FR2768749A1 - Papillomavirus virus-like particles - Google Patents

Abstract

Using human or animal papillomavirus virus-like particles (VLPs) to prepare pseudoviral vectors for introducing therapeutic genetic material into target cells of an organism, especially for gene therapy or DNA vaccination, is new. Independent claims are also included for: (1) a pseudoviral vector for introducing therapeutic genetic material into target cells of an organism for the purpose of gene therapy or DNA vaccination, comprising one or more therapeutic heterologous nucleotide sequences encapsidated in a human or animal papillomavirus VLP; (2) use of pseudoviral vectors corresponding to VLPs comprising the L1 protein and optionally the L2 protein of human or animal papillomaviruses, in which one or more heterologous nucleotide sequences (especially "nucleotide sequences coding for reporter genes") are encapsidated, the pseudoviral vectors being obtained by extracellular encapsidation of the heterologous nucleotide sequences in the VLPs, for introducing the heterologous nucleotide sequences in vitro into cells susceptible to infection by the pseudoviral vectors; (3) use of pseudoviral vectors corresponding to VLPs comprising the L1 protein and optionally the L2 protein of human or animal papillomaviruses, in which at least one reporter gene is encapsidated, the pseudoviral vectors being obtained by extracellular encapsidation of the reporter gene(s) in the VLPs, for detecting human or animal papillomavirus neutralising antibodies.

Description

ARTIFICIAL VIRUSES DERIVED FROM PAPILLOMAVIRUS AND THEIR
USES, IN PARTICULAR GENE THERAPY.

 The subject of the present invention is artificial viruses (or pseudoviruses) derived from human or animal papillomaviruses, as well as their processes for obtaining, and their uses, in particular in pharmaceutical compositions in the context of gene therapies, or for the detection of antibodies neutralizing papillomavirus.

 The efficacy, specificity and safety of vector systems used for gene transfer are important factors for their applications in humans, particularly in the context of gene therapy or immunization with HIV. DNA.

Conventional techniques for transferring plasmids within eukaryotic cells are essentially those using liposomes (Felgner et al., 1987
Felgner and Ringold, 1989), which may contain viral fusion peptides, or use whole viruses to facilitate the release of DNA into the cytoplasm (Kamata et al., 1994, Wagner et al., 1994).

 Viruses currently represent the most efficient natural system of gene transfer (II).

Adenoviruses (Berkner 1992, Kozarsky and Wilson 1993, Kremer
Perricaudet, 1995), adenovirus-associated viruses (Kremer and Perricaudet, 1995).
Kotin, 1994; Fisher et al., 1997), Epstein-Barr virus (Banerjee et al., 1995) and retroviruses (Miller, 1992; Danos and Mulligan, 1988; Naldini et al., 1996), have been modified to carry genes of therapeutic interest and to integrate their own genome within target cells.

 The use of viral vectors, however, poses problems, in particular the possible production of viruses capable of replicating in the case where recombination with wild-type viruses occurs in the target cells, the inactivation of the vectors by pre-existing neutralizing antibodies directed against these viruses in the host organism, and the immunological response induced by the virus (Gahëry-Ségard et al., 1997, Juillard et al., 1995).

 During the last ten years, it has been shown that the structural proteins of many viruses are able to self-assemble into pseudo-viral particles (hereinafter referred to as VLPs) when expressed in eukaryotic or prokaryotic expression systems.

 Papillomaviruses are members of the papovaviridae family. These are non-enveloped DNA viruses that infect humans as well as some animals. The capsid of papillomavirus consists of two proteins, L1 and L2. L1 is the major capsid protein, and has already been described as being able to self-assemble into VLPs.

 VLPs were obtained by transformation of hamster cells, carrying bovine papillomavirus type I (BPVI) genomes capable of autonomous replication, with recombinant Semliki forest virus (SVF) expressing BPVI (Roden et al., 1996). According to the authors of this article, the L1 protein expressed by the recombinant SVF is capable of selfassembling into VLPs. However, viral DNA could only be detected in the VLPs resulting from the coexpression of L1 and L2, which, again according to these same authors, indicates that the encapsidation of DNA within the VLPs, can only take place if these VLPs consist of both L1 and L2.

 The purpose of the work described in this article by Roden et al., Is to obtain infectious particles of human papillomavirus (HPV) capable of infecting mouse cells, and to study the possibility of neutralizing this infection with antibody. Thus, the authors of this article have prepared HPV type 16 VLPs which encapsidated the genome of BPV according to a process which has the characteristic of being intracellular, since this method, described above, is carried out in hamster cells. containing the genome of BPV and transformed with SVFs expressing L1 and L2 proteins of HPV-16.

 VLPs consisting of human papillomavirus L1 (HPV-1 1) L1 protein were obtained by transformation of E. coli bacteria with plasmids containing the L1 protein coding sequence (Li et al., 1997). The authors of this article have found that these VLPs obtained by transformation of E. coli are very difficult to disassemble.

