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WO2009024534A2 - Virus de l'encéphalite japonaise (jev) et peptides du virus de l'encéphalite de la taïga (tbev) stimulant des réponses de lymphocytes t humains - Google Patents

Virus de l'encéphalite japonaise (jev) et peptides du virus de l'encéphalite de la taïga (tbev) stimulant des réponses de lymphocytes t humains Download PDF

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Publication number
WO2009024534A2
WO2009024534A2 PCT/EP2008/060705 EP2008060705W WO2009024534A2 WO 2009024534 A2 WO2009024534 A2 WO 2009024534A2 EP 2008060705 W EP2008060705 W EP 2008060705W WO 2009024534 A2 WO2009024534 A2 WO 2009024534A2
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seq
peptide
cell
peptides
jev
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PCT/EP2008/060705
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WO2009024534A3 (fr
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Nicole Scharnagl
Christoph Klade
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Intercell Ag
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • JEV Japanese Encephalitis Virus
  • TBEV Tick-borne Encephalitis Virus
  • the immune system is a complex network of inter-related cell types and molecules, which can be divided into the evolutionary older innate (or natural) immunity and adaptive (or acquired) immunity.
  • the innate immune system recognizes patterns, which are usually common and essential for pathogens. For this limited number of molecular structures germ-line encoded receptors have evolved.
  • cells of the adaptive immune system - B and T lymphocytes - can recognize a huge variety of antigenic structures.
  • the receptors termed according to the cell types expressing them, B cell receptor (BCR, its soluble versions are antibodies) and T cell receptor (TCR, only cell- surface associated forms) are generated by somatic recombination and are clonally distributed. Thus, initially there are only a small number of cells with certain specificity.
  • T cells have a central role in adaptive immunity. Their receptors recognize peptides in context with major histocompatibility complex (MHC or human leukocyte antigen, HLA) molecules on the surface of cells.
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen
  • T cells CD8+ cytotoxic T cells (CTLs) restricted to MHC class I
  • Th cells CD4+ helper T cells restricted to MHC class II.
  • CTLs monitor virtually all cells of the body and have the ability to directly kill infected cells presenting foreign peptide:MHC class I complexes. Th cells are essential for many features of adaptive immunity.
  • Th cells transmit supporting signals through direct interactions and by release of cytokines to other immune cells, whereby they trigger and amplify various antigen-specific effector functions and widely determine the nature of the immune reaction [I]. Based on the cytokine profile they secrete, Th cells can be further subdivided. ThI cells are involved in the induction of a predominantly cellular immunity [2] including activation of macrophages, complement-activating and opsonising Immunoglobulin G (IgG) antibodies, antibody-dependent cell cytotoxicity (ADCC) [3] and activation of CTLs via IL-2 [4].
  • IgG Immunoglobulin G
  • ADCC antibody-dependent cell cytotoxicity
  • ThI cells are understood to lead the attack against intracellular viral and bacterial pathogens, fight cancer cells, provoke the classic delayed-type hypersensitivity (DTH) skin response and, when overreactive, generate organ-specific autoimmune disease [5].
  • Th2 cells mainly produce the cytokines IL-4, IL-5, IL-IO and IL-13 [3], thereby activating B cells, mediating Ig class switching to IgE and IgG subtypes with little complement activity and activating eosinophils.
  • Th2 responses are involved in protective immunity against bacteria and helminthes but also play a central effector role in development of allergic inflammatory reactions [6].
  • MHC class I molecules are present on virtually all nucleated cells and predominantly present peptides originating from endogenously synthesized host- or pathogen-derived proteins to circulating CTLS.
  • Class I molecules are composed of a membrane-anchored alpha-chain and a non- covalently attached beta 2 -microglobulin (beta 2 m), and form a trimeric complex with a peptide which is displayed on the cell surface.
  • the peptides are generated by protein degradation through the proteasome, and are actively transported into the endoplasmatic reticulum (ER), accomplished by the transporter associated with antigen presentation (TAP).
  • TAP transporter associated with antigen presentation
  • This ATP-dependent TAP may determine a certain degree of specificity by selecting peptides holding length and amino acid sequence optimal for MHC class I binding.
  • the peptides usually 8-11 amino acids long, interact with unloaded MHC class I molecules.
  • the formed MHC-peptide complex is thereupon allowed to exit the ER and rapidly transferred through the
  • MHC class II molecules are present only on the surface of specialized antigen-presenting cells (APC) such as dendritic cells (DC), B cells and macrophages/monocytes, and are generally loaded with peptides derived from exogenous proteins which they present to Th cells.
  • APC antigen-presenting cells
  • DC dendritic cells
  • B cells B cells
  • a class II molecule consists of two membrane-anchored proteins (alpha and beta chain), which together form a cleft, open at both ends, that can accommodate a peptide of variable length, usually from 12 to 25 amino acids long.
  • a class II alpha/beta dimer assembles in the ER and associates with the invariant chain (Ii) inserting its class II MHC-associated Ii peptide (CLIP) located in the peptide-binding groove, thus preventing premature peptide loading of the MHC class II molecule.
  • the Ii chain is also important for proper assembly and transport of MHC class II molecule to the MIIC compartment. There, the Ii chain is degraded until the MHC binding cleft is occupied by the CLIP peptide.
  • the MHC system is highly complex. It is a cluster of more than 200 genes located on the short arm of chromosome 6 and consists of 3 genetic regions termed class I, II and III. For the class I alpha-chain in humans there are three gene loci termed HLA-A, -B and -C. The class II consists of three loci coding for HLA-DR, -DQ and -DP. For each gene of the class I and class II regions, numerous alleles have been described (http://www.ebi.ac.uk/imgt/hla/stats.html). The class III region contains genes encoding soluble proteins of the complement system and cytokines like TNF-alpha [10].
  • HLA-A*0201 allele The probably best studied HLA-A*0201 allele is present in -45% of Caucasian populations. The frequency of HLA alleles varies between different populations: some are common in most populations, others are found predominantly in one or a few populations [H]. Because of HLA-confined antigen recognition and presentation, as well as ethnic variation in HLA distribution, vaccines may not be uniformly effective among individuals/ across populations [12].
  • T cell epitopes can be identified by a variety of approaches. Stimulations are typically conducted by culturing Peripheral Blood Mononuclear Cells (PBMCs) with pools of overlapping peptides. The measurement of the proliferation of lymphocytes that occurs following various stimuli is a fundamental technique for assaying T cell responses.
  • the antigens of interest are used to stimulate selectively the cell proliferation of (usually) PBMCs. After a specific incubation period, cells are pulsed with tritiated thymidine ([3H]-thymidine). The radioactivity incorporated into DNA, reflecting DNA replication by actively dividing lymphocytes, indicates the frequency of antigen- specific T cells.
  • the proliferation assay has been used widely to assess immunogen-specific T cell responses [13-24].
  • a cytokine assay also uses lymphocytes directly ex vivo for screening of (peptide) antigens.
  • the ELISPOT assay allows enumerating single cytokine-producing T cells in response to specific antigens after short- term in vitro stimulation. Antigen-specific cytokine-producing cells are visualized as spots on a nitrocellulose-membrane, each spot representing one single cell secreting the cytokine of interest.
  • the assay is very sensitive and can detect as few as one cell in 100,000, however, non-specific background reactivity may limit sensitivity of the assay [26].
  • the ELISPOT detects T cells that are pre-activated in vivo, since na ⁇ ve T cells do not secrete cytokines upon short-term stimulation.
  • the assay provides a direct determination of the precursor frequency of antigen-specific T cells in patients [27].
  • IFN-gamma has commonly been used as the cytokine of choice as it indicates activated CTLs.
  • the ELISPOT assay is widely applied in vaccine development since it is a versatile tool for accurate quantification of cytokine- secreting immune cells [14, 20, 23, 28, 29]. In order to perform a "human PBMC screening", overlapping synthetic peptides (usually 15-20 amino acids long) spanning the full sequence of the antigen of interest are used.
  • the long synthetic peptides are taken up and processed by monocytes. This allows then presentation in the context of all of the 12 or more HLA alleles of the respective donor. As a consequence, such approaches can result in the discovery of multiple novel and unpredicted epitopes [30].
  • 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 [31].
  • the spherical, mature flavivirus particle is composed of three structural proteins. 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 of multiple prM/M and E copies.
  • the genomes of flaviviruses consist of a single-stranded positive-sense RNA molecule of approximately 11,000 nucleotides in length.
  • RNA genome is translated from a single open reading frame (ORF), generating the polyprotein 5'-C-prM/M-E-NSl- NS2A-NS2B-NS3-NS4A-NS4B-NS5-3 ', which is subsequently cleaved by cellular and viral proteases to obtain the single proteins [32].
  • the NS proteins associate to form the replicase complex [33]. They are only produced during productive viral replication, thus they are not components of inactivated flavivirus vaccines.
  • the prM protein which is thought to prevent E from premature conformational changes and fusion in endosomal vesicles, is cleaved shortly before virus release to produce the mature M protein.
  • JE is characterized by an incubation period of 5-15 days, followed by an abrupt onset of a seemingly innocuous febrile, which in severe cases is complicated by the development of neurological disease.
  • the ratio of asymptomatic to symptomatic infection is estimated to be 100:1- 1000:1 [37].
  • JE is spread mainly by Culex mosquitoes, of which Culex tritaeniorhynchus is the principal vector in most of Asia [38].
  • the Biken vaccine based on the Nakayama strain is distributed under the name JE -VAX ® by Sanofi Pasteur Inc. It is prepared by homogenization of infected mouse brains followed by formalin inactivation and various purification steps. A three-dose regimen is used for travelers in Australia, the USA, and Europe (days 0, 7, 28) [36]. This schedule achieves a seroconversion rate of virtually 100% [38]. The question of when to administer a booster vaccination after the initial series has not been satisfactorily resolved. It is commonly recommended that a booster should be administered after 2 or 3 years for persons who remain at risk for JEV infection.
  • the inactivated mouse brain- derived vaccine has been proven to be efficacious; however, it has come under question due to the occurrence of allergic reactions in vaccinated travelers [38].
  • PRC People's Republic of China
  • One cell culture-derived vaccines are currently in use. One is prepared by formalin- inactivating the cell culture supernatant obtained from primary hamster kidney cells infected with the P3 strain of JEV. This has been the primary vaccine in the PRC since 1968 and approximately 70 million doses are administered per year.
  • the second vaccine used in the PRC is a primary hamster kidney cell-derived live attenuated vaccine, which gradually replaces the inactivated vaccine since it was licensed in the PRC in 1988.
  • the attenuated JEV strain used in this vaccine was produced by serial cell culture passage of a virus originally isolated from mosquitoes.
  • 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.
  • live attenuated vaccines contain agents capable of causing infection, concerns about reversion to virulence are always an issue.
  • the vaccine has been given to approximately 180 million children without any apparent side effects, and a randomized trial demonstrated that the safety of this vaccine is acceptable. Recently, this vaccine has been licensed for use in South Korea [31, 36].
  • immunization is a critical public health measure.
