MXPA99003286A - Attenuated microorganism strains expressing hpv proteins - Google Patents

Abstract

This application relates to the use of attenuated prokaryotic microorganism strains (such as Salmonella) expressing nucleic acid encoding HPV proteins as vaccines against HPV infection and the associated increased risk of cancer. In particular, the work shows that it is possible to assemble VLPs in a prokaryotic organism and that nasal immunization of mice with the strains HPV-specific conformationally dependent and neutralizing antibodies in serum and genital secretions. The experiments described herein show that it is also possible to assemble chimeric VLPs of an HPV including a fusion partner and that tumor protection can be induced.

Description

SCARS OF ATTENUATED MICROORGANISMS EXPRESSING HPV PROTEINS FIELD OF THE INVENTION The present invention relates to attenuated strains of prokaryotic microorganisms, in particular Salmonell a, transformed with the nucleic acid encoding the papillomavirus virus proteins, to the compositions comprising these microorganisms, especially for use as vaccines, and to the medical uses of these strains. In a further aspect, the present invention provides a method for producing particles similar to assembled papillomavirus viruses (VLPs).

BACKGROUND OF THE INVENTION Human papilloma virus (HPV) 16 is the major type of HPV which, in association with cofactors, can lead to cervical cancer (49). Studies on HPV have been hampered by the inability to propagate viruses in culture, by the lack of animal models and by the small number of virions in clinical lesions. This has led REF; 29967 to the development of alternative procedures for the production of antigens for immunological studies. The conformational dependence of the neutralization epitopes, as observed in the experimental animal systems of the papillomavirus (8,22), suggests that appropriately assembled HPV particles are critical for the induction and detection of clinically relevant immune reactivity. The HPV capsids are formed by 72 pentaeric capsomeres of Ll proteins arranged over an icosahedral T7 network (15). Recently, a number of researchers have demonstrated the production of HPV capsids, for example, virus-like particles (VLPs), through the use of baculovirus, vaccinia virus or yeast expression systems (15, 22, 45, 48 , 61). The potential of VLPs as subunit vaccines has been demonstrated using cottontail rabbit papillomavirus (CRPV) (4), canine oral papilloma virus (COPV) (57), and HPV11 models (45). HPV16 infects through the genital mucosa, where benign proliferative lesions are confined. Protection against infection with such a pathogen could be provided by specific secretory immunoglobulins A (anti-VLP) (slgA) or immunoglobulins G (IgG) in genital secretions. By analogy with existing animal models, VLP-specific antibodies, HPV16 in cervical secretions can help prevent sexually transmitted infection by HPV16 in women. However, this can not be formally tested in the absence of an experimental model for genital PV infection and other scenarios that require cell-mediated immunity can not be excluded. In addition, the mechanism underlying HPV infection is unclear. HPV can directly infect the basal cells of the stratified cervical epithelium in the appearance of openings. Alternatively, HPV infection could also occur either directly through the Langerhans cells in the intact epithelia or indirectly from the keratinocytes that produce HPV, and thus the neutralizing antibodies will not be functional as is shown by other viruses. . This also adds to the difficulty in providing effective vaccines against HPV infection.

Immunosuppressed individuals are more likely to develop cervical carcinoma compared to immunocompetent individuals, suggesting the possibility of using immunotherapy. Therapeutic vaccines (87) directed at the treatment of established HPV infection or the premalignant and malignant lesions associated with HPV have been investigated during the last ten years (59). The evidence for HPV antigen-directed immunotherapy against cervical cancer comes from observations that experimental tumors (13), (34), (83) and natural (82) associated with PV can be controlled by immunization with preparations of E6 and E7. These studies suggested that CTL could be the most effective immune effector mechanism. The preparations of E6 and E7 consisted of either peptides (13), bacterially prepared fusion proteins (82), eukaryotic transfected cells (83) or recombinant vaccinia virus (34). Recently, chimeric VLPs possessing the 17 D E7 protein as a fusion with L2 have been shown to induce the rejection of syngeneic tumor cells (84) genetically engineered to express Ll and / or E7 ORF (e.g. C3 cells (13 ) and TC1 cells (85)). These data demonstrate the possibility of providing prophylactic and therapeutic effects in the same vaccine preparation. Salmonells, which are attenuated but still invasive, have been proposed for the distribution of heterologous antigens to systemic mucosal immune systems (19). The antigen is administered by Salmonell to live mucus-inducing sites, where after priming, the antigen-specific B and T cells migrate from the induction site and mature in the effector cells. B cells that express Iga that migrate, are housed in different mucosal sites, including the genital tract, where they differentiate into plasma cells that secrete IgA (32) In this way, oral or nasal immunization can provide protective antibodies in genital secretions. Recently, the present inventors and others have shown that mucosal immunization with recombinant Salmonell a can elicit antibody responses in the genital mucosa of mice and humans (18, 37, 56).