 VLPs consisting of the L1 protein, and VLPs consisting of L1 and L2 proteins, human papillomavirus type 33 (HPV-33), were obtained by an intracellular process comprising the cotransformation of Cos-7 cells on the one hand with a marker plasmid encoding β-galactosidase and, on the other hand, with vaccinia virus as a vector containing the L1 coding sequence, or those coding for L1 and L2 (Unckell et al., 1997).

 The level of encapsidation by the above-mentioned intracellular method of the plasmid containing the coding sequence for .beta.-galactosidase is very low, since only 1 of the 25,000 VLPs carry this plasmid.

 Moreover, the infection rate of Cos-7 cells by the VLPs carrying said plasmid is also very low, since only 1 out of 2,000 VLPs carrying the plasmid is capable of infecting a Cos-7 cell.

Finally, the authors of this article insist on the essential nature of the protein
L2 in the context of infection of cells by VLPs.

 Given the low levels of integration of plasmid DNA into VLPs, and cell infection by these VLPs, the authors of this article have in no way mentioned the possibility of using such VLPs as as gene transfer vectors, and focused on their possible use in the context of the detection of neutralizing antibodies against papillomavirus infections.

The present invention stems from the discovery made by the inventors that the
Papillomavirus VLPs are capable of integrating plasmid DNA with a packaging rate much higher than those described above, insofar as this encapsidation is carried out by an extracellular process (that is to say which is not carried out inside host cells), in particular by in vitro disassembly-reassembly of these VLPs in the presence of plasmid.

 In addition, the inventors have also demonstrated that the infection rates of cells with plasmid DNA-carrying VLPs obtained according to the extracellular encapsidation method indicated above, are much higher than those described in the prior art. mentioned above, even when said VLPs consist of the only L1 protein.

 One of the aims of the present invention is to provide novel vectors for the transfer of genetic material into target cells, particularly in the context of gene therapy or gene immunization, these vectors being simpler to prepare and safer. to use only the vector viruses described so far.

 Another object of the present invention is to provide a simple method for preparing these transfer vectors.

 Another object of the present invention is that of providing novel pharmaceutical compositions containing said transfer vectors, and usable in gene therapy, or gene immunization.

 Another object of the present invention is to provide novel reagents for the detection of antibodies neutralizing papillomavirus, as well as new kits for the implementation of such a detection method.

 The subject of the present invention is the use of human or animal papillomavirus viral pseudo-particles (VLPs) for the preparation of vector pseudoviruses that can be used for the transfer of genetic material of therapeutic interest into target cells of the organism. especially in the context of gene therapy or gene immunization.

 Advantageously, the VLPs used in the context of the present invention contain the L1 protein, and optionally the L2 protein, papillomaviruses.

According to a preferred embodiment of the invention, the above-mentioned VLPs contain only the Lt protein (namely that said
VLPs are formed of capsomeres consisting solely of L1 proteins).

 By genetic material of therapeutic interest transferred by the pseudovirus vectors into target cells in the context of the present invention is meant any heterologous nucleotide sequence (DNA or RNA) of therapeutic interest, namely any sequence capable of encoding a protein. capable of treating a specific pathology, or of immunizing an individual against a specific pathology, said sequence being not included in the natural genome of papillomaviruses.

 In the particular case of the treatment of pathologies caused by papillomavirus infections, or of immunization against these pathologies, said heterologous sequence may consist of a fragment of the papillomavirus genome, or a nucleotide sequence derived from this fragment.

By heterologous DNA sequence of therapeutic interest, is meant in particular:
any DNA sequence capable of coding for a protein in the context of pathologies in which said protein is deficient,
any DNA sequence coding for a protein acting as a vaccine,
any DNA sequence coding for a protein acting as an antibody neutralizing pathogens,
any DNA sequence coding for a protein capable of interacting directly or indirectly with proteins involved in the development of certain pathologies,
any DNA sequence coding for an antisense RNA capable of inhibiting the production of a protein involved in the development of a pathology.

 The target cells into which the genetic material is transferred in the context of the present invention are all cells susceptible to be infected by papillomaviruses or VLPs of these papillomaviruses.

 The pseudovirus vectors of the invention are either administered in the form of pharmaceutical compositions, in particular under the conditions defined below, or used for the implementation of a process for the in vitro transformation of cells, in particular the cells of a patient, the cells thus transformed by the pseudoviruses are then readministered to the patient.

Advantageously, the VLPs used in the context of the present invention are as obtained by transformation of host cells with vectors containing a DNA sequence coding for the L1 protein of papillomaviruses and, where appropriate, a DNA sequence. coding for the protein
L2 of the papillomaviruses, said sequences being placed under the control of a transcription promoter, and recovery of the VLPs produced in said host cells by assembly of the capsomeres of L1 proteins and, where appropriate L2 proteins. The aforementioned capsomeres themselves result from the assembly of L1 proteins, at the rate of approximately 5 L1 proteins per capsomer, a VLP consisting of 7 capsomeres. In the case where the VLPs contain both the L1 protein and the L2 protein, there are approximately 30 L2 proteins per
VLPs.

 Advantageously, the VLPs used in the context of the present invention are as obtained by transformation of insect cells, as host cells, with baculoviruses used as vectors containing the DNA sequences encoding the L1 protein and, where appropriate L2 protein, said baculovirus vectors being themselves obtained by transformation of the latter with plasmids containing said DNA sequences coding for the L1 protein and, where appropriate L2, under the control of elements of regulation of their expression.