  • the beneficial role of humoral immunity and the E protein has been well characterized and is used to measure the efficacy of JEV immunization.
  • the most accepted assay to demonstrate functional antibodies able to inactivate virus is the plaque-reduction neutralization test (PRNT). It is generally accepted that a neutralizing antibody titer of 1 :10 or greater is protective.
  • Antibodies against prM and NSl have also been reported to be capable of inducing protective immunity [39].
  • Primary JEV infection triggers a rapid and potent IgM response in serum and cerebrospinal fluid (CSF). The failure to mount an IgM response is associated with a fatal outcome.
  • CSF cerebrospinal fluid
  • 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 [40].
  • JEV-specif ⁇ c Th cells and CTL cell proliferation has been demonstrated by Konishi et al. [17] using PBMCs from patients and subjects vaccinated with the mouse-brain derived inactivated JEV vaccine.
  • responses to prM and E proteins have been observed only with PBMCs from some vaccinees, but no prM and E-specific proliferation of T cells in PBMCs of JE patients has been detected.
  • the PBMCs from some vaccinees did not proliferate which has been assumed to be consistent with low levels of T memory cells induced by the inactivated JEV vaccine [17].
  • PBMCs from humans vaccinated with the mouse brain-derived inactivated vaccine also respond to WNV and DEN-I, -2, -4 virus structural antigens.
  • JEV- specif ⁇ c Th cell clones from these vaccinees showed cytotoxic activity, thus, Th cells might contribute to recovery from flavivirus infections by lysing infected cells and by supporting antibody production [13].
  • NS3 has been reported to be the most frequently targeted antigen among the 5 largest JEV proteins (prM, E, NSl, NS3, NS5), as analyzed in a healthy JE- immune cohort, eliciting the highest levels of proliferation and IFN-gamma secretion.
  • NS3 stimulates IFN-gamma production in CTLs and Th cells, however, IL-4 expression was not detected [19, 22].
  • Kumar et al. [20] demonstrated that the production of NS3-specif ⁇ c IFN-gamma by responding T cells was significantly higher in healthy donors than in patients, strongly correlating with acquired protective immunity to JEV. Furthermore, a striking inverse association between IFN-gamma levels and the severity of postencephalitic sequelae in patients implicated a role of IFN-gamma in recovery [20].
  • TBE tick-borne encephalitis
  • TBEV causes tick-borne encephalitis (TBE), a disease affecting the central nervous system. Annually approximately 10,000 to 12,000 cases of TBE are reported worldwide [42].
  • TBE tick-borne encephalitis
  • TBEV is primarily endemic in Europe, Siberia, and the Far East of Russia [31].
  • Genetic analysis shows the existence of three genetic lineages of TBEV that have been designated Central European, Far Eastern, and Siberian subtypes according to their principal geographic distribution [43].
  • the virus relies on two types of hosts for their survival, i.e. ticks acting as both biological virus vectors and reservoir hosts, and virus-amplifying vertebrates that function as a source of infection for ticks and as reservoir hosts.
  • the vector-competent ticks belong to the family Ixodidae, from which two species play a major role in human TBEV transmission: Ixodes ricinus transmitting Central European TBE, and Ixodes persulcatus transmitting the Siberian and Far Eastern TBEV subtypes [43, 44].
  • TBEV mainly uses small rodents as maintenance and amplifying hosts, humans represent dead-end hosts.
  • the current epidemiology of TBE has recently been reviewed in detail [44]. Tick bites are the primary and most effective way to transmit TBEV. Another, less frequent, route of transmission is via ingestion of raw milk [44, 45].
  • 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%). Serological surveys indicate that subclinical infections are common and the case:infection ratio can be 1 :25 or less [31].
  • the incubation period is usually 7 to 14 days but ranges from 4 to 28 days.
  • the Central European TBEV typically causes a biphasic disease. The first phase is frequently subclinical or an uncharacteristic influenza- like illness and usually resolves within one week. Following a relatively asymptomatic period of 2-10 days, approximately one-third of patients develop the second stage with symptoms and signs of meningitis or meningoencephalitis.
  • 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 [31, 47, 48]. 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 [48].
  • Cross-reactivity against various European and Asian isolates has been documented for both vaccines [42]. In the pre- vaccination era, Austria had the highest recorded morbidity of tick-borne encephalitis (TBE) in Europe. In view of this fact a mass vaccination campaign was initiated.
  • the vaccination coverage of the Austrian population increased from 6% in 1980 to 86% in 2001, exceeding 90% in some of the high-risk areas.
  • the inactivated vaccines are administered in at least two doses for primary immunization, followed by a booster at 1 year.
  • the calculated rate of protection is 96-98.7% after completing the series of three doses [49].
  • subsequent periodic boosters every 3 years were recommended for people living in, or traveling to, TBE- endemic countries.
  • the results of recently published serologic follow-up studies following primary and/or booster immunization led to the recommendation that regular boosters should be administered every 5 years for individuals younger than 60 years of age, except for the administration of the first booster, which remains unchanged [50].
  • Prophylactic TBEV vaccines have been used for more than 30 years; they have proven to be the most effective means of prevention of TBE.
  • the Austrian experience shows that high immunization coverage with a vaccine that is effective and widely accepted by the general population can reduce national incidence of a flavivirus effectively.
  • TBEV vaccines are expensive to produce and require several vaccinations to generate a protective immune response.
  • Very little information is available on human T cell responses to virus (TBEV) proteins.
  • TBEV virus
  • no human T cell epitopes have been described for TBEV yet. Detailed understanding of T cell immunity is valuable for immunological monitoring and the design of novel and improved vaccines.
  • the flaviviruses JEV and TBEV possess a sequence identity of around 40% between their structural proteins.
  • a potential cross-reactivity between JEV and TBEV could have beneficial, but also harmful effects:
  • memory T cells that were generated during an earlier infection are reactivated in response to a second, unrelated infection.
  • This heterologous immunity can be advantageous e.g. by conferring partial protection against viral infections that are otherwise lethal , but can also be associated with severe immunopathology.
  • heterologous immunity may also influence the outcome of vaccination: one vaccine might constitute a "pseudo-boost" to a second vaccine or, in the case of so-called “original antigenic sin", the immune response to a second antigen will only be directed to a similar but not identical previously encountered antigen. Accordingly, a potential cross-reactivity would be of great medical importance in sequential immunizations or infections. Thus, it would also be very important to determine the cross-reactivity of JEV and TBEV.
  • novel JEV and TBEV vaccines would be highly desirable, which are easier to manufacture, require less booster doses, and show decreased incidence of side-effects.
  • detailed information on CMI responses is indispensable.
  • novel diagnostic measures are needed to monitor especially the CMI response of JEV and TBEV vaccines.
  • the problem underlying the present invention was to provide means for the development of diagnostic assays and pharmaceutical compositions to identify, treat or prevent infections caused by JEV or TBEV. More particularly, the problem was to provide T cell epitopes, fragments or variants thereof, complexes comprising said T cell epitopes and at least one additional compound, nucleic acids encoding said T cell epitopes, vectors comprising said nucleic acids, cells comprising said T cell epitopes, and T cells specifically recognizing said T cell epitopes, which can be used for said diagnostic assays and pharmaceutical compositions.
  • a further problem was to provide pharmaceutical compositions comprising said T cell epitope, complex, nucleic acid, vector, cell, and/or T cell for the treatment or prevention of an infection with JEV and/or TBEV.
  • Yet another problem was to provide methods for immunizing an animal or human against an infection with JEV and/or TBEV, and methods for stimulating an immune response in an animal or human against an infection with JEV and/or TBEV.
  • Still another problem was to provide methods for diagnosing an infection with JEV and/or TBEV and methods for monitoring the immune response of a subject to a JEV vaccine or a TBEV vaccine.
  • the problem underlying the present invention is solved by a peptide comprising or consisting of at least one amino acid sequence selected from the group consisting of SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:7, SEQ ID NO:31, SEQ ID NO:43, SEQ ID NO:50, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:90, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO:109, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:121, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:183, SEQ ID NO:
  • peptide comprising or consisting of at least one T cell epitope according to the present invention, including each and any variant, fragment, analogue or derivative thereof, particularly as described herein.
  • the respective disclosure is also made for or in relation to any peptide according to the present invention, including each and any variant, fragment, analogue or derivative thereof, particularly as described herein.
  • any use or aspect described in connection with any of the above mentioned compounds covered by said terms according to the present invention shall be applicable also to each and any other of the above mentioned compounds covered by those terms according to the present invention.
  • T cell epitopes are understood as meaning cytotoxic T cell epitopes or T helper cell epitopes (Th, ThI or Th2).
  • noncytotoxic T cells which are likewise able to recognize MHC class I molecules, are also known such that noncytotoxic T cell epitopes are also included as variants of the present invention.
  • the T cell epitopes are T helper cell epitopes.
  • Said T helper cell epitopes mediate a T helper cell function, i.e. stimulate a T helper cell immune response.
  • T helper cell immune response may be determined by measuring the production of T helper cell specific cytokines.
  • said T helper cell function mediated by the peptides according to the invention is a ThI immune response, which may be determined e.g. by measuring the production of the cytokines IL-2, IL- 12, TNF- ⁇ , TNF-beta, and/or IFN-gamma.
  • said T helper cell function mediated by the peptides according to the invention is a Th2 immune response, which may be determined e.g. by measuring the production of the cytokines IL-4, IL-5, IL-6, IL-IO, IL-11, and/or IL- 13 .
  • the present invention also relates to variants of the peptides set forth in the Sequence Listing.
  • the variant is characterized by being derived from the peptide as defined above by one or more amino acid substitutions, insertions and/or deletions.
  • the term variant includes peptides which have at least 70% identity to such peptide according to the present invention, preferably at least 80% or 85% identity to such peptide according to the present invention, and more preferably at least 90% similarity (more preferably at least 90% identity) to such peptide according to the present invention and still more preferably at least 95%, 96%, 97%, 98%, 99% or 99.5% similarity (still more preferably at least 95%, 96%, 97%, 98%, 99%, or 99.5% identity) to such peptide according to the present invention.
  • the variant is an active variant of the peptide according to the present invention.
  • the active variant of the invention is characterized by having a biological activity which is essentially the same or which is similar to that displayed by the unaltered peptide, including the ability to stimulate an immune response as described herein.
  • the variant of a peptide is active in the context of the present invention, if the activity of the variant amounts to at least 10%, preferably at least 25%, more preferably at least 50%, even more preferably at least 70%, still more preferably at least 80%, especially at least 90%, particularly at least 95%, most preferably at least 99% of the activity of the peptide without sequence alteration(s).
  • sequence alterations of such variants can include, but are not limited to, conservative substitutions, deletions, mutations and insertions.
  • preferred variants are those that vary from a reference by conservative amino acid substitutions.
  • Conservative substitutions are those that substitute a given amino acid in a peptide according to the present invention by another amino acid of like characteristics, i.e. those substitutions that take place within a family of amino acids that are related in their side chains and chemical properties. Examples of such families are amino acids with basic side chains, with acidic side chains, with non-polar aliphatic side chains, with non-polar aromatic side chains, with uncharged polar side chains, with small side chains, with large side chains, etc.
  • conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, VaI, Leu and He; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and GIu, substitution between the amide residues Asn and GIn, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe and Tyr.
  • one conservative substitution is included in the peptide. In another embodiment, two conservative substitutions or less are included in the peptide. In a further embodiment, three conservative substitutions or less are included in the peptide.
  • conservative amino acid substitutions include, but are not limited to, those listed below:
  • variants or fragments having the amino acid sequence set forth in the Sequence Listing in which several, a few, 5 to 10, 1 to 5, 1 to 3, 2, 1 or no amino acid residues are substituted, deleted or added, in any combination.
  • silent substitutions, insertions and deletions which do not alter the properties and activities of the peptide of the present invention.
  • the variants according to the present invention also include naturally-occurring allelic variants, as well as mutants or any other non-naturally occurring variants.
  • an allelic variant is an alternate form of a peptide that is characterized as having a substitution, deletion, or addition of one or more amino acids that does essentially not alter the biological function of the peptide, as it is described herein.
  • allelic variation is the rule.
  • any viral species is usually represented by a variety of strains that differ from each other by minor allelic variations.
  • virus strains of JEV which are grouped so far into 4 genotypes [68].
  • the currently known TBEV strains are grouped into 3 genotypes (as described above). Indeed, a peptide that fulfils the same biological function in different strains can have an amino acid sequence that is not identical in each of the strains.
  • allelic variation is equally reflected at the nucleotide level.
  • the invention further relates to fragments of the peptides set forth in the Sequence Listing.
  • the fragment is characterized by being derived from the peptide as defined above or the variant as defined above by one or more amino acid deletions.
  • the deletion(s) may be C-terminally, N- terminally and/or internally.
  • the fragment is obtained by at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 amino acid deletion(s).
  • the fragment is an active fragment of the peptide according to the present invention.
  • the active fragment of the invention is characterized by having a biological activity which is essentially the same or which is similar to that displayed by the complete peptide, including the ability to stimulate an immune response as described herein.
  • the fragment of a peptide is active in the context of the present invention, if the activity of the fragment amounts to at least 10%, preferably at least 25%, more preferably at least 50%, even more preferably at least 70%, still more preferably at least 80%, especially at least 90%, particularly at least 95%, most preferably at least 99% of the activity of the peptide without sequence alteration.
  • an active fragment or active variant of the peptide according to the invention is a T cell epitope which shows similar activity in a T cell assay (e.g. a lymphoproliferation assay as described e.g. in example 1 and/or an ELISPOT assay as described e.g. in example 1 of the present invention, and/or a tetramer assay) compared to the unaltered peptide.
  • a T cell assay e.g. a lymphoproliferation assay as described e.g. in example 1 and/or an ELISPOT assay as described e.g. in example 1 of the present invention, and/or a tetramer assay
  • a similar biological activity differs from the activity of the non-fragment or the non- variant in terms of the extent of the function (e.g. the T cell activating capability), affinity, immunogenicity, stability and/or specificity.
  • the active variant or the active fragment derived from the peptide according to the present invention by amino acid substitutions, deletions or insertions may also conserve, or more preferably improve, the activity of the unaltered peptide (e.g. the T cell activating capability). Therefore, the present peptides also cover peptides, which do not contain the original sequence as derived from JEV or TBEV, but trigger the same or preferably an improved T cell response. These peptides are referred to as "heteroclitic".
  • peptide which has a similar or preferably greater affinity to MHC/HLA molecules than the original peptide, and/or which can trigger the same T cells as the original peptide, and/or which has a similar or preferably a more potent activation capacity of T cells in vivo or in vitro.
  • Heteroclitic peptides can be obtained by rational design, i.e. taking into account the contribution of individual residues for binding to MHC/HLA molecules [53], combined with a systematic exchange of residues potentially interacting with the T cell receptor (TCR) and testing the resulting sequences with T cells directed against the original peptide.
  • TCR T cell receptor
  • Another possibility for identifying epitopes and more specifically heteroclitic epitopes includes the screening of peptide libraries with T cells directed against one or several of the present epitopes.
  • a preferred way is the positional scanning of synthetic peptide libraries [52].
  • improved epitopes are their formulation or modification with substances increasing their capacity to stimulate T cells. These substances include CTL epitopes, Th cell epitopes, lipids or liposomes or preferred modifications as described in WO 01/78767 [56].
  • T cell stimulating capacity of epitopes is their formulation with immune stimulating substances, for instance cytokines or chemokines like interleukin-2, -7, -12, - 18, class I and II interferons (IFN), especially IFN-gamma, GM-CSF, TNF-alpha, flt3-ligand and others.
  • immune stimulating substances for instance cytokines or chemokines like interleukin-2, -7, -12, - 18, class I and II interferons (IFN), especially IFN-gamma, GM-CSF, TNF-alpha, flt3-ligand and others.
  • IFN interleukin-2, -7, -12, - 18, class I and II interferons
  • the fragment or variant of a peptide according to the present invention is 1) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or 2) one in which one or more of the amino acid residues includes a substituent group, or 3) one in which the peptide according to the present invention or a fragment thereof is fused with another compound, such as a compound to increase the half- life of the peptide according to the present invention or a fragment thereof such as, for example, polyethylene glycol, or 4) one in which additional amino acids are fused to the peptide according to the present invention or a fragment thereof, such as a leader or secretory sequence or a sequence which is employed for purification of said peptide according to the present invention or fragment thereof or a proprotein sequence.
  • a conserved or non-conserved amino acid residue preferably a conserved amino acid
  • the peptides according to the present invention may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions.
  • a region of additional amino acids, particularly charged amino acids may be added to the N- or C-terminus of the peptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
  • regions may be added to the peptide to facilitate purification or to enhance expression. Such regions may be removed prior to final preparation of the peptide.
  • the addition of peptide moieties to peptides to engender secretion or excretion, to improve stability, to enhance expression or to facilitate purification, among others, are familiar and routine techniques in the art.
  • Fusions also may include the peptides according to the present invention fused or coupled to moieties other than amino acids, including lipids and carbohydrates. Further, peptides of this invention may be employed in combination with other vaccinal or diagnostic agents described by the prior art, as well as with other species of vaccinal or diagnostic agents derived from other microorganisms.
  • the peptide of the invention is fused to an epitope tag which provides an epitope to which an anti-tag substance can selectively bind.
  • the epitope tag is generally placed at the amino- or carboxyl-terminus of the peptide but may be incorporated as an internal insertion or substitution as the biological activity permits.
  • the presence of such epitope-tagged forms of a peptide can be detected using a substance such as an antibody against the tagged peptide.
  • provision of the epitope tag enables the peptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
  • Various tag peptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his), poly-histidine-glycine (poly-his-gly) tags, the HA tag polypeptide, the c-myc tag, the Strep tag and the FLAG tag.
  • the peptide further comprises at least one naturally occurring amino acid(s).
  • the peptides according to the present invention further comprises 1 to 30, preferably 1 to 10, especially 1 to 6, naturally occurring amino acid residues.
  • Said naturally occurring amino acid residues may be located at the N-terminus, the C-terminus or at the N-and C-terminus.
  • naturally occurring amino acid residue relates to amino acid residues which are present in the naturally occurring protein at the specific position, relative to the peptide.
  • a "non-naturally occurring" amino acid residue is therefore any amino acid residue being different from the amino acid residue at the specific position relative to the peptide.
  • the present peptides further comprise at least one non-naturally occurring amino acid(s), preferably 1 to 100, more preferred 1 to 20, even more preferred 1 to 10, still more preferred 1 to 5 non-naturally occurring amino acid residues, especially at the N-terminus, the C-terminus or at the N-and C-terminus.
  • the present epitope may also contain modified amino acids (i. e. amino acid residues being different from the 20 "classical” amino acids, such as D-amino acids or S-S bindings of Cys) as additional amino acid residues or in replacement of a naturally occurring amino acid residue.
  • modified amino acids i. e. amino acid residues being different from the 20 "classical” amino acids, such as D-amino acids or S-S bindings of Cys
  • Said amino acid residue(s) may also be a modified or unusual amino acid.
  • those are 2- aminoadipic acid, 3-aminoadipic acid, beta-alanine, 2-amino butyric acid, 4-amino butyric acid, 6- aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2- aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-diaminopimelic acid, 2,3- diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allo -hydroxy Iy sine, 3- hydroxyproloine, 4-hydroxyproloine, isodesmosine, allo-isoleucine, N-methylglycine, N- methyliso leucine, 6-N-methyllysine, N-methylvaline, norvaline, norleucine
  • amino acid may be subject to modifications such as posttranslational modifications.
  • modifications include acetylation, amidation, blocking, formylation, gamma-carboxyglutamic acid hydroxylation, glycosilation, methylation, phosphorylation and sulfatation.
  • the peptide as defined above may be modified by a variety of chemical techniques to produce derivatives having essentially the same activity (as defined above for fragments and variants) as the unmodified peptides, and optionally having other desirable properties.
  • carboxylic acid groups of the protein whether C-terminal or side chain, may be provided in the form of a salt of a pharmaceutically acceptable cation or esterified to form an ester, or converted to an amide.
  • Amino groups of the peptide may be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or may be converted to an amide. Hydroxyl groups of the peptide side chains may be converted to alkoxy or to an ester using well recognized techniques.
  • Phenyl and phenolic rings of the peptide side chains may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with alkyl, alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids.
  • halogen atoms such as fluorine, chlorine, bromine or iodine
  • alkyl, alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids amides of such carboxylic acids.
  • Thiols can be protected with any one of a number of well recognized protecting groups, such as acetamide groups.
  • the peptides according to the invention can be prepared, for example, by means of chemical peptide synthesis, or can be recombinantly produced.
  • the peptides according to the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • the peptides according to the present invention activate T cells.
  • the activation of T cells can be determined e.g. by T cell assays as described herein.
  • the peptides according to the present invention stimulate a cytotoxic T cell response.
  • the peptides according to the present invention stimulate a T helper cell function.
  • At least 2, preferably at least 3, peptides as described above can be combined.
  • at least 4, at least 5 or at least 6 peptides may be combined.
  • the peptides may be combined by producing a fusion peptide comprising the single peptides, and optionally comprising additional amino acid residues, such as e.g. linker amino acid sequences.
  • the peptides may be combined by mixing the single peptides.
  • the present invention also encompass fusion peptides comprising at least 2, at least 3, at least 4, at least 5 or at least 6 peptides as defined above, or fragments or variants thereof.
  • fusion peptides as well as nucleic acid molecules encoding them, can readily be made using standard techniques, including standard recombinant techniques for producing and expression of a recombinant polynucleic acid encoding a fusion protein.
  • At least 2, more preferably at least 3 peptides are combined by mixing the single peptides.
