U.S. patent application number 10/610928 was filed with the patent office on 2004-10-14 for papilloma virus-like particles, fusion proteins as well as processes for their production.
This patent application is currently assigned to Loyola University of Chicago. Invention is credited to Gissmann, Lutz, Muller, Martin, Painstil, Jeanette, Zhou, Jian.
Application Number | 20040202679 10/610928 |
Document ID | / |
Family ID | 25940851 |
Filed Date | 2004-10-14 |
United States Patent
Application |
20040202679 |
Kind Code |
A1 |
Gissmann, Lutz ; et
al. |
October 14, 2004 |
Papilloma virus-like particles, fusion proteins as well as
processes for their production
Abstract
The invention relates to the recombinant production of proteins
as well as VLPs which are suitable as a vaccine for therapeutic and
prophylactic vaccination. The invention also relates to processes
for the production and purification of recombinant papilloma virus
proteins and fusion proteins.
Inventors: |
Gissmann, Lutz;
(Willowbrook, IL) ; Zhou, Jian; (Willowbrook,
IL) ; Muller, Martin; (Chicago, IL) ;
Painstil, Jeanette; (Westchester, IL) |
Correspondence
Address: |
MARSHALL, GERSTEIN & BORUN LLP
6300 SEARS TOWER
233 S. WACKER DRIVE
CHICAGO
IL
60606
US
|
Assignee: |
Loyola University of
Chicago
|
Family ID: |
25940851 |
Appl. No.: |
10/610928 |
Filed: |
July 1, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10610928 |
Jul 1, 2003 |
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09949404 |
Sep 6, 2001 |
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6599508 |
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09949404 |
Sep 6, 2001 |
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09397680 |
Sep 16, 1999 |
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6361778 |
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09397680 |
Sep 16, 1999 |
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08817335 |
Oct 2, 1997 |
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6066324 |
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Current U.S.
Class: |
424/204.1 ;
435/235.1 |
Current CPC
Class: |
C12N 2710/20034
20130101; C07K 2319/00 20130101; A61P 31/20 20180101; C12N
2710/20022 20130101; A61P 15/02 20180101; C12N 7/00 20130101; A61P
15/00 20180101; C07K 14/005 20130101; A61P 31/12 20180101; A61K
38/00 20130101; A61K 2039/5256 20130101; A61P 13/02 20180101; A61K
2039/5258 20130101; A61K 39/12 20130101; C12N 2710/20023
20130101 |
Class at
Publication: |
424/204.1 ;
435/235.1 |
International
Class: |
A61K 039/12; C12N
007/00 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 9, 1995 |
WO |
PCT/EP95/03974 |
Oct 7, 1994 |
DE |
P4435907.1 |
Jul 21, 1995 |
DE |
19526752.4 |
Claims
1-53. (canceled)
54. A papillomavirus virus-like particle (VLP) consisting of a
truncated papillomavirus L1 protein and amino acid residues from a
second protein, said truncated L1 protein consisting of an amino
acid sequence having one or more carboxy terminal amino acid
residues deleted from a full length L1 amino acid sequence, and
said amino acid residues from the second protein replacing deleted
amino acid residues in the truncated L1 protein.
55. The VLP of claim 54 wherein the papillomavirus L1 protein is
derived from a viral strain selected from the group consisting of
BPV, HPV 6, HPV 11, HPV 16, HPV 18, HPV 33, HPV 35 and HPV 45.
56. The VLP of claim 55 wherein the papillomavirus L1 protein is
derived from HPV 16.
57. The VLP of claim 56 wherein the deletion in the L1 protein
comprises from 1 to 34 carboxy terminal amino acid residues, and
wherein said deletion increases the ability of the L1 protein to
form virus-like particles compared to native L1.
58. The VLP of claim 56 wherein the truncated L1 protein consists
of an amino acid sequence having 26 amino acid residues deleted
from the carboxy terminus of the full length L1 amino acid
sequence.
59. The VLP of any one of claims 54-58 wherein the amino acid
residues from the second protein are derived from all or part of a
papilloma virus protein.
60. The VLP of claim 59 wherein the papilloma virus protein is
selected from the group consisting of E1, E2, E4, E5, E6 and
E7.
61. The VLP of claim 60 wherein the papilloma virus protein is E7.
Description
[0001] This application is a divisional of U.S. application Ser.
No. 09/949,404, filed Sep. 6, 2001, which is pending, which in turn
is a divisional of U.S. application Ser. No. 09/397,680 filed Feb.