According to another advantageous embodiment of the invention, the VLPs used in the context of the present invention are as obtained by transformation of yeasts or mammalian cells (such as
CHO), as host cells, with plasmids used as vectors, containing the DNA sequences coding for the L1 protein, and optionally the L2 protein, said DNA sequences being placed under the control of elements of regulation of their expression.

Advantageously, the VLPs produced in the host cells as part of the aforementioned methods are purified from the culture media of said host cells, generally after lysis thereof; among the techniques that can be used to purify said VLPs from these culture supernatants, mention may be made of
- ultracentrifugation in sucrose, isopycnic, or density gradient (ex CsCl),
precipitation by release to polyethylene glycol (PEG), or ammonium sulphate,
- exclusion chromatography, or ion exchange,
the use of immunoadsorbents,
Isoelectric focusing.

 The subject of the invention is also the use of papillomavirus VLPs defined above, for the preparation of a medicament comprising vector pseudoviruses as defined above, the latter corresponding to said VLPs in which one or more heterologous nucleotide sequences of therapeutic interest are encapsidated, said pseudoviruses being in association with a pharmaceutically acceptable vehicle, said medicament being intended for the treatment or the prevention of pathologies, as described above, which can be treated by gene therapy or prevented by immunization gene.

 The invention also relates to any pseudovirus vector for the transfer of genetic material into target cells of the body in the context of gene therapy, or gene immunization, characterized in that it corresponds to a VLP of human papillomavirus or animals, in which one or more heterologous nucleotide sequences of therapeutic interest are encapsidated.

 The invention more particularly relates to any pseudovirus transfer vector as defined above, characterized in that it is formed of a VLP in association with one or more heterologous nucleotide sequences encapsidated within this VLP, said VLP comprising an assembly of capsomers of L1 protein, and optionally of L2 protein, of papillomavirus (in particular in the proportions indicated above).

 Advantageously, the VLPs forming the aforementioned pseudoviruses comprise only the papillomavirus L1 protein.

 The pseudovirus vectors according to the invention are further characterized in that the heterologous nucleotide sequence or sequences of therapeutic interest are inserted into a plasmid containing the elements necessary for regulating the expression of these DNA sequences, the maximum size of said plasmid plasmid being from about 8 to about 8.5 kb.

 Advantageously, the vector pseudoviruses according to the invention are as obtained by an extracellular method of encapsidation of one or more heterologous nucleotide sequences of therapeutic interest, within said VLPs.

 Preferably, this extracellular encapsidation process is carried out by disassembly-reassembly of the VLPs defined above, in the presence of a vector, in particular a plasmid, containing the heterologous nucleotide sequence (s) (s). ) of therapeutic interest.

In this respect, the invention more particularly targets any pseudovirus vector as obtained by an in vitro disassembly-reassembly method for the aforementioned VLPs, this method comprising the following steps:
disassembly of the VLPs into capsomers, in particular by incubation of the latter with a chelating agent such as ethylene glycol tetracetate (EGTA), or ethylene diamine tetraacetate (EDTA), and a reducing agent such as dithiothreitol (DTT) ), or ss-mercaptoethanol,
mixing the plasmid containing the heterologous DNA sequence (s) of therapeutic interest, advantageously in a ratio of one plasmid molecule to one VLP molecule (ie approximately 1 μg of plasmid, for approximately 5 μg of VLP),
reassembly of capsomeres to VLPs containing the abovementioned plasmid, in particular by increasing the concentration of Ca + + ions, and addition of an oxidizing agent such as dimethylsulfoxide (DMSO) or by any other method that neutralizes the action of the reducing agent mentioned above, in particular by dialysis.

If necessary, the aforementioned method comprises a separation step, in particular by chromatographic techniques, on the one hand
VLPs in which said heterologous nucleotide sequences of therapeutic interest are encapsidated and, on the other hand, VLPs remaining empty (not containing said nucleotide sequences) after the implementation of said method.

 The subject of the invention is also any pharmaceutical composition (in particular vaccine), characterized in that it comprises a pseudovirus transfer vector containing at least one DNA sequence of therapeutic interest, and as defined above, in association with a pharmaceutically acceptable vehicle.

 Advantageously, the pharmaceutical compositions according to the invention are further characterized in that they can be administered orally, intramuscularly, intravenously, intradermally, subcutaneously, nasally, vaginally, dermally, in particular at a rate of about 10 ng to about 10 ng. yg of pseudovirus vectors per individual and per dose.

 The subject of the present invention is also the use of pseudovirus vectors corresponding to VLPs as defined above, in which one or more heterologous nucleotide sequences (in particular nucleotide sequences coding for reporter genes) are packaged, said pseudovirus vectors being obtained by an extracellular encapsidation method, as described above, of said heterologous nucleotide sequences in said VLPs, for the implementation of a method for transferring said heterologous nucleotide sequences in vitro into cells that are likely to be infected by said pseudovirus vectors.