  • Peptides can be mixed by pooling the stock solutions of the respective peptides in equal concentration. For use in an assay system, this mix may then be diluted in assay medium to the final assay concentration (e.g. 5 ⁇ g/ml per peptide) and may subsequently be used e.g. to stimulate PBMCs. For use in a pharmaceutical composition, this mix may be diluted e.g. in a formulation buffer to the final concentration.
  • the combination of peptides is selected from the group consisting of the combination of SEQ ID NO: 183 and SEQ ID NO: 184, the combination of SEQ ID NO:184 and SEQ ID NO:175, the combination of SEQ ID NO:183 and 175, and the combination of SEQ ID NO: 183, SEQ ID NO: 184 and SEQ ID NO: 175; and wherein said combination is a mixture of said single peptides.
  • the present invention relates to a complex comprising at least one peptide as described above and at least one additional compound.
  • the peptide may be coupled covalently, or by way of hydrophobic interactions, ionic bonds or hydrogen bonds to said at least one additional compound.
  • Said at least one additional compound may be e.g. another peptide, a protein, a carbohydrate, and/or a nucleic acid.
  • said additional compound is a B cell epitope.
  • Said B cell epitope may be any peptide, hapten or carbohydrate representing a selected region of an antigenic structure.
  • the T-cell epitope of the present invention can be covalently coupled to any peptide, hapten or carbohydrate representing a B-cell epitope. The coupling may be either directly by the formation of a peptide or an ester bond between free carboxyl, amino or hydroxyl groups on the T-cell epitope and corresponding groups on the peptide, hapten or carbohydrate representing a B-cell epitope or indirectly via a conventional bifunctional linking group.
  • said additional compound is an MHC molecule.
  • Said MHC molecule may be a MHC polymer, such as e.g. a tetramer, a pentamer, or an multimer.
  • MHC molecule as used herein comprise MHC class I and class II molecules, as well as MHC-like molecules.
  • MHC molecules may be selected in principle from any species, especially primates like humans (HLA, see below), chimpanzees, other mammals, e.g. macaques, rabbits, cats, dogs or rodents like hamsters, mice, rats, guinea pigs and others, agriculturally important animals like swine, cattle, horses, sheep and fish, although human (or "humanized”) molecules are of course preferred for providing diagnostic assays or vaccines for humans.
  • HLA primates like humans
  • chimpanzees other mammals, e.g. macaques, rabbits, cats, dogs or rodents like hamsters, mice, rats, guinea pigs and others, agriculturally important animals like swine, cattle, horses, sheep and fish, although human (or "humanized”) molecules are of course preferred for providing diagnostic assays or vaccines for humans.
  • said MHC molecules are selected from HLA class I molecules, HLA class II molecules, non classical MHC/HLA and MHC/HLA-like molecules, or mixtures thereof.
  • HLA class I molecules are HLA-A*0201 (in -45% of Caucasian populations), and HLA- B*07.
  • the JEV and TBEV peptides of the present invention potently induce T cell proliferation, thus Th cell binding peptides presented by MHC class II molecules are especially preferred.
  • the MHC molecules are HLA class II molecules.
  • said HLA class II molecules are selected from the group consisting of e.g. HLA-DRB 1*0101, *0301, *0401, *0404, *0701, *1101, and * 1501.
  • non-classical MHC/HLA and MHC/HLA-like molecules are comprised [69], which can specifically bind ligands, such as e.g. the peptides according to the present invention.
  • Another aspect of the present invention relates to a nucleic acid sequence encoding the peptide, fragments of variants thereof, or the complex as described above.
  • the invention also relates to nucleic acid molecules that hybridise to nucleic acid molecules encoding the peptides, fragments, variants, active variants, and active fragments, particularly those that hybridise under stringent conditions.
  • the invention relates to PCR primers for amplifying nucleic acid molecules that encode the peptides, fragments, and variants.
  • nucleic acid refers in a comprehensive manner to the nucleic acids encoding a peptide according to the present invention, including each and any variant, fragment, analogue or derivative thereof, particularly as described herein.
  • nucleic acid sequence refers in a comprehensive manner to the nucleic acids encoding a peptide according to the present invention, including each and any variant, fragment, analogue or derivative thereof, particularly as described herein.
  • the respective disclosure is also made for or in relation to any nucleic acid according to the present invention, including each and any variant, fragment, analogue or derivative thereof, particularly as described herein.
  • any use or aspect described in connection with any of the above mentioned compounds covered by said terms according to the present invention shall be applicable also to each and any other of the above mentioned compounds covered by those terms according to the present invention.
  • the nucleic acid sequence is DNA.
  • the nucleic acid sequence is RNA.
  • the nucleic acid also includes sequences that are a result of the degeneration of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Thus, nucleotide substitutions can be made which do not affect the peptide encoded by the nucleic acid. Accordingly, any nucleic acid molecule which encodes a peptide according to the present invention or a fragment or variant thereof is encompassed by the present invention.
  • any of the nucleic acid molecules encoding peptides or fragments or variants thereof provided by the present invention can be functionally linked, using standard techniques such as standard cloning techniques, to any desired regulatory sequences, whether a homologous regulatory sequence or a heterologous regulatory sequence, heterologous leader sequence, heterologous marker sequence or a heterologous coding sequence to create a fusion protein.
  • the nucleic acid sequence encoding the complex as described above may be a fusion of the nucleic acid sequence encoding the peptide and the nucleic acid sequence encoding the at least one additional compound, optionally comprising additional nucleic acids.
  • the present invention further relates to variants of the nucleic acid molecules described herein which encode fragments and derivatives of the T cell epitopes set forth in the Sequence Listing.
  • a variant of the nucleic acid molecule may be a naturally occurring variant such as a naturally occurring allelic variant, or it may be a variant that is not known to occur naturally.
  • Such non- naturally occurring variants of the nucleic acid molecule may be made by mutagenesis techniques, including those applied to nucleic acid molecules, cells or organisms.
  • variants in this regard are variants that differ from the aforementioned nucleic acid molecules by nucleotide substitutions, deletions or additions.
  • the substitutions, deletions or additions may involve one or more nucleotides.
  • the variants may be altered in coding or non- coding regions or both. Alterations in the coding regions may produce conservative or non- conservative amino acid substitutions, deletions or additions.
  • Preferred are nucleic acid molecules encoding a variant or a fragment having an amino acid sequence as set forth in the Sequence Listing, in which several, a few, 5 to 10, 1 to 5, 1 to 4, 3, 2, 1 or no amino acid(s) is substituted, deleted or added, in any combination.
  • silent substitutions, additions and deletions which do not alter the properties and activities of the T cell epitopes set forth in the Sequence Listing.
  • conservative substitutions are also especially preferred in this regard.
  • the nucleic acid molecules of the present invention may be originally formed in vitro, e.g. by chemical synthesis, or in a cell culture and subsequent isolation or purification.
  • the nucleic acids may be obtained by the manipulation of nucleic acids by endonucleases and/or exonucleases and/or polymerases and/or ligases and/or recombinases or other methods known to the skilled practitioner to produce the nucleic acids.
  • the nucleic acid molecules of the present invention may also be used as a hybridisation probe for, e.g., RNA, cDNA and genomic DNA to isolate full-length cDNAs and genomic clones encoding peptides of the present invention and to isolate cDNA and genomic clones of other genes that have a high sequence similarity to the nucleic acid molecules of the present invention.
  • Such probes generally will comprise at least 15 bases.
  • such probes will have at least 20, at least 25 or at least 30 bases, and may have at least 50 bases.
  • Particularly preferred probes will have at least 30 bases, and will have 50 bases or less, such as 30, 35, 40, 45, or 50 bases.
  • the coding region of a nucleic acid molecule of the present invention may be isolated by screening a relevant library using the known DNA sequence to synthesize an oligonucleotide probe.
  • a labeled oligonucleotide having a sequence complementary to that of a gene of the present invention is then used to screen a library of cDNA, genomic DNA or mRNA to determine to which members of the library the probe hybridizes.
  • nucleic acid molecules and peptides of the present invention may be employed as reagents and materials for the development or preparation of pharmaceutical compositions and/or diagnostics for diseases, particularly human disease, as further discussed herein.
  • the nucleic acid molecules of the present invention can be used in the methods described herein, but preferably for PCR, to determine whether or not the JEV or TBEV genes identified herein in whole or in part are present and/or transcribed in a sample obtained from an animal or human. It is recognized that such sequences will also have utility in diagnosis of the stage of infection and type of infection the pathogen has attained.
  • arrays which are known as such in the art, comprising at least one of the nucleic acids or peptides according to the present invention as described herein, may be used.
  • the nucleic acid according to the invention can, for example, also be used for producing a DNA vaccine.
  • the sequences encoding these peptides and optionally other JEV and/or TBEV epitopes can be joined together, in any arbitrary sequence, directly or with several nucleotides in between, in an open reading frame.
  • non-JEV and/or non-TBEV DNA sequences which are used, for example, for targeting the resulting peptide chain intracellularly, in particular into the endoplasmic reticulum, into the endosomes or into the lysosomes, can be added on.
  • the nucleic acid can be present as a plasmid, or as a part of a viral or nonviral vector.
  • the present invention therefore also relates to a vector, in particular an expression vector, which comprises a nucleic acid sequence according to the invention.
  • Vectors are particularly employed for a DNA vaccine.
  • An appropriate vector for delivery may be readily selected by one of skill in the art.
  • Exemplary vectors for in vivo gene delivery are readily available from a variety of academic and commercial sources, and include, e.g., adeno-associated virus, adenovirus vectors, or other viral vectors, e.g., various poxviruses, vaccinia, etc..
  • Recombinant viral vectors such as retroviruses or adenoviruses, are preferred for integrating the exogenous DNA into the chromosome of the cell.
  • the vector may additionally include nucleic acid sequences that permit it to replicate in the host cell, such as an origin of replication, one or more therapeutic genes and/or selectable marker genes and other genetic elements known in the art such as regulatory elements directing transcription, translation and/or secretion of the encoded protein.
  • the vector may be used to transduce or transform a cell, thereby causing the cell to express nucleic acids and/or proteins other than those native to the cell.
  • the vector optionally includes materials to aid in achieving entry of the nucleic acid into the cell, such as a viral particle, liposome, protein coating or the like.
  • the present invention relates to a cell which contains, and is preferably presenting, at least one peptide as described above.
  • the cell is transfected or transformed with a nucleic acid sequence or a vector according to the invention.
  • this cell expresses the peptide according to the invention under conditions which are known to the skilled person.
  • the cell containing, preferably presenting, at least one T cell epitope according to the invention can be produced by incubating a cell with at least one T cell epitope or with at least one complex as described above.
  • Antigen-presenting cells such as B cells, macrophages, dendritic cells, or fibroblasts expressing appropriate HLA alleles, are suitable for producing a cell according to the invention.
  • the cells according to the invention which present a peptide according to the invention can be used as target cells for restimulating immune cells, in particular T cells, and/or for measuring the activation of T cells.
  • a target cell is to be understood as being a cell which presents a T cell epitope by way of MHC molecules and thereby specifically elicits T cell activation, in particular a T helper cell reaction directed against the cell.
