U.S. patent application number 11/341955 was filed with the patent office on 2006-06-15 for modified vaccinia virus ankara for the vaccination of neonates.
This patent application is currently assigned to BAVARIAN NORDIC A/S. Invention is credited to Mathias Ackermann, Paul Chaplin, Marco Franchini, Hans Peter Hefti, Mark Suter, Sabine Vollstedt.
Application Number | 20060127984 11/341955 |
Document ID | / |
Family ID | 29585603 |
Filed Date | 2006-06-15 |
United States Patent
Application |
20060127984 |
Kind Code |
A1 |
Ackermann; Mathias ; et
al. |
June 15, 2006 |
Modified Vaccinia virus Ankara for the vaccination of neonates
Abstract
The invention concern the use of a virus for the preparation of
a medicament for the vaccination or treatment 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 to the same virus group
than the virus used for vaccination. Moreover, the invention
concerns the vaccination of neonates against antigens selected from
foreign antigens and tumour antigens, wherein the tumour 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 increase the level of factors which
activate dendritic cells or their precursor cells and/or to
increase the number of dendritic cells or their precursor cells
and/or to increase the production and/or cellular content of an
interferon (IFN) or IL-12.
Inventors: |
Ackermann; Mathias;
(Schwerzenbach, CH) ; Suter; Mark; (Luzern,
CH) ; Hefti; Hans Peter; (Zurich, CH) ;
Franchini; Marco; (Suhr, CH) ; Vollstedt; Sabine;
(Zurich, CH) ; Chaplin; Paul; (Munchen,
DE) |
Correspondence
Address: |
THE FIRM OF HUESCHEN AND SAGE
SEVENTH FLOOR, KALAMAZOO BUILDING
107 WEST MICHIGAN AVENUE
KALAMAZOO
MI
49007
US
|
Assignee: |
BAVARIAN NORDIC A/S
KVISTGAARD
DK
|
Family ID: |
29585603 |
Appl. No.: |
11/341955 |
Filed: |
January 27, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10418854 |
Apr 18, 2003 |
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11341955 |
Jan 27, 2006 |
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PCT/EP01/13628 |
Nov 11, 2001 |
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10418854 |
Apr 18, 2003 |
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Current U.S.
Class: |
435/69.1 ;
435/6.13 |
Current CPC
Class: |
C12N 15/86 20130101;
A61K 39/245 20130101; C12N 7/00 20130101; C12N 2710/24134 20130101;
A61K 2039/545 20130101; A61K 45/06 20130101; A61K 39/12 20130101;
C12N 2710/16643 20130101; A61K 2039/55 20130101; A61K 48/00
20130101; A61K 2039/5254 20130101; A61K 39/285 20130101; C12N
2710/24143 20130101 |
Class at
Publication: |
435/069.1 ;
435/006 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12P 21/06 20060101 C12P021/06 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 19, 2002 |
DK |
PA 2002 00590 |
Nov 23, 2000 |
DK |
PA 2000 01764 |
Claims
1. An MVA virus characterized by (i) being capable of reproductive
replication in vitro in chicken embryo fibroblasts (CEF) and by
being non-replicative in vitro in human cells, 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, for
administration to induce a general immune stimulation in an animal,
including a human, in need thereof.
2. The MVA virus of claim 1, wherein the human cells are the human
keratinocyte cell line HaCaT, the human embryo kidney cell line
293, the human bone osteosarcoma cell line (143 B), and the human
cervix adenocarcinoma cell line HeLa.
3. The MVA virus of claim 1, which is MVA-BN deposited at the
European Collection of Cell Cultures (ECACC), Salisbury (UK) under
number V00083008 or derivatives thereof exhibiting the same
replication characteristics.
4. The MVA virus of claim 1, wherein administration to the animal,
including the human, in need of general immune stimulation,
increases (i) the level of factors which activate and/or mobilize
dendritic cells or their precursor cells, (ii) the number of
dendritic cells or their precursor cells, and/or (iii) the
production and/or cellular content of an interferon (IFN) or
IL-12.
5. The MVA virus of claim 1, wherein the general immune stimulation
is characterized by stimulating a cell-mediated immune
response.
6. The MVA virus of claim 1, wherein the general immune stimulation
is characterized by an increase in the number of dendritic cells
and their precursor cells.
7. The MVA virus of claim 6, wherein the precursors of dendritic
cells are plasmacytoid dendritic cell precursors.
8. The MVA virus of claim 1, wherein the general immune stimulation
is characterized as an increase in the number of T lymphocytes.
9. The MVA virus of claim 8, wherein the T lymphocytes comprise
CD8+T cells and/or CD4+T helper cells.
10. The MVA virus of claim 1, wherein the general immune
stimulation is characterized by an at least 1.5 fold increase in
the concentration of Flt3-L two days after administration of the
virus.
11. The MVA virus of claim 1, wherein the animal, including the
human, in need of general immune stimulation is receiving antiviral
therapy.
12. The MVA virus of claim 11, wherein the antiviral therapy is for
the treatment of a virus belonging to the family Retroviridae.
13. The MVA virus of claim 12, wherein the virus belonging to the
family Retroviridae is selected from HIV-1, HIV-2, SIV,
Simian-Human immunodeficiency virus, Feline immunodeficiency virus,
Bovine immunodeficiency virus and Equine immunodeficiency
virus.
14. The MVA virus of claim 11, wherein the antiviral therapy
eliminates and/or suppresses a viral infection.
15. The MVA virus of claim 11, wherein the antiviral therapy
includes administration of nucleoside analogs, inhibitors of viral
enzymatic activity, inhibitors of viral assembly, cytokines and/or
a combination thereof.
16. The MVA virus of claim 12, wherein the antiviral therapy is
highly active antiretroviral therapy (HAART).
17. The MVA virus of claim 1, wherein the animal in need of general
immune stimulation is a human with an HIV-induced immune
deficiency.
18. The MVA virus of claim 1, wherein the animal, including the
human, in need of general immune stimulation, has a condition
linked to low CD4+levels selected from breast cancer, chronic
lymphatic leukaemia (CLL), HIV infection, HIV infection with an
opportunistic infection, Kaposi's sarcoma, and chemotherapy-induced
lymphopenia.
19. A method of inducing a general immune stimulation in an animal,
including a human, in need of general immune stimulation,
comprising the step of administering to the animal, including the
human, an effective amount of the MVA virus of claim 1.
20. The method of claim 19, wherein the MVA virus is administered
by oral, nasal, intramuscular, intravenous, intraperitoneal,
intradermal, intra-utero and/or subcutaneous application.
21. The method of claim 19, wherein the MVA virus is administered
to the animal, including the human, in need of general immune
stimulation in an amount of at least 1 01 TCID.sub.50 (tissue
culture infectious dose).
22. The method of claim 19, wherein administration to the animal,
including the human, in need of general immune stimulation,
increases (i) the level of factors which activate and/or mobilize
dendritic cells or their precursor cells, (ii) the number of
dendritic cells or their precursor cells, and/or (iii) the
production and/or cellular content of an interferon (IFN) or
IL-12.
23. The method of claim 19, wherein the general immune stimulation
is characterized by stimulating a cell-mediated immune
response.
24. The method of claim 19, wherein the general immune stimulation
is characterized by an increase in the number of dendritic cells
and their precursor cells.
25. The method of claim 24, wherein the precursors of dendritic
cells are plasmacytoid dendritic cell precursors.
26. The method of claim 19, wherein the general immune stimulation
is characterized by an at least 1.5 fold increase in the
concentration of Flt3-L two days after administration of the
virus.
27. The method of claim 19, wherein the general immune stimulation
is characterized as an increase in the number of T lymphocytes.
