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Document AM3 
Appl.No. 09/848,616 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 6 : 

C12N 15/37, 15/62, 15/74, 1/21, C07K 
14/025, A61K 39/12, 31/70, G01N 33/569 
// (C12N 1/21, C12R 1:42) 


Al 


(11) International Publication Number: WO 98/15631 
(43) International Publication Date: 16 April 1998 (16.04.98) 


(21) International Application Number: PCT/GB97/02740 

(22) International Filing Date: 7 October 1997 (07.10.97) 

(30) Priority Data: 

9621091 .9 9 October 1996 (09.10.96) GB 

(71) Applicant (for all designated States except US): FONDATION 
POUR LE PERFECTIONNEMENT ET LA RECHERCHE 
EN GYNECOLOGIE-OBSTETRIQUE [CH/CH]; CHUV, 
Avenue Pierre-Decker, CH-101 1 Lausanne (CH). 

(71) Applicant (for MN only): KIDDLE, Simon, John [GB/GBJ; 

Mewbum Ellis, York House, 23 Kingsway, London WC2B 
6HP (GB). 

(72) Inventors; and 

(75) Inventors/Applicants (for US only): HAEFLIGER, Denise, 
Nardelti [CH/CH]; Chemin de Champ-Rond 53 bis, 
CH-1010 Lausanne (CH). KRAEHENBUHL, Jean-Pierre 
[CH/CH]; Sur-la-Croix, CH-1812 Rivaz (CH). 

(74) Agents: KIDDLE, Simon, J. et al.; Mewbum Ellis, York House, 
23 Kingsway, London WC2B 6HP (GB). 


(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR, 
BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, 
GH, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, 
LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, 
NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, 
TT, UA, UG, US, UZ, VN, YU, ZW, ARIPO patent (GH, 
KE, LS, MW, SD, SZ, UG, ZW), Eurasian patent (AM, AZ, 
BY, KG, KZ, MD, RU, TJ, TM), European patent (AT, BE, 
CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU. MC, NL, 
PT, SE), OAPI patent (BF, BJ, CF, CG, Ct, CM, GA, GN, 
ML, MR, NE, SN, TD, TG). 

Published 

With international search report. 

Before the expiration of the time limit for amending the 
claims and to be republished in the event of the receipt of 
amendments. 



(54) Title: ATTENUATED MICROORGANISM STRAINS EXPRESSING HPV PROTEINS 



(57) 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. 




WO 98/15631 PCT/GB97/02740 

ATTENUATED MICROORGANISM STRAINS EXPRESSING HPV PROTEINS 

Field of the Invention 

The present invention relates to attenuated strains of 
5 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 
10 provides a method of producing assembled papillomavirus 

virus like particles (VLPs) . 

Background of the Invention 

Human papilloma virus (HPV) 16 is the major type of 

15 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 

20 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 

25 relevant immune reactivity. 

The HPV capsids are formed by 72 pentameric capsomers 
of LI 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) , 

30 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) . 

35 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 immunoglobulins A (slgA) or 
immunoglobulins G (IgG) in genital secretions. By analogy 



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2 

with existing animal models, HPV16 VLPs-specif ic 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 
5 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 

10 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 

15 difficulty in providing vaccines effective against HPV 

infection . 

Immunosuppressed individuals are more prone to develop 
cervical carcinoma as compared to immunocompetent 
individuals, suggesting the possibility of using 

20 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 

25 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 

30 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 

35 syngeneic tumour cells (84) engineered to express LI and/or 

E7 ORF (i.e. C3 cells (13) and TCI cells (85)). This data 
demonstrates the possibility of providing prophylactic and 



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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 
5 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 

10 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 

15 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 LI of HPV16 in a 

20 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- 
Ll/ Salmonella recombinant strain induces HPV16 -specific 

25 conf ormationally 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 

30 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) . 

3 5 Thus, the present invention provides a way of 

producing properly assembled papillomavirus VLPs in an 
attenuated strain of a prokaryotic microorganism such as 



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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 
5 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 

10 different types of HPV have been cloned and characterized 

(denoted HPV1 to HPV70 . . . ) , and all have an 8kb double 
stranded genome which encodes different early products and 
two late products LI and L2, and are either epitheliotropic 
or mucosatropic . LI is a major capsid protein and is 

15 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, U A Compilation 
and Analysis of Nucleic Acid and Amino Acid Sequences" , 
1994, ed. Myers et al, Theoretical and Biophysics Group T- 

20 10, Los Alamos National Laboratory. Clinically, the most 

important HPV types 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 

25 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. LI and/or L2) or may be 
chimeras of all or part of a HPV protein and a fusion 

3 0 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 LI protein 

35 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- 



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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 appealing complement to the 
induction of neutralizing antibodies, and chimeric VLPs 
have been shown to induce both functions. 

Thus, in one embodiment of the invention, Salmonella. 
strains able to induce neutralizing antibodies and CTLs by 
expressing chimeric VLPs could be therapeutic at least for 
early or pre-malignant HPV lesions in which the 
downregulation of MHC I or other factors observed in more 
advanced cancers has not yet occurred. 

Preferably, the prokaryotic microorganism is an 
attenuated strain of Salmonella. However, alternatively 
other prokaryotic microorganisms such as attenuated strains 
of Escherichia coli, Shigella, Yersinia, Lactobacillus, 
Mycobacteria, Listeria or Vibrio could be used. Examples 
of suitable strains of microorganisms include Salmonella 
typhimurium, Salmonella typhi, Salmonella dublin, 
Salmonella enteretidis, Escherichia coli, Shigella 
flexeneri, Shigella sonnei, Vibrio cholera, and 
Mycobacterium bovis (BC6) . 

Attenuated Salmonella strains are one of the best 
characterized mucosal vaccine carriers. Recombinant 
Salmonella strains that are attenuated yet invasive have 
been used as oral vaccine vectors to carry protective 
epitopes of several pathogens into the mucosal associated 
lymphoid tissue thus inducing mucosal, systemic and CTL 
immune responses against both the carrier and the foreign 
antigens (58,65,67,69,75-77) . 