26, 2001, now U.S. Pat. No. 6,361,778 issued Mar. 26, 2002, which
in turn is a divisional of U.S. application Ser. No. 08/817,335
filed on Oct. 2, 1997, now U.S. Pat. No. 6,066,324 issued May 23,
2000.
FIELD OF THE INVENTION
[0002] 1. The invention relates to recombinantly produced papilloma
virus-like particles, proteins, fusion proteins as well as
processes for the formation and purification of these particles,
proteins and fusion proteins.
BACKGROUND OF THE INVENTION
[0003] 2. Infections with certain (high-risk) types of genital
papilloma viruses in humans (HPV), e.g. HPV 16, 18 or 45 are held
to be the main risk factor for the formation of malignant tumours
of the anogenital tract. Of these, cervical carcinoma is by far the
most frequently occurring. According to an estimate aby the WHO,
about half a million new cases of this disease occur annually.
Because of this frequent occurrence, the connection between HPV
infection and cervical carcinoma has been the best
investigated:
[0004] a) Precursor lesions of cervical carcinoma (cervical
intraaepithelial neoplasia: CIN) are caused by papilloma virus
infections.
[0005] b) The genomes of certain HPV types (e.g. 16, 18, 33, 35,
45) have been proven to occur in more than 95% of tumour biopsies
as well as in cell lines derived from them. Depending on the
geographic origin of the tumours, 5-0.70% of these contain HPV
16.
[0006] c) In all subsequently examined cases the ORFs E6 and E7 are
transcribed (Wettstein et al., in Pfister H. (ed):
[0007] Papilloma viruses and human cancer, pp. 155 to 179, Boca
Raton, 1990).
[0008] d) The proteins E6 and E7 can be proven in all
cervicalcarcinoma cell-lines as well as in in vitro transformed
human keratinocytes, and the majority of patients with cervical
carcinoma have E6- or E7-specific antibodies.
[0009] e) The constitutive expression of the E6/E7 proteins is
necessary to maintain the transformed condition of HPV-positive
tumours.
[0010] f) The E6- and E7 genes of HPV 16 and HPV 18 are
biologically active in the following experimental systems:
[0011] Induction of cellular DNA synthesis in human cells;
[0012] Transformation of human keratinocytes and other cells in
culture;
[0013] Tumour formation in transgenic mice.
[0014] 3. Other HPV types (principally HPV 6 and 11) cause benign
genital warts (condylomata acuminata) and are only extremely rarely
associated with malignant tumours (low-risk types).
[0015] 4. As a rule, genital papilloma viruses in humans are
transmitted during intercourse and in most cases lead to persistent
infection in the anogenital mucous membrane. This led to the
conclusion that primary infections induce only an insufficient
immune response, or that the virus has developed possibilities of
avoiding the immune surveillance in the infected cells. On the
other hand there are good indications suggesting that the immune
system is involved during primary manifestation or during the
malignant progression of papilloma virus infections. (For an
overview see Altmann et al. (1994) in Minson A., Neil J., McCrae M.
(eds): Viruses and cancer, Cambridge University Press, pp. 71 to
80).
[0016] a) In the case of animal papilloma viruses (rabbit papilloma
virus and bovine papilloma virus), the clinical manifestation of
primary infections can be avoided by vaccination with viral
structural proteins or with wart extracts (autologous
vaccines).
[0017] b) Rodents are protected by vaccination with HPV 16 E6- or
E7-positive vaccinia recombinants or by synthetic peptides prior to
tumour formation after inoculation of HPV 16-transformed autologous
cells.
[0018] c) Regression of warts is often systemic and in the case of
animal papilloma viruses can be induced by the transfer of
lymphocytes of regressor animals.
[0019] d) In the case of immuno-suppressed patients (e.g. kidney
transplantees or HIV-infectd persons), the incidence of genital
warts, CIN and anogenital cancer is increased.
[0020] 5. This led to the conclusion that papilloma virus-specific
vaccinations aimed at preventing the primary infection and the
occurrence of genital cancer should be possible.
[0021] 6. 1. Avoidance of HPV infections is suitable by vaccination
with the structural proteins L1 and L2 of the papilloma virus
(prophylactic vaccination).