 As such, the subject of the present invention is any method for the in vitro transfer of heterologous nucleotide sequences into cells capable of being infected with pseudovirus vectors defined above, said pseudoviruses being obtained by an extracellular method of encapsidation of said sequences. heterologous nucleotides in papillomavirus VLPs, this method being as described above (in which the plasmid containing the heterologous nucleotide sequence (s) of therapeutic interest is replaced by a plasmid containing the heterologous nucleotide sequence (s)).

 The invention also relates to the use of pseudovirus vectors corresponding to VLPs as defined above, in which at least one reporter gene is packaged, said pseudoviruses being as obtained by an extracellular encapsidation method, as described herein. above, of the reporter gene (s) in said VLPs, for the implementation of a method for detecting antibodies that neutralize the infection of human or animal papillomaviruses.

The subject of the invention is also a process for the in vitro detection of antibodies neutralizing human or animal papillomaviruses, comprising the following steps:
in vitro incubation of pseudovirus vectors defined above containing a reporter gene, with cells capable of being infected by pseudovirus vectors (such as Hela or Cos-7 cells), in the presence of antibodies whose neutralizing power is tested, said pseudovirus vectors being obtained by an extracellular method of encapsidation of the reporter gene (s) in papillomavirus VLPs, this method being as described above (in which the plasmid containing the or the heterologous nucleotide sequence (s) of therapeutic interest, is replaced by a plasmid containing the reporter gene (s),
analysis of the expression or absence of the reporter gene within these cells, the absence of expression of the reporter gene in said cells being correlated with the fact that the antibodies tested neutralized the infection of the cells by said pseudoviruses, and therefore that these antibodies represent neutralizing antibodies that can be used in the context of the treatment of pathologies due to infection by papillomaviruses.

 Among the reporter genes that may be used, mention may be made of β-galactosidase, aequorin (GFP), luciferase, etc.

The invention also relates to a kit (or kit) for implementing a detection method as described above, this kit comprising
vector pseudoviruses containing at least one reporter gene, as obtained by an extracellular encapsidation process, as described above, of the reporter gene (s) in said VLPs,
cells capable of being infected by said pseudovirus vectors,
reagents necessary for the revelation of the activity encoded by the reporter gene.

The invention also relates to the method described above for the preparation of pseudoviruses as defined above of human or animal papillomaviruses that can be used as a vector for the transfer of genetic material (ie heterologous nucleotide sequences, the case therapeutically useful, as defined above) in target cells, said method being carried out by in vitro disassembly-reassembly of the aforementioned VLPs and comprises the following described steps
disassembly of the VLPs into capsomers, in particular by incubation of the latter with a chelating agent such as ethylene glycol tetracetate (EGTA), or ethylene diamine tetraacetate (EDTA), and a reducing agent such as dithiothreitol (DTT) ), or ss-mercaptoethanol,
mixing the plasmid containing the heterologous DNA sequence (s), which may be of therapeutic interest, advantageously in a ratio of one plasmid molecule to one VLP molecule (approximately 1 μg of plasmid, for approximately 5 μg of VLP),
reassembly of capsomeres to VLPs containing the abovementioned plasmid, in particular by increasing the concentration of Ca + ions and adding an oxidizing agent such as dimethylsulfoxide (DMSO) or by any other method that neutralizes the action of the abovementioned reducing agent , especially by dialysis,
where appropriate, separation, in particular by chromatographic techniques, of the VLPs in which said heterologous nucleotide sequences are encapsidated and, on the other hand, VLPs which remain empty (not containing said nucleotide sequences) after the reassembly of the VLPs. capsomeres into VLPs.

 The invention will be further illustrated by means of the following detailed description of the process for preparing VLPs as defined above, containing a heterologous DNA sequence containing the -galactosidase gene, or that of the fluorescent protein. (GFP), as reporter genes.

I - Materials and Methods
a) In vitro expression of papillomavirus VLPs
The human papillomavirus type 16 (HPV-16) VLPs are obtained by expression of the HPV-16 L1 gene in the baculovirus expression system according to the method described in the article by Le Cann et al., 1994, and summarized below
The L1 gene of HPV-16 was amplified from a pathological product and then cloned into the pTAG vector.

 The gene is then subcloned into the vector pBlue Bac III (Invitrogen) derived from a baculovirus specific for the insect Autographa californica.

 Plasmids are cotransfected with wild-type DNA in insect cells, Sf9, Sf21, or "high-five".

 The recombinant viruses are then selected by limiting dilution.

 The Spodoptera Frugiperda (Sf21) cells infected with the recombinant baculovirus are harvested 4 days after infection and lysed with a solution of NonidetB P40 in PBS for 15 mm at room temperature.

The nuclear fraction is recovered by centrifugation (10,000 xg, 15 min, 4 ° C) and subjected to 3 x 15s at maximum power (Vibra-cell,
Bioblock Scientific, Strasbourg, France).

 The nuclear lysate is deposited on a CsCl I, centrifuged at equilibrium in a Beckman B SW 28 rotor (20 h, 27,000 rpm, 4 ° C) and collected in 1.5 ml fractions.

 The density and the reactivity of the HPVs in ELISA were verified according to the method described in the article of Touzé et al., 1996.