BRIEF DESCRIPTION OF THE INVENTION In order to develop a prophylactic vaccine against HPV, the major protein of HPV16 has been expressed in a strain of Salmonel l to typhimuri um attenuated PhoPc (35). Surprisingly, the inventors found for the first time that it is possible to assemble the VLPs in a prokaryotic organism and that the nasal immunization of the mice with a recombinant strain of HPV16-L / Salmonell a induces conformationally dependent and HPV16-specific neutralizing antibodies in serum and genital secretions. The experiments described herein also show that it is possible to assemble chimeric VLPs from an HPV protein and a fusion partner. Accordingly, in a first aspect, the present invention provides an attenuated strain of a prokaryotic microorganism transformed with the nucleic acid encoding the major capsid protein of the papillomavirus virus, wherein the protein is assembled in the microorganism to form similar particles to the virus (VLPs). Thus, the present invention provides a way to produce papillomavirus VLPs, suitably assembled, in an attenuated strain of a prokaryotic microorganism such as Salmonel la, so that they can be used as a vaccine to produce an immune response in a subject . Preferably, the VLPs are distributed towards the mucosal sites, having the advantage of generating the immune response to the papillomavirus VLPs in the sites where the infection actually takes place, as well as in other mucosal surfaces. The term "papillomavirus" used herein covers human and animal PVs. Preferably, however, the papillomavirus is a human papillomavirus (HPV). Approximately 70 different types of HPV have been cloned and characterized (denoted HPV1 to HPV70 ...), and all have a double-stranded genome of 8 kb which codes for different early products and two late products Ll and L2, and they are already be epitheliotropic or mucosatropic. Ll is a major capsid protein and is relatively well conserved among the different types of HPV. For a review of the types of HPV and their nucleic and "amino acids, see Human Papillomaviruses," A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences, "1994, ed. Myers and collaborators, Theoretical and Biophysics Group T-10, Los Alamos National Laboratory.Clinically, the most important types of HPV are those that infect the anogenital tract, and that have a high oncogenic risk and a high prevalence.This group includes HPV16, 18, 31, 45 and 56, with HPV16 only representing more than 50% of the invasive cancer in the anogenital tract, as well as being the most prevalent simple type of HPV Papillomavirus proteins correspond to the major capsid proteins of wild type (for example Ll and / or L2) or they can be chimeras of all or part of an HPV protein and a partner The fusion partner can be any immunogenic protein against which the specific CTL could be directed, this protein can be an HPV protein (for example E7, E6 or E2 of any type of HPV), a prot eina from another pathogen or any specific tumor antigen. In one embodiment, the HPV protein is the Ll protein coexpressed with L2, with the fusion partner expressed so that it is linked to the L2 protein. It has been shown that chimeric VLPs can elicit anti-tumor immunity against carrier and inserted proteins in HPV16 tumor models. In this way, the chimeric VLPs which induce E7 specific CTLs directed to kill cells already infected with HPV or premalignant lesions associated with HPV. In this event, the induction of CTLs to eliminate cells already infected with HPV, therefore seems an attractive complement for the induction of neutralizing antibodies, and chimeric VLPs have been shown to induce both functions. Thus, in one embodiment of the invention, strains of Salmonella capable of inducing neutralizing antibodies and CTLs by the expression of chimeric VLPs, could be therapeutic at least for early or premalignant HPV lesions in which the HPV has not yet appeared. MHC I sub-regulation or other factors observed in more advanced cancers. Preferably, the prokaryotic microorganism is an attenuated strain of Salmonell a. However, alternatively other prokaryotic microorganisms such as attenuated strains of Escheri chia coli, Shigella, Yersinia, Lactobacillus, Mycobacteria, Li s teri or Vibri o may be used alternatively. Examples of suitable strains of microorganisms include Salmonella typhimurium, Salmonella typhi, Salmonella dublin, Salmonella enteretidi s, Escherichia coli, Shigella fl exeneri, Shigella sonnei, Vibrio cholera, and Mycobacterium bovis (BC6). Attenuated strains of Salmonell a are one of the best characterized mucosal vaccine carriers. Recombinant Salmonell a strains that are attenuated and are still invasive have been used as oral vaccine vectors to carry protective epitopes of various pathogens to mucosal-associated lymphoid tissue, thereby inducing mucosal, systemic and CTL immune responses against the carrier and the foreign antigens (58, 65, 67, 69, 75-77). The currently authorized oral vaccine against typhoid fever, S. typhi Ty21a (72) administered as a three-dose regimen of enteric-coated capsules (109 CFU / capsule) gave an efficacy of 67% over a period of 3 years. However, because S. typhi Ty21a requires high and multiple doses in liquid formulation for greater efficiency, and its mutations are not yet characterized all (63, 64, 70, 71, 78), new strains of Salmonell a have been recently developed, and tested in humans. These include nutritional auxotrophs in which the pathways for the biosynthesis of aromatic compounds (mutants? Aro) have been interrupted. The mutants? AroA,? PurA de S. Typhi have been tested in human volunteers (32) and showed that they elicit specific immune responses, mediated by cells but weak humoral responses. Other aroC mutants (aroC and aroD) were insufficiently attenuated and caused fever and bacteremia (79). A double mutant? AroC? AroD Ty2 (CVD 908) was safe and produced IgG antibodies against LPS in 80% of immunized adult volunteers (73, 80). The mutants of S. typhi were also generated, in which adenylate cyclase (cya) and cAMP receptor (cyclic AMP) (crp) genes were suppressed. These gene products are required for the transcription of many genes and operons that control transport processes, the expression of fimbrias, flagella and some outer membrane proteins. A mutant? 3927 (? Cya? Crp Ty2) was tested and shown to have immunogenic capacity, but some volunteers developed fever and bacteremia with the vaccine (79). Therefore, a new strain,? 4073, was constructed by suppressing a third gene (cdt) responsible for the colonization of deep tissue (66, 68, 74). This strain was administered to volunteers and proved completely safe at doses of up to 5 x 108 CFU and generated seroconversion in 80% of the volunteers (66). Other attenuated strains of Salmonell a include mutants in a two-component regulatory system, the phoP / phoQ genes. These genes affect the expression of a number of other genes and respond to phosphate levels and to the environmental conditions expected to be experienced by Salmonell to which it resides within macrophages. An example of these mutants is the PhoPc strain used in the examples described below. Recently, Salmonella typhi suppressed in PhoP / PhoQ (ty800) has been shown to be safe and immunogenic in humans (81). As mentioned above, the attenuated strain of the prokaryotic microorganism is transformed with a nucleic acid encoding one or more of the major capsid proteins of the papillomavirus. The inventors found for the first time that, when this nucleic acid is expressed in microorganisms, the proteins of the The capsids produced are assembled correctly to form VLPs, making them especially suitable for vaccination of subjects against papillomaviruses. Preferably, the major viral capsid p-rotein is Ll, which optionally and additionally includes the nucleic acid encoding the L2 protein. As discussed above, the capsid protein can be linked to a fusion partner such as another antigen. In a further aspect, the present invention provides a composition comprising one or more of the above attenuated prokaryotic microorganisms, optionally in combination with a physiologically acceptable carrier.