28. The method of claim 27, wherein the T lymphocytes comprise
CD8+T cells and/or CD4+T helper cells
29. The method of claim 19, wherein the animal, including the
human, is receiving antiviral therapy.
30. The method of claim 29, wherein the antiviral therapy is for
the treatment of a virus belonging to the family Retroviridae.
31. The method of claim 30, wherein the virus belonging to the
family Retroviridae is selected from HIV-1, HIV-2, SIV,
Simian-Human immunodeficiency virus, Feline immunodeficiency virus,
Bovine immunodeficiency virus and Equine immunodeficiency
virus.
32. The method of claim 29, wherein the antiviral therapy
eliminates and/or suppresses a viral infection.
33. The method of claim 29, wherein the antiviral therapy includes
administration of nucleoside analogs, inhibitors of viral enzymatic
activity, inhibitors of viral assembly, cytokines, and/or a
combination thereof.
34. The method of claim 30, wherein the antiviral therapy is highly
active antiretroviral therapy (HAART).
35. The method of claim 19, wherein the animal in need of general
immune stimulation is a human with an HIV-induced immune
deficiency.
36. The method of claim 19, wherein the animal, including the
human, in need of general immune stimulation, has a condition
linked to low CD4+levels selected from breast cancer, chronic
lymphatic leukaemia (CLL), HIV infection, HIV infection with an
opportunistic infection, Kaposi's sarcoma, and chemotherapy-induced
lymphopenia.
37. A method of inducing a general immune stimulation in an animal,
including a human, in need of general immune stimulation,
comprising the step of administering to the animal, including the
human, an effective amount of an MVA strain, wherein the general
immune stimulation is characterized as an increase in the number of
T lymphocytes.
38. The method of claim 37, wherein the MVA strain is selected from
MVA-575 (ECACC V00120707), MVA-572 (ECACC V94012707) and the MVA
virus of claim 1.
39. The method of claim 37, wherein the T lymphocytes comprise
CD8+T cells and/or CD4+T helper cells.
40. The method of claim 37, wherein the animal, including the
human, is receiving antiviral therapy.
41. The method of claim 40, wherein the antiviral therapy is for
the treatment of a virus belonging to the family Retroviridae.
42. The method of claim 41, wherein the virus belonging to the
family Retroviridae is selected from HIV-1, HIV-2, SIV,
Simian-Human immunodeficiency virus, Feline immunodeficiency virus,
Bovine immunodeficiency virus and Equine immunodeficiency
virus.
43. The method of claim 40, wherein the antiviral therapy
eliminates and/or suppresses a viral infection.
44. The method of claim 40, wherein the antiviral therapy includes
administration of nucleoside analogs, inhibitors of viral enzymatic
activity, inhibitors of viral assembly, cytokines, and/or a
combination thereof.
45. The method of claim 41, wherein the antiviral therapy is highly
active antiretroviral therapy (HAART).
46. The method of claim 37, wherein the animal in need of general
immune stimulation is a human with an HIV-induced immune
deficiency.
47. The method of claim 37, wherein the animal, including the
human, in need of general immune stimulation, has a condition
linked to low CD4+levels selected from breast cancer, chronic
lymphatic leukaemia (CLL), HIV infection, HIV infection with an
opportunistic infection, Kaposi's sarcoma, and chemotherapy-induced
lymphopenia.
Description
[0001] The invention concern thes use of a virus for the
preparation of a medicament for the vaccination or treatment 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.
[0002] In particular, the invention concerns the vaccination of
neonates against infections with viruses belonging to the same
virus group than the virus used for vaccination. Moreover, the
invention concerns the vaccination of neonates against antigens
selected from foreign antigens and tumour antigens, wherein the
tumour 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 increase the level of
factors which activate dendritic cells or their precursor cells
and/or to increase the number of dendritic cells or their precursor
cells and/or to increase the production and/or cellular content of
an interferon (IFN) or IL-12.
BACKGROUND OF THE INVENTION
[0003] The natural environment of animals and human beings contains
a large variety of infectious agents such as viruses, bacteria or
fungi. Many of these infectious agents may cause diseases in the
infected hosts. Under normal circumstances the infected host
recovers from the disease induced by the infectious agent after a
certain period of time. This recovery is due to the immune system
of an animal or a human being.
[0004] The immune system is the part of the human or animal body
that is responsible for eliminating the infectious agent. The
immune response is divided into a specific and an unspecific
(innate) reaction although both cooperate closely. The unspecific
immune response is the immediate defence against a wide variety of
foreign substances and infectious agents. In the innate immune
response against viruses, Interferon (IFN)-.alpha. and IFN-.beta.
are absolutely essential to control the initial virus replication
and to activate natural killer (NK) cells for immediate killing of
infected cells. Intracellular bacterial or parasitic pathogens
induce IL-12 that up regulates IFN-.gamma. in NK cells and/or some
T cell subsets. IFN-.gamma. activated NK cells can now kill
intracellular pathogens. Moreover, IFN-.gamma. also activates
macrophages and enables them to kill internalized pathogens.
[0005] By far the richest source of IFN-.alpha./.beta. on a per
cell basis are dendritic cells (DC), a specialized cell population
strategically distributed throughout the body. Plasmacytoid DC or
CD11c.sup.+ CD8.sup.+ DC are among the best producers of
IFN-.alpha./.beta.. CD8.sup.+DC that are infected with
intracellular non-viral pathogens are the crucial cells able to
secrete IL-12 essential for the early steps in immune defense.
[0006] A specific immune response can be induced against a
particular foreign substance (antigen) after a lag phase, when the
organism is challenged with this substance for the first time. The
initiation of the specific immune response is coordinated by DC,
too. There is a constant traffic of these cells from the periphery
to the secondary lymphoid organs, the lymph nodes or spleen where
naive T and B cells recirculate. Antigen that is carried by DC to
these organs enables activation of naive T- and B cells to become
effector T- and B cells. For this, DC not only carry the antigen,
but the plasticity of pathogen recognition allows different gene
activation in DC and thus a pathogen adjusted priming of T
cells.
[0007] The specific immune response is highly efficient and is
responsible for the fact that an individual who recovers from a
specific infection is protected against this specific infection.
Thus, a second infection with the same or a very similar infectious
agent causes much milder symptoms or no symptoms at all, since
there is already a "pre-existing specific immunity" to this agent.
Such immunity and the immunological memory, respectively, persists
for a long time, in some cases even lifelong. Accordingly, the
induction of an immunological memory can be used for vaccination,
i.e. to protect an individual against infection with a specific
pathogen.
[0008] For vaccination the immune system is challenged with a
vaccine which itself is less harmful than the pathogenic agent
against which an immune response is to be induced. The vaccine
comprises or expresses epitopes that are found in or expressed by
the agent against which the vaccination is done. The organism,
thus, is immunized against the agent containing the epitope that is
part of the vaccine.
[0009] Typical vaccines are attenuated or inactivated viruses (e.g.
the polio or small poxvirus vaccines), recombinant proteins (e.g.
recombinant Hepatitis B virus S-protein), heat inactivated
bacterial toxins (Clostridium tetani toxin) or polysaccharides of
the bacterial capsule wall (Streptococcus pneumoniae.
[0010] Since infectious diseases might lead to very critical
conditions in newborns and sucklings, there is an interest to
vaccinate children or newborn animals as early as possible.
Examples for conditions against which a vaccination is desirable
are poxvirus infections, including smallpox. However, the attempts
to successfully vaccinate newborns are hampered by the fact that
the immune system of newborn mammals is not yet mature. The immune
system of neonatal infants and mammalian animals is thought to
mature gradually over a certain period of time. For humans the
maturation occurs during the first year of life. This is the reason
for the fact that the neonatal age group is left open to various
infections during this first year (Gans et al., J. Am. Med. Assoc.
