The invention concerns the use of a virus for the preparation of a medicament for the vaccination, treatment, or protection, of a neonatal or prenatal animal, including a human, wherein the virus is capable of infecting the cells of the neonatal or prenatal animal, including a human, but not capable of being replicated to infectious progeny virus in the neonatal or prenatal animal, including a human. The virus is preferably a Modified Vaccinia Virus Ankara.In particular, the invention concerns the vaccination of neonates against infections with viruses belonging the same virus group as the virus used for vaccination. Moreover, the invention concerns the vaccination of neonates against antigens selected from foreign antigens and tumor antigens, wherein the tumor antigen and/or the foreign antigen are different from the antigens associated with the virus. The invention further concerns the use of viruses as defined above to (i) increase the level of factors which activate dendritic cells or their precursor cells, (ii) and/or to increase the number of dendritic cells or their precursor cells, (iii) and/or to increase the production and/or cellular content of an interferon (IFN) or IL-12.

OBJECT OF THE INVENTION

The object of the present invention is to provide a means to vaccinate newborn humans and animals, respectively, against foreign antigens and antigens that are associated with diseases in each group, respectively. More particularly, the object of the present invention is to provide a means for allowing the accelerated maturation of the immune system of newborn animals and humans. A further object of the present invention is to provide a means that allows vaccinating neonatal animals, including humans, against poxyirus infections, in particular against smallpox.

DETAILED DESCRIPTION OF THE INVENTION

According to the present invention, it was unexpectedly found that it is possible to safely and efficiently vaccinate, and/or treat, neonatal or prenatal animals, including humans, with viruses that are capable of infecting cells of the neonatal or prenatal animal, including a human, but not capable of being replicated to infectious progeny virus in said cells. In particular, it has been shown that the viruses used according to the present invention, such as MVA, in particular MVA-BN and its derivatives (see below), can be administered to newborns without showing any harmful effects. The vaccination of the animal with the virus leads to a specific immune response against the virus used for vaccination, and/or to a general vaccination against foreign antigens and tumor antigens, as explained below in detail. Moreover, the viruses used according to the present invention lead to an induction and/or enhancement of the maturation of the immune system, which is associated with an increase in the number of dendritic cells and factors such as interferons. Vaccination with the viruses used according to the present invention is possible even if the formulation administered to the animal does not include an adjuvant.

In summary, the viruses used according to the present invention (i) elicit an effective immune response in neonates, (ii) can be administered without the need of an adjuvant, and (iii) do not bear the risk of overwhelming the organism.

According to the present invention, the protective effect is exerted for at least 5 days, preferably for at least 7, 14, or 28 days after the first vaccination.

Viruses that are "capable of infecting cells" harbor structures on the viral surface that are capable of interacting with host cells to such an extent that the virus, or at least the viral genome, becomes incorporated into the host cell. Although, the viruses used according to the present invention are capable of infecting the host cell, they are not capable of being replicated to infectious progeny virus in the infected cells. In the context of the present invention, the term "virus not capable of being replicated to infectious progeny virus in said cells" refers to viruses with a genome that is at least partially transcribed and translated into viral proteins, or even replicated; however, not packaged into infectious viral particles. Thus, the viruses used according to the present invention, are viruses leading to abortive infections in the host. Abortive infections may occur for two reasons. According to the first alternative, a cell may be susceptible to infection but it may not permit multiplication of the virus; e.g., due to not having all the necessary viral genes for multiplication expressed and/or present in the viral genome. An example of this type of virus according to the present invention in human cells is Modified Vaccinia Virus Ankara (MVA), which is explained in more detail below. According to the second alternative, an abortive infection may also result from infection of cells with defective viruses, which lack a full complement of viral genes. An example of such a virus according to the present invention in human cells is DISC-HSV1 (disabled single-cycle Herpes simplex virus), i.e., a Herpes simplex virus, which is restricted to a single cycle of infection (Dilloo et al., Blood 1997, 89: 119 127). This virus lacks the gene for the essential glycoprotein H (gH), but can be grown to high titer in a complementing cell line expressing gH. In noncomplementing cell lines that are permissive for herpesvirus growth it is restricted to a single cycle of replication, leading to the release of noninfectious virus. The term "not capable of being replicated" refers preferably to viruses that do not replicate in the cells of the vaccinated animal. However, viruses showing minor residual replication activity that is controlled by the immature immune system of the neonate are within the scope of the present invention.