  • the peptide can then be isolated and purified from this cell.
  • the present invention is drawn to T cells, a T cell clone, or a population (preparation) of T cells, specifically recognizing any JEV or TBEV peptide according to the present invention.
  • a preferred application of such T cells is their expansion in vitro and use for therapy of patients, e. g. by adoptive transfer [51]. Therefore, the present invention also provides the use of T cells, a T cell clone or a population (preparation) of T cells for the preparation of a composition for the therapy of JEV and/or TBEV infected animals or humans.
  • T cells clones or lines
  • JEV or TBEV peptides are also useful for the identification of heteroclitic epitopes, which are distinct from the originally identified epitopes but trigger the same T cells (as described above).
  • a pharmaceutical composition preferably a vaccine, comprising at least one peptide, or at least one active fragment or active variant thereof, as defined above, or at least one complex as defined above, or at least one nucleic acid molecule as defined above, or at least one vector as defined above, or at least one cell as defined above, or at least one T cell as defined above, and optionally a pharmaceutically acceptable carrier or excipient.
  • compositions preferably a vaccine, comprising at least one peptide, or at least one active fragment or active variant thereof, as defined above, or at least one complex as defined above, or at least one nucleic acid molecule as defined above, or at least one vector as defined above, or at least one cell as defined above, or at least one T cell as defined above, and optionally a pharmaceutically acceptable carrier or excipient for the treatment or prevention of an infection with JEV or TBEV.
  • the pharmaceutical composition may comprise one, preferably at least 2, more preferably at least 3, or more of said peptides, fragments or variants thereof, complexes, nucleic acid molecules, vectors, cells, T cells, all as defined above, or any combination thereof.
  • Said combination may be a cocktail, i.e. a simple mixture, of the single components according to the invention, optionally mixed with other peptides of other pathogens.
  • said pharmaceutical composition is a vaccine for preventing or treating an infection with JEV and/or TBEV.
  • the pharmaceutically acceptable carriers and/or excipients useful in this invention are conventional and may include buffers, stabilizers, diluents, preservatives, and solubilizers.
  • Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, PA, 15th Edition, 1975 [54] describes compositions and formulations suitable for pharmaceutical delivery of the peptides herein disclosed.
  • the nature of the carrier or excipients will depend on the particular mode of administration being employed.
  • 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.
  • 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.
  • solid compositions e.
  • non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • 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. Since said peptides according to the present invention may be broken down in the stomach, they are preferably 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 anti-oxidants, 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.
  • the vaccine formulation may also include adjuvant systems for enhancing the immunogenicity of the formulation, such as oil-in-water systems and other systems known in the art. The dosage will depend on the specific activity of the vaccine and can be readily determined by routine experimentation.
  • the pharmaceutical composition of the present invention further comprises at least one immunostimulatory substance, preferably polycationic polymers, especially polycationic peptides, preferably peptides containing at least two LysLeuLys motifs, especially KLKLLLLLKLK, immunostimulatory oligo-deoxynucleotides (ODNs), especially Oligo(dIdC)13, neuroactive compounds, especially human growth hormone, alum, Freund's complete or incomplete adjuvants, or combinations thereof.
  • at least one immunostimulatory substance preferably polycationic polymers, especially polycationic peptides, preferably peptides containing at least two LysLeuLys motifs, especially KLKLLLLLKLK, immunostimulatory oligo-deoxynucleotides (ODNs), especially Oligo(dIdC)13, neuroactive compounds, especially human growth hormone, alum, Freund's complete or incomplete adjuvants, or combinations thereof.
  • ODNs immunostimulatory oligo-de
  • Oligo(dIdC)13 as used in the present invention means a phosphodiester backboned single- stranded DNA molecule containing 13 deoxy (inosine-cytosine) motifs, also defined by the term [oligo-d(IC)13].
  • Oligo(dIdC)13 can also be defined by the terms (oligo-dIC26); oligo-dIC26-mer; oligo-deoxy IC, 26-mer; or oligo-dIC, 26-mer, as specified for example in WO 01/93903 [57] and WO 01/93905 [58].
  • the polycationic compound(s) to be used according to the present invention may be any polycationic compound, which shows the characteristic effects according to the WO 97/30721 [59].
  • Preferred polycationic compounds are selected from basic polypeptides, organic poly cations, basic polyamino acids or mixtures thereof. These polyamino acids should have a chain length of at least 4 amino acid residues [59].
  • Other preferred polycations and their pharmaceutical compositions are described in WO 97/30721 [59] (e.g. polyethyleneimine) and WO 99/38528 [60].
  • these polypeptides contain between 20 and 500 amino acid residues, especially between 30 and 200 residues.
  • polycationic compounds may be produced chemically or recombinantly or may be derived from natural sources.
  • Cationic (poly)peptides may also be anti-microbial with properties as reviewed in Ganz, T., 1999 [55]. These (poly)peptides may be of prokaryotic or animal or plant origin or may be produced chemically or recombinantly [61]. Peptides may also belong to the class of defensins [61]. Sequences of such peptides can be, for example, found in the Antimicrobial Sequences Database under the following internet address: http://www.bbcm.univ.trieste.it/ ⁇ tossi/pag2.html
  • Such host defence peptides or defensives are also a preferred form of the polycationic polymer according to the present invention.
  • a compound allowing as an end product activation (or down-regulation) of the adaptive immune system, preferably mediated by APCs (including dendritic cells) is used as polycationic polymer.
  • Polycationic compounds derived from natural sources include HIV-REV or HIV-TAT (derived cationic peptides, antennapedia peptides, chitosan or other derivatives of chitin) or other peptides derived from these peptides or proteins by biochemical or recombinant production.
  • Other preferred polycationic compounds are cathelin or related or derived substances from cathelin.
  • mouse cathelin is a peptide, which has the amino acid sequence NH 2 - RLAGLLRKGGEKIGEKLKKIGOKIKNFFQKLVPQPE-COOH.
  • Related or derived cathelin substances contain the whole or parts of the cathelin sequence with at least 15-20 amino acid residues.
  • Derivations may include the substitution or modification of the natural amino acids by amino acids, which are not among the 20 standard amino acids. Moreover, further cationic residues may be introduced into such cathelin molecules. These cathelin molecules are preferred to be combined with the T cell epitope. These cathelin molecules surprisingly have turned out to be also effective as an adjuvant for an antigen without the addition of further adjuvants. It is therefore possible to use such cathelin molecules as efficient adjuvants in vaccine formulations with or without further immunactivating substances.
  • Another preferred polycationic substance to be used in accordance with the present invention is a synthetic peptide containing at least 2 KLK-motifs separated by a linker of 3 to 7 hydrophobic amino acids [62].
  • the pharmaceutical composition of the present invention may further comprise immunostimulatory nucleic acid(s).
  • Immunostimulatory nucleic acids are e.g. neutral or artificial CpG containing nucleic acids, short stretches of nucleic acids derived from non-vertebrates or in form of short oligonucleotides (ODNs) containing non-methylated cytosine-guanine di-nucleotides (CpG) in a certain base context (e.g. described in WO 96/02555 [63]).
  • ODNs long oligonucleotides
  • CpG non-methylated cytosine-guanine di-nucleotides
  • nucleic acids based on inosine and cytidine as e.g.
  • deoxynucleic acids containing deoxy-inosine and/or deoxyuridine residues may preferably be used as immunostimulatory nucleic acids in connection with the present invention.
  • the mixtures of different immunostimulatory nucleic acids may be used according to the present invention.
  • any of the aforementioned polycationic compounds is combined with any of the immunostimulatory nucleic acids as aforementioned.
  • such combinations are according to the ones as described in WO 01/93905 [58], WO 02/32451 [62], WO 01/54720 [65], WO 01/93903 [57], WO 02/13857 [61], WO 02/095027 [64], and WO 03/047602 [66].
  • such vaccine composition may further comprise a neuroactive compound.
  • the neuroactive compound is human growth factor as, e.g. described in WO 01/24822 [67].
  • the neuroactive compound is combined with any of the polycationic compounds and/or immunostimulatory nucleic acids as afore-mentioned.
  • the immunostimulatory substance is a combination of a polycationic polymer and immunostimulatory deoxynucleotides, preferably a peptide containing at least two LysLeuLys motifs and immunostimulatory deoxynucleotides, especially a combination of KLKLLLLLKLK and Oligo(dIdC)13.
  • the polycationic polymer is a polycationic peptide, especially polyarginine.
  • Still another aspect of the present invention provides a peptide, or an active fragment or an active variant thereof, as defined above, or a complex as defined above, or a nucleic acid molecule as defined above, or a vector as defined above, or a cell as defined above, or a T cell as defined above for the treatment or prevention of an infection with JEV or TBEV.
  • Another preferred embodiment of the invention relates to the use of a peptide, or an active fragment or an active variant thereof, as defined above, or a complex as defined above, or a nucleic acid molecule as defined above, or a vector as defined above, or a cell as defined above, or a T cell as defined above for the preparation of a pharmaceutical composition, especially for the preparation of a vaccine, for treating or preventing infections with JEV or TBEV.
  • the pharmaceutical composition is a vaccine composition.
  • such vaccine composition is conveniently in injectable form.
  • Conventional adjuvants may be employed to enhance the immune response.
  • a suitable unit dose for vaccination with a peptide is for adults between 0.02 to 3 ⁇ g peptide / per kg of body weight and for children between 0.2 to 10 ⁇ g peptide / per kg body weight, and such dose is preferably administered 1-3 times and with an interval of 2 to 24 weeks.
  • An especially preferred unit dose is from 3.5 to 7 ⁇ g peptide / per kg of body weight.
  • the present invention relates to diagnostic and pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
  • the ingredient(s) can be present in a useful amount, dosage, formulation or combination.
  • 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 invention relates to a method for immunizing an animal or human against infection with a JEV or a TBEV, comprising the step of administering to said animal or human an effective amount of at least one peptide according to the invention, or at least one complex according to the invention, or at least one nucleic acid sequence according to the invention, or at least one vector according to the invention, or at least one cell according to the invention, or at least one T cell according to the invention, or a pharmaceutical composition as described above, wherein the effective amount is suitable to elicit a protective immune response in said animal or human.
  • Said protective immune response is preferably a cell mediated T cell response, either cytokine- producing T cells or cytotoxic T cells, which protects said animal or human from the disease, i.e. an infection with JEV and/or TBEV.
  • Yet another aspect relates to a method for stimulating an immune response to a JEV or a TBEV in an animal or human in need thereof, which method comprises administering to said animal or human an effective amount of at least one peptide according to the invention, or at least one complex according to the invention, or at least one nucleic acid sequence according to the invention, or at least one vector according to the invention, or at least one cell according to the invention, or at least one T cell according to the invention, or a pharmaceutical composition as described above, whereupon an immune response to said JEV or a TBEV is stimulated in said animal or human.
  • the term "stimulating an immune response" as used herein is further defined below.
  • an “effective amount” or “therapeutically effective amount” of a peptide, complex, nucleic acid, vector, cell, T cell, or a pharmaceutical composition of the invention 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 JEV or TBEV. Such amounts may be determined by one of skill in the art.