[0022] 7. Because papilloma viruses cannot be propagated to
adequate titres in cell cultures or other experimental systems, the
viral proteins can only be produced by means of recombinant
vectors. Recently, virus-like particles (VLPs) which, after
expression of the viral structure proteins L1 and L2 (or L1 on its
own), are formed in recombinant vaccinia or baculo virus, have been
described. Purification of the VLPs can be achieved very simply by
means of centrifugaton in CsCl-- or sucrose gradients.
[0023] 8. WO 93/02184 describes a method which provides papilloma
virus-like particles (VLPs), which are used for diagnostic
applications or as a vaccine against infections caused by the
papilloma virus.
[0024] 9. WO 94/00152 describes a recombinantly produced L1 main
capsid protein which mimics the conformational neutralising epitope
on human and animal papilloma virions. These recombinant proteins
can be used as vaccines which protect against papilloma
virus-infections.
[0025] 10. 2. Treatment of cervical carcinoma or precursor lesions
by immunotherapy assisted by early papilloma virus-proteins
(principally E6 or E7) which are expressed in the persistently
infected cells (therapeutic vaccination).
[0026] 11. It is assumed that by this vaccination, cytotoxic
T-cells are activated against persistently infected genital
lesions. The target population are patients with HPV associated
pre-malignant or malignant genital lesions.
[0027] 12. Early HPV proteins are produced by expression in E. coli
or eukaryotic vectors (e.g. baculo virus or yeast). Purification is
however rendered difficult by the low solubility and as a rule
requires a combination of ion exchange chromatography, gel
filtration and affinity chromatography.
[0028] 13. PCT patent application WO 93/20844 discloses that the E7
protein of the papilloma virus from HPV or BPV is therapeutically
effective in the regression (not however in the prevention) of
papilloma virus-tumours in mammals. In addition, preferred
antigenic proteins fragment sequences are described.
[0029] 14. So far, however, no VLPs were described which are
suitable both for prophylactic and therapeutic vaccination. The
last-mentioned processes have the disadvantage that, for example,
early HPV proteins, because of their low solubility, can only be
cleaned with difficulty.
[0030] 15. A high particle production would be particularly
desirable, in particular in view of a vaccine for prophylactic and
therapeutic vaccination.
[0031] 16. A disadvantage for the process described so far was that
it was not possible to produce VLPs after expression of L1 in E.
coli.
DESCRIPTION OF THE INVENTION
[0032] 17. It is therefore the object of the present invention, to
make available recombinantly produced proteins as well as VLPs
which are suitable as a vaccine for prophylactic and therapeutic
vaccination, as well as processes for the production of these
proteins and VLPs. Equally, simple purification of the recombinant
proteins obtained should be possible. Also production of VLPs after
expression of L1 in E. coli should be possible.
[0033] 18. The present invention accomplishes this task according
to VLPs stated in the independent claims 1 and 12; the proteins
stated in the independent claim 36; the fusion proteins stated in
the independent claims 8 and 38; the processes stated in the claims
42 and 43; and the use according to claims 55 and 56. Further
preferred embodiments, aspects and details of the invention are
disclosed in the dependent claims of the description as well as in
the preferred embodiments.
[0034] 19. According to the present invention, VLPs are produced
which consist of fusion proteins of late and early HPV proteins (or
fragments thereof) (HVLP) and which can be used for prophylactic or
therapeutic vaccination. Such a vaccine offers the following
advantages when compared with conventional preparations:
[0035] a) In the case of prophylactic vaccination, HVLPs, through
induction of L1/L2-specific antibodies not only prevent entry of
the virus into the cell but they also eliminate already infected
cells (through induction of cytotoxic T-cells) if an infection has
taken place earlier or if the humoral immune response was in
adequate.
[0036] b) In the case of therapeutic vaccination, HVLPs eliminate
persistently infected cells (e.g. in patients with CIN or cervical
carcinoma), and above all they prevent re-infection in female
patients with CIN lesions.
[0037] c) Purification of the HVLPs is simple, in a similar way to
purification of the VLPs without early HPV proteins.
[0038] 20. According to the present invention, VLPs of the bovine
papilloma virus (BPV) type 1 and the human papilloma viruses 11 and
16 after expression of L1 plus L2, or L1 on its own, can be
produced in vaccinia or baculo virus. Experiments show that parts
of the L1 protein can be deleted (amino acid sequence 311-351,
331-371, 391-431 of BPV 1; 306-315 of HPV 16), without the ability
to form VLPs being lost. Such sections exist in the L1 proteins of
all papilloma viruses so that the deleted section of L1 can be
replaced by other proteins (of papilloma viruses or of other
origin) and that in this way, hybrid virus-like particles can be
produced. In the same way, parts of the papilloma virus protein L2
are deleted and replaced by other (early HPV or other) proteins, so
that HVLPs can also be formed from the complete L1 protein plus an
L2 fusion protein.