Preferably, the composition is a vaccine, especially a vaccine for mucosal immunization, for example for administration via the oral, rectal, nasal, vaginal or genital routes. The first studies using recombinant Salmonella expressing the viral hepatitis B antigen (18) showed that vaccination via any of these routes produces a slgA response in mucosal secretions and other sites.

Advantageously, for prophylactic vaccination, the compositions comprise one or more strains of Salmonell to which they express a plurality of different VLPs, for example VLPs from different types of papillomavirus. This has the advantage of improving the protective effect of the vaccine against a range of challenges due to the different types of papillomavirus. For therapeutic vaccination, the constructions of subsequent chimeric VLPs may comprise fusion products of various HPV type L capsids with the same L2 fusion partner. In a further aspect, the present invention provides an attenuated strain of a prokaryotic microorganism described above for use as a medicament, especially as a vaccine. In a further aspect, the present invention provides the use of an attenuated strain of a prokaryotic microorganism transformed with the nucleic acid encoding the major capsid protein of the papilloma virus virus, wherein the protein is assembled into the microorganism to form particles similar to the virus, in the preparation of a medicament for the prophylactic or therapeutic treatment of papillomavirus infection or anogenital cancer, especially cervical cancer In general, the microorganisms or VLPs according to the present invention are provided in a form isolated and / or purified, for example substantially pure This can include that it is in a composition where it represents at least about 90% of the active ingredient, more preferably at least about 95%, and still more preferably at least 98%. , however, include inert carrier materials or other pharmaceutical excipients eutically and physiologically acceptable. A composition according to the present invention may include in addition to the microorganisms or VLPs as described, one or more other active ingredients for therapeutic use, such as an antitumor agent. The compositions of the present invention are preferably administered to an individual in a "prophylactically effective amount" or a "therapeutically effective amount" (as the case may be, although prophylaxis may be considered therapy), this being sufficient to demonstrate benefit to the patient. individual. The effective amount administered, and the proportion and course in time of administration, will depend on the nature and severity of the treatment. The prescription of treatment, for example, decisions about dosage, etc., is within the responsibility of general practitioners and other physicians. A composition can be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the condition to be treated. The pharmaceutical compositions according to the present invention, and for use in accordance with the present invention, may include, in addition to the active ingredient, an excipient, buffer carrier, stabilizer or other pharmaceutically acceptable materials, well known to those of experience in the art. technique. Such materials must be non-toxic and must not interfere with the effectiveness of the active ingredient. The precise nature of the carrier or other material will depend on the route of administration. Examples of techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 16th Edition, Osol, A. (ed.), 1980. In a further aspect, the present invention provides a method for producing particles similar to the assembled papillomavirus virus, which comprises culturing an attenuated strain of a prokaryotic microorganism transformed with the nucleic acid encoding the major capsid protein of the papillomavirus virus, wherein the protein is expressed and assembled in the microorganism to form virus-like particles. Preferably, the method further comprises the step of recovering the VLPs of the prokaryotic microorganism. In a further aspect, the present invention provides the use of a papillomavirus VLP as is obtainable by transforming an attenuated prokaryotic microorganism with the nucleic acid encoding the VLPs, and expressing the nucleic acid to produce the assembled VLPs, in a diagnostic method. In one embodiment, the present invention provides a method for detecting the presence of anti-papillomavirus antibodies in a sample from a subject, comprising immobilizing the HPV VLPs on a solid support, exposing the support to the sample and detecting the presence of the antibodies, for example, using ELISA. Preferred embodiments of the present invention will be described by way of example and not limitation, with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE FIGURES Figure 1. Ll expression of HPV16 in the PhoPc / HPV strain. Salmonell ace was developed overnight and prepared as indicated in Materials and Methods. A. Commassie blue staining of a 10% SDS-PAGE gel; M band: molecular weight marker; Lane 1: total use of PhoP strain Band 2: total lysate of the PhoPc / HPV strain, a single 57 kDa protein is indicated by an arrow. B. Immunostaining using the anti-HPV16-Ll monoclonal antibody from the total and fractionated PhoPc / HPV lysate. Tot band: total lysate, Band 1 to 25: different fractions obtained after fractionation of the PhoPc / HPV lysate through a sucrose gradient of 10 to 40%, the heaviest fraction of the gradient is in Band 1. The band of 57 kDa protein identified as Ll is indicated (arrow) Figure 2. Ll of HPV16 is assembled in the VLPs. Electron micrographs of (A) PhoPc / HPV16 VLPs and (B) HPV16 VLPs derived from vaculovirus. In A, fractions 7 to 13 of the PhoPc / HPV lysate (Figure 1) were combined. The samples were negatively stained with the phosphotungstic acid. The bar represents 53 nm.