 ELISA-positive fractions which have a density between 1.27-1.30 were pooled, diluted in PBS and subjected to ultracentrifugation in an SW 28 rotor (3 h, 28,000 rpm, 4 ° C).

 The pellet is resuspended in 0.15M NaCl.

Protein content was determined using the MicroBCA PierceB assay.
b) Plasmids
Two plasmids carrying different reporter genes were used. pGreenLantern (Life Technologies, Eragny, France) containing the gene encoding the fluorescent protein (GFP) of Aequoria victoria under the control of the cytomegalovirus promoter. pCMV-ssgal containing the gene coding for E. coli s-galactosidase. Plasmid pCMV-HBc was used for immunization studies. This plasmid contains the truncated gene at the 3 'end of the hepatitis nucleus antigen
B under the control of CMV promoter.
c) Disassembly and reassembly of HPV-16 VLPs
The disassembly and reassembly of the recombinant HPV-16 VLPs was carried out according to a method adapted from that described in the article by Colomar et al., 1993, as part of the SV40 virions.

 In summary, purified HPV-16 VLPs at a concentration of 500 μg / ml are incubated in 50 mM Tris-HCl buffer (pH 7.5) containing 150 mM NaCl, 1 mM EGTA, and 20 mM DTT in a final volume of 50, ul at room temperature for 30 minutes.

At this stage, 1 μg of plasmid containing the reporter gene (GFP or ss-gal) in a 50 mM Tris-HCl buffer (pH 7.5) and 150 mM NaCl is added to the
VLPs disassembled.

 To encapsidate the DNA in the viral particles, the CaCl 2 concentration is then gradually increased to 5mM and DMSO is added (1% final) (final volume 100 ass1). This solution is incubated for 1 h at 20 ° C.

The reconstituted VLPs are treated with 10 U Benzonase (Merck,
Darmstadt, Germany) for 1 hour at 20 ° C to verify that the plasmid DNA is integrated into the VLPs, and not absorbed at their surface.
d) Transfection / infection experiments
Cells grown in monolayer in DMEM / Glutamax (Life Technologies) supplemented with fetal calf serum (FCS), 10%, penicillin 100 IU / ml and streptomycin 100 μg / ml, were seeded on 6-well plates (Nunc, Life Technologies) and grown up to 80% confluency. Cells were washed twice with DMEM / Glutamax.

 The pCMV-ssgal-VLPs or pGreenLantern-VLPs viral pseudo-particles in 1 ml of culture medium were added to the wells and incubated for 4 h at 37 ° C.

 At this stage, the VLPs were removed, and 3 ml of DMEM / Glutamax supplemented with 10% FCS was added.

As a positive control, cells were transfected using the same amount of DNA (1, ug) with 10, a1 of a liposome, lipofectamineX (Life).
Technologies) according to the manufacturer's instructions.

 After 4 hours of incubation at 37 ° C, 2 ml of culture medium containing fetal bovine serum was added and the cells were incubated for 48 h at 37 ° C.

 At this point, the GFP or β-galactosidase activities of the cells were analyzed.

For the neutralization experiments, artificial viruses were preincubated for 30 mm at 37 ° C with PBS containing anti-HPV16-VLP mouse antiserum diluted 1/100. This antiserum was obtained by subcutaneous immunization of mice with , ug of HPV-16 VLPs absorbed on ALOI3 as an adjuvant.
e) Cellular lines
Eight cell lines were used for these experiments.

 Three human cell lines including MRC5 (lung), Hela (cervix) and CACO2 (colon).

NIH 3T3 embryonic cells from mice and 4 other animal cell lines including Vero and Cos-7 cells (monkey, kidney),
MDCK (dog, kidney) and CHO (hamster, ovary), were used.
f) Detection of reporter gene activity
For the determination of ss-galactosidase activity in situ, the cells were washed with PBS and fixed for 10 mm at room temperature with 0.2% formalin-2-glutaraldehyde solution in PBS for the detection of P-galactosidase.

 After three washes with PBS, the cells were exposed to 1 ml of a PBS solution containing 4mM K3Fe (CN) 6, K4Fe (CN) 6 3H2O 4 mM, 2mM MgCl 2, 100mM Tris (pH 7.4 and 1 mg / ml X-gal at 37 ° C for the expression of β-galactosidase.

 After development of staining (24 h), the cells were washed with PBS and observed.

Quantitative determination of β-galactosidase produced in transfected cell lines was tested by ELISA-β-gal (Boehringer,
Mannheim, Dassel, Germany) according to the manufacturer's instructions.

 In summary, the cells were washed 3 times with PBS 48 h after transfection. The cells were lysed and, after centrifugation, the total protein content of the supernatants was determined using the Bradford method and adjusted to a concentration of 500 μg / ml prior to testing for the presence of β-galactosidase.

 Samples of the supernatant were then added to the wells of a 96-well microplate pre-coated with an anti-β-galactosidase antibody.

 After one hour of incubation at 37 ° C, the wells were washed and an anti-β-galactosidase immunoglobulin of E. coli conjugated to digoxigenin was added.