[0039] 21. Fusion proteins comprising deleted L1 or L2 protein from
different HPV types (principally HPV 6, 11, 16, 18, 33, 35, 45) and
the respective early proteins E1, E2, E4, E5, E6, E7 (or parts
thereof) are produced by expression in vaccinia recombinants which
can be constructed in a very short time. The formation of VLPs,
consisting either of a L1 fusion protein or of the complete L1
protein plus an L2 fusion protein, is monitored by electron
microscopy, and the presence of the early. HPV protein is tested by
Western blot analysis by means of specific antisera. For
large-scale production of HPLVs the expression of the proteins is
carried out in viral or eukarytic systems, preferably in baculo
virus or in yeast.
[0040] 22. Respective experiments for producing fusion proteins can
be carried out with proteins of other origin.
[0041] 23. Essential for the present invention are recombinantly
produced virus-like particles (VLPs) which are formed after
expression of the viral structural proteins L1 and/or L2, whereby
sections of the L1 and/or L2 protein are deleted, without the
ability of forming VLPs being lost.
[0042] 24. According to the present invention, the deleted section
in the L1 protein of the bovine papilloma virus type 1 preferably
concerns the amino acid sequences 311-351, 331-371, 391-431. In the
case of L1 proteins of the human papilloma virus 16, it
advantageously concerns the amino acid sequence 306-315.
[0043] 25. In a preferred embodiment of the present invention the
deleted section of L1 and/or L2 proteins are replaced by other
proteins or protein fragments, whereby fusion proteins are
obtained. The share of L1 or L2 proteins is advantageously approx.
50 to 99%, preferably approx. 60 to 90%, particularly preferred
approx. 80%.
[0044] 26. However, according to the present invention, even if
this is not explicitly stated below, more than one section of the
L1 and/or L2-protein should also be deleted and preferably be
replaced by other proteins or protein fragments.
[0045] 27. It is particularly preferred to replace the deleted
section in the L1 or L2 protein by other proteins of papilloma
viruses and/or proteins of other origin, whereby hybrid virus-like
particles (HVLPs) can be produced.
[0046] 28. It has been shown to be particularly favourable
according to the present invention, if the formation of the VLPs is
from an L1 fusion protein or, according to a further embodiment,
from a complete L1 protein and a L2 fusion protein.
[0047] 29. The fusion proteins, in particular for the formation of
hybrid virus-like particles according to a further embodiment of
the present invention, preferably consist of a deleted L1 and/or L2
protein of different HPV types (human papiloma virus), particularly
preferred are HPV 6, 11, 16, 18, 33, 35 and 45, and other proteins
or protein fragments. Preferably these other proteins or protein
fragments concern respective early proteins or fragments thereof,
such as for example the early proteins E1, E2, E4, E5, E6 and/or
E7.
[0048] 30. According to the process covered in the invention, the
expression of the fusion proteins and proteins is carried out in
viral or eukaryotic vectors; particularly preferred in baculo
viruses or in yeasts.
[0049] 31. According to a further embodiment of the process
according to the invention, the fusion proteins are produced
through expression in vaccinia recombinants.
[0050] 32. According to the present invention, the application of
the fusion proteins or the hybrid virus-like particles for the
production of a prophylactic and therapeutic vaccine preferably
takes place after adding further components.
[0051] 33. Up to now, for the production of VLPs, such as for
example of VLPs from HPV 16, the L1 (ORF) was expressed by means fo
eukaryotic vectors, such as for example baculo virus. Formation of
the VLPs (assembly) takes place in the karyon of infected
cells.
[0052] 34. Essential to the present invention are therefore in
particular recombinant papilloma virus-like particles which are
formed after expression of the viral structural proteins L1 and/or
L2, in which one or several sections of the L1 and/or L2 proteins
are deleted, whereby the ability to form virus-like particles is
increased in comparison to the native formation and/or in vitro
production.
[0053] 35. According to the present invention, at least one of the
deleted sections in the L1 and/or L2 protein of a papilloma virus
is a deletion, advantageously in the C-terminal amino acid
sequence, preferably approximately 1 to 34 amino acids in length,
preferably from 1 to 26 amino acids in length, in particular 26
amino acids in length.