Figure 3. Responses of the systemic antibody and mucosal anti-HPV16 and anti-LPS after nasal immunization with the PhoPc / HPV strain. Three female Balb / c mice of 6 weeks of age were immunized with 5 x 107 CFU, samples were taken at the indicated weeks, sacrificed and bled at week 27. The data are expressed as the geometric means of the reciprocal dilutions of Specific IgG in serum and specific IgA in serum per microgram of IgA or total IgG per microgram of total IgG in secretions. The error bars indicate the standard errors of the means.

Figure 4. HPV viral cycle and vaccination strategies. In the left portion of the figure, the productive viral cycle of HPV is drawn schematically during the differentiation of keratinocytes (early infection CIN I). A late stage of infection (CIN III tumor) in which the HPV DNA is integrated into the host genome, shown on the right. Different protective immune mechanisms are shown with the arrows indicating the sites of action. Antibody-dependent cellular toxicity mechanisms (ADCC) which require viral antigens to be expressed on the surface of cells are not indicated.

Figure 5. Neutralization in vi tro of infection by HPV16 pseudotype virus of mouse C127 cells. (A) without added virus. (B-H) Equal aliquots of an HPV16 pseudotype virus (BPV1) containing the extract were added. The aliquots were incubated with (B) without antibodies, (C) monoclonal antibody (MoAb) B1.A1 neutralizer of BPV1, (D) monoclonal antibody (MoAb) H16.E70 neutralizer of HPV16 (BPV1), (E) preimmune serum of mouse, (F) immune sera. { week 27) of mouse # 4, (G) immune sera (week 2), mouse # 5, immune sera (week 27) of mouse # 4.

Figure 6. Shows the results of the tumor development experiments in mice immunized with strains of Salmonell to HPV producer. Nasal immunizations were performed three times weekly with either 20 μl of PBS (A) or 5 μg of purified HPV16 VLPs plus 5 μg of cholera toxin (CT) (E) and twice in week 0 and in the week 2 with 10 CFU of PhoPc / HPV16 Ll (b),? 4550 / pYA34Ll (C) and? 4550 / pYA32Ll (D). All mice were challenged with 5 X 10 5 C3 cells on the flank two weeks after the last immunization. The average volume of tumors in each group is shown, while the number of mice harboring a tumor / number of injected mice is indicated on day 17.

Figure 7 shows the coexpression of Ll and L2 in L1-L2 of PhoPc / HPV16. Spot A was developed with an anti-L2 antibody, while spot B was revealed with an anti-Ll antibody (Camvir).

Figure 8 shows the expression of Ll in E. coli BL12 pET 3D-L1. Identical amounts of bacteria were loaded (3 X 106 CFU) after 3 hours of incubation with IPTG and the spot was developed with an anti-Ll antibody.

DETAILED DESCRIPTION Materials and methods Plasmidic construction and bacterial strains used.

Plasmid pFS14nsd HPV16-L1 was constructed by exchange in plasmid pFS14 NSD (54), the nucleocapsid gene of hepatitis B (HBcAg, NcoI-HindIII fragment) for a fragment NcoI-HindIII coding for the open reading structure HPV16-L1. The NcoI-HindIII fragment of HPV16-L1 was generated by the Polymerase Chain Reaction (PCR) using the baculovirus expression plasmid pSynwtVI "HPVl 6 114 / B-L1 + L2 (23) as a template with a 28mer containing an Ncol site: 5'-GGGCCATGGCTCTTTGGCTGCCTTAGTGA-3 'and a 27mer containing a HindIII site 5'-GGGAAGCrTCAATACTTAAGCTTACG-3' The final construct containing the Tac promoter places the HPV16-Ll ATG at the +8 position relative to the Shine-Dalgarno sequence and introduces a change in the second amino acid, which becomes an alanine instead of the serine encoded by the original sequence.The sequence of the open reading structure Ll, complete, was carried out (MycrosynthAG) and no additional nucleotide change was observed Plasmid pFS14nsd HPV16-L1 was amplified in E. coli JM105 and then subjected to electroporesis as previously described (50) in bacterial strain CS022.This strain is derived from strain ATCC 14028 , within which the pho-24 mutation was introduced by P22 transduction, resulting in attenuation in virulence and survival within macrophages in vi tro (PhoPc, (35)). The resulting recombinant strain is referred to hereinafter as PhoPc / HPV.

Expression of HPV16-L1 in Salmonell a and Purification of VLPs.

After an overnight development at 37 ° C, the recombinant bacteria were used by boiling in Lae mli buffer containing 5% SDS. The used ones were separated on 10% SDS / PAGE gels and L expression was analyzed by Western blotting using the monoclonal antibody for HPV16-L1 CAMVIR-1 (33) as primary antibody, an anti-mouse IgG, loading , conjugated to alkaline phosphatase (Sigma) as secondary antibody and BCIP / NBT (Boehringer) as a substrate. To prepare the VLPs, the bacteria were used by sonication and the lysate was fractionated on a gradient of 10% -40% sucrose in Phosphate-buffered Saline (PBS) containing 1 M sodium chloride for 1 hour at 40 Krpm using a rotor TST1.14. The fractions of the gradient were then analyzed for the presence of the Ll protein by Western blotting. The high sedimentation fractions containing the Ll protein were combined, and dialysed against PBS / 0.5 M NaCl. The VLPs were concentrated by sedimentation for 1 hour at 50 Krpm using a TST65.1 rotor, adsorbed to carbon-coated grids, they stained negatively with phosphotungstic acid and examined with a Philips electron microscope.