 After a second incubation of one hour at 37 ° C and three washes, the specifically bound antibodies were detected by incubation for one hour at 37 ° C with an anti-digoxigenin immunoglobulin conjugated to horseradish peroxidase.

 After three washes, a solution of ABTS was added and the optical densities were read at 405 nm after a 20 min incubation at room temperature. The concentration of β-galactosidase (μg / ml) was determined relative to a calibration curve.

For the determination of GFP activity, the number of fluorescent and non-fluorescent cells was determined using flow cytometry
FAC Scan to measure the expression of GFP protein.

For this purpose, the cells were treated with trypsin, centrifuged and resuspended in 1 ml of culture medium. Cells were analyzed using a FAC Sort flow cytometer (Becton-Dickinson, Montain View, CA, USA). In addition, the cells were directly observed using a fluorescence microscope and photographed directly.
g) Immunization study
Female BALB / c (H2d) mice, 12-16 weeks old, were kept under pathogen-free conditions during the immunization protocol.

 BALB / c mice received two injections of 5 g of reconstituted HPV-16 VLPs containing plasmid pCMV-HBc into Tibialis anterior muscle two weeks apart.

Two groups of control mice received 100 μg of plasmid pCMV
HBc in the same place, with or without cardiotoxin, 5 days before the vaccinating injection, and a group of control mice received 5 g of purified recombinant HBcAg.

 The intramuscular injection of 100, t41 of 10 mM cardiotoxin was carried out so as to increase the capture of the plasmid DNA by the muscle cells.

 Blood samples were collected at each vaccinating injection and 15 days after the second.

Recombinant HBcAg assembled to VLPs was purified from infected insect cells with a recombinant baculovirus encoding the HBc gene.
h) Detection of anti-HBc and anti-HPV16 antibodies
Antibodies directed against HBcAg and HPV-16 VLPs were studied using ELISA techniques.

 VLPs of HPV-16 or HBcAg (100 mg / well) in PBS (pH 7.4) were incubated overnight at 40C.

 After 4 washes with 0.1% PBS-Tween 20, nonspecific binding sites were blocked by incubation for 30 min at 37 ° C with 1% PBS-NBS (bovine newborn serum).

 The blocking solution was replaced with 100 μl of mouse serum diluted twice in SxPBS containing 10% NBS and 2% Tween 20.

 After incubation at 45 ° C for 90 mm, and 4 washes, the bound antibodies were detected with anti-mouse immunoglobulin covalently bound to horseradish peroxidase (Sigma, Saint-Quentin Fallavier, France).

 After incubation at 45 ° C for 90 mm, and 4 washes, the substrate solution containing o-phenylene diamine and H 2 O 2 was added.

 The reaction is stopped after 30 minutes by addition of 4N H 2 SO 4 and the optical densities (492 nm) were read with an automatic reader (Microplate reader EL 311, Bioteck Instruments).

 The titers or antibodies correspond to the inverse of the last dilution of serum giving a positive result (OD greater than 0.1).

 The results are expressed as a geometric mean of titles calculated from the different titles obtained from each mouse of each group.

Il - Results
a) Disassembly - reassembly of papillomavirus VLPs
HPV-16 VLPs were produced in Sf21 insect cells using a recombinant baculovirus.

 The VLPs were purified by ultracentrifugation to sucrose followed by a CsCl gradient (Le Cann et al., 1994).

 Fractions of the CsCl gradient containing the VLPs were pooled, diluted in PBS and then ultracentrifuged.

 The pellet is then resuspended in a buffer containing EGTA and DTT and incubated at room temperature for 30 minutes. Under these conditions, the VLPs are completely disintegrated into capsomer-like structures.

The plasmid DNA is then added and the preparation is diluted in a buffer containing a high concentration of calcium and 1% DMSO so as to reform the VLPs. About 75% of L1 proteins appear to reassemble into VLPs under these conditions.
b) In vitro transfer of plasmid DNA via papillomavirus VLPs
Hela cells have been studied for their sensitivity to VLPspGreenLantern. The fluorescence of the GFP protein produced in the transfected cells was analyzed by flow cytometry. 70% of the Hela cells transfected with the plasmid pGreenLantern packaged in the VLPs are fluorescent (Table 1).

 In comparison, no fluorescent cells were found when the cells were incubated with the plasmid DNA alone, or with the plasmid and the L1 protein assembled into capsomers, and 40% of the cells were fluorescent when treated with the plasmid pGreenLantern and lipofectamine8.

 The specificity of VLP transfer has further been demonstrated by the fact that HPV-16 anti-VLP antibodies completely block gene transfer and that benzonase treatment does not affect the transfer. Free capsomeres can not act as vectors for gene transfer. In addition, no gene transfer could be identified with 0.5 μg VLPs-pGreenLantern when Hela cells were incubated with 10 μg (20-fold excess) of empty HPV-16 VLPs (Table 1). 1).