  • the present invention relates in another aspect to a method for diagnosing an infection with a JEV or a TBEV comprising the steps of isolating lymphocytes from a sample obtained from a subject, incubating said lymphocytes with at least one peptide according to the invention, or with at least one pool of peptides according to the invention, and determining the proliferation of the lymphocytes in comparison to at least one reference sample.
  • the at least one pool of peptides may comprise a combination of at least 2, preferably at least 3 peptides as described above.
  • the pool comprises a combination of SEQ ID NO: 183 and SEQ ID NO: 184, the combination of SEQ ID NO: 184 and SEQ ID NO: 175, the combination of SEQ ID NO: 183 and 175, or the combination of SEQ ID NO: 183, SEQ ID NO: 184 and SEQ ID NO: 175, optionally comprising at least one further peptide according to the invention.
  • the pool of peptides may comprise at least 4, 5, 6, 7, 8, 9, or at least 10 peptides according to the invention.
  • the pool may be prepared by mixing the single peptides as described herein.
  • the proliferation of the lymphocytes can be determined e.g. by [3H]-Thymidine incorporation by PBMCs. Therefore, the peptides according to the invention can be used to stimulate selectively the cell proliferation of PBMCs. After a specific incubation period, cells are pulsed with tritiated thymidine ([3H]-thymidine).
  • the radioactivity incorporated into DNA, reflecting DNA replication by actively dividing lymphocytes, indicates the frequency of peptide-specific T cells.
  • the ratio of the mean counts incorporated by PBMCs in the presence of a peptide or a pool of peptides according to the invention and that of at least one reference sample (e.g. a control peptide pool) may be expressed in SI values. For example, SI values of at least 4, or more preferably of more than 4 may be considered to be positive.
  • Another aspect of the invention relates to a method for diagnosing an infection with a JEV or a TBEV comprising the steps of isolating lymphocytes from a sample obtained from a subject, incubating said lymphocytes with at least one peptide according to the invention, or with at least one pool of peptides according to the invention, determining the cytokine production of the lymphocytes in comparison to at least one reference sample.
  • the cytokine production can be determined e.g. by an ELISPOT assay.
  • the ELISPOT assay allows enumerating single cytokine-producing T cells in response to specific peptides according to the invention after short-term in vitro stimulation. Peptide-specific cytokine-producing cells can be visualized as spots on a nitrocellulose-membrane, each spot representing one single cell secreting the cytokine of interest.
  • the ELISPOT assay detects T cells that are pre-activated in vivo, since na ⁇ ve T cells do not secrete cytokines upon short-term stimulation.
  • the assay provides a direct determination of the precursor frequency of peptide- specific T cells in subjects.
  • IFN-gamma has commonly been used as the cytokine of choice as it indicates activated CTLs (and/or ThI cells).
  • further cytokines may be selected from the group consisting of IL-2, IL-4, IL-5, IL-6, IL-IO, IL-I l, IL-12, IL-13, TNF- ⁇ , and TNF-beta.
  • Said cytokines may also be determined in other cytokine production assays, such as e.g. an ELISA assay, a cytokine array for simultaneous detection of multiple cytokine expression in array format of a sandwich ELISA assay, flow cytometry combining intracellular cytokine staining, cytokine mRNA quantification techniques including real-time PCR, RNase protection assay etc., or electrochemiluminescence assays using magnetic beads linked with cytokine antibody for cytokine detection.
  • cytokine production assays such as e.g. an ELISA assay, a cytokine array for simultaneous detection of multiple cytokine expression in array format of a sandwich ELISA assay, flow cytometry combining intracellular cytokine staining, cytokine mRNA quantification techniques including real-time PCR, RNase protection assay etc., or electrochemiluminescence assays using magnetic beads linked with cytokine antibody for cytokine detection
  • Yet another aspect is related to a method for diagnosing an infection with a JEV or a TBEV comprising the steps of isolating lymphocytes from a sample obtained from a subject, incubating said lymphocytes with a fluorescently labeled complex comprising at least one peptide and at least one MHC molecule as described above to obtain a T-cell-MHC-peptide-complex; and determining T-cell-MHC-peptide-complex obtained in comparison to at least one reference sample.
  • the peptide according to invention is bound to synthetic (e.g. tetrameric) forms of fluorescently labeled MCH molecules. Since T cells recognize an antigen, e.g.
  • a peptide or T cell epitope in the form of short peptides bound to MHC molecules, cells with the appropriate T cell receptor will bind to the labeled tetramers and can be quantified.
  • said T cells are CTLs and said MHC molecules are MHC class I molecules.
  • Said T-cell- MHC-peptide-complex can be determined or quantified by e.g. a flow-cytometric method or any other method suitable for the detection of fluorescence emissions, such as e.g. fluorescence microscopy or fluorescence scanning procedures.
  • a further aspect of the present invention relates to a method for monitoring the immune response of a subject to a JEV vaccine or a TBEV vaccine comprising the steps of isolating lymphocytes from a sample obtained from a vaccinated subject, incubating said lymphocytes with at least one peptide according to the invention, or with at least one pool of peptides according to the invention, and determining the proliferation of the lymphocytes in comparison to at least one reference sample.
  • Still another aspect relates to a method for monitoring the immune response of a subject to a JEV vaccine or a TBEV vaccine comprising the steps of isolating lymphocytes from a sample obtained from a vaccinated subject, incubating said lymphocytes with at least one peptide according to the invention, or with at least one pool of peptides according to the invention, determining the cytokine production of the lymphocytes in comparison to at least one reference sample.
  • Yet another aspect of the invention is related to a method for monitoring the immune response of a subject to a JEV vaccine or a TBEV vaccine comprising the steps of isolating lymphocytes from a sample obtained from a vaccinated subject, incubating said lymphocytes with a fluorescently labeled complex comprising at least one peptide and at least one MHC molecule as described above to obtain a T-cell-MHC-peptide-complex; and determining T-cell-MHC-peptide-complex obtained in comparison to at least one reference sample.
  • vaccinated comprises the administration of any JEV or TBEV antigenic preparation, or any JEV or TBEV vaccine to a subject, preferably, the administration of at least one peptide, complex, nucleic acid, vector, cell, T cell, or pharmaceutical composition according to the present invention.
  • the sample obtained from a subject described above for the several methods for diagnosis or for the methods for monitoring an immune response is preferably a blood sample.
  • PBMCs can be isolated from said blood samples. Methods for isolating PBMCs from blood samples are well known in the art.
  • the sample obtained from a subject may also be a sample of spleen cells, lymph node cells, or T cells from cerebrospinal fluid.
  • Suitable methods for the determination or quantification of the proliferation, the cytokine production, and/or the T-cell-MHC-peptide-complex, as well as suitable cytokines to be measured are described above.
  • the at least one reference sample may be any negative control, such as medium without peptides, a reference peptide or a pool of reference peptides which do not contain T cell epitopes, such as e.g. the HSV peptides described in the example section.
  • the at least one reference sample has been obtained from the subject.
  • the at least one reference sample obtained from the subject has been obtained from the subject before the first vaccination.
  • the at least one reference sample has been obtained from the subject after a previous vaccination but before the latest vaccination. In a preferred example, the at least one reference sample has been obtained after the first vaccination and/or the second vaccination, but before the latest vaccination. In another preferred example, the at least one reference sample has been obtained after the first and/or the second and/or the third vaccination, but before the latest vaccination. In an example, the latest vaccination may be a booster vaccination.
  • two or more reference samples can be employed in the methods for diagnosing an infection with JEV or a TBEV and in the methods for monitoring the immune response of a subject to a JEV vaccine or a TBEV vaccine.
  • the effect of the at least one peptide, complex, nucleic acid, vector, cell, T cell, or pharmaceutical composition i.e. the capability of stimulating an immune response, may be determined in comparison to said at least one reference sample. If more than one reference samples are employed, said effect may be determined in comparison to each single reference sample or in comparison to the average of the reference samples, such as e.g. the arithmetic mean or the median of the reference samples.
  • the at least one peptide, complex, nucleic acid, vector, cell, T cell, or pharmaceutical composition is considered to stimulate an immune response if the stimulation index measured in a proliferation assay is positive, preferably at least 1, 2, 3, especially preferred at least 4, or most preferred greater than 4.
  • the stimulation index may be calculated by dividing the median stimulated culture counts by the median of at least one reference.
  • the at least one peptide, complex, nucleic acid, vector, cell, T cell, or pharmaceutical composition is considered to stimulate an immune response if the median spot number as measured in an ELISPOT assay is at least 2 times, preferably at least 3 times greater than the median spot number of at least one reference. Results may also be considered positive, if the spot number is 2-5 spots per million PBMCs, or at least 25 spots per million PBMCs.
  • the lymphocytes are incubated with a combination of at least 2 of the peptides according to the present invention as described above, more preferably with a combination of at least 3 of the peptides according to the present invention.
  • the combination may be either a fusion peptide comprising at least 2, more preferably at least 3 peptides as described herein, or a mixture of at least 2, more preferably at least 3 peptides as described herein.
  • said method is a method for diagnosing an infection with TBEV or for monitoring the immune response of a subject to a TBEV vaccine, and the lymphocytes are incubated with a combination of SEQ ID NO: 183 and SEQ ID NO: 184, the combination of SEQ ID NO:184 and SEQ ID NO:175, the combination of SEQ ID NO:183 and 175, or the combination of SEQ ID NO:183, SEQ ID NO:184 and SEQ ID NO:175, or fragments or variants thereof, as defined above.
  • Still another aspect is related to the use of at least one peptide according to the invention, or at least one complex according to the invention, or at least one nucleic acid sequence according to the invention, or at least one vector according to the invention, or at least one cell according to the invention, or at least one T cell according to the invention, or a pharmaceutical composition as described above, for stimulating, or for determining an immune response.
  • stimulating an immune response comprises the meaning of inducing an immune response for the first time i.e. a subject had no previous immune response to said at least one peptide, complex, nucleic acid, vector, cell, T cell, or pharmaceutical composition, as well as the meaning of re-stimulating an immune response, i.e. the immune response is stimulated for the second, third, or further time in a subject, i.e. said subject had a previous immune response to said at least one peptide, complex, nucleic acid, vector, cell, T cell, or pharmaceutical composition, which is re-stimulated.
  • the stimulation of an immune response can be determined e.g. by measuring the T cell response to the at least one peptide, complex, nucleic acid, vector, cell, T cell, or pharmaceutical composition as described above, and comparing the T cell response to the T cell response of at least one reference sample as described above.
  • the T cell response can be measured, e.g. by determining the proliferation and/or the cytokine production of lymphocytes, or by determining the T-cell-MHC- peptide-complex as described above.
  • the term "determining an immune response" as used herein comprises the determination of an immune response in comparison to at least one reference sample as described above, especially a T cell response.
  • the immune response can be determined as described above.
  • FIG. 1 Example of a positive lymphoproliferative response of PBMCs from a JEV-vaccinated donor to a pool of JEV peptides PBMCs.
