GB2467437A - Phosphoantigen for use in the treatment of tularemia - Google Patents

Abstract

The present invention relates to a phosphoantigen for use as a medicament; particularly in the prophylaxis and/or treatment of infection byFrancisella, e.g. Francisella tularensis, or tularemia; and a pharmaceutical composition comprising such phosphoantigens.

Description

Treatment The present disclosure relates to phosphoantigens for use iii the treatment and/or prophylactic protection against tulareniia and pharmaceutical compositions comprising said phosphoantigen for the same.

Franciseila ruicirensis is a small plciornorphic Gram negative coccobacillus which causes the disease tularemia. Tularemia is a zoonotic infection. It circulates in populations of rodents and lagomorphs, and outbreaks in humans often parallel outbreaks in animal populations.

However, it is not clear whether these animal species are the true reservoir of the bacterium in the environment. A wide ra.nge of arthropod vectors have been implicated in the transmission of tularemia between mammalian hosts including mosquitoes, ticks, and deer flies. These vectors can also transmit the pathogen to man. F. rukirensis can also be acquired by contact with, or ingestion of, contaminated material including food and water, and by inhalation of infectious particles. Rural populations and especially those individuals who spend periods of tinie in endemic areas such as farmers, hunters, walkers aiid forest workers are most at risk of contracting tularaeniia. Outbreaks associated with contaniinatecl water supplies can involve large numbers of cases, but usually the incidence of the disease is low.

These water-associated outbreaks are mainly caused by subspecies hoiarcrica; subspecies rularensis has never been linked to water-borne infections.

The type and severity of disease is dependent on strain, dose and route of infection. F. rularensis subspecies rularensis and holarctica cause the majority of reported cases, with subspecies tularensis causing the more severe disease of the two. Although tularernia can be a severely debilitating or even fatal disease, especially when caused by F. tulcirensis subspecies tuiarensi.c, many cases of disease caused by lower virulence strains are undiagnosed due to the non-specific nature of the symptoms. The incubation period is 3-5 days normally (range 1-21), and patients develop flu-like symptoms which may be protracted and relapsing if untreated.

Infection through the skin results in ulceroglandular tularemia. This is the most common presentation of the disease and can arise following the bite of an infected vector or through *:: direct contact with the flesh of inFected animal. Less commonly, infection can occur through the conjunctiva. This is termed oculoglandular tularemia and arises following direct *.: contamination of the eye. ingestion of infected meat or water can result in oropharyngeal or gastrointestinal tularemia. lnhaltioii of F. tukirensis results in respiratory oi. pneumonic tularemia. Respiratory tularemia has been reported in farmers following activities such as making hay, where infectious dusts can be generated. Other high-risk activities in endemic : ... 40 areas are lawn-mowing and bush cLitting. For example in Martha's Vineyard, Massachusetts, the majority of investigated cases were respiratory, in landscapers undertaking these types of * work. Pneumonia can also arise following haematogenous spread in other forms of tularaemia. Symptoms can be variable and depend on the virulence of the strain involved.

Infection with the most highly virulent strains can have a case fatality rate of up to 30% if untreated, but antibiotic therapy has reduced this to around 2%. Presentation can range from a mild pneumonia to an acute infection with high fever, malaise, chills, cough, delirium and pulse-temperature dissociation. Radiological examination may reveal parenchymal infiltrates, most commonly in one lobe, and hilar lymphadenopathy may be present. The infectious dose required to cause disease by this route is very low.

s Tularemia responds well to antibiotic therapy. As described above, the mortality rate of the more acute forms of the disease is reduced significantly if the patient receives suitable antibiotics. Historically aminoglycosides have been drugs of choice. Although clinically effective, streptomycin is rarely used now due to problems of ototoxicity and nephrotoxicity.

Similarly, although chloramphenicol has been used historically for treatment, it would be unlikely to be used as a first choice due to the possibility of irreversible effects on haematopoiesis. Gentamicin is a suitable alternative aminoglycoside, and has been used for treatment of pneumonic tularemia. Due to the requirement for parenteral dosing and monitoring of serum levels, aminoglycosides are now only used for the most serious cases.

