data not shown).

Baseline TBE vaccination status

A total of 108 subjects (13%) in the ITT population, 54 in both the IC51 (13%) and JE-VAX^® (12%) groups, reported to have received a basic immunization and/or a booster vaccination against TBE within the previous 10 years (data not shown). Thus, the proportion of TBE ELISA-positive IC51 subjects (19%) was in a similar order of magnitude as the proportion of IC51 subjects who reported a TBE vaccination within the previous 10 years (13%). Additionally, detailed TBE vaccination history was assessed in a subgroup of 58 subjects who were treated with IC51 in a single study site. Sixteen subjects had received the most recent TBE vaccination within 1 year before start of this clinical trial, 10 subjects had the most recent TBE vaccination within 1 to 3 years preceding the trial start, 19 subjects between 3 to 5 years preceding the trial start, and 7 subjects more than 5 to 10 years preceding the trial. One subject's last TBE vaccination was more than 10 years before, one subject had received a single TBE vaccination with date unknown. One subject's TBE vaccination status was unknown, and three subjects had not received a previous TBE vaccination.

Immunogenicity stratified by previous TBE vaccination

The supportive analysis of SCR and PRNT50 in subjects with and without previous TBE vaccination confirmed the advantage of a previous exposure to TBEV for the anti-JE antibody response after JE vaccination, especially for subjects immunized with IC51 (Table 9): At Day 28, the anti-JE SCR after a single IC51 vaccination was similar to the anti-JE SCR after 2 JE -VAX^® vaccinations for subjects who had received a previous TBE vaccination (91% for IC51 subjects and 94% for JE -VAX^® subjects). For subjects without previous TBE vaccination, however, the percentage of anti-JE seroconverted subjects was higher after 2 JE- VAX^® vaccinations (81%) than after a single IC51 vaccination (49%). At Day 56, anti-JE SCRs were comparable for all subjects, ranging from 89% for JE-VAX^® subjects without previous TBE vaccination to 98% for IC51 subjects with a previous TBE vaccination.

The PRNT50 analysis also showed an advantage of a previous TBE vaccination for IC51 subjects at 28 days after the first vaccination (Table 9): relative to baseline, the increase in GMT at Day 28 was greater in those IC51 subjects with a previous TBE vaccination (from 7.6 at baseline to 34.1 at Day 28), than in subjects with no previous TBE vaccination (from 5.1 to 16.0). Higher GMTs were obtained for JE-VAX^® subjects than for IC51 subjects in both subpopulations at Day 28, due to the additional vaccination these subjects had received at Day 7; however, the difference between JE- VAX^® subjects with and without a previous TBE vaccination (95.1 versus 65.4) was less pronounced than for IC51 subjects. At Day 56, i.e., after 2 IC51 injections or 3 JE-VAX^® injections, no major differences were seen in the GMTs between subjects with or without previous TBE vaccination receiving IC51 (230.3 versus 182.7) or JE-VAX^® (475.6 versus 453.5).

JEV PRNT at baseline in TBE Elisa positive Subjects

A total of 36 subjects (20.8%) of the TBE ELISA positive subjects at baseline were also JEV PRNT positive (i.e. PRNT50 > 10), whereas only one TBE ELISA negative subject (0.2%) and none of the 166 subjects in the TBE borderline group was JEV PRNT positive at baseline. In TBE ELISA positive subjects who had a high TBE titer (> 1000 VIE Units), a higher JEV SCR of 28.7% was found compared to 7.7% in TBE ELISA positive subjects with a lower TBE titer (156-1000 VIE Units). However, the JEV specific GMTs did not exceed the cut-off of 10 used in the PRNT test; GMT was 7 in TBE ELISA positive subjects (6 and 8 in subjects with a lower or high TBE titer) and 5 in TBE ELISA negative or borderline subjects.

JEV PRNT at baseline in TBE vaccinated Subjects

About one quarter of the subjects with a TBE vaccination within the previous 10 years (28% of IC51 subjects and 26% of JE-VAX^® subjects) had already been anti-JE positive at baseline compared to only 1% (IC51 and JE-VAX^®; p<0.0001 χ² test) of subjects who did not have a previous TBE vaccination. Table 9: Immunogenicity results stratified by previous TBE vaccination (ITT population)

Time point IC51 JE-VAX^®

TBE No TBE TBE No TBE

Vaccination vaccination Vaccination vaccination

N=54 N=376 N=54 N=383

SCR [n (%)]

Day 28 49 (91%) 182 (48%) *** 51 (94%) 310 (81%)*

Day 56 53 (98%) 344 (92%) 52 (96%) 341 (89%)

PRNT₅₀ [GMT (SD)]

Day 0 (baseline) 7.6 (12.9) 5.1 (7.7) 8.0 (19.8) 5.1 (6.5)

Day 28 34.1 (41.8) 16.0 (146.3) 95.1 (584.0) 65.4 (661.4)

Day 56 230.3 (469.5) 182.7 (1135.3) 96.0 (475.6) 88.1 (453.5)