  • [3H]-Thymidine incorporated by PBMCs from JEV- vaccinated subjects was measured during the last 16-18 h of 6 days culture with pools of JEV peptides.
  • 5 x 10 4 cells/well of 2,4 x 10 7 thawed PBMCs were plated and stimulated with medium as background control, with a pool of 10 HSV-2-derived as irrelevant peptide pool, with Con A as non-specific positive control, with Tetanus Toxoid (TT) as antigen-specific positive control, or with a pool of JEV peptides.
  • TT Tetanus Toxoid
  • SI values denote ratio of mean counts incorporated by PBMCs in the presence of antigen to that of control HSV-2 peptide pool plated in duplicates. SI values >4 were considered to be positive.
  • FIG. 2 Secondary screen. Single JEV and 20-mers contained in positive pools after primary screening were tested with PBMCs of the respective patient.
  • FIG. 3 Example of a positive lymphoproliferative response to a pool of TBEV peptides.
  • [3H]- Thymidine incorporated by PBMCs was measured during the last 16-18 h of 6 days culture with pools of TBEV peptides.
  • 5 x 10 4 cells/well of 2,4 x 10 7 thawed PBMCs were plated and stimulated with medium as background control, with a pool of 10 HSV-2-derived as irrelevant peptide pool, with Con A as non-specific positive control, with Tetanus Toxoid (TT) as antigen-specific positive control, or with a pool of JEV peptides. All antigens were used at final concentration of 5 ⁇ g/ml.
  • SI values denote ratio of mean counts incorporated by PBMCs in the presence of antigen to that of control HSV-2 peptide pool plated in duplicates. SI values >4 were considered to be positive.
  • FIG. 4 Secondary screen. Single TBEV and 20-mers contained in positive pools after primary screening were tested with PBMCs of the respective patient. [3H]-Thymidine incorporated by PBMCs was measured during the last 16-18 h of 6 days culture with single TBEV peptides. 5 x 10 4 cells/well of 2,4 x 10 7 thawed PBMCs were plated and stimulated with medium as background control, with HSV-2-derived peptide 1 as irrelevant peptide, with Con A as non-specific positive control, with Tetanus Toxoid (TT) as antigen-specific positive control, or with a single TBEV peptide. All antigens were used at final concentration of 5 ⁇ g/ml. SI values denote ratio of mean counts incorporated by PBMCs in the presence of antigen to that of control HSV-2 peptide 193 plated in duplicates. SI values >4 were considered to be positive.
  • FIG. 5 Example of a positive IFN-gamma ELISPOT response to a pool of TBEV peptides.
  • 2 x 10 5 cells/well of 2,4 x 10 7 thawed PBMCs were plated and stimulated with medium as background control, with a pool of 10 HSV-2-derived as irrelevant peptide pool, with Con A as non-specific positive control, with a Recall Pool containing HLA class I A*0201 restricted peptides from human cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza virus (FLU) as antigen-specific positive control, or with a pool of TBEV peptides. All antigens were used at final concentration of 5 ⁇ g/ml. Results were considered positive when the median spot number was at least three times greater than the median spot number obtained in the negative-control wells and at least 25 spots per million PBMCs.
  • CMV human cytomegalovirus
  • EBV Epstein-Barr virus
  • FLU influenza
  • Figure 6 Secondary screen. Single TBEV and 20-mers contained in positive pools after primary screening were tested with PBMCs of the respective patient. 2 x 10 5 cells/well of 2,4 x 10 7 thawed PBMCs were plated and stimulated with medium as background control, with a HSV-2-derived peptide 193 as irrelevant peptide, with Con A as non-specific positive control, with a Recall Pool containing HLA class I A*0201 restricted peptides from human cytomegalovirus (CMV), Epstein- Barr virus (EBV) and influenza virus (FLU) as antigen-specific positive control, or with a single TBEV peptide. All antigens were used at final concentration of 5 ⁇ g/ml. Results were considered positive when the median spot number was at least three times greater than the median spot number obtained in the negative-control wells and at least 25 spots per million PBMCs.
  • CMV human cytomegalovirus
  • EBV Epstein- Barr virus
  • FLU influenza virus
  • Figure 7 Amino acid sequence alignments of structural proteins from JEV strain SA- 14- 14-2 and TBEV strain Neudoerfl.
  • Table 1 JEV and TBEV derived peptides and peptide pools used in Examples 1 to 3. The peptides which have been tested positive in Example 1 are marked in bold (see also Table 3).
  • Table 3 T cell epitope containing JEV and TBEV peptides identified in the present invention. 29 out of 151 JEV derived peptides and 28 out 41 TBEV derived peptides were found reproducibly (2 - 3 independent experiments) positive by lymphoproliferation. Numbering of peptides corresponds to Table 1. See Table 1 or the attached Sequence Listing for amino acid sequences. Right columns show frequency of recognition among 10 responders.
  • Table 4 Combinations of frequently targeted (immunodominant) TBEV peptides.
  • Numbering of peptides corresponds to Table 1. See Table 1 or the attached Sequence Listing for amino acid sequences. Right columns show frequency of recognition among 10 responders.
  • Table 6 Proliferation and cytokine secretion data from the clinical trial. T cell responses to specific antigens (live JE virus SA- 14- 14-2 and peptide TBEV 184), tetanus toxoid (TT), Pokeweed mitogen (PWM) were assayed by ELISA for secretion IFN-gamma and IL-4. Medium was used as negative control.
  • the sample code is as in Table 5.
  • these subjects were randomized into two vaccine groups and received either two vaccinations of the Vero cell-derived, alum- formulated JE-PIV (IC51) on days 0 and 28 or three vaccinations of JE-V AX ® on days 0, 7 and 28.
  • PBMCs were examined using [3 H] -thymidine lymphoproliferation assay as well as IFN-gamma ELISPOT.
  • JEV protein C was covered by 23 peptides, divided into 2 pools; JEV protein prM/M was represented by 31 peptides and 3 pools.
  • JEV protein prM/M was represented by 31 peptides and 3 pools.
  • TBEV peptides 1 peptide of protein C and 9 peptides of prM/M were put into one pool, 31 peptides of protein E were distributed to 3 pools (see Table 1 "Pools of peptides (mainly 20-mers), derived from JEV and TBEV antigens").
  • HSV-2 derived peptides known to not contain any T cell epitopes were also synthesized for using in lymphoproliferation and ELISPOT assays as background control (see Table 2 "Peptides used as controls").
  • a "Recall-Pool” was prepared containing three peptides with known HLA-A*0201 T cell epitopes, Epstein-Barr virus (EBV)-derived BMLF- 1(280-288) (GLCTLVAML; SEQ ID NO: 203), influenza virus (FLU)-derived Matrix peptide(58-66) (GILGFVFTL; SEQ ID NO: 204) and human cytomegalovirus (CMV)-derived pp65(495-503) (NLVPMVATV; SEQ ID NO: 205), for using as positive control in lymphoproliferation and ELISPOT assays and was expected to give a signal in -45% of the subjects (see above, HLA frequency).
  • EBV Epstein-Barr virus
  • GLCTLVAML Epstein-Barr virus
  • FLU influenza virus
  • GILGFVFTL GILGFVFTL
  • CMV human cytomegalovirus
  • NLVPMVATV human cytomegalovirus
  • TBEV peptides In order to select TBEV peptides from regions with increased homology between JEV and TBEV structural antigens, amino acid sequences were aligned (http://www.ebi.ac.uk/clustalw/). Synthesized TBEV peptides were chosen from regions indicated with black boxes (see Figure 7 which shows amino acid sequence alignments of structural proteins from JEV strain SA- 14- 14-2 and TBEV strain Neudoerfl). T cell proliferation assay and IFN-gamma ELIspot assay were carried out with cryo -preserved PBMCs. These assays allow reliable measurements of epitope-specif ⁇ c T cell responses induced by either vaccine. The proliferation assay was defined as the primary outcome variable.
  • PBMCs blood anticoagulated with acid citrate dextrose (1 :9) was processed within 1 h after sample collection.
  • PBMCs were isolated on Lymphoprep (PAA Laboratories GmbH, Linz, Austria) using Leuco-sep tubes (Greiner, Frickenhausen, Germany), washed 3x with PBS (Gibco BRL Div. of Invitrogen Life Technologies, Carlsbad, CA, USA) and resuspended at a concentration of 2x 10 7 InF 1 in freezing medium consisting of nine parts foetal bovine serum (FCS; from PAA Laboratories GmbH, Linz, Austria) and one part DMSO (SIGMA, Deisenhofen, Germany).
  • FCS foetal bovine serum
  • SIGMA Deisenhofen, Germany
  • the T cell proliferation assay was used to analyze JEV and TBEV peptide-specific T helper cell responses.
  • Cryo-preserved PBMCs were thawed quickly in a 37°C water bath, washed Ix with assay medium (RPMI 1640 supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50 ⁇ M 2-mercaptoethanol, 1% antibiotic/antimycotic (all from Invitrogen Life Technologies) and 5% human serum type AB (PAA, Linz, Austria)) and incubated overnight (37°C, 5% CO 2 ).
  • assay medium RPMI 1640 supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50 ⁇ M 2-mercaptoethanol, 1% antibiotic/antimycotic (all from Invitrogen Life Technologies) and 5% human serum type AB (
  • HSV-2-derived peptides or a single HSV-2-derived peptide were used as negative controls (see Table 2: "Peptides used as controls”).
  • Concanavalin A Con A; Amersham Biosciences, UK
  • Tetanus toxoid Statens Serum Institut, Copenhagen, Denmark
  • Stimulation index was calculated by dividing the median stimulated culture counts by the median of the negative-control (HSV-2 peptides) culture counts. A positive stimulation index was considered when SI was >4.
  • the ELISPOT was used to screen for peptide-specific IFN-gamma production by PBMCs from JEV-vaccinated subjects. Briefly, Multi Screen 96-well filtration plates MAIP S4510 (Millipore, Bedford, MA) were coated with 10 ⁇ g/ml (0.75 ⁇ g/well) anti-human IFN-gamma monoclonal antibody (Mab) clone B140 (Bender Med Systems, Vienna, Austria) over night at 4°C.
  • PBMCs were thawed quickly in a 37°C water bath, washed Ix with ELIspot medium and incubated overnight (37°C, 5% CO 2 ).
  • Streptavidin-alkaline phosphatase (DAKO, Glostrup, Denmark) was added at 1.2 ⁇ g/ml for 1 h at 37°C.
  • the assay was developed by addition of 100 ⁇ l/well BCIP/NBT alkaline phosphatase substrate (SIGMA).
  • SIGMA BCIP/NBT alkaline phosphatase substrate
  • a pool of HSV-2-derived peptides or a single HSV-2-derived peptide (SSAGGAGGSVASASGAGERR; SEQ ID NO: 193), all known to not contain T cell epitopes, or medium without peptide served as negative controls (see Table 2: "Peptides used as controls").