The tetracyclines have been associated with high relapse rates on withdrawal. Doxycycline is effective in treatment of tularemia, but should be avoided for use in young children due to possible effects on developing teeth. Ciprofloxacin has been shown to be highly effective in oral therapy of tularemia, and can be considered the current drug of choice for uncomplicated tularemia. It has shown to be effective in treating tularemia in children, and may he suitable for use in pregnant women.

No licensed vaccine is available for prophylaxis of tularaemia. A live vaccine strain (LVS) was developed in the 1950s. and used extensively to vaccinate at-risk workers under Investigational New Drug Status which resulted in a significant decrease in laboratory acquired infections. Although LVS appears to be effective, there have been problems with the strain, such as reversion to virulence, mixed colony morphology and variable immunogenicity, and thus the LVS strain has failed to achieve licensing for human use.

Given the debilitating nature of tulareniia that no prophylactic treatmenUvaccine is available which is suitable for use in the general public and the fact that the disease in some instances is difficult to treat, an effective prophylactic therapy and/or treatment of the disease would be useful. * .*S

*** The present disclosure provides a phosphoantigen for the prophylaxis and/or treatment of tularemia. * *

: Whilst phosphoantigens have been suggested previously as potential adjuvants, there has * * been no suggestion that such compounds can be used in the treatment of a patient in their own right, that is, it has not been previously suggested that a treatment against any of the pathogens described herein could involve a phosphoantigen in the absence of an antigen, or if an antigen is present, that the antigen is not pharmaceutically active in the prophylaxis and/or * treatment of tularemia, or it is not present in sufficient amounts to he pharmaceutically active in the prophylaxis and/or treatment of tularemia. As used herein, "adjuvant" refers to a non-antigenic substance or substantially non-anrigenic substance that is used in combination with an antigen for enhancing the immune response against the antigen. The present invention is not intended to cover use of a phosphoantigen as an adjuvant in a medicament, such as a rnedicament against any of the diseases mentioned herein. As used herein, "treatment" can mean prophylactic treatment (i.e. pre-treating) or treatment post-infection with a pathogen of the type disclosed herein. A treatment is considered successful if the symptoms of an infection are ameliorated or prevented. As used herein, "pharmaceutically active" means a substance that has a statistically significant measureable effect on the material/host to which is administered. As used herein, "antigen specific to the pathogen" refers to an attenuated or avirulent strain of the pathogen or a subunit of the said pathogen that is peculiar to that particular type of pathogen.

Use of phosphoantigens for the treatment of a number of diseases, such as tumours, HIV, malaria and other viral and bacterial infections has been suggested. Clinical trials have been performed in relation to the treatment of tumours. However, there is little or no clinical support to show any efficacy in other clinical indications.

Surprisingly it seems that treating an individual with phosphoantigen provides beneficial therapeutic effects in relation to prevention of infection, or amelioration of symptoms after infection with F. itilarensis.

Thus there is provided a phosphoantigen for use in the treatment or prophylaxis of F. rularensis including subspecies thereof such as tuiarensis.

In one aspect there is provided a method of treating a patient in need thereof comprising administering a therapeutically effective amount of a phosphoantigen.

In another aspect there is provided a use of a phosphoantigen for the manufacture of a medicarnent for the treatment of infection with F. ruia.rensis.

Most bacteria including F. titlareits is produce phosphoantigens as intermediates of the DOXP metabolic pathway. Phosphoantigens are small molecular weight molecules with phosphorylated structures that selectively activate human or iion-hurnan primate T cells expressing Vy9V2 T cell receptor.

Phosphoantigens have been isolated from bacterial species such as TUBag1-4 from /t'!, * :: tuberculosis. Certain other phosphoantigens are known such as BrHPP and IHPP examples I and 2 respectively in US application publication No. 2004/0087555. The application relates to compounds of the following formula: OF! 0 0 0 *. I II II II : R:i OCat OCat' OCat1 wherein X is a halogen selected from I, Br or Cl, R' is selected from methyl amid ethyl, and Cat is a cation, and ii is au integer between 2 and 20.