[0054] 36. Advantageously, after insertion of the C-terminal
deletion into the L1 and/or L2 protein, the production of VLPs is
increased many times, preferably at least 10 times, and in
particular approx. 10 to 100 times.
[0055] 37. In a preferred embodiment of the present invention, the
deleted sections in the L1 and/or L2 protein, in particular of the
bovine papilloma virus, concern 26 C-terminal amino acids.
Particularly preferred is the C-terminal deletion, 26 amino acids
in length,
(Gly-Ala-Gly-Cys-Ser-Thr-Val-Arg-Lys-Arg-Arg-Ile-Ser-Gln-Lys-Thr-Ser-Ser--
Lys-Pro-Ala-Lys-Lys-Lys-Lys-Lys) (SEQ ID NO:2) corresponding to the
nucleotide positions 7016 to 7093 GGGGCAGGAT GTTCAACTGT GAGAAAACGA
AGAATTAG CC AAAAAACTTC CAGTAAGCCT GCAAAAAAAA AAAAAAAA (SEQ ID NO:1)
is inserted into the L1 ORF of the bovine papilloma virus type 1
(BPV 1). Advantageously, after inserting the C-terminal deletion
into the L1 and/or L2 protein, the production of VLPs is increased
at least ten times.
[0056] 38. According to a further embodiment of the present
invention, the deletion, of which there is at least one, in the L1
and/or L2 protein concerns a homologous amino acid sequence of the
human papilloma virus 16 or of other papilloma viruses.
[0057] 39. According to a further preferred embodiment, the deleted
sections in the L1 and/or L2 protein concern 34 C-terminal amino
acids of the human papilloma virus type 16 (HPV 16); preferably the
amino acid sequence
(Ala-Gly-Leu-Lys-Ala-Lys-Pro-Lys-Phe-Thr-Leu-Gly-Lys-Arg-Lys-Ala-
-Thr-Pro-Thr-Thr-Ser-Ser-Thr-Ser-Thr-Thr-Ala-Lys-Arg-Lys-Lys-Arg-Lys-Leu)
SEQ ID NO:4) corresponding to the nucleotide positions 7052 to 7153
GCAGGATTGA AGGCCAAACC AAAATTTACA TTAGGAAAAC GAAAAGCTAC ACCCACCACC
TCATCTACCT CTACAACTGC TAAACGCAA AAACGTAAGC TG (SEQ ID NO:3), which
is inserted into the L1 ORF of the HPV 16.
[0058] 40. It is particularly preferred if the deletion of the L1
and/or L2 protein comprises the nuclear localisation signal (NLS).
Particle production from the L1 proteins or the L1 proteins and L2
proteins takes places in particular in the cytoplasm. Preferably,
the particles are secreted into the supernatant liquid;
particularly preferred is a secretion of approx. 5 to 10% of the
particles.
[0059] 41. The expression of L1 proteins or L1 proteins and L2
proteins in E. coli takes place according to a further preferred
embodiment. In this, at the C-terminal deletion in the L1 protein,
in particular in addition 6-histidines are inserted. Advantageously
the production of VLPs takes place after expression of L1 proteins
or L1 and L2 proteins in E. coli.
[0060] 42. According to the present invention, the further deleted
sections in the L1 protein of the bovine papilloma virus type 1
preferably concern the amino acid sequences 311-351, 331-371,
391-431. Advantageously the L1 proteins of the human papilloma
virus 16 concern the amino acid sequence 306-315.
[0061] 43. In a preferred embodiment of the present invention, the
further deleted section of L1 and/or L2 proteins is replaced by
other proteins or protein fragments, whereby proteins are obtained
which in this document are called fusion proteins. Advantageously,
the content of L1 or L2 protein is approx. 50 to 99%, preferably
approx. 60 to 90%, particularly preferred approx. 80%.
[0062] 44. However, according to the present invention, even if
this is not explicitly stated, more than one further section of the
L1 and/or L2 protein should be deleted and preferably be replaced
by other proteins or protein fragments.
[0063] 45. It is particularly preferred if the deleted section of
L1 or L2 protein is replaced by other proteins of papilloma viruses
and/or proteins of other origin, whereby hybrid virus-like
particles (HVLPs) can be produced.