(1998) 280, 527-532). More particularly, the neonatal infants have
impaired B-cell function, deficiencies in primary antigen
presentation by dendritic cells and limited T-cell proliferation
(Gans et al., J. Am. Med. Assoc. (1998) 280, 527-532). Shortly
after birth the levels of T cells in the spleen are 1,000 fold
lower than in adults. In order to achieve at least a weak
immunization it was suggested to use either replicating viruses or
formulations comprising an adjuvant for immunization. However, with
replication viruses there is always the risk that the immature
immune system may become overwhelmed by virus infection or live
viral vaccines since T cells are necessary for viral clearance
(Hassett et al., J. Virol. (1997) 71, 7881-7888). Since there is a
reduced production of cytokines by Th-1 helper T cells in neonates,
the response by the infants is predominantly Th-2. Consequently,
cytotoxic T cells are not recruited and viral clearance is not
achieved.
[0011] The situation in mammalian animals is very similar to the
situation in humans, i.e. the immune system after birth is not yet
mature. In newborn mice, the number of splenic CD4+T cells is
80.000 and that of CD8+T cells 1000 fold lower than in spleens of
adults. Moreover, the Interferon (IFN) producing system is immature
in these mice. Therefore, neonatal mice are unable to efficiently
control the expansion of intracellular pathogens by IFN at the site
of infection. In addition, the low number and possibly inadequate
activation stage of immune cells are too limited to cope with the
rapidly expanding pathogens or replicating viruses used for
vaccination.
[0012] Due to the risk associated with live viral vaccines it is
not recommended to vaccinate neonatal animals, including humans,
with replicating viruses. E.g. it is recommended not to vaccinate
newborns against smallpox with the vaccinia virus strains that have
been used until the eradication of smallpox, such as strains
Elstee, Copenhagen and NYCBH. According to recent recommendations
in the USA, babies younger than 12 months of age should not get the
smallpox vaccines commercialized so far.
[0013] The vaccination of neonates with formulations comprising an
adjuvant has the disadvantage that numerous harmful substances are
introduced into the body. Thus, a vaccination in human neonates is
only done in emergency cases, e.g. in case of the Hepatitis B virus
infection.
[0014] In summary, it is to be noted that the immune system is not
mature at birth. Since the vaccination with replication competent
viruses or formulations comprising an adjuvant have significant
disadvantages, infants are not vaccinated before the age of 2
months in Germany (Empfehlung der Standigen Impfkommission STICO,
2001) or 6 weeks in the USA (ACIP "Recommended Childhood
Immunization Schedule, United States").
[0015] The delay in the development of the immune system is
compensated in part by the transfer of maternal antibodies from the
mother to the suckling during pregnancy or by breastfeeding.
However, not all infants are breasffeed due to various reasons.
Thus, there is a very critical period of time of about 6-8 weeks in
humans during which the infant having an immature and thus a not
fully functional immune system does not receive maternal antibodies
and during which a vaccination is usually not successful or too
dangerous.
[0016] The situation is very similar in mammalian animals, in
particular for economically important animals such as cows or
companion animals such as cats and dogs. To reduce costs the amount
of milk the calf receives from the mother is often drastically
reduced. Instead the calf receives a mixture of milk powder,
starter and specific concentrated feed, sometimes already in the
first week after birth. Consequently, the calf does not receive the
necessary amount and variety of maternal antibodies so that the
immature immune system is very susceptible to infections.
Furthermore, farmers who breed calves and those who raise them for
meat production are often not the same. At 4 to 6 weeks of age
calves from different breeder farms are pooled and shipped to other
farms for meat production. At this time maternal antibodies are low
and the immune system is not fully developed but the animals are
exposed to new infectious agents under stress conditions. This
increases the risk for infections that could be prevented by
vaccination. A similar situation can be found in catteries or dog
breeding facilities where the infectious pressure is high.
OBJECT OF THE INVENTION
[0017] It is the object of the present invention to provide means
to vaccinate newborn humans and animals, respectively, against
foreign antigens and antigens that are associated with diseases in
humans and animals, respectively. More particularly, it is the
object of the present invention to provide means allowing the
accelerated maturation of the immune system of newborn animals and
humans. It is a further object of the present invention to provide
means that allow vaccinating neonatal animals, including humans,
against poxvirus infections, in particular against smallpox.
DETAILED DESCRIPTION OF THE INVENTION
[0018] 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 in
said cells to infectious progeny virus. 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 tumour
antigens as explained below in more 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. The vaccination with the viruses used
according to the present invention is possible even if the
formulation that is administered to the animal does not comprise an
adjuvant.
[0019] In summary, the viruses that are 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.
[0020] 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.
[0021] Viruses that are "capable of infecting cells" are viruses
harbouring on the viral surface structures capable of interacting
with the 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 the genome of which 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 be nonpermissive for
multiplication of the virus, e.g. due to the fact that not all
necessary viral genes for multiplication of the virus in said cell
are expressed and/or present in the viral genome. An example for
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 for such a virus according to the present invention for
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 at all in the cells of the vaccinated animal.
However, also those viruses are within the scope of the present
application that show a minor residual replication activity that is
controlled by the immature immune system of the neonate.
[0022] 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 that is 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. E.g., for humans MVA-BN and
its derivatives (see below) are viruses that are capable of
infecting cells of the human but that are not capable of being
replicated to infectious progeny virus in human cells. The same
viruses are very efficiently replicated in chickens, i.e. in
chicken MVA-BN is not a virus that is capable of infecting cells of
the chicken but not capable of being replicated to infectious
progeny virus in said cells. It is known to the person skilled in
the art which virus has to be chosen for a specific animal species.
A test that allows to determine whether a virus is capable or not
capable of being replicated in a neonatal or prenatal animal is
disclosed in WO 02/42480 and uses the AGR129 mice strain. The
results obtained in this mice 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 cells 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 CEF cells but that is a virus that 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 the properties of
this preferred embodiment since inactivated viruses are capable of
infecting the 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, but
these viruses are not capable of replicating in at least one type
of cells of at least one animal species.
[0023] 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 Vaccinia virus Ankara
(MVA).
[0024] Modified Vaccinia Ankara (MVA) virus is related to Vaccinia
virus, a member of the genera Orthopoxvirus in the family of
Poxviridae. MVA has been generated by 516 serial passages on
chicken embryo fibroblasts of the Ankara strain of vaccinia virus
(CVA) (for review see Mayr, A., et aL Infection 3, 6-14 [1975]). 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 host cell restricted to avian 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 vaccine to immunize against the human smallpox disease
(Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167, 375-390 [1987],
Stickl etal., 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
immunogenicity.
[0025] Preferred strains according to the present invention are MVA
575, deposited at the European Collection of Animal Cell Cultures
(ECACC) with the deposition number V00120707 and MVA-BN, deposited
at the same institution with the deposition number V000083008, and
derivatives thereof, in particular if it is intended to
vaccinate/treat humans. Most preferred for humans is MVA-BN and its
derivatives.
[0026] 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:
[0027] (i) capability of reproductive replication in chicken embryo
fibroblasts (CEF) and in the cell line BHK, but no capability of
reproductive replication in the human cell line HaCaT,
[0028] (ii) failure to replicate in vivo,
[0029] (iii) induction of a higher immunogenicity compared to the
known strain MVA 575 (ECACC V00120707) in a lethal challenge model
and/or
[0030] (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.