A particularly suitable structure therein is: OH 0 0

I II II

X -Cf12-C-(CI-12)1-0---P--0---P-O-R3 RI OCat' OCat wherein X, RI and Cat+ is as defined above and R3 is selected from:

OH

X-CI-12-C-(CH2)-

RI

CH-,-0 \_ / and CH2 C-(CH2)1-

RI

Below is a table of a number of phosphoantigens from said application. * * ** *

** * S. * S ** I * S S * S. * 5w * S * *i.

*..S.. * S

MOLECULE

Name Abbreviation Structure isopentenyl 1PP Cl!2 p yrop hosphate II Cll1-C----(Cl-12).,-OPI' 3-(ch ioioaiethvi)-CII-! PP cu1ci 3-hutanol-i -1 I d phosphate cI-l1-c-(CI-I1)2Oii' O t-I 3-(bromonietlivi)-13iI'IPI' CI-l.,Br 3-hutanoi-I-yi I diphosphale CI-l-C--(CH2)2-OPP

OH

3-(iodoniethvi)-3-l-li'P CFI2I butanoi-1-vi I d iphosphate CIh-C-(Cl-I2)-Ol'i' OF! 3-(b 0 momeihv I)-13 rI-I I' Pt' Cl-h 13i 3-butanol-:1 -vi I triphosphate Cl-!---C (C!-1.2)2-OPPP OIl 3-(iodoinethyi)-3-IFIPPP Cu2! butanoi-1-yl I triphosphate Cl-I3----C-(CH2)2 -OPPI' 0!-l a. y di-3-di-I3iHTP CI-j,Br Cl-I-,13r (hromonsethvl)-3--I -butanoi-i -vi l-I2C-C-(CI-1.).2-Ol'I'PO-CiI2)2-C-Cl-I3 tripho.sphate I I 01-I 0!-! a, y di-3-di-Il-IT1' Cl-!1 Cl-!2! (iodoniethyi)-3-I I butanot-1-ri H3C-C-(CH2)2-Ol'PPO-CH2)2-C-CF!3 triphosphate I OH 01-1 wherein P is phosphate.

* Other compounds explicitly disclosed in the case include: 3-(brornomethy!)-3-butanol-I -yl . : triphosphate (BrHPPP); 3-(iodomethyl)-3-butanol-l-yl triphosphate (IHPPP); uridine 5'- * triphosphate gamma43-methyl-3butene-I -yll; alpha, beta cli I3-bronlomethyl-3-hLllanol-I -yl]diphosphate.

An alternative phosphoantigen is C-IPP 0 0 HO CH2-P-O-P--ONH4 0NH44 ONH4 (3-methylbut-3-enyl pyrophosphonate); H oO--O--ONH4 ONH4 ONH4 HIPP; HOCoQH HTigIyIPP

H OO+OH

HAngeIylPP A particularly potent phosphoantigen is CHDMAPP: (LO4O4OH

OH OH OH * .10 a...

Certain phosphoantigens are described in published patent application publication number *:*. US200810207568 (Innate Pharma) which describes certain phosphoantigeiis of the formula: I..

S * .**

S

*.SS..

S S (1) r0 1

I lii ii R5WCCABP (11) R6 R3 [a 1 a \ I ill I II CC__C_AP_B_1__P_Y; lb Ri R4 L QCat j OCit Ill (III) R3 [o 1 0

I III I II

R5-NCCAP-B P-Y; or R7 R4 [OCat CYCat (V) R3 ro 1 I Ill I II R5-WC-C-A--I--P-BJ_..P--Y.

I I [I] I CF[ R4 0Cat OCa( wherein Cat+ represents one or more cations in is an integer I to 3, B is 0, NH, CHF, CF2 or CH2 or any other isosteric group, W is C-R6 or N, R7 is a C1.3 alkyl group or any other isosteric group such as CF1, R3, R4 and R6 are independently selected from H, C1.3 alky or any other isosteric group such as CF3, R5 is selected from C2.3acyl, aldehyde, a Ci3 alcohol or a C2.3 ester, and Y=0Cat is as defined therein.