[0064] 46. It has been shown particularly advantageous according to
the present invention, that the formation of the VLPs takes place
from an L1 protein, an L1 fusion protein, an L1 protein and L2
protein, an L1 fusion protein and an L2 protein, an L1 protein, and
an L2 fusion protein or an L1 fusion protein and an L2 fusion
protein.
[0065] 47. According to a further embodiment of the present
invention, at least one of the deleted sections in the L1 and/or L2
protein of a papilloma virus concerns N-terminal amino acid
sequences.
[0066] 48. According to the present invention, in a further
embodiment at least one of the deleted sections in the L1 protein
and/or L2 protein of a papilloma virus concerns amino acid
sequences in the middle section of the protein.
[0067] 49. Also essential for the invention are proteins, in
particular for the formation of hybrid papilloma virus-like
particles, whereby one or several sections of the L1 and/or L2
protein are deleted. In particular, at least one of the deleted
sequences in the L1 and/or L2 protein concerns the deletion of a
C-terminal amino acid sequence.
[0068] 50. The fusion proteins, in particular for the formation of
hybrid papilloma virus-like particles according to a further
embodiment of the present invention, advantageously consist of a
deleted L1 and/or L2 protein of different papilloma viruses,
specially preferred are HPV 6, 11, 16, 18, 31, 33, 35 and 45, and
other proteins or protein fragments of papilloma viruses or of
other origin. Preferably, these other proteins or protein fragments
concern the respective early papilloma virus proteins or fragments
concern the respective early papilloma virus proteins or fragments
thereof, such as for example the early proteins E1, E2, E4, E5, E6,
and/or E7.
[0069] 51. According to the process covered in the invention, the
expression of the proteins and/or fusion proteins and the
production of papilloma virus-like particles is carried out in
viral, eukaryotic or prokaryotic vectors, especially advantageous
in vaccinia recombinants, in baculo viruses, in yeasts or in
bacteria, in particular in E. coli.
[0070] 52. Preferably particle production occurs in cytoplasm. In a
particularly preferred manner, the particles are secreted into the
supernatant liquid; it is particularly preferred if approx. 5 to
10% of the particles are secreted into the supernatant liquid.
[0071] 53. In particular, according to the present invention, by
inserting a C-terminal deletion, 25 amino acids in length, into the
nucleotide positions 7016 to 7093 in the L1 ORF of the bovine
papilloma virus type 1 (BPV 1), the production of VLPs is increased
more than tenfold. Thus with the same quantity of L1 protein, as
can be demonstrated for example in a Western blot, an increase in
the particle number can be demonstrated in the electron microscope.
Since deletion preferably comprises the nuclear localisation signal
(NLS), the particle production takes place in the cytoplasm, a
significant part of the particles is secreted into the supernatant
liquid. This is particularly advantageous because it significantly
facilitates purification.
[0072] 54. Proteins, preferably with the mentioned deletion with
additional 6 histidines (His L1 proteins), according to the present
invention are expressed in E. coli. The proteins, in particular His
L1 proteins, are preferably purified by way of Ni affinity
chromatography, whereby according to an advantageous embodiment, at
this point in time the proteins are present in a denaturation
buffer, for example 6M guanidine hydrochloride. Renaturation takes
place for example in 150 mM NaCl, 1 mM CaCl.sub.2, 0.01% Triton-X
100, 10 mM Hepes (N-2-hydroxyethyl piperazine-N'-2 ethane sulfonic
acid), pH 7.4.
[0073] 55. According to a preferred embodiment of the present
invention, production (assembly) of the VLPs takes place after
dialysis of the proteins, preferably after dialysis against 150 mM
NaCl, 25 mM Ca.sup.2+, 10% DMSO (dimethyl sulfoxide), 0.1% Triton-X
100, 10 mM tris [tris-(hydroxymethyl) aminomethane] acetic acid
with a pH value of 5.0.
[0074] 56. The deletion of sequence in the L1 protein of all
papilloma viruses which prevent the premature assembly fo the VLPs
leads to a higher yield during VLP production.
[0075] 57. As far as in these cases the L1 NLS is concerned, the
assembly takes place in the cytoplasm. Consequently, according to
the invention, purification of the VLPs is possible from the
cytoplasm, instead of, as up to now, from the karyon. According to
the invention, shorter deletions are also possible. According to
the present invention, deletions of up to one amino acid and/or
substitutions of up to one amino acid are carried out. In this it
is advantageous that with short deletions or substitutions of up to
one or only a few amino acids, the antigenic properties of the
proteins and the LVPs formed thereof, are changed as little as
possible when compared to the native antigenic properties of the
proteins or VLPs.