Table 1:
Transfer of the GFP gene by the viral pseudo-particles of HPV-16
highlighting and specificity

<tb><SEP><SEP> Hela <SEP> cells treated <SEP> with <SEP>% <SEP> fluorescent <SEP> cells
<tb> 5 <SEP>, ag <SEP> from <SEP> VLP-pGreenLantern <SEP> 70 <SEP>%
<tb> 5 <SEP>, ug <SEP> of <SEP> VLP-pGreenLantern <SEP> + <SEP> Benzonase <SEP> 70 <SEP>%
<tb> 5 <SEP> yg <SEP> of <SEP> VLP-pGreenLantern <SEP> + <SEP> AC <SEP> anti-VLPs <SEP> of HPV-16 <SEP> 0 <SEP><SEP> % <SEP>
<tb> 5 <SEP> yg <SEP> of <SEP> capsomeres <SEP> + <SEP> pGreenLantern <SEP> 0 <SEP>% <SEP>
<tb> pGreenLantern <SEP> + <SEP> LipofectamineB <SEP> 40 <SEP>%
<tb> pGreenLantern <SEP> 0% <SEP>
<Tb>
Transfer of pCMV-ssgal DNA has also been studied.

 The results show that both plasmids were transfected with the same efficiency and the proportion of transfected cells is similar to that evaluated by flow cytometry.

Expression of β-galactosidase was measured by ELISA in the cell lysate. The results indicate that the level of expression corresponds with the amount of pCMV-ssgal -VLPs used. An increase in the expression level of β-galactosidase of about 100 was observed between vector concentrations
VLPs from 1 to 10, ug.

 Eight cell lines were studied for their ability to be transfected with the plasmid pCMV-ssgal packaged in HPV-16 VLPs.

 The results (Table 2) show that all the cell lines can be transfected with this vector and that the level of ss-gal expression obtained is generally 3 to 6 times higher than that observed after transfection of the same cells by lipofection. However, the level of expression of β-galactosidase ranges from 102 μg / ml in Vero cells to 2253 μg / ml in CaCo2 cells.

Table 2:
Effectiveness of gene transfer mediated by pseudo-viral particles in
different animal and human cell lines

<tb> Cell lines <SEP><SEP> ss-galactosidase <SEP> produced <SEP> (pg / ml)
<tb><SEP> studied <SEP> Lipofectamine <SEP> Pseudo-particles
<tb><SEP> MRC5 <SEP> 79 <SEP> 265
<tb><SEP> Hela <SEP> 234 <SEP> 852
<tb><SEP> CaCo2 <SEP> 320 <SEP> 2253
<tb><SEP> NIH3T3 <SEP> 65 <SEP> 275
<tb><SEP> Cos-7 <SEP> 487 <SEP> 3142
<tb><SEP> Vero <SEP> 41 <SEP> 102
<tb><SEP> MDCK <SEP> 5 <SEP> 145
<tb><SEP> CHO <SEP> 43 <SEP> 150
<Tb>
c) Humoral immune response in mice Immunized with pCMV-HBc DNA packaged inside HPV-16 VLPs
DNA immunization was studied in BALB / c mice using plasmid pCMV-HBc encoding hepatitis B core antigen as a possible application of gene transfer using a packed gene in HPV-16 VLPs.

Results of two intramuscular injections of 5 μg of pCMV-HBc
HPV-VLPs in a two-week interval were compared with those obtained by intramuscular injection of 100 μg of plasmid DNA alone, and those obtained by intramuscular injection of 5 g of HBc VLPs produced in Sf21 cells with a baculovirus. recombinant.

 Anti-HBc antibodies were observed in all immunized animals. However, the anti-HBc titer (20, 138) is higher in mice immunized with pCMV-HBc packaged inside HPV VLPs, than in mice immunized with pCMV-HBc alone (1,000 to 1,600), and above. high than the titre (6,400) observed by intramuscular injection of HBc capsids.

 Anti-HPV-16 antibodies were not detected in mice immunized with HBc VLPs or with HBc DNA, but are present at a titre of 800 in mice immunized with HPV VLPs containing the HBc DNA after the second injection. The level of anti-HPV-16 antibody is however much lower than that observed in the mice immunized with 5 μg of HPV VLPs absorbed on Al (OH) 3 as an adjuvant, in which the anti-VLP titers reach 30,000 to 70,000.

III- Conclusion
HPV-16 L1 protein VLPs, obtained by expression of the major capsid protein of human papillomavirus type 16 in insect cells, can encapsulate a plasmid carrying either a gene for the GFP protein, or a gene for β-galactosidase during the in vitro disassembly-reassembly of
VLPs.

 The introduction of the plasmid into different cells by these pseudovirions or artificial viruses, is easily detected by the accumulation of the gene product, and the number of cells carrying the gene is a function of the concentration of HPV-16 VLPs in the transfer experience.

 The demonstration of plasmid encapsidation rather than an association between plasmid and L1 protein is based on Benzonase B resistance of VLP-mediated DNA transfer within the cells.

 The efficiency of gene transfer by VLPs greatly exceeds that achieved by calcium phosphate or lipid transfection.

 In contrast to results obtained by other teams (Zhou et al., 1994, Roden et al., 1996 and Unckell et al., 1997), the results presented here indicate that HPV L2 protein is not necessary. for efficient encapsidation of the DNA, nor for its transfer into a host cell. In addition, the transfer of the gene is 103 to 104 times more efficient than with the systems described by these authors.