The currently licensed oral vaccine against typhoid 
fever S. typhi Ty21a (72) administered as a three-dose 
regimen of enteric-coated capsules (10 9 CFU/capsule) 
provided a 67% efficacy over a 3 year period. However, 
because the S. typhi Ty21a requires high and multiples 



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doses in liquid formulation for higher efficacy, and its 
mutations are not yet all characterised (63,64,70,71,78), 
new attenuated Salmonella strains have recently been 
developed and tested in humans. These include nutritional 
5 auxotrophs in which pathways for biosynthesis of aromatic 

compounds have been interrupted {Aaro mutants) . The AaroA, 
ApurA mutants of S. typhi have been tested in human 
volunteers (32) and were shown to elicit specific cell- 
mediated immune responses but weak humoral responses . 

10 Other aro mutants (aroC and aroD) were insufficiently 

attenuated and caused fever and bacteremia (79) . A double 
mutant AaroC AaroD Ty2 (CVD 908) was safe and elicited IgG 
antibodies against LPS in 80% of the immunized adult 
volunteers (73,80). S. typhi mutants were also generated 

15 in which the adenylate cyclase (cya) and the cAMP receptor 

(crp) genes were deleted. These gene products are required 
for the transcription of many genes and operons that 
control transport processes, expression of fimbriae, 
flagella and some outer membrane proteins. One mutant 

20 X3 927 (Acya Acrp Ty2) was tested and shown to be 

immunogenic but some volunteers developed fever and vaccine 
bacteremia (79). Therefore, a novel strain, *4073, was 
constructed by deleting a third gene (cdt) responsible for 
colonization of deep tissue (66,68,74) . This strain was 

25 administered to volunteers and proved to be completely safe 

at doses up to 5xl0 8 CFU and generated a seroconversion in 
80% of the volunteers (66) . 

Other attenuated Salmonella strains include mutants in 
a two-component regulatory system, the phoP/phoQ genes. 

30 These genes affect expression of a number of other genes 

and are responsive to phosphate levels and to enviromental 
conditions expected to be experienced by Salmonella 
residing within macrophages. One example of these mutants 
is the PhoP c strains used in the examples described below. 

35 Recently, a PhoP/PhoQ-deleted Salmonella typhi (ty800) has 

been shown to be safe and immunogenic in humans (81) . 

As mentioned above, the attenuated strain of the 



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prokaryotic microorganism is transformed with a nucleic 
acid encoding one or more major papillomavirus capsid 
proteins. The inventors found for the first time that, 
when this nucleic acid is expressed in the microorganisms, 
5 the capsid proteins produced assemble correctly to form 

VLPs, making them especially suitable for the vaccination 
of subjects against papillomaviruses. Preferably, the 
major viral capsid protein is LI, optionally additionally 
including nucleic acid encoding L2 protein. As discussed 

10 above, the capsid protein may 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 above attenuated 
prokaryotic microorganisms, optionally in combination with 

15 a physiologically acceptable carrier. Preferably, the 

composition is a vaccine, especially a vaccine for mucosal 
immunization, e.g. for administration via the oral, rectal, 
nasal, vaginal or genital routes. Our earlier studies 
using recombinant Salmonella expressing hepatitis B virus 

2 0 antigen (18) showed that vaccination via any of these 

routes produces a slgA response in the mucosal secretions 
at other sites. Advantageously, for prophylactic 

vaccination, the compositions comprises one or more strains 
of Salmonella expressing a plurality of different VLPs, 

25 e.g. VLPs from different papillomavirus types. This has 

the advantage of improving the protective effect of the 
vaccine to a range of challenges by the different 
papillomavirus types. For therapeutic vaccination, 

subsequent chimeric VLP constructs can comprise fusion 

30 products of various HPV type LI 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. 

3 5 In a further aspect, the present invention provides 

the use of an attenuated strain of a prokaryotic 
microorganism transformed with nucleic acid encoding 



WO 98/15631 PCT/GB97/02740 

8 

papillomavirus virus major capsid protein, wherein the 
protein assembles in the microorganism to form virus like 
particles, in the preparation of a medicament for the 
prophylactic or therapeutic treatment of papillomavirus 
5 infection or anogenital cancer, especially cervical cancer. 

Generally, the microorganisms or VLPs according to the 
present invention are provided in an isolated and/or 
purified form, i.e. substantially pure. This may include 
being in a composition where it represents at least about 

10 90% active ingredient, more preferably at least about 95%, 

more preferably at least about 98%. Such a composition 
may, however, include inert carrier materials or other 
pharmaceutical ly and physiologicaly acceptable excipients. 
A composition according to the present invention may 

15 include in addition to the microorganisms or VLPs as 

disclosed, one or more other active ingredients for 
therapeutic use, such as an ant i- tumour agent. 

The compositions of the present invention are 
preferably given to an individual in a "prophylactically 

20 effective amount" or a "therapeutically effective amount" 

(as the case may be, although prophylaxis may be considered 
therapy) , this being sufficient to show benefit to the 
individual. The actual amount administered, and rate and 
time-course of administration, will depend on the nature 

25 and severity of what is being treated. Prescription of 

treatment, e.g. decisions on dosage etc, is within the 
responsibility of general practioners and other medical 
doctors . 

A composition may be administered alone or in 
3 0 combination with other treatments, either simultaneously or 

sequentially dependent upon the condition to be treated. 

Pharmaceutical compositions according to the present 
invention, and for use in accordance with the present 
invention, may include, in addition to active ingredient, 
35 a pharmaceutical^ acceptable excipient, carrier, buffer, 

stabiliser or other materials well known to those skilled 
in the art. Such materials should be non-toxic and should 



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9 

not interfere with the efficacy 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 
5 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 assembled papillomavirus virus like 
particles comprising culturing an attenuated strain of a 

10 prokaryotic microorganism transformed with nucleic acid 

encoding papillomavirus virus major capsid protein wherein 
the protein is expressed and assembles in the microorganism 
to form virus like particles. Preferably, the method 
additionally comprises the step of recovering the VLPs from 

15 the prokaryotic microorganism. 