Any suitable phosphoanligen may be employed to for the prophylaxis or treatment of *....: tularemia, including a synthetic and/or natural phosphoantigen. Particularly suitable * 5 phosphoantigens are described above, such as CHDMAPP and IPP, which appear to be very *: * suitable for the treatment of tularemia.

Prophylaxis as employed herein is intended to refer to wherein an individual has a reduced risk of infection i.e. invasion and/or multiplication with the relevant pathogen compared to an *20 individual who has not had any prophylactic treatment against the relevant pathogen.

In one embodiment there is a reduced risk of developing severe infection, for example caused by subspecies tularensis.

Reduced risk as employed herein refers to a 20, 30, 40, 50, 60, 70, 80% or more reduced risk of developing the infection or a severe form thereof (such as developing the infection).

In one aspect the phosphoantigens employed are not compounds of the following formula: 01-I 0 0 0

I II II II

X-CH-C-(CU)O-P-0-P0P0Ca4 RI OCat OOf OCat wherein X is a halogen selected from 1, Br or Cl, R' is selected from methy and ethyl, and Cat is a cation, and n is an integer between 2 and 20.

In one aspect the pliosphoantigen is employed in combination with a cytokine, for example human recombinant interleukin-2 (IL-2) which may be co-administered or co-formulated with the phosphoantigen.

In one embodiment, for example when a patient has been exposed to F. lu/arensis or inoculated by the same (or alternatively is going into an area with a high risk of infection) the phosphoantigen or formulation thereof, for example as defined herein, niay be employed in combination with an antibiotic treatment, for example selected from aminoglycosides, gentamicin, chloramphenicol, doxycycline and ciprofloxacin, particularly ciprofloxacin.

Thus in one aspect. the disclosure provides a method of treating tulareniia comprising administering a therapeutically effective amount of the phosphoantigen prophylactically or to a patient infected by 1 lularensis.

innoculation or exposure to the relevant pathogen in the environment is not prophylaxis or treatment with an antigen specific to the pathogen, as employed herein. Nor is it to be ****3O considered to be treatment or prophylaxis by administration of a pharmaceutical composition comprising au antigen specific to the pathogen.

. : In one embodiment the phosphoantigen is administered before infection, for eXaml)IC 1, 2 or * 3 days or a week or a month before exposure. e 35

In one embodiment the phosphoantigen in given shortly after exposure, inoculation or infection, for example I hour to 3 days, such as 2 to 24 hours after.

:: In one embodiment the phosphoantigen is administered before exposure and shortly after exposure, for example as per the time frames above.

In one embodiment the phosphoantigen is employed for the treatment of chronic infection of tularemia.

The in vitro data with the relevant pathogens indicates that the bacterial load in a human monocytic cell line is reduced 10 to 100 fold in the presence of human blood or purified yö T cells treated with phosphoantigen and human recombinant IL-2 cx vivo. Given that the cells employed in the in vitro assay aie retrieved from the blood of healthy individuals' significant confidence can be gained that the phosphoantigens will work by the same mechanism in vivo.

In one embodiment the disclosure relates to a pharmaceutical composition comprising phosphoantigen in the presence or absence of IL-2, along with a pharmaceutically acceptable excipient for the treatment or prophylaxis of F. lu/c/re/isis infection. The composition may be employed as described above for the phosphoantigen.

In one embodiment the phosphoantigen is employed in combination with IL-2 and/or a further cytokine such as IL-15, IL-2l, interieron-y and/or interferon-u.

The active components of the combinations of the disclosure may, for example, be co-formulated if they are stable when mixed together. Alternatively, the components may be formulated separately and co-administered, that is administered at the same time or approximately the same time, for example one imniediately after the other. In further alternative embodiments the components may be administered sequentially, that is to say with a delay between the administration of each component, for example a delay of I to 12 hours.

Formulations The phosphoantigens or compositions according to the present disclosure maybe administered orally, topically, parenterally, transdermally, as a suppository or by any other pharmaceutically appropriate route.