The virus, according to the present invention, may be any virus that is capable of infecting cells of the animal, but not capable of being replicated to infectious progeny virus in said cells. It is to be understood, that a virus capable of infecting cells of a first animal species, but not capable of being replicated to infectious progeny virus in said cells, may behave differently in a second animal species. In humans, for example, MVA-BN virus and its derivatives (see below) are capable of infecting human cells, but are not capable of being replicated to infectious progeny virus in said human cells. The same viruses, however, are very efficiently replicated in chickens, i.e., MVA-BN virus is capable of infecting chicken cells, and replicating to infectious progeny virus in chicken cells. One skilled in the art understands which virus to choose for a specific animal species. WO 02142480 discloses a test using murine strain AGR129, that allows determination of whether a virus is capable, or not, of being replicated in a neonatal or prenatal animal. The results obtained in this murine model are indicative for humans. Thus, the term "not capable of being replicated to infectious progeny virus" as used in the present application, corresponds to the term "failure to replicate in vivo" as used for mice in WO 02/42480. More details on this test are given below. The viruses according to the present invention are preferably capable of being replicated in at least one type of cell of at least one animal species. Thus, it is possible to amplify the virus prior to administration to the animal that is to be vaccinated and/or treated. By way of example, reference is made to MVA-BN that can be amplified in chicken embryonic fibroblast (CEF) cells, but is not capable of being replicated to infectious progeny virus in the neonatal or prenatal human. In this context, it is to be noted that chemically or physically inactivated viruses do not have all of the properties of this preferred embodiment. Inactivated viruses are capable of infecting cells of the neonatal or prenatal animal, including a human, and not capable of being replicated to infectious progeny virus in the neonatal or prenatal animal, including a human. However, inactivated viruses are not capable of replicating in at least one type of cell of at least one animal species. Preferably, the virus is a DNA virus. More preferably, for mammalian cells, in particular for human cells, the DNA virus is selected from DISC-Hepesviruses and Modified Vacciniavirus Ankara (MVA).

Modified Vaccinia Ankara (MVA) virus is related to Vaccinia virus, a member of the genera Orthopoxyirus in the family of Poxyiridae. MVA has been generated by 516 serial passages of the Ankara strain of vaccinia virus (CVA) (for review see Mayr, A., et al. Infection 3, 6 14 [1975]) in CEF. As a consequence of these long-term passages, the resulting MVA virus deleted about 31 kilobases of its genomic sequence and, therefore, was described as highly restricted to avian host cells (Meyer, H. et al., J. Gen. Virol. 72, 1031 1038 [1991]). It was shown, in a variety of animal models that the resulting MVA was significantly avirulent (Mayr, A. & Danner, K. [1978] Dev. Biol. Stand. 41: 225 34). Additionally, this MVA strain has been tested in clinical trials as a vaccine to immunize against the human smallpox disease (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167, 375 390 [1987], Stickl et al., Dtsch. med. Wschr. 99, 2386 2392 [1974]). These studies involved over 120,000 humans, including high risk patients, and proved that, compared to Vaccinia based vaccines, MVA had diminished virulence or infectiousness while it maintained good induction of immunity.

Preferred strains, according to the present invention, are MVA 575, deposited on Dec. 7, 2000, at the European Collection of Animal Cell Cultures (ECACC) with the deposition number V00120707; and MVA-BN, deposited on Aug. 30, 2000, at ECACC with the deposition number V000083008, and derivatives thereof, in particular if it is intended to vaccinate/treat humans. MVA-BN and its derivatives are most preferred for humans.

The properties of particularly preferred MVA strains, preferably the most preferred strains for humans, such as MVA-BN and its derivatives, can be summarized as follows: (i) capability of reproductive replication in chicken embryo fibroblasts (CEF) and in baby hamster kidney cells (BHK), but no capability of reproductive replication in the human keratinocyte cell line, HaCat, (ii) failure to replicate in vivo, (iii) induction of a higher level of immunity compared to the known strain MVA 575 (ECACC V00120707) in a lethal challenge model and/or (iv) induction of at least substantially the same level of immunity in vaccinia virus prime/vaccinia virus boost regimes when compared to DNA-prime/vaccinia virus boost regimes.

The preferred MVA strains according to the present invention possess property (ii) above, i.e., failure to replicate in the organism, which is to be vaccinated or treated and/or in the corresponding test system, as explained below. The preferred MVA strains preferably have two of the above properties. More preferably, the preferred MVA strains have three of the above properties. Most preferred are MVA strains having all of the above properties. An example of an MVA strain having all of the above properties in humans is MVA-BN. Preferred derivatives of MVA-BN are derivatives having in addition to feature (ii), at least one of the above properties, and more preferably at least two of the above properties. Most preferred are MVA-BN derivatives having all of the above properties.

Reference is made to WO 02/42480 for detailed information regarding assays used to determine whether a MVA strain has one, or more, of the above features (i) to (iv). The publication also discloses how viruses having the desired properties can be obtained. In particular, WO 02/42480 provides: a detailed definition of the features of MVA-BN and a derivative thereof; a detailed description of biological assays used to determine whether a MVA strain is MVA-BN or a derivative thereof; and methods to obtain MVA-BN or a derivative thereof. In other words, the features of MVA-BN; the description of biological assays allowing to evaluate whether a MVA strain is MVA-BN or a derivative thereof; and methods describing how to obtain MVA-BN, or a derivative thereof, are disclosed in WO 02/42480.