In a further aspect, the present invention provides 
the use of a papillomavirus VLP as obtainable by 
transforming an attenuated prokaryotic microorganism with 
nucleic acid encoding the VLPs and expressing the nucleic 

20 acid to produce assembled VLPs, in a diagnostic method. in 

one embodiment, 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 

25 and detecting the presence of the antibodies, e.g. using 

ELISA. 

Preferred embodiments of the present invention will 
now be described by way of example and not limitation with 
reference to the accompanying drawings. 

30 

Brief Description of the Figures 

Figure 1. HPV16 LI expression in the PhoP c /HPV strain. 
Salmonella were grown overnight and prepared as indicated 
in Material and Methods. A. Commassie blue staining of a 
35 10%SDS PAGE gel; Lane M : Molecular weight marker; Lane 1: 

total lysate of the PhoP c strain Lane 2 : total lysate of the 
PhoP c /HPV strain, a unique 57kDa protein is indicated with 



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10 

an arrow. B. Immunoblot using anti-HPV16-Ll mAb of . the 
total and fractionatd PhoP c /HPV lysate. Lane tot: total 
lysate, Lane 1 to 25: different fractions obtained after 
fractionation of the PhoP c /HPV lysate through a 10-40% 
5 sucrose gradient, the heavier fraction of the gradient 

being in lane 1. The 57kDa protein band identified as being 
LI is indicated (arrow) . 

Figure 2. HPV16 LI assemble into VLPs . Electron 
micrographs of (A) PhoP c /HPV16 VLPs and (B) Baculo-derived 

10 HPV16 VLPs. In A, fractions 7 to 13 of PhoP c /HPV lysate 

(Fig 1) were pooled. The samples were negatively stained 
with phosphotungstic acid. Bar represents 53 nm. 

Figure 3. Anti-HPV16VLP and anti-LPS systemic and 
mucosal antibody responses after nasal immunization with 

15 the PhoP c /HPV strain. Three 6 -week-old BALB/c female mice 

were immunized with 5xl0 7 CFU, sampled at the indicated 
weeks, sacrificed and bled at week 27. Data are expressed 
as the geometric means of the reciprocal dilutions of 
specific IgG in serum and specific IgA per microgram of 

20 total IgA or IgG per microgram of total IgG in secretions. 

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 HPV productive viral 
cycle during keratinocyte differentiation is schematically 

25 drawn (early inf ection-CIN I) . A late stage of infection 

(CIN Ill-tumour) in which the HPV DNA is integrated into 
the host genome is shown on the right. Different 
protective immune mechanisms are shown with arrows 
indicating the sites of action. Antibody-dependent 

30 cellular cytotoxicity (ADCC) mechanims which require viral 

antigens to be expressed at the surface of cells are not 
indicated. 

Figure 5. In vitro neutralization of HPV16 pseudotype 
virus infection of mouse C127 cells. (A) no virus added. 
35 (B-H) Equal aliquots of an HPV16 (BPV1) pseudotype virus 

containing extract was added. The aliquots were 

preincubated with (B) no antibodies, (C) BPV1 neutralizing 



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MoAb B1.A1, (D) HPV16(BPV1) neutralizing MoAb H16.E70, (E) 
mouse preimmune sera, (F) mouse#4 immune sera {week27) , (G) 
mouse#5 immune sera (weck 27) ; (H) mouse#4 immunes sera« week 27) . 
Figure 6 shows the results of tumour growth 
5 experiments in mice immunized with Salmonella HPV producing 

strains. Nasal immunisations were performed three times 
weekly with either 20/il PBS (A) or 5/xg of purified HP VI 6 
VLPs + 5/xg of cholera toxin (CT) (E) and two times at weekO 
and week2 with 10 CFU of PhoPc/HPV16 LI (b) , x4550/pYA34Ll 
10 (C) and x4550/pYA32Ll {D) . A11 mice were challenged with 

5 105 C3 cells into the flank two weeks after the last 
immunisation. The mean volume of the tumours in each group 
are shown, while the number of mice harbouring a 
tumour/number of mice injected is indicated at Day 17. 

15 Figure 7 shows coexpression of LI and L2 in PhoP c /HPV16 

L1-L2. Blot A was revealed with an anti-L2 antibody, while 
blot B was revealed with'anti-Ll antibody (Camvir) . 

Figure 8 shows the expression of LI in E. coli BL12 
pET 3D-L1. Identical amounts of bacteria were loaded (3 

20 x 10 6 CFU) after 3 hours incubation with IPTG and the blot 

was revealed with an ant i -LI antibody. 

Detailed Description 
Materials and Methods 

25 Plasmid construction and bacterial strains used. 

Plasmid pFS14nsd HPV16-L1 was constructed by 
exchanging in the plasmid pFS14 NSD (54) the hepatitis B 
nucleocapsid gene (HBcAg , Ncol-Hindlll fragment) for a 
Ncol-Hindlll fragment encoding the HPV16-L1 open reading 

30 frame. The HPV 16-L1 Ncol-Hindlll fragment was generated 

by Polymerase Chain Reaction (PCR) using the baculovirus 
expression plasmid pSynwtVI*HPV16 114/B-L1+L2 (23) as a 
template with a 28mer containing a Ncol site : 5'- 
GGG CCA TGGCTCTTTGGCTGCCTTAGTGA- 3 ' and a 27mer containing a 

35 Hindi 1 1 site 5 ' -GGGAAGCTTCAATACTTAAGCTTACG-3 ' . The final 

construct containing the Tac promoter places the HPV16-L1 
ATG at position +8 relative to the Shine -Dalgarno sequence 



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and introduces a change in the second amino acid which 
becomes an alanine instead of the serine encoded by the 
original sequence. Sequencing of the entire LI open 
reading frame was carried out (MycrosynthAG) and no further 
5 nucleotide change was observed. Plasmid pFS14nsd HPV16-L1 

was amplified in E. co2i JM105 and then electroporated as 
described previously (50) into bacterial strain CS022. 
This strain is derived from the ATCC 14028 strain, into 
which the pho-24 mutation was introduced by P22 
10 transduction, resulting in attenuation in both virulence 

and survival within macrophages in vitro (PhoP c , (35)). The 
resultant recombinant strain is called PhoP c /HPV hereafter. 