Typical delivery routes include parenreral administration, e.g., intradermal, intramuscular or by subcutaneous delivery. Other routes include oral administration, intranasal, intravaginal *1*S* routes, intradermal and transdernial administration. S...

**35 In one embodiment the phosphoantigen according to the disclosure is provided optionally in either as a lyophihized formulation for reconstitution later or as a liquid formulation. * S

.: Transdermal administration, such as by iontophoiesis, may also be an effective method to deliver phosphoantigen to muscle. Epidermal administration may also be employed. Thus the disclosure also extends to delivery by a transderrnal patch, which may be occlusive or non- occlusive.

S

*5**SS * The actives can also be formulated for administration via the nasal passages. Formulations suitable for nasal administration, wherein the. carrier is a solid, include a coarse powder having a particle size, for example, in the range of about 10 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid for administration as, for example, nasal spray, nasal drops, or by aerosol administration by nebulizer, include aqueous 01. oily solutions of the active ingredient. For fLirther discussions of nasal administration of AIDS-related vaccines, references are made to the following patents, U.S. Pat. Nos. 5,846,978, 5,663,169, 5,578,597, 5,502,060, 5,476,874, 5,413,999, 5,308,854, 5,192,668, and 5,187,074.

Compositions of use in the disclosure include liquid preparations, for an orifice, e.g., oral, nasal, anal, vaginal, etc. administration, such as suspensions, syrups or elixirs; and, preparations for parentetal, subcutaneous, intradermal, intramuscular or intravenous administration (e.g., injectable administration) such as sterile suspensions or emulsions.

In compositions of the disclosure the relevant active ingredient may be in admixture with a suitable carrier, diluent, or excipient such as sterile water, physiological saline, glucose or the like.

The active ingredients can be incorporated, if desired, into liposomes, microspheres or other polymer matrices (Feigner et al., U.S. Pat. No. 5,703,055; Gregoriadis, Liposome Technology, Vols. Ito III (2nd ed. 1993), each of which is incorporated herein by reference).

Liposornes, for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

Liposome carriers may serve to target a particular tissue or infected cells, as well as increase the half-life of the active. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the vacciiie to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which hinds to, e.g., a receptor prevalent aniong lymphoid cells, such as monoclonal antibodies, or with other therapeutic or immunogenic compositions.

Thus, liposomes either filled or decorated with a desired immunogen of (he disclosure can be directed to the site of lymphoid cells, where the liposomes then deliver the immunogen(s).

Liposomes may be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing *: liposomes, as described in, e.g., Szoka, et al., Anti. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

The liposomes generally contain a neutral lipid, for example phosphatidylcholine, which is usually non-crystalline at room temperature, for example egg yolk phosphatidylcholine, dioleoyl phosphatidylchol me ot dilauryl phosphatidylcholine.

* * Optionally the formulation may comprise an adjuvant, for example a known adjuvant :: formulation may be used to reconstitute the formulation.

Phosphoantigens employed in the disctosLlre may be mixed or adsorbed with adjuvants, which include but are not limited to alum, inuramyl dipeptide and saponins such as Quil A. This may further boost the immune system's ability to deal with the infection.

Particular adjuvants are those selected from the group of metal salts, oil in water emulsions, Toll like receptors agonist, (in particular Toll like receptor 2 agonist, Toll like receptor 3 agonist, Toll like receptor 4 agonist, Toll like receptor 7 agonist, Toll like receptor 8 agonist and Toll like receptor 9 agonist), saponins or combinations thereof. The level of free antigen in a given formulation may be increased by, for example, formulating the composition in the presence of phosphate ions, such as phosphate buffered saline, or by increasing the ratio of antigen to metal salt. In one embodiment the adjuvant does not include a metal salt as sole adjuvant. In one embodiment the adjuvant does not include a metal salt.

In an embodiment the adjuvant is a Toll like receptor (TLR) 4 ligand, for example an agonist such as a lipid A derivative, in particular monophosphoryl lipid A or more specifically 3-deacylated monophoshoryl lipid A (3D-MPL).