  • HLA-A2 restricted peptides from human cytomegalovirus (CMV), Epstein-Barr virus (EBV) and influenza virus (FLU) served as specific positive control (see Table 2: "Peptides used as controls"), Concanavalin A (Amersham Biosciences, UK) as non-specific positive control. Results were considered positive when the median spot number was at least three times greater than the median spot number obtained in the negative-control wells (HSV peptide, or a pool of 10 HSV peptides); 0-5 spots per million PBMCs in most assays) and at least 25 spots per million PBMCs.
  • PBMCs of healthy, JEV-vaccinated subjects were initially stimulated with peptide mixtures, and positive pools were subsequently confirmed in 1-2 independent experiments (depending on availability of PBMCs) with individual 20-mers.
  • Cells and peptides were screened for specific lymphoproliferation and in an enzyme-linked immunospot (ELISPOT) assay for specific IFN-gamma secretion.
  • ELISPOT enzyme-linked immunospot
  • JEV-specific and TBEV-specific T-cell responses were detected in an equal frequency of about 50% of the subjects by the proliferation assay.
  • the analysis failed to identify dominant responses against individual single JEV peptides by IFN-gamma ELISPOT analysis, whereas some TBEV-specific IFN-gamma- production could be observed (see Figure 5 and 6).
  • a number of single TBEV-peptides were frequently recognized (by up to 7 subjects), whereas no prevalent single JEV peptides were identified (see Table 3).
  • JEV and TBEV peptides specifically stimulating human T cell responses was successfully applied with pools of synthetic JEV- and TBEV peptides which span the entire antigenic content of inactivated JEV vaccines and partly cover the antigenic content of inactivated TBEV vaccines, respectively.
  • the improved understanding of JEV-specif ⁇ c T lymphocytes may have important implications for the treatment of JEV infection and for the development of safe and effective vaccines.
  • PBMC peripheral blood mononuclear cells
  • a subgroup of 40 healthy subjects underwent blood sampling for the isolation of PBMCs at screening (day -7 to -1), visit 2 (day 7), visit 3 (day 28), visit 3a (day 35), visit 4 (day 56), visit 5 (1 year) and visit 6 (2 years) after first vaccination.
  • the objective of this analysis was to investigate cellular immune response to the JEV vaccines IC51 and JE -V AX ® . T-cell proliferation with different antigens for the stimulation of the PBMCs were evaluated.
  • a selected assay format was qualified following ICH (Q2 Rl) and FDA/CDER Guidance for Industry, Biological method validation.
  • Cells were stimulated with JEV and TBEV peptide pools, a single TBEV peptide or a live JEV SA- 14- 14-2 preparation.
  • Pokeweed mitogen served as unspecific positive control, Tetanus toxoid (TT) as antigen-specific positive control (which in general is expected to give a positive response in a large proportion of subjects due to the high tetanus vaccination coverage in the human population).
  • TT Tetanus toxoid
  • a pool of 8 irrelevant HSV-2 derived 20-meric peptides with physicochemical properties comparable to JEV and TBEV peptides or assay medium without antigen served as negative control.
  • PBMC samples from ⁇ 40 healthy subjects available for days 0 baseline, day of first vaccination
  • day 7, day 28, day 35, day 56, and for a subset of subjects also for 1 and 2 years after vaccination were analyzed.
  • Changes in cell mediated immune responses to baseline were evaluated by stimulation with a live JEV SA- 14- 14-2 preparation.
  • 12 peptide pools prepared out of 97 synthetic 20-meric peptides overlapping by 15 amino acids which comprehensively span the E protein from a Japanese Encephalitis Virus (JEV) consensus sequence (strain SA- 14- 14-2) listed in the right hand column of Table 1 designated with "JEV Pool #" were used.
  • Table 5 summarizes lymphoproliferation data from 38 evaluable subjects in this trial.
  • the first column ,,Sample” specifies the PBMC sample tested.
  • the code identifies the subject number, the last two digits indicate the time-point of blood take:
  • the following antigens were tested: live JE virus (supernatant of SA- 14- 14-2 infected Vero cells, designated with "JE virus"), JEV peptide pools #1-#12, TBEV peptide pools #13-#15, individual peptides JEV 61, TBEV 175 and TBEV 184, tetanus toxoid (TT) and Pokeweed mitogen (PWM) as positive controls, medium as negative control for the live JE virus stimulation and a Herpes simplex virus (HSV) derived peptide pool (see Table 2) as negative control for all others.
  • JE virus live JE virus
  • JEV peptide pools #1-#12 JEV peptide pools #1-#12
  • TBEV peptide pools #13-#15 individual peptides JEV 61, TBEV 175 and TBEV 184
  • TT tetanus toxoid
  • PWM Pokeweed mitogen
  • medium negative control for the live JE virus
  • Values in the table are median values from replicate stimulations (5-10 replicates). Units are cpm (counts per minute), higher cpm indicate stronger proliferation.
  • peptide pools in general values >700 are deemed to be positive, for live virus and the two positive controls >1200.
  • the signal needs to be >3x over background (cpm of negative control). Positive data are marked with a star.
  • the described peptide pools and even more selected individual peptides are useful for sensitive high-resolution analysis of JEV or TBEV T cell reactivity. This has applicability in diagnosis, immune monitoring (e.g. response to vaccination, longevity of immunity), also in development of immunotherapeutics and immunoprophylactics including vaccines.
  • IFN-gamma interferon-gamma
  • IL-4 interleukin-4
  • Table 6 summarizes lymphoproliferation and cytokine secretion data from a subset of 25 evaluable subjects in this trial.
  • TBEV 184 showed lymphoproliferation in 19 of the 25 subjects, in 11 of these 19 cases (58%) IFN- gamma secretion was detected, in 6 cases (32%) IL-4 secretion was detected, the latter being present mostly in IFN-gamma positive samples.
  • peptide TBEV 184 proved to be useful for delineating type-1 (IFN-gamma), type-2 (IL-4) or mixed immune responses in this cohort of humans.
  • T cell responses to specific antigens live JE virus SA- 14- 14-2 and peptide TBEV 184 were assayed for secretion IFN- gamma and IL-4.

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Abstract

La présente invention porte sur des moyens pour le développement de dosages de diagnostic et sur des compositions pharmaceutiques pour identifier, traiter ou prévenir des infections provoquées par JEV ou TBEV. Plus particulièrement, l'invention porte sur des épitopes de lymphocytes T, des fragments ou variants de ceux-ci, des complexes comprenant lesdits épitopes de lymphocytes T et au moins un composé supplémentaire, des acides nucléiques codant pour lesdits épitopes de lymphocytes T, et sur des lymphocytes T reconnaissant spécifiquement lesdits épitopes de lymphocytes T, qui peuvent être utilisés pour lesdits dosages de diagnostic et lesdites compositions pharmaceutiques. L'invention porte également sur des compositions pharmaceutiques comprenant ledit épitope de lymphocyte T, un complexe, un acide nucléique et/ou un lymphocyte T pour le traitement ou la prévention d'une infection par JEV et/ou TBEV. De plus, la présente invention porte sur des procédés pour immuniser un animal ou un être humain contre une infection par JEV et/ou TBEV, et sur des procédés pour stimuler une réponse immunitaire chez un animal ou un être humain contre une infection par JEV et/ou TBEV. Dans d'autres aspects, l'invention porte sur des procédés pour diagnostiquer une infection par JEV et/ou TBEV et sur des procédés pour surveiller la réponse immunitaire d'un sujet à un vaccin JEV ou d'un vaccin TBEV.
PCT/EP2008/060705 2007-08-17 2008-08-14 Virus de l'encéphalite japonaise (jev) et peptides du virus de l'encéphalite de la taïga (tbev) stimulant des réponses de lymphocytes t humains WO2009024534A2 (fr)

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WO2011133112A1 (fr) * 2010-04-20 2011-10-27 National University Of Singapore Peptide de pénétration cellulaire dérivé de la protéine pré-membranaire de flavivirus
WO2013050048A3 (fr) * 2011-10-07 2013-07-18 Skau Aps Identification et atténuation des domaines immunosuppresseurs dans des protéines de fusion de virus à arn enveloppés
WO2016037985A1 (fr) * 2014-09-08 2016-03-17 Ruprecht-Karls-Universität Heidelberg Construction pour l'administration d'une molécule dans le cytoplasme d'une cellule
CN108314708A (zh) * 2017-01-17 2018-07-24 南京农业大学 一种具有促进疫苗免疫反应的法氏囊活性九肽及其应用

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DE3853693D1 (de) * 1987-03-20 1995-06-08 Immuno Ag DNA- und RNA-Moleküle des westlichen Subtyps des FSME-Virus, Polypeptide, die von diesen Molekülen codiert werden, und deren Verwendung.
CU22683A1 (es) * 1997-01-15 2001-07-20 Inst De Medicina Tropical Pedro Kouri Epítopes de la proteína pre-m/m del virus del dengue, péptidos sintéticos, proteínas quiméricas y sus usos
US7227011B2 (en) * 1998-06-04 2007-06-05 United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention Nucleic acid vaccines for prevention of flavivirus infection
US7425335B2 (en) * 2001-01-05 2008-09-16 The Secretary Department Of Biotechnology Chimeric T helper-B cell peptide vaccine for Japanese encephalitis virus
GB2372991B (en) * 2001-03-09 2004-11-17 Fiocruz Fundacao Oswaldo Cruz Flavivirus expression vector
AU2003267943C1 (en) * 2002-02-26 2009-05-21 Altravax, Inc. Novel flavivirus antigens
AU2003263853A1 (en) * 2002-08-16 2004-03-03 Board Of Regents The University Of Texas System Compositions and methods related to flavivirus envelope protein domain iii antigens
ES2562456T3 (es) * 2003-03-24 2016-03-04 Valneva Austria Gmbh Uso de un adyuvante que induce una respuesta inmune Th1 para mejorar las respuestas inmunes

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011133112A1 (fr) * 2010-04-20 2011-10-27 National University Of Singapore Peptide de pénétration cellulaire dérivé de la protéine pré-membranaire de flavivirus
WO2013050048A3 (fr) * 2011-10-07 2013-07-18 Skau Aps Identification et atténuation des domaines immunosuppresseurs dans des protéines de fusion de virus à arn enveloppés
US10961279B2 (en) 2011-10-07 2021-03-30 Isd Immunotech Aps Identification and attenuation of the immunosuppressive domains in fusion proteins of enveloped RNA viruses
WO2016037985A1 (fr) * 2014-09-08 2016-03-17 Ruprecht-Karls-Universität Heidelberg Construction pour l'administration d'une molécule dans le cytoplasme d'une cellule
CN107073135A (zh) * 2014-09-08 2017-08-18 麦考拉·阿恩特 用于将分子递送至细胞的细胞质中的构建体
US10442863B2 (en) 2014-09-08 2019-10-15 Lutana Gmbh Construct for the delivery of a molecule into the cytoplasm of a cell
CN108314708A (zh) * 2017-01-17 2018-07-24 南京农业大学 一种具有促进疫苗免疫反应的法氏囊活性九肽及其应用

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