Purification of HPV16 VLPs expressed in insect cells from a recombinant vaculovirus.

The transfer vector pSynwtVI "HPVl 6 114 / B-L1 + L2 (23) was cotransfected with the linearized genome of the baculovirus (Baculo-Gold, Pharmingen) using the calcium phosphate method in SF9 cells.The recombinant baculoviruses were purified in plates and propagated by standard methods (39). HPV16 VLPs derived from baculovirus were purified as previously described (23).

Immunization and sample taking of mice.

Six-week old BALB / c female mice were immunized on day 0 and day 14 by the nasal route with 5 X 107 CFU inoculum.

Samples of blood, saliva and genitalia were taken as previously described (18). All samples were stored at -70 ° C.

ELISA The amount of total antibodies IgA, Iga anti-LPS and IgG in the samples, were determined by the immunosorbent assay linked to enzyme (ELISA) as previously described (18). For him Anti-HPV16 VLP, the ELISA plates were coated with 10 ng of a preparation of the HPV16 VLPs derived from baculovirus in PBS (the total protein content was determined 'with a BioRad protein assay with BSA as standard). This amount of VLP was saturating the ELISA test. The endpoint dilutions of the samples were carried out. The specific amounts of IgA or IgG are expressed as the reciprocal of the highest dilution that produced a 0D 92 four times that of the pre-immune samples. These reciprocal dilutions were normalized in the amount of IgA or total IgG in saliva and genital washings. The ELISA plates were also coated with 10 ng of HPV16 VLPs derived from baculovirus in 0.2 M carbonate buffer pH 9.5, to determine the titre of the antibodies that recognize unfolded VLPs (14).

Neutralization test of HPV16 in vi tro.

Infectious pseudovirions consisting of the HPV capsid made of Ll and L2 surrounding the bovine papillomavirus genome type 1 (BPV1) designated HPV16 (BPV1), were generated as recently described (43). In summary, the BPHE-1 hamster cells harboring the autonomously replicating BPV1 genomes were coinfected with the defective, recombinant Semliki forest viruses that expressed the HPV16 Ll and L2 virion capsid genes. The HPV16 virus of the infectious pseudotype in cell extracts was quantified by the induction of the transformed foci in monolayers of mouse C127 cells. The neutralizing activity was measured after preincubation of the cell extracts with mouse sera diluted 1:50 (final volume of 1.0 ml) in culture medium. The mouse monoclonal antibodies H16.E70 and B1A1 were generated against the VLPs of HPV16 Ll expressed in recombinant baculoviruses, and the VLPs of BPV16 respectively, and were used at a dilution of 1: 100. H16.E70 and B1.A1 served as positive and negative controls for the neutralization of HPV16 (BPV1), respectively.

Results HPV16-L1 is expressed in the set of PhoPc and VLP.

The open reading frame of the major HPV16 protein Ll was cloned into the plasmid pFS14 NSD (53). Ll is constitutively expressed under the control of the Tac promoter in S. typhimuri um. A single 57 kDa protein detected in the lysate of overnight PhoPc / HPV cultures (Figure 1A), was identified as HPV16 L1 by Western immunostaining using an anti-HPV1 6-L1 monoclonal antibody (CAMVIR, ( 33), Figure IB). To determine whether the Ll protein expressed by PhoPc / HPV was assembled within VLP, the bacterial lysate was fractionated through a 10-40% sucrose gradient and the heavier fractions containing the Ll protein (Figure IB) were analyzed. by electron microscope. The spherical particles typical of the PV capsids were coated from the bacterial preparation (Figure 2A) but the bacterial VLPs appeared more polymorphic in sizes with diameters in the range of 40 to 55 nm (Figure 2A) when compared to the VLPs of approximately 55 nm expressed in insect cells (Figure 2B).

Nasal immunization with the PhoPc / HPV strain induces systemic and mucosal antibody responses.

Since nasal immunization using the Salmonell a recombinant showed that it provokes strong responses of vaginal SIgA against a foreign expressed antigen (18), mice were immunized nally with the PhoPc / HPV strain (5 X 107 CFU). Blood samples, saliva and vaginal washings were taken at 0, 2, 4 and 6 weeks after immunization. The immune responses against the carrier, for example, anti-LPS and the carried antigen, for example, anti-HPV16 VLP, were also determined. The serum HPV16 VLP specific IgG (Figure 3) was detected after 2 weeks in one mouse and after 4 weeks in all mice. The response peaked after 6 weeks at relatively low titres and persisted at least until week 14. At that time, no specific HPV16 VLP antibodies were detected in vaginal secretions, whereas a mouse had low IgA titers in the saliva The systemic and mucosal immune responses against LPS were relatively low (Figure 3), but similar to those elicited by the PhoPc / HBc strain (18) suggesting normal uptake of Salmonel I to PhoPc / HPV by the mice. The low anti-LPS response observed after nasal immunization prompted the present inventors to perform a booster immunization. In this way, a second nasal immunization was performed at week 14 and samples were taken 5 and 10 weeks later (week 19 and 24 respectively). The second immunization induced, 5 weeks later (week 19), a 15-fold increase in anti-HPV16 VLP IgG in serum, as well as an anti-HPV16 VLP IgA in the vaginal washings (Figure 3) from three mice . The IgG anti-HPV16 VLP was also found in vaginal washings but only in two mice in week 19, and the titers were again almost undetectable in week 24 (Figure 3). The IgA and IgG anti-HPV16 VLP were also found in the saliva of the three mice in amounts comparable or slightly higher than those found in vaginal washings.