[0031] The preferred MVA strains according to the present invention
have the property (ii) failure to replicate in the organism, which
is to be vaccinated or treated and/or in the corresponding test
system as explained below and preferably one additional of the
above properties, more preferably two additional of the above
properties. Most preferred are MVA strains having all of the above
properties. An example for 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, more preferably at least two of the above
properties. Most preferred are MVA-BN derivatives having all of the
above properties.
[0032] For detailed information regarding to the assays used to
determine whether a MVA strain has one or more of the above
features (i) to (iv) reference is made to WO 02/42480. This
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 of a derivative
of MVA-BN and discloses in detail the biological assays that are
used to determine whether an MVA strain is 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 allowing to obtain
MVA-BN or a derivative thereof are disclosed in WO 02/42480. In the
following it is shortly summarized how a person skilled in the art
arrives in MVA strains having one or more of the above features and
how he can test whether a given MVA strain has one or more of said
features and is thus a most preferred virus according to the
present invention. The following summary is not to be understood as
to limit the relevance of WO 02/42480 for the present application
to the following information. Instead, WO 02/42480 is herewith
incorporated in its entirety by reference.
[0033] 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) ("amplification
ratio"). 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" than the deposited
virus are viruses that replicate with similar amplification ratios
than the deposited strain in CEF cells and the cell lines BHK,
HeLa, HaCaT and 143B and that show a similar replication in vivo as
determined in the AGR129 transgenic mouse model (see below).
[0034] The term "failure to replicate in vivo" is used in the
present application as defined in WO 02/42480. Thus, said term
refers to viruses that do not replicate in humans and in the mice
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, most preferably within 90 days after the infection
of the mice with 10.sup.7 pfu virus administered intra peritonealy.
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 the infection of the mice with
10.sup.7 pfu virus administered intra peritonealy. Instead of the
AGR129 mice any other mouse strain can be used that is incapable of
producing mature B and T cells and as such is severely immune
compromised and highly susceptible to a replicating virus.
[0035] 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 the
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 the vaccination with
10.sup.2 TCID.sub.50/ml virus the ovary virus titers are reduced by
at least 70%, preferably by at least 80%, more preferably by at
least 90% compared to unvaccinated mice.
[0036] 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 harbouring heterologous
sequences, can be used to efficiently first prime and then boost
immune responses in native animals as well as in animals with a
pre-existing immunity to poxviruses. 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.
[0037] A vaccinia virus, in particular an 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 the "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.
[0038] 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 for a
non-recombinant vaccinia virus is MVA-BN and its derivatives.
Alternatively the virus may be a recombinant virus, such as a
recombinant MVA that contains additional nucleotide sequences,
which are heterologous to the virus.
[0039] The term "heterologous" as used in the present application
refers to any combination of nucleic acid sequences that is not
normally found intimately associated with the virus in nature, such
virus is also called "recombinant virus".
[0040] 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.
[0041] 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 tumour antigen and
foreign antigen, wherein the tumour antigen and the foreign antigen
is different from the antigens associated with the virus.
Alternatively and more particulary the "beneficial proteins" are
active in increasing the level of factors which activate dendritic
cells and/or active in increasing the number of dendritic cells
and/or active in increasing the production and/or cellular content
of an interferon (IFN) or IL-12. Thus, examples for such beneficial
proteins are interferons such as IFN-alpha or IFN-beta, IL-12,
Flt-3-L and or GM-CSF.
[0042] The antigenic epitopes may be any epitopes to which it makes
sense to induce an immune response. Examples for antigenic epitopes
are epitopes from Plasmodium falciparum, Mycobacteria, Influenza
virus, from viruses selected of the family of Flaviviruses,
Paramyxoviruses, Hepatitis viruses, Human immunodeficiency viruses
or from 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.
[0043] 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.
[0044] The insertion of 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 how to insert heterologous
sequences into the viral genome such as a poxviral genome are known
to a person skilled in the art.
[0045] 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.
[0046] 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.
[0047] For the preparation of vaccines, the virus or its
recombinants is converted into a physiologically acceptable form.
Such methods are known to the person skilled in the art. For MVA
and other poxviruses the vaccine can be prepared based on the
experience in the preparation of poxvirus 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, the vaccine shots can be produced by stepwise
freeze-drying of the virus in a formulation. This formulation can
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.
[0048] 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 parenterally, intramuscularly or any other path of
administration know 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.
[0049] 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 performed parenterally or nasaly, whereas in larger
animals or humans a subcutaneous, intramuscular or oral inoculation
is preferred.
[0050] 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.
[0051] As pointed out 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").
[0052] In the context of the present invention the term "animal"
covers also human beings. More generally, the animal is a
vertebrate animal, preferably a mammalian animal including a human.
Specific examples for animals are pets such as dogs, cats,
economically important animals such as calves, cattle, sheep,
goats, horses, pigs and other animal such as mice, rats. For these
animal species and for humans MVA and DISC-HSV are particularly
preferred viruses. The invention may also be used for economically
important birds such as turkeys, ducks, goose and hens if viruses
are used that are capable to infect the bird's cells but not
capable of being replicated to infectious progeny virus in said
cells.
[0053] 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, deer.
[0054] 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
than 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 poxvirus infections, in
particular against smallpox. MVA, in particular MVA-BN and its
derivatives, may also be used to vaccinate vertebrate animals
against poxvirus 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. Examples for this kind of genes are cytokine genes and
interferon genes.
[0055] 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 for 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)
tumour antigens, (iii) bacteria, (iv) fungi. If the antigen
expressed from the recombinant virus is e.g. an 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.
[0056] According to a third alternative it has been unexpectedly
found that viruses that are 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 tumour
antigen and foreign antigen, wherein the tumour antigen and/or the
foreign antigen are different from the antigens associated with the
virus.
[0057] 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, i.e. 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 in the
critical time span until a functional and mature immune system is
established.
[0058] According to the present invention "the tumour 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 a
immune response or at least a general immune stimulation that
protects the host against foreign antigens and tumour antigens,
respectively, 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.
[0059] In the context of this embodiment the term "foreign
antigens" refers to any 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,
adenoviruses; bacteria such as Salmonella, Mycoplasm,
Meningicoccus, Hemophilus; prions or fungi.
[0060] The invention is not only of interest to vaccinate animals
against foreign antigens but, in an alternative embodiment, is also
suitable to vaccinate against tumour antigens. "Tumour antigens"
are antigens associated with certain tumoural diseases. Tumour
antigens are most often antigens encoded by the genome of the host
that develops the tumour. Thus, in a strict sense tumour antigens
are not foreign antigens. However, tumour antigens are found in
significant amounts in tumours, whereas the amount of tumour
antigens in normal tissues is significantly lower and most often no
tumour antigens are found at all in normal tissue. Examples for
tumour antigens are known to the person skilled in the art and
include e.g. the MAGE antigens. MVA is effective against these
tumour antigens since the vaccination of animals leads to an
activation and/or accelerated maturation of the immune system which
then may lead to the destruction of tumour cells.
[0061] The term "protecting against an antigen" refers to the
development of an immune response, which is directed against the
foreign or tumour antigen. If the foreign antigen is an infectious
agent the host is protected against said agent, i.e. the host
develops an immune response against said antigen. Consequently, the
infection with the infectious agent leads to a less severe disease
or to 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 tumour antigen. Instead, the term
"protection" as used in the present application refers to any
beneficial effect that helps the animal to deal with the foreign
antigen and the tumour antigen, respectively.
[0062] 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 said antigen after 5, 7, 14
and 28 days, respectively.
[0063] 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 the maturation
of the immune system" refers inter alia to the accelerated increase
of dendritic cells or their precursors in vaccinees relative to
controls. The terms "acceleration of the maturation" of the immune
system an "enhancement of the maturation" of the immune system are
used interchangeably in this description.