The procedures disclosed in WO 02/42480 are summarized below. This summary does not limit the relevance of this disclosure, the full extent of which is incorporated by reference.

The term "not capable of reproductive replication" in the cell line HaCAT (Boukamp et al. 1988, J Cell Biol 106(3): 761 71) is used in the present application as defined in WO 02/42480. Thus, a virus that is "not capable of reproductive replication" in the cell line HaCat, is a virus that shows an amplification ratio of less than 1 in the human cell line HaCat. Preferably, the amplification rate of the virus used as a vector according to the invention is 0.8, or less, in the human cell line HaCat. The "amplification ratio" of a virus is the ratio of virus produced from an infected cell (Output) to the amount originally used to infect the cells in the first place (Input). A ratio of "1" between Output and Input defines an amplification status wherein the amount of virus produced from the infected cells is the same as the amount initially used to infect the cells. The term "derivatives" of the viruses as deposited under ECACC V00083008 refers preferably to viruses showing essentially the same replication characteristics as the deposited strain, but showing differences in one, or more, parts of its genome. Viruses having the same "replication characteristics" as the deposited virus replicate with similar amplification ratios as the deposited strain in CEF cells and the cell lines BHK, HeLa, HaCat, and 143B. These viruses also show a similar replication in vivo, as determined in the AGR129 transgenic mouse model (see below).

The term "failure to replicate in vivo" is used in the present application as defined in WO 02/42480. Thus, the term refers to viruses that do not replicate in humans and in the murine model, as explained in WO 02/42480. The mice used in WO 02/42480 are incapable of producing mature B- and T-cells (AGR 129 mice). In particular, MVA-BN and its derivatives, do not kill AGR129 mice within a time period of at least 45 days, more preferably within at least 60 days, and most preferably within 90 days, after infection of the mice with 10.sup.7 pfu virus administered via intra peritoneum. Preferably, the viruses that show "failure to replicate in vivo" are further characterized in that no virus can be recovered from organs or tissues of the AGR129 mice 45 days, preferably 60 days, and most preferably 90 days after infection with 107 pfu virus administered via intra peritoneum. Instead of AGR129 mice, another mouse strain may be used which is incapable of producing mature B and T cells and, as such, is severely immune compromised and highly susceptible to a replicating virus.

The details of the lethal challenge experiment used to determine whether a MVA strain has "a higher immunogenicity compared to the known strain MVA 575" are explained in WO 02/42480. In such a lethal challenge model, unvaccinated mice die after infection with replication competent vaccinia strains such as the Western Reserve strain L929 TK+ or IHD-J. The infection with replication competent vaccinia viruses is referred to as "challenge" in the context of description of the lethal challenge model. Four days after the challenge, the mice are usually killed and the viral titer in the ovaries is determined by standard plaque assays using VERO cells. The viral titer is determined for unvaccinated mice and for mice vaccinated with MVA-BN and its derivatives. More specifically MVA-BN and its derivatives are characterized in that, in this test after vaccination with 102 TCID.sub.50/ml virus, ovarian virus titers are reduced by at least 70%, preferably by at least 80%, and more preferably by at least 90%, compared to unvaccinated mice.

In a preferred embodiment, the viruses according to the present invention, such as MVA, in particular MVA-BN and its derivatives, are useful for prime/boost administration. The viruses, in particular MVA strains that are most preferably used in the present invention, such as MVA-BN and its derivatives, as well as, corresponding recombinant viruses harboring heterologous sequences, can be used to efficiently first prime, and then boost immune responses in naive animals, as well as, in animals with a pre-existing immunity to poxyiruses. Thus, the most preferred virus according to the present invention induces at least substantially the same level of immunity in vaccinia virus prime/vaccinia virus boost regimes compared to DNA-prime/vaccinia virus boost regimes.

A vaccinia virus, in particular a MVA strain, is regarded as inducing at least substantially the same level of immunity in vaccinia virus prime/vaccinia virus boost regimes when compared to DNA-prime/vaccinia virus boost regimes, if the CTL response as measured in one of "assay 1" and "assay 2" as disclosed in WO 02/42480, preferably in both assays, is at least substantially the same in vaccinia virus prime/vaccinia virus boost regimes when compared to DNA-prime/vaccinia virus boost regimes. More preferably, the CTL response after vaccinia virus prime/vaccinia virus boost administration is higher in at least one of the assays, when compared to DNA-prime/vaccinia virus boost regimes. Most preferably the CTL response is higher in both assays.

The virus used according to the present invention, may be a non-recombinant virus, such as MVA, i.e., a virus that does not contain heterologous nucleotide sequences. An example of a non-recombinant vaccinia virus is MVA-BN and its derivatives. Alternatively, the virus may be a recombinant virus, such as a recombinant MVA containing additional additional nucleotide sequences that are heterologous to the virus.

The term "heterologous" as used in the present application, refers to any combination of nucleic acid sequences that are not normally found intimately associated with the virus in nature; such virus is also called a "recombinant virus".