Expression of HPV16-L1 in Salmonella and VLPs purification. 

15 After overnight growth at 37 °C the recombinant 

bacteria were lysed by boiling in Laemmli buffer containing 
5%SDS. The lysates were separated on 10% SDS/PAGE gels and 
expression of LI was analyzed by Western blot using HPV16- 
Ll mAb CAMVIR-1 (33) as primary antibody, an alkaline- 

20 phosphatase conjugated goat ant i- mouse IgG (Sigma) as 

secondary antibody and BCIP/NBT (Boehringer) as substrate. 

To prepare VLPs, bacteria were lysed by sonication and 
the lysate f ractionnated on a 10%-40% sucrose gradient in 
Phosphate Buffer Saline (PBS) containing 1M NaCl for 1 hour 

25 at 40Krpm using a TST41.14 rotor. Fractions of the 

gradient were then analysed for the presence of the LI 
protein by Western blot. The fractions of high 

sedimentation containing the LI protein were pooled, 
dialyzed against PBS/0. 5M NaCl . VLPs were pelleted for lh 

30 at SOKrpm using a TST65.1 rotor, adsorbed to carbon-coated 

grids, negatively stained with phosphotungstic acid and 
examined with a Philips electron microscope. 

Purification of HP VI 6 VLPs expressed in insect cells from 
3 5 a recombinant baculovirus. 

The transfer vector pSynwtvrHPV16 114/B-L1+L2 (23) was 
cotransf ected with the linearized genome of baculovirus 



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(Baculo-Gold, Pharmingen) using the calcium-phosphate 
method into SF9 cells. The recombinant baculoviruses were 
plaque-purified and propagated by standard methods (39) . 
Baculo-derived HP VI 6 VLPs were purified as described 
5 previously (23) . 

Immunization and sampling of mice. 

Six-week-old female BALB/c mice were immunized at day 
0 and at week 14 by the nasal route with 5xl0 7 CFU of 
10 inoculum. Blood, saliva and genital samples were taken as 

described previously (18) . All samples were stored at - 
70°C. 

ELISA. 

15 The amount of total IgA, anti-LPS IgA and IgG 

antibodies in samples were determined by enzyme-linked 
immunosorbent assay (ELISA) as described previously (18) . 
For the anti-HPV16 VLP, ELISA plates were coated with 10 ng 
of a preparation of baculo-derived HPV16 VLPs in PBS (total 

2 0 protein content was determined with a BioRad Protein assay 

with BSA as standard) . This amount of VLP was saturating 
in our ELISA test. Endpoint dilutions of samples were 
carried out. The specific IgA or IgG amounts are expressed 
as reciprocal of the highest dilution that yielded an OD 492 

25 four times that of preimmune samples. These reciprocal 

dilutions were normalized to the amount of total IgA or IgG 
in saliva and genital washes. ELISA plates were also 
coated with 10 ng of baculo-derived HPV16 VLPs in 0 . 2M 
carbonate buffer pH9.5 to determine the titer of antibodies 

30 recognizing unfolded VLPs (14) . 

In vitro HPV16 neutralization assay. 

Infectious pseudovirions consisting of HPV capsid made 
of LI and L2 surrounding the bovine papillomavirus type 1 
35 (BPV1) genome, designated HPV16(BPV1), were generated as 

recently described (43) . Briefly, BPHE-1 hamster cells 
harbouring autonomously replicating BPV1 genomes were co- 



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infected with defective recombinant Semliki forest viruses 
that expressed LI and L2 virion capsid genes of HPV16. 
Infectious pseudotype HPV16 virus in cell extracts was 
quantitated by the induction of transformed foci in 
5 monolayers of mouse C127 cells. Neutralizing activity was 

measured after preincubation of the cell extracts with 
mouse sera diluted 1:50 (1.0ml final volume) in culture 
medium. Mouse monoclonal antibodies H16.E70 and B1A1 were 
generated against recombinant baculovirus expressed HP VI 6 
10 LI VLPs and BPV16 VLPs respectively, and used at a 1:100 

dilution. H16.E70 and B1.A1 served as positive and 
negative controls for HPV16 (BPV1) neutralization, 
respectively. 

15 Results 

HPV16-L1 is expressed in PhoP c and VLP assemble. 

The open reading frame of the major protein LI of 

HPV16 was cloned in the plasmid pFS14 NSD (53). LI is 

constitutively expressed under the control of the Tac 
20 promoter in S. typhimurium. A unique 57kDa protein 

detected in the lysate of PhoP c /HPV overnight cultures (Fig. 

IA) , was identified as HPV16 LI by Western immunoblot using 
an anti-HPV16-Ll monoclonal antibody (CAMVIR, (33) , Fig 

IB) . To determine whether the LI protein expressed by 
25 PhoP c /HPV assembled into VLP, the bacterial lysate was 

fractionated through a 10-40% sucrose gradient and the 
heavier fractions containing the LI protein (Fig. IB) were 
analyzed by electron microscopy. Spherical particles 
typical of PV capsids were recovered from the bacterial 
30 preparation (Fig. 2A) but the bacterial VLPs appeared more 

polymorphic in size with diameters ranging from 4 0 to 55 nm 
(Fig. 2A) when compared to -55 nm VLPs expressed in insect 
cells (Fig. 2B) . 