3-Deacylated monophosphoryl lipid A is known froni US patent No. 4,912,094 and UK patent application No. 2,220,2 II (Ribi) and is available from Ribi Immunochem, Montana, USA.

3D-MPL is sold under the trademark MPL� by Corixa Corporation and primarily promotes CD4+ T cell responses with an IFN-g (Th1) phenotype. It can be produced according to the methods disclosed in GB 2220211 A. Chemically it is a mixture of 3-deacylated monophosphoryl lipid A with 3, 4, 5 or 6 acylated chains. Generally in the compositions of the present disclosure small particle 3D-MPL is used. Small particle 3D-MPL has a particle size such that it may be sterile-filtered through a 0.22tm filter. Such preparations are described in International Patent Application No. WO 94/2 1292.

Synthetic derivatives of lipid A are known and thought to be TLR 4 agonists including, but not limited to: OM 174 (2-deoxy-6-O-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylam inol-4-o- phosphono-3D-glucopyranosyl]-2[(R)-3-hydroxytetradecanoyl aminoj-ct-D-glucopyranosyldihydrogenphosphate), (WO 95/14026).

OM 294 DP (3S, 9 R)-3-[(R)-dodecanoyloxytetradecanoylaminoI-4-oxo-5-aza-9(R)-LI(R)-3 -hydroxytetradecanoylarni nodecan-1, 1 0-diol, I, I 0-bis(dihydrogenophosphate) (WO 99/64301 and WO 00/0462).

OM 197 MP-Ac DP ( 3S-, 9R) -3-[(R) -dodecanoyloxytetradecanoylaniinoll-4-oxo-5-aza-9- * [(R)-3 -hydroxyteradecanoylaniino} decan-I, 10-diol, I -dihydrogenophosphate lO-(6-aminohexanoate) (WO 01/46127).

Typically when 3D-MPL is used the antigen and 3D-MPL are delivered with alum or * presented in an oil in water emulsion or multiple oil in water emulsions. The incorporation of *:: 3D-MPL is advantageous since it is a stimulator of effector T-cell responses. Alternatively the 3D-MPL may be formulated as liposomes.

Other TLR4 ligands which may be Lised are alkyl Glucosaminide phosphates (AGPs) such as those disclosed in WO 98(50399 or US 6303347 (processes for preparation of AGPs are also disclosed), or pharmaceutically acceptable salts of AGPs as disclosed in US 6764840. Some AGPs are TLR4 agOniSts, and some are TLR4 antagonists. Both are thought to be usefLil aS adjuvants.

Another immunostimulant for use in the present disclosure is Quil A and its derivatives. Quil A is a saponin preparation isolated from the Soitth American tree Quilaja Saponaria Molina and was first described as having adjuvant activity by Dalsgaard et al. in 1974 (Saponin adjuvants", Archiv. fur die gesamte Virusforschung, Vol. 44, Springer Verlag, Berlin, p243- 254). Purified fragnients of Quil A have been isolated by HPLC which retain adjuvant activity without the toxicity associated with Quil A (EP 0 362 278), for example QS7 and QS2I (also known as QA7 and QA2I). QS2 is a natural saponin derived from the bark of Quihlaja saponaria Molina which induces CD8+ cytotoxic T cells (CTLs), Th cells and a predominant lgG2a antibody response.

Particular formulations of QS2I have been described which further comprise a sterol (WO 96/33739). The ratio of QS2 I: sterol will typically be in the order of I: 100 to 1:1 weight to weight.

Generally an excess of sterol is present, the ratio of QS2I:sterol being at least 1:2 w/w.

Typically for human administration QS2I and sterol will be present in a vaccine in the range of about I ig to about 100 g, such as about 10 ig to about 50 pg per dose.

A formulation comprising QS2I and liposomes may be prepared, for example containing a charged lipid, which increases the stability of the lipsome-QS2 I structure for liposomes composed of saturated lipids. In these cases the amount o Charge(l lipid is often 1-20% w/w, such as 5-10%. The ratio of sterol to phospholipid is 1-50% (mol/mol), such as 20-25%.