The heterologous nucleic acid sequence is preferably selected from a sequence coding for at least one antigen, antigenic epitope, beneficial proteins, and/or therapeutic compound.

The term "beneficial proteins" as used in the present application refers to any proteins that are helpful in protecting an animal against an antigen selected from tumor antigen and foreign antigen, wherein the antigen is different from the antigens associated with the virus. Alternatively and more particularly, the "beneficial proteins" are active in (i) increasing the level of factors that activate dendritic cells; and/or (ii) increasing the number of dendritic cells; and/or (iii) increasing the production and/or cellular content of an interferon (IFN) or IL-12. Examples of such beneficial proteins are interferons such as IFN-alpha and IFN-beta, IL-12, Flt-3-L and GM-CSF.

The antigenic epitopes may be any epitope to which it makes sense to induce an immune response. Examples of antigenic epitopes are epitopes from Plasmodium falciparum, Mycobacteria, Influenza virus, viruses selected from the family of Flaviviruses, Paramyxoviruses, Hepatitis viruses, and Human immunodeficiency viruses, or viruses causing hemorrhagic fever such as Hantaviruses or Filoviruses, i.e., Ebola or Marburg virus. Thus, if e.g., a recombinant MVA expressing heterologous epitopes is used to vaccinate neonates according to the present invention, the result of this treatment is not only a general vaccination due to the accelerated maturation of the immune system, but also a specific vaccination against the heterologous epitope expressed from the heterologous MVA.

A "therapeutic compound" encoded by the heterologous nucleic acid in the recombinant virus can be, e.g., a therapeutic nucleic acid such as an antisense nucleic acid, or a peptide or protein with desired biological activity.

The insertion of a heterologous nucleic acid sequence is preferably into a non-essential region of the virus genome. Alternatively, the heterologous nucleic acid sequence is inserted at a naturally occurring deletion site of the viral genome (for MVA disclosed in PCT/EP96/02926). Methods of how to insert heterologous sequences into the viral genome, such as a poxyiral genome, are known to a person skilled in the art.

The present invention also provides a pharmaceutical composition and a vaccine comprising a virus according to the present invention, such as MVA, e.g., for inducing an immune response in a living animal body, including a human.

The pharmaceutical composition may generally include one or more pharmaceutical acceptable and/or approved carriers, additives, antibiotics, preservatives, adjuvants, diluents and/or stabilizers. Such auxiliary substances can be water, saline, glycerol, ethanol, wetting or emulsifying agents, pH buffering substances, or the like. Suitable carriers are typically large, slowly metabolized molecules such as proteins, polysaccharides, polylactic acids, polyglycollic acids, polymeric amino acids, amino acid copolymers, lipid aggregates, or the like.

For the preparation of vaccines, the virus or its recombinants are converted into a physiologically acceptable form. A person skilled in the art is familiar with such methods. For MVA and other poxyiruses, the vaccine can be prepared based on the experience in the preparation of poxyirus vaccines used for vaccination against smallpox (as described by Stickl, H. et al. [1974] Dtsch. med. Wschr. 99, 2386 2392). For example, the purified virus is stored at -80.degree. C. with a titre of 5.times.10.sup.8 TCID.sub.50/ml formulated in about 10 mM Tris, 140 mM NaCl pH 7.4. For the preparation of vaccine shots, e.g., 10.sup.1 10.sup.8 particles of the virus, such as MVA, are lyophilized in 100 ml of phosphate-buffered saline (PBS) in the presence of 2% peptone and 1% human albumin in an ampoule; preferably a glass ampoule.

Alternatively, vaccine shots may be produced by stepwise, freeze-drying of the virus in a formulation. This formulation may contain additional additives such as mannitol, dextran, sugar, glycine, lactose or polyvinylpyrrolidone, or other additives such as antioxidants or inert gas, stabilizers or recombinant proteins (e.g. human serum albumin) suitable for in vivo administration. The glass ampoule is then sealed and can be stored between 4.degree. C. and room temperature for several months. However, as long as no need exists, the ampoule is stored preferably at temperatures below -20.degree. C.

For vaccination or therapy, the lyophilisate can be dissolved in 0.1 to 0.5 ml of an aqueous solution, preferably physiological saline or Tris buffer, and administered either systemically or locally, i.e., by parenteral, intramuscular, or any other path of administration known to the skilled practitioner. The mode of administration, the dose, and the number of administrations, can be optimized by those skilled in the art in a known manner.

The virus according to the present invention, in particular MVA, can be administered by oral, nasal, intramuscular, intravenous, intraperitoneal, intradermal, intra-utero and/or subcutanous application. In small animals the inoculation for immunization is preferably given by parenteral, or nasal administration; whereas, in larger animals or humans, a subcutaneous, intramuscular, or oral inoculation is preferred.

MVA is administered preferably in a dose of 10.sup.1 TCID.sub.50 (tissue culture infectious dose) to 10.sup.9 TCID.sub.50.