[0076] 58. The introduction of a C-terminal deletion or
substitution in L1 and/or L2 fusion proteins, as carried out
previously, also leads to an increase in the production of hybrid
VLPs. In this, those VLPs should also be included which only
contains L1 fusion proteins, as well as hybrid VLPs which contain
an L1 or L2 fusion protein and a L2 or L1 protein.
[0077] 59. For this, VLPs are produced which comprise fusion
proteins of late and early HPV proteins (or fragments thereof)
(HVLPs) and which can be used for prophylactic or therapeutic
vaccination. Such a vaccine offers the following advantages when
compared with conventional preparations:
[0078] a) In the case of prophylactic vaccination, HVLPs, through
induction of L1/L2-specific antibodies not only prevent entry of
the virus into the cell but they also eliminate already infected
cells (through induction of cytotoxic T-cells) if an infection has
taken place earlier or if the humoral immune response was
inadequate.
[0079] b) In the case of therapeutic vaccination, HVLPs eliminate
persistently infected cells (e.g. in patients with CIN or cervical
carcinoma), and above all they prevent reinfection in female
patients with CIN lesions.
[0080] c) Purification of the HVLPs is simple, in a similar way to
purification of the VLPs without early HPV proteins.
[0081] 60. VLPs of the bovine papilloma virus (BPV) type 1 and the
human papilloma viruses 11 and 16 after expression of L1 plus L2,
or of L1 on its own, can be produced in vaccinia or baculo virus.
Experiments show that parts of the L1 protein can be deleted (amino
acid sequences 311-351, 331-371, 391-431, of BPV 1; 306-315 of HPV
16), without the ability to form VLPs being lost. Such sections
exist in the L1 proteins of all papilloma viruses so that the
deleted section of L1 can be replaced by other proteins (of
papilloma viruses or of other origin) and that in this way, hybrid
virus-like particles can be produced. In the same way, parts of the
papilloma virus protein L2 are deleted and replaced by other (early
HPV or other) proteins, so that HVLPs can also be formed from the
complete L1 protein plus an L2 fusion protein.
[0082] 61. Fusion proteins comprising deleted L1 or L2 protein from
different HPV types (mainly HPV 6, 11, 16, 18, 33, 35, 45) and the
respective early proteins E1, E2, E4, E5, E6, E7 (or parts thereof)
are produced by expression in vaccinia recombinants which can be
constructed in a very short time. The formation of VLPs, consisting
either of an L1 fusion protein or of the complete L1 protein plus
an L2 fusion protein, is monitored by electron microscopy, and the
presence of the early HPV protein is tested by Western blot
analysis by means of specific antisera. For large-scale production
of HPLVs the expression of the proteins in viral eukaryotic or
prokaryotic systems, preferably in baculo virus, in yeast, or in E.
coli, is carried out.
[0083] 62. According to the present invention, the application of
the fusion proteins or the hybrid virus-like particles for the
production of a prophylactic and therapeutic vaccine preferably
takes place after adding further components.
[0084] 63. Respective experiments for producing fusion proteins can
be carried out with proteins of other origin.
Sequence CWU 1
1
4 1 26 PRT Papillomavirus sylvilagi 1 Gly Ala Gly Cys Ser Thr Val
Arg Lys Arg Arg Ile Ser Gln Lys Thr 1 5 10 15 Ser Ser Lys Pro Ala
Lys Lys Lys Lys Lys 20 25 2 78 DNA Papillomavirus sylvilagi 2
ggggcaggat gttcaactgt gagaaaacga agaattagcc aaaaaacttc cagtaagcct
60 gcaaaaaaaa aaaaaaaa 78 3 34 PRT Papillomavirus sylvilagi 3 Ala
Gly Leu Lys Ala Lys Pro Lys Phe Thr Leu Gly Lys Arg Lys Ala 1 5 10
15 Thr Pro Thr Thr Ser Ser Thr Ser Thr Thr Ala Lys Arg Lys Lys Arg
20 25 30 Lys Leu 4 102 DNA Papillomavirus sylvilagi 4 gcaggattga
aggccaaacc aaaatttaca ttaggaaaac gaaaagctac acccaccacc 60
tcatctacct ctacaactgc taaacgcaaa aaacgtaagc tg 102
* * * * *