 Gene transfer was specifically inhibited either by preincubation of VLPs with anti-VLP antisera, or by competition with empty HPV-16 VLPs. Therefore, the HPV pseudovirions described herein also represent a useful system for the detection of anti-papillomavirus neutralizing antibodies.

 The use of VLPs for gene transfer has advantages over the use of recombinant viruses. The production of such pseudovirions is much easier than that of the usual viral vectors, and the risk of induction of neutralizing antibodies is lower. In addition, these pseudovirions are safer to use because no recombination with a wild-type virus is possible since they do not contain a papillomavirus DNA sequence.

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Claims (11)

 1. Use of viral pseudo-particles (VLPs) of human or animal papillomaviruses, for the preparation of vector pseudoviruses that can be used for the transfer of genetic material of therapeutic interest into target cells of the organism, in particular within the framework of gene therapy, or gene immunization.  2. The use according to claim 1, of VLPs containing the protein L1, and where appropriate the L2 protein, papillomaviruses.  3. Use of papillomavirus VLPs defined in claims 1 or 2, for the preparation of a medicament comprising pseudovirus vectors to said VLPs in which one or more heterologous nucleotide sequences of therapeutic interest are encapsidated, said pseudoviruses being in association with a pharmaceutically acceptable carrier, said medicament for the treatment or prevention of pathologies, as described above, which can be treated by gene therapy or to be prevented by gene immunization.  4. Pseudovirus vector for the transfer of genetic material into target cells of the organism in the context of gene therapy, or of gene immunization, characterized in that it corresponds to a VLP of human or animal papillomaviruses, in which one or more heterologous nucleotide sequences of therapeutic interest are encapsidated.  5. Pseudovirus vector according to claim 4, characterized in that the VLPs contain the L1 protein and, where appropriate, the L2 protein, papillomavirus.  6. Pseudovirus vector according to claim 4 or 5, as obtained by an extracellular method of encapsidation of one or more heterologous nucleotide sequences of therapeutic interest within the VLPs, in particular by a process of disassembly-reassembly in vitro VLPs, in the presence of a plasmid containing the (or) heterologous DNA sequence (s) of therapeutic interest.  7. A vector pseudovirus according to claim 6, as obtained by an in vitro disassembly-reassembly process of VLPs comprising the following steps  disassembly of the VLPs into capsomers, in particular by incubation of the latter with a chelating agent such as ethylene glycol tetracetate (EGTA), or ethylene diamine tetraacetate (EDTA), and a reducing agent such as dithiothreitol (DTT) ), or β-mercaptoethanol,  mixing the plasmid containing the heterologous DNA sequence (s) of therapeutic interest, advantageously in a ratio of one plasmid molecule to one VLP molecule (ie approximately 1 μg of plasmid, for about 5% of VLP),  reassembly of capsomeres to VLPs containing the abovementioned plasmid, in particular by increasing the concentration of Ca + + ions and adding an oxidizing agent such as dimethylsulfoxide (DMSO) or by any other method that neutralizes the action of the abovementioned reducing agent , especially by dialysis.  8. Pharmaceutical composition, characterized in that it comprises a vector pseudovirus according to one of claims 4 to 7, in association with a pharmaceutically acceptable vehicle.  9. Use of pseudovirus vectors corresponding to VLPs containing L1 protein and, where appropriate, L2 protein, human or animal papillomaviruses, in which one or more heterologous nucleotide sequences (in particular nucleotide sequences coding for reporter genes) are encapsidated, said pseudovirus vectors being obtained by an extracellular method of encapsidation of said heterologous nucleotide sequences in said VLPs, for the implementation of a method of in vitro transfer of said heterologous nucleotide sequences in cells susceptible to be infected by said pseudoviruses vectors.  10. Use of pseudovirus vectors corresponding to VLPs containing L1 protein and, where appropriate, L2 protein, human or animal papillomaviruses, in which at least one reporter gene is encapsidated, said pseudoviruses being as obtained by a method extracellular packaging of the gene (s) reporter (s) in said VLPs, for the implementation of a method for detecting antibodies neutralizing the infection of human or animal papillomaviruses.  11. A method for preparing human and animal papillomavirus pseudoviruses, said pseudoviruses being capable of being used as a vector for transferring genetic material into target cells, characterized in that it comprises the following steps  disassembly of VLPs into capsomers, in particular by incubation of the latter with a chelating agent such as ethylene glycol tetracetate (EGTA) or ethylene diamine tetraacetate (EDTA), and a reducing agent such as dithiothreitol (DTT ), or ss-mercaptoethanol,  mixing the plasmid containing the heterologous DNA sequence (s), where appropriate of therapeutic interest, advantageously in a ratio of one plasmid molecule to one VLP molecule (approximately 1% plasmid, for approximately 5% of VLP),  reassembly of capsomeres to VLPs containing the abovementioned plasmid, in particular by increasing the concentration of Ca + + ions and adding an oxidizing agent such as dimethylsulfoxide (DMSO) or by any other method that neutralizes the action of the abovementioned reducing agent , especially by dialysis.