As indicated above, the virus according to the present invention, in particular MVA, such as MVA-BN and its derivatives, may be administered in a therapeutically effective amount in a first inoculation ("priming inoculation") and in a second inoculation ("boosting inoculation").

In the context of the present invention the term "animal" also includes human beings. More generally, the animal is a vertebrate animal, preferably a mammalian animal, including a human. Specific examples of animals are pets, such as dogs, and cats; economically important animals such as calves, cattle, sheep, goats, horses, and pigs; and other animals such as mice and rats. MVA and DISC-HSV are particularly preferred viruses for these animal species, and humans. The invention may also be used for economically important birds such as turkeys, ducks, geese, and hens, if the viruses used are capable of infecting avian cells, but not capable of being replicated to infectious progeny virus in said cells.

The term "domestic animals" as used in the present description refers preferably to mammalian domestic animals; more preferably to dogs, cats, calves, cattle, sheep, goat, pigs, horses, and deer.

According to a first alternative, the viruses according to the present invention, in particular MVA-BN and its derivatives, may be used as specific vaccines, i.e., to elicit an immune response that protects the vaccinated newborn against diseases caused by a virulent virus belonging to the same virus group, family or genus as the virus that was used for vaccination. By way of example, MVA as defined above, in particular MVA-BN and its derivatives, can be used to vaccinate newborn humans against poxyirus infections, in particular against smallpox. MVA, in particular MVA-BN and its derivatives, may also be used to vaccinate vertebrate animals against poxyirus infections of veterinary importance. According to this first alternative, the virus used for vaccination may be a non-recombinant virus, such as MVA-BN or its derivatives, or a recombinant virus harboring genes in the viral genome that are not naturally found in said genome. Preferably, the recombinant virus harbors additional genes that are helpful in stimulating the immune response; e.g., cytokine genes and interferon genes.

According to a second, but related alternative, neonates are vaccinated with a recombinant virus harboring a heterologous nucleic acid sequence, as defined above, to induce an immune response against the amino acid sequence expressed from the heterologous nucleic acid sequence. By way of example, the nucleic acid sequence may code for an antigen or an antigenic epitope, as defined above. Examples of a recombinant virus according to this embodiment are recombinant MVA, in particular recombinant MVA-BN or a derivative thereof, comprising a heterologous nucleic acid coding for antigens from (i) viruses other than MVA, such as HIV-1, HIV-2, Denguevirus, West-Nile Virus, Japanese Encephalitis virus, measles virus, (ii) tumor antigens, (iii) bacteria, and (iv) fungi. If the antigen expressed from the recombinant virus is, e.g., a HIV antigen, it is possible to use the recombinant virus to induce an immune response in the vaccinated neonate against HIV and to prevent AIDS. In a broader sense, the recombinant virus expressing the antigen or antigenic epitope is used to induce an immune response against the agent from which the heterologous sequence is derived, and/or against the agent that comprises the antigen or antigenic epitope.

According to a third alternative, it has been unexpectedly found that viruses capable of infecting the cells of the neonatal or prenatal animal, including a human, but not capable of being replicated to infectious progeny virus in the neonatal or prenatal animal, including a human, can be used for the preparation of a medicament for protecting an animal, in particular a newborn animal, including a human, against an antigen selected from tumor antigens and foreign antigens, wherein the antigen is different from antigens associated with the virus.

According to this third alternative, newborns vaccinated with the viruses according to the present invention, in particular with MVA, such as MVA-BN and its derivatives, are protected against a challenge with foreign antigens such as infectious agents. Thus, the viruses according to the present invention, in particular MVA, are a general vaccine for newborns. That is, by vaccinating newborns with the viruses according to the present invention, in particular MVA, the immune system of the newborns becomes more competent to deal with foreign antigens such as viruses. In the Example section, this is exemplified for vaccination with MVA and a subsequent challenge with Herpes simplex virus type 1. Thus, if the virus according to the present invention, in particular MVA, is used for the vaccination of newborns, the vaccinated animals are more protected against foreign antigens than unvaccinated animals during the critical time span until a functional and mature immune system is established.

According to the present invention, "the tumor antigen and/or the foreign antigen is different from the antigens associated with virus". This term is to be interpreted, in that according to this embodiment, the invention is not primarily intended to use a virus, such as MVA, to induce an immune response against the virus itself.

Instead, the virus is used to induce an immune response, or at least a general immune stimulation, that protects the host against foreign antigens and tumor antigens that are not associated with the virus. The term "antigens associated with the virus" refers to epitopes and antigens of the virus particle, and to antigens and epitopes on the surface of a cell infected with the virus that are the result of the expression of the viral genome.

In the context of this embodiment, the term "foreign antigens" refers to antigens and epitopes that are not naturally a part, or a component, of the animal body. Foreign antigens are especially antigens and epitopes from infectious agents and toxins. Typical infectious agents are viruses such as herpesviruses, retroviruses, rabiesviruses, rhabdoviruses, and adenoviruses; bacteria such as Salmonella, Mycoplasm, Meningicoccus, Hemophilus; prions; or fungi.