These compositions may contain MPL (3-deacylated mono-phosphoryl lipid A, also known as 3D-MPL).

The saponins may be separate in the form of micelles, mixed micelles (generally, but not exclusively with bile salts) or may be in the form of ISCOM matrices (EP 0109942), liposomes or related colloidal structures such as worm-like or ring-like multimeric complexes or lipidic/layered structures and lamellae when formulated with cholesterol and lipid, or in the form of an oil in water emulsion (for example as in WO 95/172 10). The saponins may often be associated with a metallic salt, such as aluminium hydroxide or aluminium * phosphate (WO 98/15287).

Usually, the saponin is presented in the form of a liposome, ISCOM or an oil in water emulsion.

*.ê**ê * Imrnunostiinulatory oligonuclcotides may also be used. Examples of oligonucleotides for use in adjuvants of the present disc'osure include CpG containing oligonucleotides, generally containing two or more dinucleotide CpG motifs separated by at least three, more often at least six or more nucleotides. A CpG motif is a cytosine nucleotide followed by a guanine nucleotide. The CpG oligonucleotides are typically deoxynucleotides. In one embodiment the internucleotide in the oligonucleotide is phosphorodithioate, or more preferably a phosphorothioate bond, although phospliodiester and other internucleotide bonds are within the scope of the disclosure. Also included within the scope of the disclosure are oligonucleotides with mixed internucleotide linkages. Methods for producing phosphorothioate oligonucleotides or phosphorodithioate are described in US5,666,l53, US5,278,302 and WO 95/26204.

Examples of oligonucleotides are as follows: TCC ATO ACG TTC CTG ACG TT (CpG 1826) TCT CCC AGC GTG CGC CAT (CpG 1758) ACC OAT GAC GTC 0CC GGT GAC GGC ACC ACG TCG TCG TTT TOT CGT TTT GTC GTT (CpG 2006) TCC ATG ACG TTC CTG ATG CT (CpG 1668) TCG ACG TTT TCG GCG CGC GCC G (CpG 5456), the sequences may contain phosphorothioate modified internucleotide linkages.

Alternative CpG oligonucleotides may comprise one or more sequences above in that they have inconsequential deletions or additions thereto.

The CpG oligonucleotides may be synthesized by any method known in the art (for example see EP 468520). Conveniently, such oligonucleotides maybe synthesized utilising an automated synthesizer.

Examples of a TLR 2 agonist include peptidoglycan or lipoprotein. lmidazoquinolincs, such as Imiquimod and Resiquimod are known TLR7 agonists. Single stranded RNA is also a known TLR agonist (TLR8 in humans and TLR7 in mice), whereas double stranded RNA and po1y IC (polyinosinic-polycytidylic acid -a commercial synthetic minletic of' viral RNA) are exemplary of TLR 3 agoniss. 3D-MPL is an example of a TLR4 agonist whilst CpG is an example of a TLR9 agonist.

An immunostimulant may alternatively or in addition be included. In one embodiment this immunostimulant will be 3-deacylated monophosphoryl lipid A (3D-MPL). S...

*:: Adjuvants combinations include 3D-MPL and QS21 (EP 0 671 948 BI), oil in water emulsions comprising 3D-MPL and QS2I (WO 95/17210, WO 98/56414), or 3D-MPL : formulated with other carriers (EP 0 689 454 BI) including liposomes. Other adjuvant * systems comprise a combination of 3D-MPL, QS2I and a CpG oligonucleotide as described in US 6558670 and US 6544518.

:. In one aspect the adjuvant comprises 3D-MPL. In one aspect the adjuvant comprises QS2I.

In one aspect the adjuvant comprises CpG. In one aspect the adjuvant comprises QS2I and *..*.

* * 3D-MPL. In one aspect the adjuvant comprises QS2 I, 3D-MPL and CpG In one aspect the adjuvant is formulated as an oil in water emulsion.

In one aspect the adjuvant is formulated as liposomes.