GMT = geometric mean titre; ITT = intention-to-treat; N = maximum number of subjects; n = number of subjects with available data; PRNT₅₀ = serum dilution giving a 50% plaque reduction compared to plaque formation in virus-only controls; SCR = seroconversion rate; SD = standard deviation; TBE = tick-borne encephalitis. *p<0.05 and ***p<0.001 between subjects with and without previous TBE vaccination

3. Discussion:

After one vaccination with IC51 SCR and GMT were statistically significant higher in subjects with positive anti-TBE immune status, compared to subjects with a negative baseline TBE status. This effect of enhanced immunogenicity of IC51 was evened out after the second vaccination of the primary immunization with IC51. This promotive effect was more pronounced in persons that were younger than 50 years of age: persons with positive TBE immune status had a higher SCR after one IC51 vaccination than those aged 50 years and older. After two vaccinations the difference between the age groups was cleared. The effect of baseline anti-flavi virus immune status on SCR after a single IC51 vaccination in subjects aged >50 years was smaller, i.e. 15% which may be attributed to the small sample size of TBE positives in this group. GMT at day 28 was similar in IC51 subjects with positive immune status regardless of age, but was lower in subjects aged <50 years at day 56. The benefit of preexisting TBE immunity is predominant in vaccinees younger than 50 years and in European subjects. The western subtype of TBEV is endemic in central, eastern and northern Europe, with a high disease prevalence [26]. In earlier clinical trials it was observed that prior exposure to JEV increases the SCR upon vaccination with JEV vaccine SA₁₄-5-3, a strain that is less immunogenic than SAi4-14-2. In endemic areas, vaccination with SAi4-5-3 rendered a SCR of 85%, in non-endemic areas the SCR was only 61% in subjects who lacked prior flavivirus antibodies [27, 28, 29]. Sequential exposure to two or more flaviviruses induces rapid onset of very broad anamnestic responses with high-titer heterotypic antibodies following the second infection [30]. Yellow fever virus priming broadens the antibody response to monovalent dengue virus vaccination. Eight of 69 (12%) healthy adult volunteers vaccinated with monovalent live-attenuated dengue virus vaccine candidates had atypical antibody responses, with high-titer hemagglutination- inhibiting and neutralizing antibodies, and a typical reduced viraemia. Six of the eight volunteers harboured yellow fever antibodies before dengue virus vaccination [31]. A hamster model showed that prior exposure to other flaviviruses leads to increased response upon yellow fever challenge. Hamsters were singly or sequentially infected with JEV, St. Louis encephalitis, West Nile, and/or dengue- 1 viruses, and then challenged with a virulent strain of yellow fever virus. In contrast to control hamsters, which appeared clinically ill or died after YFV infection, the flavivirus-immune animals remained asymptomatic. The flavivirus-immune hamsters also had a reduced viremia [32]. The results presented here indicate that pre-existing anti-TBE immunity enhances the neutralizing antibody response to the novel, Vero-cell derived JEV vaccine IC51.

REFERENCES

The following references which have been recited in the present specification with their reference numbers are incorporated herein by reference in their entirety.

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The features of the present invention disclosed in the specification, the claims and/or the drawings may both separately and in any combination thereof be material for realizing the invention in various forms thereof.

Claims

1. A pharmaceutical composition comprising a cell culture derived, inactivated JEV for use in the treatment or prevention of an infection with JEV in a human subject with at least 40 years of age.

2. A pharmaceutical composition according to claim 1, wherein the human subject is from 40 to 50 years of age.

3. A pharmaceutical composition according to claim 1, wherein the human subject is at least 65 years of age.

4. A pharmaceutical composition according to claim 3, wherein the human subject is from 65 to 80 years of age.

5. A pharmaceutical composition according to one of claims 1 to 4, wherein the cell culture derived, inactivated JEV comprises the JEV strain SAi 4- 14-2.

6. A pharmaceutical composition according to one of claims 1 to 5, wherein the cell culture derived, inactivated JEV is produced in Vero cells.

7. A pharmaceutical composition according to one of claims 1 to 6, wherein the cell culture derived, inactivated JEV is formalin inactivated.

8. A pharmaceutical composition according to one of claims 1 to 7, wherein the cell culture derived, inactivated JEV is adsorbed to 0.1% aluminum hydroxide.

9. A pharmaceutical composition according to one of claims 1 to 8, wherein the pharmaceutical composition is a liquid formulation

10. Use of a cell culture derived, inactivated JEV for the preparation of a pharmaceutical composition for the treatment or prevention of an infection with JEV, wherein the pharmaceutical composition is administered to a human subject with at least 40 years of age.

11. Use according to claim 10, wherein the pharmaceutical composition is administered to a human subject from 40 to 50 years of age.

12. Use according to claim 10, wherein the pharmaceutical composition is administered to a human subject with at least 65 years of age.

13. Use according to claim 12, wherein the pharmaceutical composition is administered to a human subject from 65 to 80 years of age.