Anti-HPV16 VLP antibodies recognize only the folded VLP.

In order to examine whether the immune responses induced by the PhoPc / HPV strain generated conformational antibodies directed against the native but deployed VLPs, the binding of the antibodies was measured by ELISA (Table 1), in the samples from the immunized mice, for baculovirus-derived VLPs in PBS (native form) or in carbonate buffer (pH 9.5, expanded VLP, (14)). The specific IgG or IgA produced by the PhoPc / HPV strain recognizes very poorly the unfolded VLPs, suggesting that most of Ll was folded into highly ordered structures when expressed in PhoPc / HPV (Table 1).

Neutralization activity in vi tro of immune sera.

In previous studies of baculovirus-derived VLPs, the neutralizing activity and the protection against experimental infection correlated in general with the reactivity in ELISA to native VLPs. It was therefore desired to determine whether the conformationally dependent anti-VLP antibodies produced by the Salmonell a viva vaccine were also neutralizers. Although no assay or source of virus infectivity currently exists for authentic HPV16, it has recently been shown that HPV16 capsid proteins can encapsidate the genomes of BPV1 that replicate autonomously, resulting in virions of the HPV16 pseudotype (BPV1) whose Infectivity can be verified periodically by focal transformation of cultured fibroblasts from mice (43). The HPV16 infectivity assay (BPV1) was therefore used to examine the neutralizing activity of the mouse sera generated above. Each of the three immune sera showed strong neutralizing activity against HPV16 (BPV1) (Figure 5), but did not neutralize the virions of BPV1 (data not shown). The pre-immune sera did not have neutralizing activity. The neutralizing activities of the immune sera appeared to correlate with the titers in the native VLP ELISA, although the sera were only tested at a single dilution.

Tumor protection assay in the mouse tumor model, HPV16.

It has recently been shown that the development of syngeneic tumor cells (C3) 'injected into the flank of C57BL / 6 mice, was inhibited by a subcutaneous immunization with the purified HPV16 cell (84). It has been tested whether nasal immunization with purified VLPs and recombinant strains of Salmonell a / HPV were able to induce the same effect. Specifically, the following strains were tested: PhoPc / HPV16 Ll (86) and the? 4550 (56) expressing either high levels (? 4550 / pYA34Ll) or low levels (? 4550 / pYA32Ll) HPV16 Ll. The tumor growth in the different groups of mice is shown in Figure 6. The present preliminary results demonstrate that nasal immunization with purified VLPs is effective, and that all strains of Salmonell a / EPV tested induced partial tumor protection. Of interest, it is strain? 4550 / pYA34L1 that prevented the complete tumor development in 4/10 mice.

Coexpression of the L2 protein in PhoPc / HPV16 L2 0R17 was cloned downstream of the Ll ORF by PCR into plasmid pPSnsdHPV16 Ll (86). The PCR reaction included a 5 'specific oligonucleotide containing a synthetic Shine-Dalgarno sequence, in order to allow the 'translation of 12 of a polyistronic L1-L2 RNA. The resulting PhoPc / HPV16 L1 + L2 recombinant strain expressed Ll and L2 and the VLPs assembled in similar amount to the parental PhoPc / HPV16 strain Ll, as assessed by a sandwich ELISA assay. This suggests that by fusing E7 ORF to L2 ORF, in strain PhoPc / HPV16 L1 + L2, a chimeric VLPs could also be assembled, and such a recombinant strain of Salmonella be used to induce HPV16 E7-CTLS.

High level of expression of Ll in the PET expression system of E. col i inducible.

The Ll ORF was cloned into plasmid pET3 (Novagen). The expression of Ll driven by a T7 promoter was evaluated in strain BL21apLysS (which expresses T7 polymerase after IPTG induction). After the induction of IPTG, a 10-fold higher level of expression of Ll / bacteria compared to the Salmonell strain was achieved to PhoPc (see Figure 8). The lysate of this recombinant from E. coli formed a band at a density of VLPs in a cesium chloride density gradient, suggesting that the VLPs self-assembled into this bacterium.

Discussion In this study, it was demonstrated that an attenuated Salmonell strain expressing the capsid protein major of HPV16 is a promising candidate vaccine against HPV16 infection, since the VLPs that are assembled by this recombinant bacterium can induce specific conformational antibodies of serum VLPs as well as genitalia. The above results also show that the antibodies are capable of neutralizing the HPV16 viruses. These results could easily be extrapolated by the person skilled in the art to other types of HPV or to other papillomaviruses, or to other prokaryotic microorganisms. The life cycle of the papillomavirus is intimately associated with the differentiation of epithelial cells in the skin or in the oral and genital mucosa (5, 19, 40, 62). It is believed that viruses have access to basal epithelial cells through mucosal abrasions (21). After infection of the cervical epithelium for example, the "viral DNA released into the cytoplasm of the basal cells migrates to the nucleus, where it remains episomal and the early genes are transcribed leading to a low rate of cell proliferation and thickening of the cell. basal layer (cervical intraepithelial neoplasia type I, CIN I) As the infected epithelial cells migrate through the suprabasal layer and undergo differentiation, the episomal viral genome replicates reaching approximately 1000 copies per cell (29). Viral DNA, late genes are expressed and capsids are assembled into terminally differentiated keratinocytes (Figure 4), facilitating a new round of infection. In high-grade lesions (CIN III and carcinoma) the entire epithelium consists of undifferentiated basal cells in which viral DNA has been integrated into cellular DNA. In these cells, the E6 / E7 gene products constitute the major HPV proteins expressed, and the viruses are no longer produced. Based on the present knowledge of the pathogenesis of HPV, it seems that two immunity arms (humoral and cellular) have to be effective to prevent viral infection, to decrease local viral load, or to cure tumors (Figure 4, see also (59)). A local immune or humoral immune response with neutralizing antibodies is likely to block early infection, whereas a cellular response may contribute to the elimination of infected, non-transformed or transformed cells. An ideal vaccine could trigger both types of response, although the immunological correlation of protection and cure has not yet been identified. Prophylactic vaccines that induce type-specific neutralizing conformational antibodies (anti-VLP) have been shown to prevent CRPV or COPV infections in cottontail rabbit (4) or dog (57), respectively. In both cases the serum neutralizing antibodies were generated by vaccination with self-assembled PV capsids. By analogy, neutralizing antibodies to the HPV16 capsid in cervical secretions are expected to prevent infection. Since the precise mucosal site where early HPV infection occurs is not known, it is difficult to predict whether the sIgA antibodies acting from the lumenal site or the circulating IgG antibodies reach the basal layers will be the most efficient.