35 



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

Since nasal immunization using recombinant Salmonella 



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was shown to elicit strong vaginal slgA responses against 
an expressed foreign antigen (18) , we immunized mice 
nasally with the PhoP c /HPV strain (5xl0 7 CFU) . Samples of 
blood, saliva and vaginal washes were taken 0, 2, 4, and 6 
5 weeks after immunization. The immune responses against 

both the carrier, i.e. anti-LPS and the carried antigen, 
i.e. anti-HPV16 VLP, were determined. Serum HPV16 VLP 
specific IgG (Fig. 3) were detected after 2 weeks in one 
mouse and after 4 weeks in all mice. The response peaked 

10 after 6 weeks at relatively low titers and persisted at 

least until week 14. At that time, no HPV16 VLP specific 
antibodies were detected in vaginal secretions, while one 
mouse had low titers of IgA in the saliva. The systemic 
and the mucosal immune responses against LPS were 

15 relatively low (Fig. 3) , but similar to those elicited by 

the PhoP c /HBc strain (18) suggesting a normal take of 
PhoP c /HPV Salmonella, by the mice. The low anti-LPS response 
observed after nasal immunization incited us to perform a 
booster immunization. Thus, a second nasal immunization 

20 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 of anti-HPV16 VLP IgG in serum, as well as anti- 
HPV16 VLP IgA in the vaginal washes (Fig. 3) from the three 

25 mice. Anti-HPV16 VLP IgG were also found in vaginal washes 

but only in two mice at week 19 and titers were again 
almost undetectable at week 24 (Fig. 3) . Anti-HPV16 VLP 
IgA and IgG were also found in the saliva of the three mice 
in amounts comparable or slightly higher to those found in 

3 0 vaginal washes. 

Anti-HPV16 VLP antibodies recognize only folded VLP. 

In order to examine whether the immune responses 
induced by the PhoP c /HPV strain generated conformational 
35 antibodies directed against native but not unfolded VLPs, 

we measured by EL ISA (Table 1) the binding of antibodies, 
in the samples from the immunized mice, to baculo-derived 



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VLPs in PBS (native form) or in carbonate buffer (pH 9.5, 
unfolded VLP, (14)). The specific IgG or IgA elicited by 
the PhoP c /HPV strain very poorly recognizes unfolded VLPs 
suggesting that the majority of LI were folded into highly 
5 ordered structures when expressed in PhoP c /HPV (Table 1) . 

In vitro neutralizing activity of the immune sera. 

In previous studies of baculo-derived VLPs, 
neutralizing activity and protection from experimental 

10 infection generally correlated with EL ISA reactivity to 

native VLPs. We therefore wished to determine if the 
conformational ly dependent anti-VLP antibodies elicited by 
the live Salmonella vaccine were also neutralizing. 
Although no infectivity. assay or source of the virus 

15 currently exists for authentic HPV16 , it has recently been 

demonstrated that HP VI 6 capsid proteins can encapsidate 
autonomously replicating BPV1 genomes resulting in 
HPVIS(BPVI) pseudotype virions whose infectivity can be 
monitored by focal transformation of cultured mouse 

20 fibroblasts (43). We therefore used the HPV16(BPV1) 

infectivity assay to examine the neutralizing activity of 
the mouse sera generated above. Each of the three immune 
sera displayed strong neutralizing activity against 
HPV16(BPV1) (Fig. 5), but did not neutralize BPV1 virions 

25 (data not shown) . The preimmune sera had no neutralizing 

activity. The neutralizing activities of the immmune sera 
appeared to correlate with the titers in the native VLP 
ELISA, although the sera were only tested at a single 
dilution. 

30 

Tumour protection assay in the HPV16 mouse tumour model . 

It has been recently shown that the growth syngeneic 
tumour cells (C3) injected into the flank of C57BL/6 mice 
was inhibited by a subcutaneous immunization with purified 
3 5 HPV16 cell (84) . We have tested whether nasal immunization 

with purified VLPs and recombinant Salmonella /RW strains 
was able to induce the same effect. Specifically, we have 



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tested the following strains: PhoPc/HPV16 LI (86) and the 
X4550 (56) expressing either high levels (x4550/pYA34Ll) or 
low levels (x4550/pYA32Ll) HPV16 Ll . Tumour growth in the 
different groups of mice is shown in figure 6. Our 
5 preliminary results demonstrate that nasal immunization 

with purified VLPs is effective and that all the 
Salmonella/RW strain tested induced partial tumour 
protection. Of interest, is the strain x4 550/pYA34Ll that 
prevented complete tumour growth in 4/10 mice. 

10 

Coexpression of the L2 protein into PhoPc/HPVl6 

The L2 OR17 was cloned downstream of the Ll ORF by PCR 
into the plasmid pPSnsdHPV16 Ll (86) . The PCR reaction 
included a 5' specific oligonucleotide that contained a 

15 synthetic Shine -Dalgarno sequence in order to allow 

translation of 12 from a polycistronic L1-L2 RNA. The 
resultant PhoPC/HPVI6 L1+L2 recombinant strain expressed 
both Ll and L2 and VLPs assembled in amount similar to the 
parent PhoPC/HPV16 Ll strain as assessed by a sandwich 

20 ELISA. This suggests that by fusing the E7 ORF to the L2 

ORF, in the PhoP c /HPV16 L1+L2 strain, a chimeric VLPs would 
also assemble and such recombinant Salmonella strain used 
to induce HPV16 E7-CTLS. 

25 High level expression of Ll in the inducible E. coli PET 

expression system. 

The Ll ORF was cloned in the plasmid pET3 (Novagen) . 
Ll- expression driven by a T7 promoter was assessed in the 
strain BL21apLysS (expressing T7 polymerase upon IPTG 

30 induction) . After IPTG induction, a 10 fold higher level 

of Ll expression/bacteria was achieved in comparison to the 
Salmonella PhoP c strain (see figure 8) . The lysate of this 
recombinant E. coli formed a band at a density of VLPs in 
a CsCl density gradient, suggesting that the VLPs self- 

35 assembled in this bacteria. 



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Discussion 

In this study, we demonstrate that an attenuated 
Salmonella strain expressing the major capsid protein of 
HPV16 is a promising vaccine candidate against HPV16 
5 infection, as the VLPs that are assembled by this 

recombinant bacteria can induce serum as well as genital 
VLP-specific conformational antibodies. The results above 
also show that the antibodies are able to neutralize HPV16 
viruses. These results could be readily extrapolated by 

10 the skilled person to other types of HPV or other 

papillomaviruses, or other prokaryotic microorganisms. 