[0064] The "activation of the immune system" is characterized by
the expression on the surface of cells of molecules and hormones
that ease cell/cell interaction or trafficking and/or by the
secretion of said molecules and hormones by the cells. Specific
receptors take up these signals and respond. Activation markers are
inter alia Flt3-L, IL-12, IFN-alpha, MHC-II and CD8, in particular
CD8alpha (see below).
[0065] The accelerated development/maturation of the immune system
is correlated with an increase of the level of factors activating
and or mobilizing dendritic cells (DC) or their precursor cells
and/or an increase in the number of dendritic cells and their
precursor cells and/or an increase in the production and/or
cellular content of an interferon or IL-12. An example for DC
precursor cells that are induced by the virus according to the
present invention, in particular by MVA, are plasmacytoid DC
precursors that are very important for the defence against viral
infections and that seem to produce IFN .alpha./.beta..
[0066] More specifically, the enhancement of the maturation of the
immune system is preferably defined by an at least 2-fold increase
in surface markers found on DC, such as MHC-II, CD40 and/or
CD80/86. Preferably such an increase may be measured in the blood.
Further markers to characterize an enhancement of the maturation of
the immune system are Flt3-L, IL-12, IFN-alpha, MHC-II and CD8a
(see below). Moreover, the accelerated maturation of the immune
system is preferably correlated to an at least 1.5 fold increase,
preferably an at least 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 compared to control
animals that have not received MVA-BN. Moreover, the enhancement of
maturation of the immune system may preferably be correlated with
an at least 1.5 fold increase, more preferably an at least 2.0 fold
increase of 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.
[0067] 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 characterised by their surface phenotype
(Hochrein et al. 2002. Hum. Immunol. 63: 1103). DC in the blood can
be detected using flow cytometry using a range of surface markers
(MacDonald et al. 2002. Blood. 100:4512) that also allow specific
populations of DC, such as the plasmactoid 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).
[0068] According to the present invention the viruses as defined
above might also be used to treat neonatal or prenatal animals to
increase the level of factors activating and or mobilizing
dendritic cells (DC) or their precursor cells and/or an increase in
the number of dendritic cells and their precursor cells and/or an
increase in the production and/or cellular content of an interferon
or IL-1 2. It has been demonstrated that following vaccination with
MVA-BN the plasmacytoid dendritic cells make significantly more
IL-12 and have an increased IFN-alpha production and upregulation
of MHC-II and CD8a. The increase of IL-12 after the administration
of the viruses used according to the present invention is
preferably 2 times, more preferably 100 times, 500 times, 1000
times, 2500 times or 5000 times. The increase of the concentration
of FIt3-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.
[0069] 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 stimuli. The
intermediates of DC are termed precursors. These immature DC reach
the periphery. Different (antigenic) stimuli activate DC.
Activation markers, which are upregulated in activated dendritic
cells are inter alia Flt3-L, IL-12, IFN-alpha, MHC-II and CD8a (see
below).
[0070] 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.
[0071] Consequently, the vaccination of animals including a human
is especially useful, if it is intended to increase the level of
factors activating dendritic cells (DC) or their precursor cells
and/or to increase the number of dendritic cells or their precursor
cells and/or to increase the production and/or cellular content of
an interferon or IL-12.
[0072] Factors that activate dendritic cells comprise inter alia
Flt3-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.
[0073] In the context of the present application a newborn animal
or human is defined as a 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 an IFN production which is low
compared to adults and by the fact that the secondary lymphoid
organs 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,
most preferably at least 20,000 fold lower than the average number
of splenic CD4+cells in adults.
[0074] In mice the immune system is mature at the age of 4 weeks.
In humans maturity is probably 6 month to 1 year. In cats and dogs
the immune system is mature usually at the age of 6 month, in
calves, sheep and pigs at the age of 4-12 weeks. The vaccination
with the virus according to the present invention, in particular
with MVA is preferably done during 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 not 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
preferably within 4 weeks after birth, more preferably within 2
weeks after birth, even more preferably within 1 week after birth,
most preferably within 3 days after birth. These general terms are
applicable to all important domestic animals, in particular to all
important domestic mammalian animals, including humans. The 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 that, thus, the 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, most preferably within 3 days after birth.
[0075] 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 might 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 lets through the
virus the prenate 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 having a placenta heamochorialis, such as primates including
humans. In animals having a placenta chorionepithelialis, such as
cattle and sheep or 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.
[0076] Since the viruses according to the present invention, in
particular MVA lead to an accelerated maturation of the immune
system and since the viruses according to the present invention, in
particular MVA 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,
especially in fattening pigs.
[0077] As pointed out it is a preferred embodiment to use the
viruses according to the present invention, in particular MVA, in
newborns or prenatal animals to protect said animal against a
foreign antigen and/or a tumour antigen, wherein the tumour 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 tumour
antigen and foreign antigen, wherein the tumour antigen and/or the
foreign antigen is different from the antigens associated with the
virus. As pointed out 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.
[0078] In contrast to newborns, the immune system of adult animals
has already matured. Nevertheless, it might be that the immune
system is weakened due to certain diseases or simply due to the age
of the animal. Especially in immune-compromised people and in
elderly people the administration of the viruses according to the
present invention, in particular MVA to the animal may have a
beneficial effect inter alia by increasing the level of factors
activating and/or mobilizing dendritic cells (DC) or their
precursor cells and/or by increasing the number of dendritic cells
or their precursor cells and/or 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 antigens and/or tumour
antigens. In other words, the viruses used according to the present
invention are useful for the activation of the immune system in
general.
[0079] The invention further concerns the 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 increases the level of factors which
activate dendritic cells and/or increases the number of dendritic
cells and/or increases the production and/or cellular content of an
interferon (IFN) or IL-12. All definitions given 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 tumour
antigens. Instead, this embodiment is aimed at treating conditions
and diseases characterized by a low level of factors which activate
dendritic cells and/or by a insufficient or too low number of
dendritic cells and/or by a low production and/or cellular content
of an interferon (IFN) or IL-12. Thus, according to this embodiment
the treatment with the 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.
[0080] 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
defence against infectious agents.
[0081] The invention further concerns a method for protecting an
animal, including a human, against an antigen selected from tumour
antigen and foreign antigen, by administration of a virus according
to the present invention, in particular Modified Vaccinia virus
Ankara (MVA), wherein the tumour antigen and/or the foreign antigen
is different from the antigens associated with the virus.
[0082] In a further embodiment the invention concerns a method for
the treatment of an animal, including a human, to increase the
level of factors which activate dendritic cells and/or to increase
the number of dendritic cells and/or increase the production and/or
cellular content of an interferon (IFN) or IL-12, comprising the
administration of a Modified Vaccinia virus Ankara (MVA).