The elimination of cells infected with HPV or tumor cells requires a cellular immune response with cytotoxic T lymphocytes (CTL) that recognize the viral antigens presented by the MHC class I molecules on the infected cells. The therapeutic vaccines aimed at eliminating HPV-induced tumors have been generated using either peptides corresponding to the T cell epitopes from the E6 / E7 oncogenes or the vaccinia viruses expressing E6 / E7. Both showed that they produce CTLs and in some cases the regression of the tumor was observed (3, 6, 7, 12, 13, 34). One of the main problems, however, is that MHC class I molecules are sub-regulated in differentiated keratinocytes that produce viruses or in tumor cells (9). Since humoral and cellular immunity is believed to control HPV infection and since local and systemic responses are desirable, an efficient vaccine should reach the inductive sites associated with the mucosal surface and / or the peripheral lymph nodes. They are known "live bacterial vaccines that cross mucosal surfaces and that elicit humoral or cellular responses." 41 Recombinant and attenuated enteropathogenic bacteria, such as Salmonell a, represent ideal antigen delivery systems, because they efficiently cross all the antigens. mucosal surfaces to access organized or mucosal lymphoid tissue (MALT) or draining lymph nodes, which exploit the two basic sampling systems that mediate the uptake of mucosally administered antigens including M cells in simple epithelia, and dendritic cells in simple and stratified epithelia (38). An attenuated Typhimurim Salmonell strain has been selected for survival in macrophages, because the long-term antibody responses were caused by a simple, nasal, oral, rectal or vaginal administration of the recombinant bacteria that express a foreign antigen or (18) In this study, the best genital responses were obtained after nasal immunization. In the respiratory tract, antigen uptake occurs through the M cells found in NALT, the lymphoid tissue associated with the nose (25), and BALT, the lymphoid tissue associated with the bronchi (55). The lymphocytes that express primed IgA then migrate to the cervical and uterine tissues where they produce polymeric IgA antibodies, which are transported through the epithelium by the receptor, of polymeric Ig (26-28). Dendritic intraepithelial cells in the bronchial epithelium also play a major role in the presentation of the antigen by uptake of the antigens in the respiratory epithelium and into the distant drusen lymph nodes, where the priming occurs (17). This probably explains why nasal immunization is so efficient in triggering the nasal and systemic antibody responses. Antigens expressed in Salmonell a strains can also elicit cellular responses with specific CTLs (1, 16, 58). Depending on which viral antigen is expressed, specific CTLs that recognize infected cells in different stages of differentiation could also be generated (Figure 4). For example, E7-specific CTLs were generated by immunization of mice with recombinant Salmonel l a that expresses HPV16 E7 epitopes (31). To release the neutralized antibodies using recombinant Salmonella, it is essential that the antigen retains its native conformation. For HPV, this requires that Ll proteins form VLPs. Papilloma VLPs have been shown to assemble in eukaryotic cells (15, 22, 45, 48, 61), but not in prokaryotes. In bacteria the fusion proteins were expressed mainly to Ll (2, 20, 24) and when Ll proteins were expressed in good faith, the VLP assembly was not examined (11). As shown in this document, the VLP assembly of HPV16 in Salmonel l probably due to the level of expression achieved in the present experiments, was high and the capsid assembly does not require glycosylation (60). The production of the capsid in bacteria has also been reported for other viruses such as the nucleocapsid of the Hepatitis B virus (52) and the polyomavirus capsid (30, 46). The polyomavirus VP1 major capsid protein, analogous to HPV ll, forms capsomeres when expressed in E. coli, which are subsequently self-assembled in VLPs in vi tro (46). The fact that only the capsomeres but not the VLPs are recovered is probably due to the reducing agents present during the purification, which are known to break the capsids (47).