14. Use according to one of claims 10 to 13, wherein the cell culture derived, inactivated JEV comprises the JEV strain SAi ₄- 14-2.

15. Use according to one of claims 10 to 14, wherein the cell culture derived, inactivated JEV is produced in Vero cells.

16. Use according to one of claims 10 to 15, wherein the cell culture derived, inactivated JEV is formalin inactivated.

17. Use according to one of claims 10 to 16, wherein the cell culture derived, inactivated JEV is adsorbed to 0.1% aluminum hydroxide.

18. Use according to one of claims 10 to 17, wherein the pharmaceutical composition is a liquid formulation.

19. A method for treating or preventing an infection with JEV, comprising the step of administering a therapeutically effective amount of a pharmaceutical composition comprising a cell culture derived, inactivated JEV to a human subject with at least 40 years of age.

20. A method according to claim 19, wherein the human subject is from 40 to 50 years of age.

21. A method according to claim 19, wherein the human subject is at least 65 years of age.

22. A method according to claim 21, wherein the human subject is from 65 to 80 years of age.

23. A method according to one of claims 19 to 22, wherein the cell culture derived, inactivated JEV comprises the JEV strain SAi 4- 14-2.

24. A method according to one of claims 19 to 23, wherein the cell culture derived, inactivated JEV is produced in Vero cells.

25. A method according to one of claims 19 to 24, wherein the cell culture derived, inactivated JEV is formalin inactivated.

26. A method according to one of claims 19 to 25, wherein the cell culture derived, inactivated JEV is adsorbed to 0.1% aluminum hydroxide.

27. A method according to one of claims 19 to 26, wherein the pharmaceutical composition is a liquid formulation.

28. A pharmaceutical composition comprising a JEV for use in the treatment or prevention of an infection with JEV in a subject which has previously been vaccinated with a TBEV vaccine.

29. A pharmaceutical composition according to claim 28, wherein the subject which has previously been vaccinated with a TBEV vaccine is a human subject younger than 50 years of age.

30. A pharmaceutical composition according to claim 29, wherein the pharmaceutical composition is administered once.

31. A pharmaceutical composition according to one of claims 28 to 30, wherein the JEV is a cell culture derived, inactivated JEV.

32. A pharmaceutical composition according to one of claims 28 to 31, wherein the JEV comprises the JEV strain SAi ₄- 14-2.

33. A pharmaceutical composition according to one of claims 28 to 32, wherein the JEV and is produced in Vero cells.

34. A pharmaceutical composition according to one of claims 28 to 33, wherein the JEV is formalin inactivated.

35. A pharmaceutical composition according to one of claims 28 to 34, wherein the JEV is adsorbed to 0.1% aluminum hydroxide.

36. A pharmaceutical composition according to one of claims 28 to 35, wherein the pharmaceutical composition is a liquid formulation.

37. Use of a JEV for the preparation of a pharmaceutical composition for the treatment or prevention of an infection with JEV, wherein the pharmaceutical composition is administered to a subject which has previously been vaccinated with a TBEV vaccine.

38. Use according to claim 37, wherein the subject which has previously been vaccinated with a TBEV vaccine is a human subject younger than 50 years of age.

39. Use according to claim 38, wherein the pharmaceutical composition is administered once.

40. Use according to one of claims 37 to 39, wherein the JEV is a cell culture derived, inactivated JEV.

41. Use according to one of claims 37 to 40, wherein the JEV comprises the JEV strain

42. Use according to one of claims 37 to 41, wherein the JEV is produced in Vero cells.

43. Use according to one of claims 37 to 42, wherein the JEV is formalin inactivated.

44. Use according to one of claims 37 to 43, wherein the JEV is adsorbed to 0.1% aluminum hydroxide.

45. Use according to one of claims 37 to 44, wherein the pharmaceutical composition is a liquid formulation.

46. A method for treating or preventing an infection with JEV, comprising the steps of a. administering at least once a therapeutically effective amount of a TBEV vaccine to a subject, and b. subsequently administering at least once a therapeutically effective amount of a pharmaceutical composition comprising a JEV to said subject.

47. A method according to claim 46, wherein the therapeutically effective amount of a TBEV vaccine is administered three times.

48. A method according to one of claims 46 to 47, wherein the subject is a human subject younger than 50 years of age.

49. A method according to claim 48, wherein the therapeutically effective amount of a pharmaceutical composition comprising a JEV is administered once.

50. A method according to one of claims 46 to 49, wherein the JEV is a cell culture derived, inactivated JEV.

51. A method according to one of claims 46 to 50, wherein the JEV comprises the JEV strain

52. A method according to one of claims 46 to 51, wherein the JEV and is produced in Vera cells.

53. A method according to one of claims 46 to 52, wherein the JEV is formalin inactivated.

54. A method according to one of claims 46 to 53, wherein the JEV is adsorbed to 0.1% aluminum hydroxide.

55. A method according to one of claims 46 to 54, wherein the pharmaceutical composition is a liquid formulation.