SUMMARY OF THE INVENTION
[0083] The invention inter alia comprises the following, alone or
in combination:
[0084] An MVA virus characterized by
[0085] (i) being capable of reproductive replication in vitro in
chicken embryo fibroblasts (CEF) and by being non-replicative in
vitro in human cells, and
[0086] (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,
for administration to induce a general immune stimulation in an
animal, including a human, in need thereof, such a
[0087] MVA virus, wherein the human cells are the human
keratinocyte cell line HaCaT, the human embryo kidney cell line
293, the human bone osteosarcoma cell line (143 B), and the human
cervix adenocarcinoma cell line HeLa, such a
[0088] MVA virus, which is MVA-BN deposited at the European
Collection of Cell Cultures (ECACC), Salisbury (UK) under number
V00083008 or derivatives thereof exhibiting the same replication
characteristics, such a
[0089] MVA virus, wherein administration to the animal, including
the human, in need of general immune stimulation, increases (i) the
level of factors which activate and/or mobilize dendritic cells or
their precursor cells, (ii) the number of dendritic cells or their
precursor cells, and/or (iii) the production and/or cellular
content of an interferon (IFN) or IL-12, such a
[0090] MVA virus, wherein the general immune stimulation is
characterized by stimulating a cell-mediated immune response, such
a
[0091] MVA virus, wherein the general immune stimulation is
characterized by an increase in the number of dendritic cells and
their precursor cells, such a
[0092] MVA virus, wherein the precursors of dendritic cells are
plasmacytoid dendritic cell precursors, such a
[0093] MVA virus, wherein the general immune stimulation is
characterized as an increase in the number of T lymphocytes, such
a
[0094] MVA virus, wherein the T lymphocytes comprise CD8+T cells
and/or CD4+T helper cells, such a
[0095] MVA virus, wherein the general immune stimulation is
characterized by an at least 1.5 fold increase in the concentration
of Flt3-L two days after administration of the virus, such a
[0096] MVA virus, wherein the animal, including the human, in need
of general immune stimulation is receiving antiviral therapy, such
a
[0097] MVA virus, wherein the antiviral therapy is for the
treatment of a virus belonging to the family Retroviridae, such
a
[0098] MVA virus, wherein the virus belonging to the family
Retroviridae is selected from HIV-1, HIV-2, SIV, Simian-Human
immunodeficiency virus, Feline immunodeficiency virus, Bovine
immunodeficiency virus and Equine immunodeficiency virus, such
a
[0099] MVA virus, wherein the antiviral therapy eliminates and/or
suppresses a viral infection, such a
[0100] MVA virus, wherein the antiviral therapy includes
administration of nucleoside analogs, inhibitors of viral enzymatic
activity, inhibitors of viral assembly, cytokines and/or a
combination thereof, such a
[0101] MVA virus, wherein the antiviral therapy is highly active
antiretroviral therapy (HAART), such a
[0102] MVA virus, wherein the animal in need of general immune
stimulation is a human with an HIV-induced immune deficiency, such
a
[0103] MVA virus, wherein the animal, including the human, in need
of general immune stimulation, has a condition linked to low
CD4+levels selected from breast cancer, chronic lymphatic leukaemia
(CLL), HIV infection, HIV infection with an opportunistic
infection, Kaposi's sarcoma, and chemotherapy-induced lymphopenia,
such a
[0104] method of inducing a general immune stimulation in an
animal, including a human, in need of general immune stimulation,
comprising the step of administering to the animal, including the
human, an effective amount of the MVA virus, such a
[0105] method wherein the MVA virus is administered by oral, nasal,
intramuscular, intravenous, intraperitoneal, intradermal,
intra-utero and/or subcutaneous application, such a
[0106] method wherein the MVA virus is administered to the animal,
including the human, in need of general immune stimulation in an
amount of at least 10.sup.1 TCID.sub.50 (tissue culture infectious
dose), such a
[0107] method wherein administration to the animal, including the
human, in need of general immune stimulation, increases (i) the
level of factors which activate and/or mobilize dendritic cells or
their precursor cells, (ii) the number of dendritic cells or their
precursor cells, and/or (iii) the production and/or cellular
content of an interferon (IFN) or IL-12, such a
[0108] method wherein the general immune stimulation is
characterized by stimulating a cell-mediated immune response, such
a
[0109] method wherein the general immune stimulation is
characterized by an increase in the number of dendritic cells and
their precursor cells, such a
[0110] method wherein the precursors of dendritic cells are
plasmacytoid dendritic cell precursors, such a
[0111] method wherein the general immune stimulation is
characterized by an at least 1.5 fold increase in the concentration
of Flt3-L two days after administration of the virus, such a
[0112] method wherein the general immune stimulation is
characterized as an increase in the number of T lymphocytes, such
a
[0113] method wherein the T lymphocytes comprise CD8+T cells and/or
CD4+T helper cells, such a
[0114] method wherein the animal, including the human, is receiving
antiviral therapy, such a
[0115] method wherein the antiviral therapy is for the treatment of
a virus belonging to the family Retroviridae, such a
[0116] method wherein the virus belonging to the family
Retroviridae is selected from HIV-1, HIV-2, SIV, Simian-Human
immunodeficiency virus, Feline immunodeficiency virus, Bovine
immunodeficiency virus and Equine immunodeficiency virus, such
a
[0117] method wherein the antiviral therapy eliminates and/or
suppresses a viral infection, such a
[0118] method wherein the antiviral therapy includes administration
of nucleoside analogs, inhibitors of viral enzymatic activity,
inhibitors of viral assembly, cytokines, and/or a combination
thereof, such a
[0119] method wherein the antiviral therapy is highly active
antiretroviral therapy (HAART), such a
[0120] method wherein the animal in need of general immune
stimulation is a human with an HIV-induced immune deficiency, such
a
[0121] method wherein the animal, including the human, in need of
general immune stimulation, has a condition linked to low
CD4+levels selected from breast cancer, chronic lymphatic leukaemia
(CLL), HIV infection, HIV infection with an opportunistic
infection, Kaposi's sarcoma, and chemotherapy-induced lymphopenia,
such a
[0122] method of inducing a general immune stimulation in an
animal, including a human, in need of general immune stimulation,
comprising the step of administering to the animal, including the
human, an effective amount of an MVA strain, wherein the general
immune stimulation is characterized as an increase in the number of
T lymphocytes, such a
[0123] wherein the MVA strain is selected from MVA-575 (ECACC
V00120707), MVA-572 (ECACC V94012707) and the MVA virus, such a
[0124] method wherein the T lymphocytes comprise CD8+T cells and/or
CD4+T helper cells, such a
[0125] method wherein the animal, including the human, is receiving
antiviral therapy, such a
[0126] method wherein the antiviral therapy is for the treatment of
a virus belonging to the family Retroviridae, such a
[0127] method wherein the virus belonging to the family
Retroviridae is selected from HIV-1, HIV-2, SIV, Simian-Human
immunodeficiency virus, Feline immunodeficiency virus, Bovine
immunodeficiency virus and Equine immunodeficiency virus, such
a
[0128] method wherein the antiviral therapy eliminates and/or
suppresses a viral infection, such a
[0129] method wherein the antiviral therapy includes administration
of nucleoside analogs, inhibitors of viral enzymatic activity,
inhibitors of viral assembly, cytokines, and/or a combination
thereof, such a
[0130] method wherein the antiviral therapy is highly active
antiretroviral therapy (HAART), such a
[0131] method wherein the animal in need of general immune
stimulation is a human with an HIV-induced immune deficiency, such
a
[0132] method wherein the animal, including the human, in need of
general immune stimulation, has a condition linked to low
CD4+levels selected from breast cancer, chronic lymphatic leukaemia
(CLL), HIV infection, HIV infection with an opportunistic
infection, Kaposi's sarcoma, and chemotherapy-induced
lymphopenia.
SHORT DESCRIPTION OF THE FIGURES
[0133] FIG. 1A: Newborn mice were injected once within 24-48 h of
birth with 10.sup.6 p.f.u. of MVA or DISC HSV-1 or treated with
physiological saline (NaCl) as controls. At 7 days of age, CD11c, a
pan DC marker was used to determine these cells in peripheral blood
by flow cytometry. Mean and standard deviation of 3 to 5
experiments are shown.