The invention is not only of interest to vaccinate animals against foreign antigens but, in an alternative embodiment, is also suitable to vaccinate against tumor antigens. "Tumor antigens" are antigens associated with certain tumor-causing diseases. Tumor antigens are most often antigens encoded by the genome of the host that develops the tumor. Thus, in a strict sense, tumor antigens are not foreign antigens. However, tumor antigens are found in significant amounts in tumors; whereas, the amount of tumor antigens in normal tissue is significantly lower, and most often not found at all. Examples of tumor antigens are known to a person skilled in the art and include, e.g., MAGE antigens. MVA is effective against these tumor antigens since the vaccination of animals leads to an activation and/or accelerated maturation of the immune system that may then lead to the destruction of tumor cells.

The term "protecting against an antigen" refers to the development of an immune response, which is directed against the foreign or tumor antigen. If the foreign antigen is an infectious agent, the host is protected against the agent, i.e., the host develops an immune response against the antigen. Consequently, infection with the infectious agent leads to a less severe disease, or no disease at all. The term "protecting" is not to be understood in the sense that there is always a 100% protection against the foreign or tumor antigen. Instead, the term "protection" as used in the present application, refers to any beneficial effect that helps the animal deal with the foreign antigen and the tumor antigen, respectively.

According to the present invention such a protective effect is exerted for at least 5 days, preferably for at least 7, 14, or 28 days, after the first vaccination. In other words, the vaccinated and/or treated animal is protected, e.g., against a foreign antigen if the animal comes into contact with the antigen after 5, 7, 14, and 28 days, respectively.

In the context of the present invention, the effect of the vaccination of newborns with the virus according to the present invention, in particular with MVA, may be explained by the induction or enhancement of maturation of the immune system, and/or the activation of the immune system. In the context of the present invention, the term "induction or enhancement of the maturation of the immune system" refers inter alia to the accelerated increase of dendritic cells or their precursors in vaccines, relative to controls. The terms "acceleration of the maturation" of the immune system and "enhancement of the maturation" of the immune system are used interchangeably in this description.

The "activation of the immune system" is characterized by the secretion and/or cell surface expression of molecules and hormones that facilitate cell/cell interaction or trafficking. Specific receptors take up these signals and respond accordingly. Activation markers are inter alia Flt3-L, IL-12, IFN-alpha, MHC-II and CD8, in particular CD8alpha (see below).

The accelerated development/maturation of the immune system is correlated with (i) an increase in the level of factors activating and or mobilizing dendritic cells (DC) or their precursor cells; and/or (ii) an increase in the number of dendritic cells and their precursor cells; and/or (iii) an increase in the production and/or cellular content of an interferon or IL-12. An example of DC precursor cells that are induced by a virus according to the present invention, in particular by MVA, are plasmocytoid DC precursors which are very important in defense against viral infections, and which seemingly produce IFN .alpha./.beta..

More specifically, the enhancement of the maturation of the immune system is preferably defined by at least a 2-fold increase in surface markers found on DC, such as MHC-II, CD40 and/or CD80/86. Preferably, such an increase can be measured in the blood. Additional markers that characterize enhancement of the maturation of the immune system are Flt3-L, IL-12, IFN-alpha, MHC-II and CD8a (see below). Moreover, accelerated maturation of the immune system is preferably correlated to at least a 1.5 fold increase, preferably at least a 2.0 fold increase, in the number of CD11c positive cells in the blood, and/or the spleen, 7 days after the administration of MVA-BN to newborn animals, when compared to control animals that have not received MVA-BN. Moreover, the enhancement of maturation of the immune system may preferably be correlated with at least a 1.5 fold increase, more preferably at least a 2.0 fold increase, in the concentration of Flt3-L two days after the vaccination of neonates with viruses according to the present invention, when compared to age matched controls.

In this context, it is to be noted that there is an association between the phenotype and function of murine and human DC populations that can be characterized by their surface phenotype (Hochrein et al. 2002. Hum. Immunol. 63: 1103). Dendritic cells in the blood can be detected by flow cytometry using a range of surface markers (MacDonald et al. 2002. Blood. 100:4512) that allow specific populations of DC, such as plasmaytoid DC, to be identified (Dzionek et al. 2002. Hum Immunol. 63: 1133; Dzionek et al 2000. J. Immunol. 165: 6037). Using similar techniques, DC can also be detected in other human tissues (Summers et al. 2001. Am. J. Pathol. 159: 285).