Nasal immunization with the PhoPc / HPV strain induced systemic and mucosal antibodies against native but not denatured HPV16 VLPs. In contrast, the recombinant vaccinia expressing the HPV1 capsid protein fired serum antibodies that recognize the folded and unfolded VLP, probably reflecting in different mode of expression of the viral protein, and low titers of HPV specific genital IgA antibody (14 ), as expected with a non-mucosal route of administration. The antibody titers against the foreign antigen induced by PhoPc / HPV compared to PhoPc / HBc from Salmonell a, were approximately 10 times lower (18). This could reflect differences in immunogenicity between the two viral antigens (51) or, more likely, differences in the stability of the plasmid. In contrast to the DNA of HBc, the plasmid which possesses the HPV16-L1 DNA was unstable in Salmonell a vi in the absence of selective pressure, since less than 1% of the Salmonella recovered from the different tissues two weeks after the immunization still harbored the plasmid containing Ll (data not shown). To increase the stability of the plasmid, the Ll gene is currently reclining in vectors based on (β-aspartate-semialdehyde-dehydrogenase) which maintain selective pressure in vi (36, 56). The previous work also demonstrates the following points: a) that purified VLPs and strains of Salmonell a /? 'PV are able to provide tumor protection in a mouse tumor model HPVl 6. b) that the chimeras of an HPV protein and a fusion partner are assembled into prokaryotes to form VLPs. c) that the high expression levels of the HPV proteins that are assembled to form VLPs were obtained in E. coli, demonstrating that the invention is applicable in prokaryotes other than Salmonella. In conclusion, a recombinant Salmonell strain has been constructed that expresses the capsid proteins HPV16-L1 and the assembly of the VLPs that induce the conformational antibodies serum IgG and vaginal sIgA, which recognize the VLPs. The neutralizing activities of these antibodies were tested and showed that they have strong neutralizing activity in an HPV16 infectivity assay (BPV1).

Table 1: Titers of IgG (in serum) or IgA (in vaginal washings) against native HPV16 VLP and displayed in mice immunized with PhoPc / HPV Samples VLP anti-HPV16 VLP anti-HPV16 deployed3 titles of IgGb # 4 SUerO (week 27) 60,000 100 # 5 SUERO (week 27) 80,000 200 # 6 SUERO (week 27) 20,000 100 Camvirc 16,000 80,000 titles of IgA # 4 vaginal washes (smana is »40 <1 # 5 vaginal washes (week is) 20 <1 # 6 vaginal washes week 40 40 1 ELISA plates were coated with VLP in buffer carbonate pH 9.5.

The titres are expressed as the reciprocal of the highest dilution of the sample that produced an OD g2 four times that of the pre-immune sample. • c Monoclonal IgG anti-HPV16 Ll (35 μg / ml), 30) used as a positive control.

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It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (21)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. An attenuated strain of a prokaryotic microorganism transformed with the nucleic acid encoding the major capsid protein of the papillomavirus virus, characterized in that the protein is assembled in the microorganism to form virus-like particles (VLPs).

2. The attenuated microorganism strain according to claim 1, characterized in that it is an attenuated strain of Salmonell a.

3. The attenuated microorganism strain according to claim 2, characterized in that the Salmonella strain is Salmonella typhimurium, Salmonella typhi, Salmonella dublin, or Salmonella enteretidis.

4. The attenuated microorganism strain according to claim 1, characterized in that it is an attenuated strain of Escheri chi a coli, Shigella, Yersinia, Lactobacillus, Mycobacteria or Listeria.

5. The attenuated microorganism strain according to any of the preceding claims, characterized in that the nucleic acid encodes a capsid protein greater than the human papillomavirus virus.

6. The attenuated microorganism strain according to claim 5, characterized in that the HPV strain is HPV16, 18, 31, 45 or 56.

7. The attenuated microorganism strain according to any of the preceding claims, characterized in that the major capsid protein of the papillomavirus virus is the Ll protein.

8. The attenuated microorganism strain according to any of claims 1 to 7, characterized in that the major capsid protein of the papillomavirus virus is expressed as a chimera with a fusion partner.

9. The attenuated microorganism strain according to claim 8, characterized in that the major capsid prqtein of the papillomavirus is coexpressed with the L2 protein, the L2 protein being fused to the fusion partner.

10. The attenuated microorganism strain according to claim 8 or claim 9, characterized in that the fusion partner is the HPV E6, E7 or E2 protein, an immunogenic protein derived from a non-pathogenic antigen for HPV or tumor-specific.

11. The attenuated microorganism strain according to any of the preceding claims, characterized in that the microorganism is transformed with the nucleic acid encoding two or more major capsid proteins of the papilloma virus virus.

12. A composition, characterized in that it comprises one or more of the attenuated microorganisms according to claims 1 to 11, in combination with a physiologically acceptable carrier.

13. A vaccine, characterized in that it comprises one or more of the attenuated microorganisms according to claims 1 to 11, in combination with a physiologically acceptable carrier.

14. The vaccine according to claim 13, characterized in that it is formulated for mucosal immunization.

15. The vaccine according to claim 14, characterized in that the mucosal immunization is by means of the oral, rectal, nasal, or genital routes.

16. The vaccine according to any of claims 3 to 15, characterized in that the vaccine provides protection against infection by papillomavirus or against cancer of the anogenital tract.

17. An attenuated strain of a prokaryotic microorganism according to any one of claims 1 to 11, for use in a medical treatment method.

18. The use of an attenuated strain of a prokaryotic microorganism according to any one of claims 1 to 11, in the preparation of a medicament for the prophylactic or therapeutic treatment of papillomavirus infection.

19. The use of an attenuated strain of a prokaryotic microorganism according to any one of claims 1 to 11, in the preparation of a medicament for the treatment of cancer of the anogenital tract.

20. A method for producing papillomavirus-like particles, assembled, characterized the method because it comprises culturing an attenuated microorganism strain according to any of claims 1 to 11, and recovering the assembled virus-like particles, produced from this way.

21. A method to detect the presence of anti-papillomavirus antibodies in a sample "from a subject, the method is characterized in that it comprises the immobilization of the HPV VLPs on a solid support, exposing the support to the sample and detecting the antibodies that bind to the immobilized HPV VLPs, where the HPV VLPs are produced by an attenuated microorganism strain according to any one of claims 1 to 11.