[0134] FIG. 1B: Experiment as in FIG. 1A. However, CD11c cells were
determined in spleen by flow cytometry
[0135] FIG. 1C: Experiment as in FIG. 1A. However, CD11c cells were
determined in peritoneal fluid by flow cytometry
[0136] FIG. 2: Mice were vaccinated with MVA-BN as indicated in the
left column. After two weeks the percentage of CD11c.sup.+single
and CD11c.sup.+/CD8.sup.+double positive cells in spleen and in
blood were determined by flow cytometry.
[0137] FIG. 3: Newborn mice were injected with MVA or NaCl as
control at day one and 5 of age. At day 8, murine Flt3-L was
determined in serum of these mice by ELISA and the values are given
as pg/ml.
[0138] FIG. 4: Newborn mice were injected once within 24-48 h of
birth with 106 p.f.u. of MVA or treated with NaCl as controls. At 7
days of age, all mice were exposed to 100.times.LD.sub.50 of HSV-1
strain F. The number of surviving animals was monitored for 21
days.
[0139] FIG. 5: Mice were treated as in FIG. 4. The data represent 9
different challenge experiments with 100 LD.sub.50 of HSV-1. None
of the control animals survived the challenge.
[0140] FIG. 6: Survival of adult mice vaccinated on the first day
of life with MVA-BN following a lethal vaccinia challenge. Three
litters of 6 1-day-old pups (18 mice) were vaccinated with MVA-BN
(2.5 .times.10.sup.7 TCID.sub.50) and at 4 weeks (adult mice)
challenged with a lethal dose of vaccinia. MVA-BN vaccination
clearly induced a protective immunity in neonatal mice that lasted
until adulthood.
EXAMPLES
[0141] The following examples will further illustrate the present
invention. It will be well understood by a person skilled in the
art that the provided examples in no way may be interpreted in a
way that limits the applicability of the technology provided by the
present invention to this examples.
EXAMPLE 1
(I) MVA-BN and DISC-HSV induces DC of the CD11c.sup.+and
CD8.sup.+phenotype in newborn animals
[0142] First set of experiments: Newborn mice are naturally
immunodeficient because the IFN system is not mature. The number
and activation state of DC, the best producers of IFN know today
has not been analyzed. DC can be induced in vitro as well as in
vivo by a variety of stimuli. In these studies it was tested
whether a controlled MVA-BN replication could induce DC and
analyzed their phenotype. Groups of mice were injected with
10.sup.6 plaque forming units (p.f.u.) of MVA-BN or saline only
within 1-2 days after birth and in some cases 5 days after birth.
Blood and spleen cells from individual mice of both groups were
analyzed by FACS and the data compared.
[0143] Data from 7 to 8 individual mice indicated that treatment
with MVA-BN increased CD11c.sup.+cells 2-3 fold accompanied with
increased expression of MHC II and increased presence of T cells of
the CD4 or CD8 type Interestingly, CD19/54, a marker for mature B
cells decreased indicating that these cells emigrated in organs
other than spleen or that precursor of B cells were recruited early
to other lineages notably DC of the plasmacytoid phenotype that
carries early B cell markers (B220).
[0144] Data from three different experiments indicated
reproducibility and significant differences. Experiments with
DISC-HSV-1, a different replication controlled viral vaccine,
induces similar amounts of CD11c+cells after neonatal priming.
[0145] The results are summarized in FIG. 1A-C.
[0146] To further investigate subpopulations of DC in blood and
spleen and analyze long-term effect of treatment with MVA-BN, cells
in blood and spleen were analyzed at 2 weeks of age. At this time
point, treated animals had about twice the number of
CD11c.sup.+cells in spleen than at one week of age but a single
treatment with the virus at birth lead to a 3 fold elevated number
of these cells in spleen 2 weeks later (FIG. 2). Similar effects
were seen in blood with the exception that
CD11c.sup.+/CD8a.sup.+were about 4 times higher. A single treatment
with MVA-BN at 7 days of birth lead to an increase of
CD11c.sup.+/CD8a.sup.+from 13 to 40 fold with a less dramatic
effect on the CD11c.sup.+cells. As expected, two vaccinations at
birth and day 7 had a significant effect on CD11c.sup.+cells. The
various effects are shown in FIG. 2.
[0147] Second set of experiments: One-week-old mice that were
vaccinated at birth with 2.5.times.10.sup.7 TCID.sub.50 of MVA-BN
showed a different composition of immunologically relevant cell
populations in spleen and blood than control mice (Table 1). In
blood there was an increase in the CD8 positive lymphocyte
population as well as an increase in the number of NK cells. The
number of CD11c positive cells was about 3 times higher than in
controls and the percent of B-cells (B220 and CD19 double positive
cells) was significantly decreased. In the spleen the total number
of cells did not differ between immunised animals and controls. In
contrast to the blood, the spleen of vaccinated animals had more
CD4 positive T lymphocytes than controls and the number of NK cells
was not increased. Similar to blood the relative number of CD8
positive lymphocytes were increased and the number of B-cells
decreased. The percentage of CD11c positive cells was about 3 times
higher than in controls. We first recognised a difference in the
percentage of dendritic cells at day 5 following vaccination with
MVA-BN, when the number of CD11c positive cells in the spleen of 4
untreated controls were 3.6 %, compared to 4.8% in 4 MVA-BN
vaccinated mice. The same amount of UV-inactivated MVA-BN did not
cause any significant change in the cell populations after
vaccination of neonatal mice compared to controls (data not shown).
The initial vaccination dose was chosen arbitrarily. After
titration of the inoculum we selected a standard dose of
2.5.times.10.sup.6 TCID.sub.50 for vaccination (10 time less than
in the initial experiment). At this dose maximal numbers of DC were
induced (Table 2). TABLE-US-00001 TABLE 1 Changes induced in blood
and spleen cells in newborn mice 1 week after immunisation with 2.5
.times. 10.sup.7 TCID.sub.50 MVA-BN Blood Spleen Parameter % NaCl
MVA-BN P* NaCl MVA-BN P* Total cells .times. 10.sup.6 17.9 .+-. 1.9
24.1 .+-. 2.6 0.105 % CD11c 5.4 .+-. 1.3 18.6 .+-. 1.5 0.001 2.8
.+-. 0.1 7.9 .+-. 0.8 0.001 % CD11c/CD8.alpha. 0.5 .+-. 0.1 2.7
.+-. 0.3 0.001 1.1 .+-. 0.1 4.6 .+-. 0.7 0.002 % CD4/CD3 16.9 .+-.
1.1 16.1 .+-. 1.5 0.999 4.8 .+-. 0.3 8.1 .+-. 1.5 0.004 %
CD8.alpha./CD3 6.0 .+-. 0.9 10.3 .+-. 0.9 0.002 4.7 .+-. 0.3 8.4
.+-. 1.1 0.002 % NK1.1/DX5 16.4 .+-. 1.2 24.4 .+-. 3.3 0.032 2.5
.+-. 0.3 2.4 .+-. 0.2 0.862 % CD19/B220 22.3 .+-. 0.5 8.4 .+-. 0.8
0.001 16.2 .+-. 1.3 8.6 .+-. 0.9 0.004 *Mann-Whitney U-Test
[0148] TABLE-US-00002 TABLE 2 Induction of CD11c positive cells in
the spleen of 1-ay-old wt mice and mice with gene-targeted
disruptions within 7 days after MVA-treatment. Mouse MVA dose
controls MVA-BN strain (TCID.sub.50) % CD11c % CD11c ratio wt.sup.a
2.5 .times. 10.sup.7 2.8 7.9 2.8 wt 2.5 .times. 10.sup.6 2.1 11.9
5.6 wt 2.5 .times. 10.sup.5 2.5 6.6 2.6 RAG.sup.b 2.5 .times.