The life cycle of papillomavirus is intimately 
associated with the differentiation of the epithelial cells 
in skin or the oral and genital mucosa (5,19,40,62). It is 

15 believed that viruses gain access to the basal epithelial 

cells through mucosal abrasions (21) . Upon infection of 
the cervical epithelium for instance, the viral DNA 
released in the cytoplasm of the basal cells migrates into 
the nucleus where it remains episomic and early genes are 

20 transcribed leading to a low rate of cell proliferation and 

the thickening of the 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 

25 reaching -1000 copies per cell (29) . Concomitant to viral 

DNA amplification, late genes become expressed and capsids 
assemble in terminally differentiated keratinocytes (Fig. 
4), thus facilitating a new round of infection. In high 
grade lesions (CIN III and carcinoma) the entire epithelium 

3 0 consists of undifferentiated basal cells in which the viral 

DNA has been integrated into cellular DNA. In these cells, 
the E6/E7 gene products constitute the major HPV proteins 
expressed and viruses are no longer produced. 

Based on our knowledge of HPV pathogenesis, it appears 

35 that two arms of immunity (humoral and cellular) have to be 

effective to prevent viral infection, to decrease the local 
viral load, or to cure tumors (Fig. 4, see also (59)). A 



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local or systemic humoral immune response with neutralizing 
antibodies is likely to block early infection, while a 
cellular response may contribute to the elimination of 
untransformed or transformed infected cells. An ideal 
5 vaccine should trigger the two types of response, although 

the immunological correlate of protection and of cure have 
not been identified so far. 

Prophylactic vaccines inducing type-specific 
neutralizing conformational (anti-VLP) antibodies have been 

10 shown to prevent CRPV or COPV infections in cottontail 

rabbit (4) or dog (57) , respectively. In both cases serum 
neutralizing antibodies where generated by vaccination with 
self -assembled PV capsids. By analogy, neutralizing 
antibodies to HPV16 capsid in cervical secretions are 

15 expected to prevent infection. Since the precise mucosal 

site where early HPV infection takes place is not known, it 
is difficult to predict whether slgA antibodies acting from 
the lumenal site or circulating IgG antibodies reaching the 
basal layers will be most efficient. 

20 The elimination of HPV- infected cells or tumor cells 

requires a cellular immune response with cytotoxic T 
lymphocytes (CTL) recognizing viral antigens presented by 
MHC class I molecules on the infected cells. Therapeutic 
vaccines aimed at eliminating HPV- induced tumors have been 

25 generated using either peptides corresponding to T cell 

epitopes from the E6/E7 oncogenes or E6/E7 expressing 
vaccinia viruses. Both were shown to elicit CTLs and in 
some cases tumor regression was observed (3,6,7,12,13,34). 
One of the major problems, however, is that MHC class I 

30 molecules are down -regulated in the differentiated 

keratinocytes that produce viruses or in tumour cells (9) . 

Since both humoral and cellular immunity are believed 
to control HPV infection and since local and systemic 
responses are desirable, an efficient vaccine should reach 

35 inductive sites associated with mucosal surface and/or 

peripheral lymph nodes. Live bacterial vaccines are known 
to cross mucosal surfaces and elicit humoral or cellular 



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responses (41) . Recombinant arid attenuated 

enteropathogenic bacteria, such as Salmonella, represent 
ideal antigen delivery systems, because they efficiently 
cross all mucosal surfaces to gain access to both mucosal 
organized lymphoid tissue (MALT) or draining lymph nodes. 
They exploit the two basic sampling systems mediating 
uptake of mucosally administered antigens including M cells 
in simple epithelia and dendritic cells both in simple and 
stratified epithelia (38) . We have selected a Salmonella 
typhlmurium strain attenuated for macrophage survival, 
because long lasting antibody responses were elicited by a 
single nasal, oral, rectal or vaginal administration of 
recombinant bacteria expressing a foreign antigen (18) . In 
that study, the best genital responses were obtained after 
nasal immunization. In the airways, antigen uptake occurs 
through M cells found in NALT, the nasal associated 
lymphoid tissue (25) and BALT, the bronchial associated 
lymphoid tissue (55) . The primed IgA-expressing 

lymphocytes then migrate into cervical and uterine tissues 
where they produce polymeric IgA antibodies, which are 
transported across the epithelium by the polymeric Ig 
receptor (26-28) . Intraepithelial dendritic cells in the 
bronchial epithelium also play a major role in antigen 
presentation by taking up the antigens in the respiratory 
epithelium and carrying them to distant draining lymph 
nodes where priming occurs (17) . This probably explains 
why nasal immunization is so efficient in triggering both 
local and systemic antibody responses. 

Antigens expressed in Salmonella strains can also 
elicit cellular responses with specific CTLs (1,16,58). 
Depending on which viral antigen is expressed, specific 
CTLs recognizing infected cells at different stages of 
differentiation could be generated (Fig. 4) . For instance, 
E7- specific CTLs were generated by immunizing mice with 
recombinant Salmonella expressing HPV16 E7 epitopes (31) . 

To trigger neutralizing antibodies using recombinant 
Salmonella, it is essential that the antigen retains its 



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native conformation. For HPV, this requires that the LI 
proteins form VLPs. Papilloma VLPs have been shown to 
assemble in eukaryotic cells (15,22,45,48,61), but not in 
prokaryotes. In bacteria mainly Ll-fusion proteins were 
5 expressed (2,20,24) and when bona fide LI proteins were 

expressed, VLP assembly was not examined (11) . As shown in 
this paper, HPV16 VLP assemble in Salmonella probably 
because the level of expression achieved in our experiments 
was high and capsid assembly does not require glycosylation 

10 (60) . Capsid production in bacteria has also been reported 

for other viruses such as the nucleocapsid of Hepatitis B 
virus (52) and the capsid of Polyomavirus (30,46). 
Polyomavirus VP1 major capsid protein, analogous to HPV LI, 
forms capsomers when expressed in E.coli which subsequently 

15 self -assembled into VLPs in vitro (46) . The fact that only 

capsomers but no VLPs were recovered is probably due to the 
reducing agents present during purification, which are 
known to disrupt capsids (47) . 

Nasal immunization with the PhoP c /HPV strain induced 

20 systemic and mucosal antibodies against native but not 

denatured HPV16 VLPs. In contrast, recombinant vaccinia 
expressing HPV1 capsid protein triggered serum antibodies 
recognizing both folded and unfolded VLP, probably 
reflecting different mode of viral protein expression, and 

25 low HPV-specific genital IgA antibody titers (14) , as 

expected with a non-mucosal route of immunization. 

