Attenuated microorganism strains and their uses

Details Attenuated microorganism strains and their uses Attenuated microorganism strains and their uses

1999-10-07

2001-06-26

Salimi, Ali R. (Department: 1648)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Bacterium or component thereof or substance produced by said...

C424S204100, C424S192100, C424S234100, C424S093100, C424S093200, C435S005000, C435S007100, C435S235100, C435S320100

Reexamination Certificate

active

06251406

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to attenuated strains of prokaryotic microorganisms, in particular Salmonella, transformed with nucleic acid encoding papillomavirus virus proteins, to 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 of producing assembled papillomaavirus Virus like particles (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 the virus in culture, the lack of animal models and the paucity of virions in clinical lesions. This has led to the development of alternative approaches of antigen production for immunological studies. The conformational dependency of neutralizing epitopes, as observed in experimental animal papillomavirus systems (8, 22) suggests that properly assembled HPV particles are critical for the induction and detection of clinically relevant immune reactivity.
The HPV capsids are formed by 72 pentameric capsomers of L1 proteins arranged on a T7 icosahedral lattice (15). Recently, a number of investigators have demonstrated the production of HPV capsids, i.e. virus like particles (VLP), by utilizing baculovirus, vaccinia virus or yeast expression systems (15,22,45,48,61). The potential of VLPs as subunit vaccines has been demonstrated using the cottontail rabbit papillomavirus (CRPV) (4), the canine oral papillomavirus (COPV) (57), and the 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 (anti-VLP) secretory immunoglobulin A (sIgA) or immunoglobulins G (IgG) in genital secretions. By analogy with existing animal models. HPV16 VLPs-specific antibodies in cervical secretions might help to prevent sexually transmitted infection by HPV16 in women. However, this cannot be formally proven in the absence of an experimental model for genital PV infection and other scenarios requiring cell-mediated immunity cannot be excluded.
Moreover, the mechanism underlying HPV infection is unclear. HPV may directly infect the basal cells of the stratified cervical epithelium at the occurrence of breaches. Alternatively, HPV infection could also occur either directly through Langerhans cells in intact epithelia or indirectly from an HPV-producing keratinocyte, and thus neutralizing antibodies will not be functional as shown for other viruses. This further adds to the difficulty in providing vaccines effective against HPV infection.
Immunosuppressed individuals are more prone to develop cervical carcinoma as compared to immuncocompetent individuals, suggesting the possibility of using immunotherapy. Therapeutic vaccines (87) aimed to the treatment of established HPV infection or HPV associated premalignant and malignant lesions have been investigated during the last ten years (59). Evidence for HPV-antigen-directed immunotherapy against cervical cancer comes from the observations that experimental (13),(34),(83) and natural (82) PV-associated tumours can be controlled by immunization with E6 and E7 preparations. These studies suggested that CTL might be the most effective immunological effector mechanisms. E6 and E7 preparations consisted in either peptides (13), bacterially prepared fusion proteins (82), eukaryotic transfected cells (83) or recombinant vaccinia viruses (34).
Recently, chimeric VLPs carrying the 17kD E7 protein as a fusion with L2 have been shown to induce rejection of syngeneic tumour cells (84) engineered to express L1 and/or E7 ORF (i.e. C3 cells (13) and TC1 cells (85)). This data demonstrates the possibility of providing prophylactic and therapeutic effects in the same vaccine preparation. Salmonella that are attenuated, yet invasive, have been proposed for the delivery of heterologous antigens to the mucosal and systemic immune systems (10). The antigen is delivered by the live Salmonella to mucosal inductive sites, where after priming, antigen-specific B and T cells migrate from the site of induction and mature into effector cells. The migrating IgA-expressing B cells home to different mucosal sites, including the genital tract where they differentiate into IgA secreting plasma cells (32). Thus, oral or nasal immunization can provide protective antibodies in genital secretions Recently, we and others have shown that mucosal immunization with recombinant Salmonella can elicit antibody responses in the genital mucosa of mice and humans (18,37,56).
SUMMARY OF THE INVENTION
In order to develop a prophylactic vaccine against HPV, we have expressed the major protein L1 of HPV16 in a PhoP
c
(35) attenuated strain of
Salmonella typhimurium
. Surprisingly, the inventors found for the first time that it is possible to assemble VLPs in a prokaryotic organism and that nasal immunization of mice with an HPV16-L1/Salmonella recombinant strain induces HPV16-specific conformationally dependent and neutralizing antibodies in serum and genital secretions. The experiments described herein also show that it is possible to assemble chimeric VLPs of a HPV protein and a fusion partner.
Accordingly, in a first aspect the present invention provides an attenuated strain of a prokaryotic microorganism transformed with nucleic acid encoding papillomavirus virus major capsid protein wherein the protein assembles in the microorganism to form virus like particles (VLPs).
Thus, the present invention provides a way of producing properly assembled papillomavirus VLPs in an attenuated strain of a prokaryotic microorganism such as Salmonella so that they can be used as a vaccine to raise an immune response in a subject. Preferably, the VLPs are delivered to mucosal sites, having the advantage of generating the immune response to the papillomavirus VLPs at the locations where infection actually takes place, as well as at other mucosal surfaces.
The term “papillomavirus” used herein covers both human and animal PVs. However, preferably, the papillomavirus is a human papillomavirus (HPV). About 70 different types of HPV have been cloned and characterized (denoted HPV1 to HPV70 . . . ), and all have an 8 kb double stranded genome which encodes different early products and two late products L1 and L2, and are either epitheliotropic or mucosatropic. L1 is a major capsid protein and is relatively well conserved among the different HPV types. For a review of the HPV types and their nucleic and amino acid sequences, see Human Papillomaviruses, “A Compilation and Analysis of Nucleic Acid and Amino Acid Sequences”, 1994, ed. Myers et al, Theoretical and Biophysics Group T-10 Los Alamos National Laboratory. Clinically, the most important HPV types a are those that infect the anogenital tract, and that have high oncogenic risk and a high prevalence. This group includes HPV16, 18, 31, 45 and 56, with HPV16 alone accounting for more than 50% of invasive cancer in the anogenital tract, as well as being the most prevalent single type of HPV.
The papillomavirus proteins correspond to wild type major capsid proteins (e.g. L1 and/or L2) or may be chimeras of all or part of a HPV protein and a fusion partner. The fusion partner may be any immunogenic protein against which specific CTL would be targeted. This protein may be an HPV protein (e.g. E7, E6 or E2 of any HPV type), a protein from another pathogen or any tumour specific antigen. In one embodiment the HPV protein is L1 protein coexpressed with L2, with the fission partner expressed so that it is linked to the L2 protein.
It has been shown that chimeric VLPs can elicit anti-tumour immunity against carrier and inserted proteins in HPV16 tumour models. Thus, chimeric VLPs which induce E7-specific CTLs aimed to the killing of already HPV infected cells or HPV-associated premalignant lesions. In this event, induction of CTLs to eliminate already HPV infected cells appears therefore an

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