According to the present invention, the viruses as defined above might also be used to treat neonatal or prenatal animals to (i) increase the level of factors activating and/or mobilizing dendritic cells (DC) or their precursor cells; and/or (ii) to increase the number of dendritic cells and their precursor cells; and/or (iii) to increase the production and/or cellular content of an interferon or IL-12. It has been demonstrated that following vaccination with MVA-BN, the plasmocytoid dendritic cells upregulate MHC-II and CD8a, and produce significantly more IL-12 and IFN-alpha. The increase of IL-12 after the administration of the viruses used according to the present invention is preferably 2-fold, more preferably 100-fold, 500-fold, 1000-fold, 2500-fold or 5000-fold. The increase of the concentration of Flt3-L two days after the vaccination of neonates with viruses according to the present invention, most preferably with MVA-BN or its derivatives, is preferably 1.5-fold, more preferably 2.0-fold, when compared to age matched controls.

The term "activation of dendritic cells or their precursors" refers to the maturation of DC to antigen presenting cells through ill-defined cell stages driven by hormones and different antigenic stimuli. Intermediates of DC are termed precursors. These immature DC reach the periphery. Activation markers that are upregulated in activated dendritic cells are inter alia Flt3-L, IL-12, IFN-alpha, MHC-11 and CD8a (see below).

It was noted that hormones such GM-CSF, lead to more immature DC in the periphery. Because GM-CSF leads to more DC precursor in bone marrow, blood and peripheral organs (and the cells have to move there), this phenomenon has been termed "mobilization of dendritic cells or their precursors". This definition is also used in the present description.

Consequently, the vaccination of animals, including a human, is especially useful if it is intended to (i) increase the level of factors activating dendritic cells (DC) or their precursor cells; and/or (ii) increase the number of dendritic cells or their precursor cells; and/or (iii) increase the production and/or cellular content of an interferon or IL-12.

Factors that activate dendritic cells include inter alia FIt3-L (Lyman et al., Cell 1993, 75: 1157 1167) and GM-CSF. Typical interferons according to the present invention are IFN-alpha and IFN-beta. The viruses used according to the present invention induce Flt3-L and it is assumed that some of the beneficial effects observed are due to said induction.

In the context of the present application, a newborn animal, or human, is defined as an animal or human, not yet having a mature immune system. Throughout this specification, the terms "newborn animal" and "neonatal animal" are used synonymously. A mature immune system is characterized by the ability to fully activate the innate immune system, and by the fact that all known T and B cell functions and products are in place; in particular immunoglobulin isotypes such as IgA and IgE. Thus, an immature immune system is characterized by a low number of T cells, B cells, and dendritic cells, relative to adults; by low IFN production compared to adults; and by secondary lymphoid organs that are not fully mature. More specifically, a "neonatal" or "newborn" in the context of the present invention may be defined as an infant animal having a number of splenic CD4+ cells being preferably at least 2-fold, more preferably at least 20-fold, more preferably at least 200-fold, more preferably at least 2,000-fold, and most preferably at least 20,000-fold lower than the average number of splenic CD4+cells in adults.

In mice, the immune system is mature at the age of 4 weeks. In humans, maturity is probably achieved at 6 month to 1 year of age. In cats and dogs, the immune system is mature usually at the age of 6 month; and in calves, sheep and pigs at the age of 4 12 weeks. Vaccination with the virus according to the present invention, in particular with MVA, is preferably done before the immune system is mature. However, since maturity develops almost exponentially after birth, it is most preferred to vaccinate with the virus according to the present invention, in particular with MVA, as early after birth as possible. Since in all relevant domestic animals, and in humans, the immune system is mature no earlier than 4 weeks after birth, it is generally preferable that vaccination with the virus according to the present invention, in particular with MVA, is done within 4 weeks after birth, more preferably within 2 weeks after birth, even more preferably within 1 week after birth, and most preferably within 3 days after birth. These general terms are applicable to all important domestic animals, in particular to important domestic mammalian animals, and humans. A person skilled in the art will be aware of the fact that even older animals may be regarded as newborns/neonatals in the context of the present invention; and therefore, vaccination may also be successfully carried out with older animals, when the immune system is not yet mature 4 weeks after birth. Thus, in humans, the vaccination may be carried out within 6 month after birth, more preferably within 3 month after birth, more preferably within 2 month after birth, more preferably within 4 weeks after birth, more preferably within 2 weeks after birth, even more preferably within 1 week after birth, and most preferably within 3 days after birth.

Since the best effects of the virus according to the present invention, in particular MVA, as a general vaccine are observed if the virus is administered to an immature immune system, it may be preferred to vaccinate even prenatal animals, including humans. Prenatal vaccination may be desirable in economically important animals such as cattle or pigs. If the placenta allows passage of the virus, the prenadte can be vaccinated simply by vaccinating the mother animal. Thus, the vaccination of the mother animal to vaccinate the prenate is particularly promising in an animal having a placenta endotheliochorialis, such as dogs, cats, rats, and mice; or animals having a placenta heamochorialis, such as primates, including humans. In animals having a placenta chorionepithelialis, such as cattle and sheep, or animals having a placenta syndesmochorialis, such as pigs and horses, the vaccination of prenates can be preferably done by in-utero administration. Of course, this mode of administration can be also done for animal having a placenta endotheliochorialis or haemochorialis.