Antibody titers against the foreign antigen induced by 
PhoP c /HPV compared to PhoP /HBc Salmonella were about 10 
times lower (18) . This could reflect differences in 

30 immunogenic ity between the two viral antigens (51) or, more 

likely,, differences in plasmid stability. In contrast to 
the HBc DNA, the plasmid carrying the HPV16-L1 DNA was 
unstable in Salmonella in vivo in the absence of selective 
pressure, since less than 1% of the Salmonella recovered 

35 from different tissues two weeks after immunization still 

harboured the LI -containing plasmid (data not shown) . To 
increase the stability of the plasmid we are currently 



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recloning the LI gene in ^-aspartate semialdehyde 
dehydrogenase (asd) -based vectors which maintain selective 
pressure in vivo (36,56). 

The above work also demonstrates the followinng 
5 points: 

(a) that purified VLPs and Salmonella/UPV strains are 
capable of providing tumour protection in a HPV16 mouse 
tumour model . 

(b) that chimeras of a HPV protein and a fusion 
10 partner assemble in prokaryotes to form VLPs. 

(c) that high levels of expression of HPV proteins 
that assemble to form VLPs can be obtained in E. coli, 
demonstrating that the invention is applicable in 
prokaryotes other than Salmonella. 

15 In conclusion, we have constructed a recombinant 

Salmonella strain expressing HPV 16 -LI capsid proteins and 
assembling VLPs that induce conformational serum IgG and 
vaginal slgA antibodies recognizing VLPs. Neutralizing 
activities of these antibodies were tested and shown to 

20 display strong neutralizing activity in an HPV16(BPV1) 

infect ivity assay. 



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TABLE 1: Titers of IgG (in serum) or IgA (in vaginal 
washes) against native and unfolded HPV16 VLP in mice 
immunized with PhoP c /HPV 



Samples anti-HPV16 VLP anti-unf olded a 

HPV16 VLP 

IgG titers 6 

#4 serum (week 27 , 60, 000 100 

#5 serum (week 27 , 80,000 200 

#6 serum (week 27) 20,000 100 

Camvir c 16,000 80,000 

IgA titers 

#4 Vaginal Washes (weeJc 19 , 40 <l 

#5 Vaginal Washes (week 19 , 20 <l 

#6 Vaginal Washes (week 19) 4 0 l 



a ELISA plates were coated with VLP in Carbonate buffer 
pH 9.5 

b Titers are expressed as the reciprocal of the highest 
sample dilution that yielded an OD 492 four times that of 
pre immune sample 

c Anti-HPV16 LI monoclonal IgG (35/xg/ml) , 30) used as 
positive control 



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53. Schodel et al, 1993. Hybrid hepatitis B virus 
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477. 



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Claims: 

1. 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) . 

2. The attenuated microorganism strain of claim 1 which 
is an attenuated strain of Salmonella. 

3. The attenuated microorganism strain of claim 2 wherein 
the Salmonella strain is Salmonella typhimurium, Salmonella 
typhi, Salmonella dublin, or Salmonella enteretidis . 

15 4. The attentuated microorganism strain of claim 1 which 

is an attenuated strain of Escherichia coli, Shigella, 
Yersinia, Lactobacillus, Mycobacteria or Listeria . 

5. The attenuated microorganism strain of any one of the 
20 preceding claims wherein the nucleic acid encodes a human 

papillomavirus virus major capsid protein. 

6 . The attenuated microorganism strain of claim 5 wherein 
the HPV strain is HPV16, 18, 31, 45 or 56. 

25 

7. The attenuated microorganism strain of any one of the 
preceding claims wherein the papillomavirus virus major 
capsid protein is LI protein. 

30 8. The attenuated microorganism strain of any one of 

claims 1 to 7 wherein the papillomavirus virus major capsid 
protein is expressed as a chimera with a fusion partner. 

9. The attenuated microorganism strain of claim 8, 
35 wherein the papillomavirus major capsid protein is 

coexpressed with L2 protein, the L2 protein being fused to 
the fusion partner. 



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10. The attenuated microorganism strain of claim 8 or 
claim 9 wherein the fusion partner is E6, E7 or E2 HPV 
protein, an immunogenic protein from a non-HPV pathogen or 
a tumour specific antigen. 

11. The attenuated microorganism strain of any one of the 
preceding claims wherein the microorganism is transformed 
with nucleic acid encoding two or more papillomavirus virus 
major capsid proteins. 

12. A composition comprising one or more of the attenuated 
microorganisms of claims 1 to 11, in combination with a 
physiologically acceptable carrier. 

13. A vaccine comprising one or more of the attenuated 
microorganisms of claims 1 to 11, in combination with a 
physiologically acceptable carrier. 

14 . The vaccine of claim 13 formulated for mucosal 
immunization . 

15. The vaccine of claim 14 wherein the mucosla 
immunization is via oral, rectal, nasal, or genital routes. 

16. The vaccine of any one of claim 13 to 15 wherein the 
vaccine provides protection against papillomavirus 
infection or cancer of the anogenital tract. 

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

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



WO 98/15631 



PCT/GB97/02740 



30 

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

5 

20. A method for producing assembled papillomavirus virus 
like particles comprising culturing an attenuated 
microorganism strain of any one of claims 1 to 11 and 
recovering the assembled virus like particles thus 

10 produced. 

21. A method of detecting the presence of anti- 
papillomavirus antibodies in a sample from a subject, the 
method comprising immobilizing the HPV VLPs on a solid 

15 support, exposing the support to the sample and detecting 

the antibodies binding to the immobilized HPV VLPs, wherein 
the HPV VLPs are produced by an attenuated microorganism 
strain of any one of claim 1 to 11. 

20 



25 




SUBSTITUTE SHEET (RULE 26) 



WO 98/15631 PCT/GB97/02740 




SUBSTITUTE SHEET (RULE 26) 



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PCT/GB97/02740 



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SUBSTITUTE SHEET (RULE 26) 



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Fig.6. 