10.sup.7 4.2 5.4 1.3 AG129.sup.c 2.5 .times. 10.sup.3 2.6 2.7 1.0
.sup.aWt = either C57BL/6 or 129 Sv/Ev mice. .sup.bRAG mice
deletion in recombination activating gene (i.e no functional T and
B cells). .sup.cAG129 gene targeted disruptions of IFN receptor
Type I (IFN-.alpha. and -.beta.) and Type II (IFN-.gamma.)
[0149] (ii) MVA-BN induces preferentially plasmacytoid dendritic
cells (pDC).
[0150] According to other authors CD1 1c positive cells that also
expressed CD45RA or CD45R were considered as pDC (Asselin-Paturel,
et al. 2001, Nat Immunol, 12: 1144). It was asked whether MVA-BN
induced an increase of pDC. A further experiment was performed in
which also CD45RA or CD45R on CD11 c positive were analysed. The
percentage of CD11c and CD45R double positive cells was
significantly higher in MVA-BN treated mice (5.6.+-.0.7%) than in
both control groups (untreated 3.0.+-.0.3%, p=0.01; UV-inactivated
MVA-BN 3.0.+-.0.2%, p=0.006. Mann-Whitney U-test).
[0151] (iii) Neonatal mice treated with MVA-BN have elevated levels
of serum Flt3-L
[0152] Flt3-L is a hematopoetic factor that leads to increased
levels of DC in adult animals. In human and possibly mice the
richest source of this factor are activated T cells. To determine
whether the elevated numbers of DC could be the results of induced
Flt3-L, serum of MVA-BN treated mice was compared to mock treated
animals for the presence of this factor. Animals treated at day 2
and 5 had twice the levels of Flt3-L in the serum when compared to
serum of mock treated animals. Hence, Flt3-L is one of the factors
that could be made responsible for elevated numbers of DC (FIG.
3)
[0153] The time course of the Flt3-L induction in newborn mice was
assessed after administration of MVA-BN. In newborns, MVA-BN
vaccination induced an increase in Flt3-L concentration within 24
hours. The induction reached a maximum after 48 hours and was still
present at day 7, the time when spleen cells were usually analyzed
and resistance against HSV-1 was tested (see below). In the
vaccinated mice the Flt3-L concentration in the serum was two-fold
increased 24 hours and 48 hours after the vaccination, compared
with age matched control animals.
EXAMPLE 2
[0154] (i) MVA-BN treated neonatal mice survive a challenge with
100 to 500 LD50 of HSV-1
[0155] Groups of mice were treated with the standard dose of MVA-BN
one or 2 days after birth and challenged at 7-8 days of age with
100 to 500 LD.sub.50 of Herpes simplex virus 1 (HSV-1) (FIG. 4).
MVA BN treated mice survived the challenge with HSV 1, whereas all
the control mice died within 5-6 days after inoculating the
challenge virus.
[0156] To further support these observations, 9 challenge
experiments were performed with 40 MVA BN treated and 45 control
mice. More than 80 % of the virus treated mice survived the
challenge, whereas all the control mice died (FIG. 5).
[0157] In a separate set of experiments the mice were treated at
birth with MVA-BN (2.5.times.10.sup.6 TCID.sub.50 mouse). At day 8
a challenge with either 10.sup.3 (1 LD.sub.50) or 10.sup.5 (100
LD.sub.50) PFU of HSV-1 was performed. Following MVA-BN vaccination
65% of the mice survived a viral dose that killed 100% of the
control mice (100 LD.sub.50) and 90% survived a dose that killed
45.5% of the controls (1 LD.sub.50). In additional experiments a
group of 7 mice vaccinated with UV-inactivated MVA-BN were infected
with HSV-1. Five of them died within 7 days. The remaining 2
animals ceased to grow and died at day 22 and 29. Therefore, mice
treated with MVA-BN reached a state of increased resistance against
HSV-1 that was associated with live MVA-BN, but not inactivated
MVA-BN.
[0158] In control experiments done with mice that do not have
functional T-cells it was determined that the protection against
HSV-1 after vaccination with MVA-BN was not due to cross-reacting
cytotoxic T- lymphocytes induced by MVA-BN.
[0159] It was tested whether DC cells were responsible for the
protection of mice from HSV-1 after vaccination with MVA-BN. To
this end naive 8-day-old mice were challenged with 5.times.10.sup.4
PFU HSV-1 4 hr after transfer of cells from MVA-treated mice. In a
first experiment splenocytes from 8-days-old mice treated at 1 day
of life with MVA-BN were separated in DC rich (low-density) and DC
poor (high-density) fractions. Mice receiving 5.times.10.sup.6
cells from the DC rich fraction survived the challenge to 50%
whereas all the mice receiving 10 times less DC rich suspension or
untreated mice died within 5 days. A second approach was done by
transferring positively isolated CD11c positive cells from
8-days-old mice treated at 1 day of life with MVA-BN to naive age
matched mice. A suspension of 2 .times.106 splenocytes containing
more than 80% CD11c positive cells from MVA-BN treated mice
protected naive mice from HSV-1 infection. In contrast, 4 untreated
littermates as well as 8 additional untreated animals died after
the challenge. Furthermore, mice receiving the same amount of
spleen cells or mice receiving one spleen equivalent (50
.times.10.sup.6 cells) from the negative fraction did not show
increased resistance against HSV-1. Thus CD11 c positive cells are
able to protect mice from HSV-1.
[0160] After administration of MVA short-term protective effects in
the range of about 24 hours were described in the prior art
(Vilsmeier, B., Berl. Munch. Tierarztl. Wschr. 112 (1999),
329-333). Although the viruses used in said publication are not
viruses that are not capable of being replicated to infectious
progeny virus in the neonatal or prenatal animal used, it was
tested whether the mode of action as disclosed in Vilsmeier is
similar to the mode of action described in the present application.
More particularly, Vilsmeier discloses that MVA, in particular
inactivated MVA, induces a paramunity for about 24 hours. To test
whether the paramunity effect counts also for the protective
effects as disclosed in the present application mice 24 hours of
birth were vaccinated either with MVA-BN or with inactivated
MVA-BN. At 7 days of age the mice were challenged with a lethal
dose of HSV-1 (10 .sup.5PFU HSF-1f). Unvaccinated control mice died
6 days after challenge. Also the mice vaccinated with inactivated
MVA-BN were not protected against a challenge with HSV-1. The
number of DC cells in these mice was not elevated. In contrast, the
mice vaccinated with non-inactivated MVA-BN were significantly
protected against a challenge with HSV-1. 30 days after the
challenge more than 80% of the mice were still alive. Two days
after vaccination elevated serum Flt3-L was found in the serum.
Elevated numbers of DC were found in the spleen. The enhanced
Flt3-L was associated with elevated numbers of DC. This confirms
that paramunity effects are not responsible for the observed
protection.
[0161] (ii) MVA-BN induces a specific immunity in neonates that
lasts until adulthood
[0162] One-day-old C57BI/6 mice (group size of 18) were vaccinated
(i.p) with MVA-BN (2.5.times.10.sup.7 TCID.sub.50). Four weeks
after vaccination, when the mice were considered adults there where
challenged with a lethal dose (1.times.10.sup.4 TCID.sub.50) of
vaccinia Western Reserve (VV-WR). With the exception of one animal
all other MVA-BN vaccinated animals survived. In contrast, all
placebo vaccinated animals died within 7 days and demonstrated
severe clinical symptoms such as ruffled fur, weight loss and
reduced activity. Clearly this is a clear demonstration that MVA-BN
vaccination is not only safe in neonatal animals, but is capable of
inducing a protective immune response against a lethal vaccinia
(related virus to MVA-BN) infection.
* * * * *