Since the viruses according to the present invention, in particular MVA, lead to an accelerated maturation of the immune system and are thus, useful as a general vaccine, the vaccinated animals are protected against a variety of diseases. More specifically, the viruses according to the present invention, in particular MVA, can be used to vaccinate cats for general well being and against feline herpes or feline infectious peritonitis. The viruses according to the present invention, in particular MVA, may be used in dogs for general well being and against respiratory tract associated (viral) diseases. The viruses according to the present invention, in particular MVA, may be used in pigs for general well being and against Hemophilus or Mycoplasm infections; and especially in fattening pigs.

As previously indicated, it is a preferred embodiment to use the viruses according to the present invention, in particular MVA, in newborns or prenatal animals to protect the animal against a foreign antigen and/or a tumor antigen, wherein the tumor antigen is different from the antigens associated with the virus used for vaccination. However this embodiment is not restricted to newborn and prenatal animals. Instead, in an alternative embodiment, the invention can be carried out for animals of all ages, since a beneficial effect can be observed also in adult animals. Thus, according to this embodiment, the viruses as defined above, in particular MVA-BN and its derivatives, are useful to protect an animal, including a human, against an antigen selected from tumor antigens and foreign antigens, wherein the antigen is different from the antigens associated with the virus. As indicated above, the viruses used according to the present invention are capable of infecting cells of the animal, but not capable of being replicated to infectious progeny virus in said cells. All information, definitions, including the definition of the duration of the protective effect, examples, as well, as the preferred, more preferred and most preferred embodiments given above for neonates, also apply for the present embodiment according to which the virus may also be administered to adults.

In contrast to newborns, the immune system of adult animals has already matured. Nevertheless, it may be that the immune system is weakened due to certain diseases, or simply due to the age of the animal. Especially in immune-compromised or elderly individuals, the administration of the viruses according to the present invention, in particular MVA, may have a beneficial effect inter alia by (i) increasing the level of factors activating and/or mobilizing dendritic cells (DC) or their precursor cells; and/or (ii) by increasing the number of dendritic cells or their precursor cells; and/or (iii) by increasing the production and/or cellular content of an interferon or IL-12. Thus, even in adult animals, the administration of the viruses according to the present invention, in particular MVA, may lead to an increased competence of the immune system to deal with foreign and/or tumor antigens. In other words, the viruses used according to the present invention are useful for the activation of the immune system, in general.

The invention further concerns viruses according to the present invention, in particular MVA, for the preparation of a medicament to be administered to an animal, including a human, wherein said medicament (i) increases the level of factors which activate dendritic cells; and/or (ii) increases the number of dendritic cells; and/or (iii) increases the production and/or cellular content of an interferon (IFN) or IL-12. All definitions provided above for the other embodiments are also applicable for the present embodiment. According to this embodiment, the invention does not aim primarily at inducing a protection against foreign antigens and/or tumor antigens. Instead, this embodiment is aimed at treating conditions and diseases characterized by (i) a low level of factors which activate dendritic cells; and/or (ii) insufficient or too low number of dendritic cells; and/or (iii) low production and/or cellular content of an interferon (IFN) or IL-12. Thus, according to this embodiment the treatment with viruses according to the present invention, in particular MVA, could protect against allergies or autoimmune diseases. Again, this treatment is particularly promising if the viruses according to the present invention, in particular MVA, are administered to newborn animals.

Additionally, according to a further embodiment, the virus according to the present invention, such as MVA, in particular MVA-BN and its derivatives, is particularly useful to induce immune responses in immuno-compromised animals, e.g., monkeys (CD4<400 .mu.l of blood) infected with SIV, or in immuno-compromised humans. The term "immuno-compromised" describes the status of the immune system of an individual, which shows only incomplete immune responses or has a reduced efficiency in the defense against infectious agents.

The invention further concerns a method for protecting an animal, including a human, against an antigen selected from tumor antigen and foreign antigen, by administration of a virus according to the present invention, in particular Modified Vaccinia virus Ankara (MVA), wherein the tumor antigen and/or the foreign antigen is different from the antigens associated with the virus.

In a further embodiment the invention concerns a method for the treatment of an animal, including a human, comprising the administration of a Modified Vaccinia virus Ankara (MVA) to (i) increase the level of factors which activate dendritic cells; and/or (ii) increase the number of dendritic cells; and/or (iii) increase the production and/or cellular content of an interferon (IFN) or IL-12.
 

Claim 1 of 26 Claims

1. An MVA derived vaccinia virus characterized by (i) being capable of reproductive replication in chicken embryo fibroblasts (CEF) and the Baby hamster kidney cell line BHK but not capable of reproductive replication in the human cell line HaCaT, and (ii) by a failure to replicate in a mouse strain that is incapable of producing mature B and T cells and as such is severely immune compromised and highly susceptible to a replicating virus, wherein the virus induces a general immune stimulation and/or an immune response directed against foreign antigens and/or an immune response against antigens associated with the virus when administered to a neonatal animal, including a human.
 

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