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INTERNATIONAL SEARCH REPORT 



Int itlonaJ Application No 

PCT/GB 97/02740 



A. CLASSIFICATION OF SUBJECT MATTER 

IPC 6 C12N15/37 C12N15/62 C12N15/74 C12N1/21 C07K14/025 
A61K39/12 A61K31/70 G01N33/569 //(C12N1/21 .C12R1 :42) 

According to International Patent Classification (IPC) or to both national classification and IPC 

B. FIELDS SEARCHED 

Minimum documentation searched (classification system followed by classification symbols) 

IPC 6 C12N C07K A61K G01N 



Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched 



Electronic data base consulted during the international search (name of data base and, where practical, search terms used) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category * 


Citation of document, with indication, where appropriate, of the relevant passages 


Relevant to claim No. 


Y 


L0ND0N0 L.P. ET AL.: "Immunization of 


1-21 




mice using Salmonella typhlmurlum 






expressing human papillomavirus type 16 E7 






epitopes inserted into hepatitis B virus 






core antigen. " 






VACCINE, 






vol. 14, no. 6, April 1996, 






pages 545-552, XP002054898 






cited in the application 






see the whole document 




Y 


WO 96 11274 A (US HEALTH) 18 April 1996 


1-21 




see the whole document 






-/~ 





Further documents are listed in the continuation of box C. 



0 



Patent family members are listed in annex. 



* Special categories of cited documents : 

"A" document defining the general state of the art which is not 

considered to be of particular rolevance 
"E" earlier document but published on or after the international 

filing date 

"L" document which may throw doubts on priority claim(s) or 
which is cited to establish the publication date ot another 
citation or other special reason (as specified) 

"O" document referring to an oral disclosure, use, exhibition or 
other means 

"P" document published prior to the international fifing date but 
later than the priority date claimed 



T later document published after the international filing date 
or priority date and not in conflict with the application but 
cited to understand the principle or theory underlying the 
invention 

"X" document of particular relevance; the claimed invention 
cannot be considered novel or cannot be considered to 
involve an inventive step when the document is taken alone 

"Y" document of particular relevance: the claimed invention 

cannot be considered to Involve an inventive step when the 
document is combined with one or more other such docu- 
ments, such combination being obvious to a person skilled 
in the art. 

'&" document member of the same patent family 



Date of the actual completion of the international search 



9 February 1998 



Date of mailing of the international search report 



25/02/1998 



Name and mailing address of the ISA 

European Patent Office, P.B. 5818 Patenllaan 2 

NL-2280HVRIJswi|k 

Tel. (+31-70) 340-2040, Tx. 31 651 epo nl. 

Fax: (+31-70) 340-3016 



Authorized officer 



Mandl, B 



Foim PCT71SA/210 (second shoot) (July 1992) 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 


Inti lional Application No 

PCT/GB 97/02740 


C.(Contlnuatlon) DOCUMENTS CONSIDERED TO BE RELEVANT 


Category * 


Citation of document, with indication.where appropriate, of the relevant passages 


Relevant to claim No. 


A 
n 


HOPKINS S. ET AL.: "A recombinant 
Salmonella typhlmurlum vaccine induces 

local immunitv bv four dlffprpnt rnntpc nf 

immunization. " 
INFECTION AND IMMUNITY. , 
vol. 63, no. 9, 1995, 
Danes 327Q-1?86 XPflO?n^dftQ7 

cited In the application 
see the whole document 




1-21 


A 


WO 92 08486 A (UNIV WASHINGTON) 29 May 
1992 

see page 12, line 13 - page 13, line 19 




1-21 


A 


WO 94 03615 A (MEDEVA HOLDINGS BV ;KHAN 
MOHAMMED ANJAM (GB); HORMAECHE CARLOS EST) 
it reuruary lyyn 




1-21 


A 


KRUL M.R.L. ET AL.: "Induction of an 
antibody response in mice against human 
papillomavirus (HPV) type 16 after 
immunization with HPV recombinant 
Salmonella strains." 
CANCER IMMUNOL. IMMUNOTHER. , 

41 ^ontomhor 1 QQA 
VUI . HO, OcJJLclllUcf lsro, 

pages 44-48, XP002054899 
see the whole document 




1-21 


A 


HORMAECHE C.E. ET AL.: "Salmonella 
Vaccines: Mechanisms of immunity and their 
use as carriers of recombinant antigens." 
1995 , JOHN WILEY & SONS LTD. , 
CHICHESTER, UK; PAGES 119-153; 
XP002054901 

see page 129 - page 140 




1-21 


P,X 


NARDELLI-HAEFLIGER D. ET AL.: "Human 
papillomavirus type 16 virus-like 
particles expressed in attenuated 
Salmonella typhimurium elicit mucosal and 
systemic neutralizing antibodies in mice." 
INFECTIONA AND IMMUNITY, 
vol. 65, no. 8, August 1997, 
pages 3328-3336, XP002054900 
see the whole document 




1-20 



! — 

Form PCT7ISA/21 0 (continuation of second shoet) (July 1992) 



page 2 of 



2 



INTERNATIONAL SEARCH REPORT 

Information on patent (amity members 



Inti Atonal Application No 

PCT/GB 97/02740 



Patent document 
cited in search report 



Publication 
date 



Patent family 
member(s) 



Publication 
date 



WO 9611274 A 



WO 9208486 A 



18-04-96 



29-05-92 



US 5618536 A 
AU 3828495 A 
EP 0789766 A 



W0 9403615 A 



17-02-94 



AU 
AU 
CA 
CN 
EP 
JP 
NZ 
US 
US 



AU 
CA 
EP 
FI 
JP 
NO 



666108 B 
9120491 A 
2095534 
1063416 
0556333 
6501849 

240538 
5387744 
5294441 



4719393 
2141427 
0652962 

950396 
8503602 

950348 



08-04-97 

02- 05-96 
20-08-97 

01-02-96 

11- 06-92 
10-05-92 

12- 08-92 

25- 08-93 

03- 03-94 

26- 01-94 
07-02-95 
15-03-94 



03-03-94 
17-02-94 
17-05-95 
30-01-95 
23-04-96 
28-03-95 



Foim PCTrlSA/210 (patent tamily annex) (July 1992)