The amount of 3D-MPL used is generally small, but depending on the vacciiie formulation may be in the region of I to I000ig per dose, generally I to 500tg per dose, and more such as between I to l00tg per dose (10, 20, 30, 40, 50, 60, 70, 80 or 90ig per dose).

The amount of CpG or immunostimulatory oligonucleotides in the adjuvants or vaccines of the present disclosure is generally small, but depending on the vaccine formulation maybe in the region of I to iO00ig per dose, generally I to S00ig per dose, and more such as between 1 to l00tg per dose (10, 20, 30, 40, 50, 60, 70, 80 or 90tg per dose).

The amount of saponin for use in the adjuvants of the present disclosure may be in the region of I to 1O001g per dose, generally I to 500g per dose, more such as I to 2501g per dose, and more specifically between 1 to l00tg per dose (10, 20, 30, 40, 50, 60, 70, 80 or 90tg per dose).

Thus in one embodiment there is provided a formulation comprising phosphoanligen and MPL.

In one embodiment there is provided a formulations comprising phosphoantigen and QS21.

In one embodiment there is provided a formulation comprising phosphoantigen and CpG.

Thus in one crnbodimcnt there is provided a formulation comprising phosphoantigen and MPL and QS2 1.

Thus in one embodiment there is provided a formulation comprising phosphoantigen and MPL and CpG.

Thus in one embodiment there is provided a formulation comprising phosphoantigen and QS2I andCpG.

Thus in one embodiment there is provided a formulation comprising phosphoantigen and MPL,QS2I and CpG. * .35

The above formulations may optionally comprise an antigen/immunogen provided that the antigen or immunogen is not specific to F. tularensis.

In one embodiment the phosphoantigen/formulation/composition is a vaccine.

:... Vaccine preparation is generally described in New Trends and Developments in Vaccines, edited by Voller er a!., University Park Press, Baltimore, Maryland, U.S.A., 1978.

* a Encapsulation within liposomes is described, for example, by Fullerton, US 4,235,877.

In one embodiment the formulation is provided as a formulation for topical administrations including inhalation.

Suitable inhalable preparations include inhalable powders, metering aerosols containing propellant gases or inhalable solutions free from propellant gases. Inhalable powders according to the disclosure containing the active substance may consist solely of the abovementioned active substances or of a mixture of the abovementioned active substances with physiologically acceptable excipient.

These inhalable powders may include monosaccharides (e.g. glucose or arahinose), disaccharides (e.g. lactose, saccharose, maltose), oligo-and polysaccharides (e.g. dextranes), polyalcohols (e.g. sorhitol, mannitol, xylitol), salts (e.g. sodium chloride, calcium carbonate) or mixtures of these with one another. Mono-or disaccharides are preferably used, the use of lactose or glucose, particularly but not exclusively in the form of their hydrates.

Particles for deposition in the lung require a particle size less than 10 microns, such as 1-9 microns suitably from 0. 1 to 5 j.tm, particularly preferably from I to 5 urn. The particle size of the active (that is the antigen is of primary importance).

The propellent gases which can he used to prepare the inhalable aerosols are known from the prior art. Suitable propellent gases are selected from among hydrocarbons such as n-propane, n-butane or isobutane and halohydrocarbons such as chlorinated andlor fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane. The abovernentioned propellent gases may be used on their own or in mixtures thereof.

Particularly suitable propellent gases are halogenated alkane derivatives selected from among TGI1, TG 12, TG 134a and TG227. Of the abovernentioned halogenated hydrocarbons, TG134a (1,1, 1,2-tetrafluoroethane) and TG227 (1,1,1,2,3,3,3-heptafluoropropaiie) and mixtures thereof are preferred according to the invention.

The propellant-gas-containing inhalable aerosols may also contain other ingredients such as cosolvents, stabilisers, surface-active agents (surfactants), antioxidants, lubricants and means for adjusting the pH. All these ingredients are known in the art.

The propellant-gas-containing inhalable aerosols according to the invention may contain up to 5 % by weight of active substance. Aerosols according to the invention contain, for ::35 example, 0.002 to 5 % by weight, 0.01 to 3 % by weight, 0.015 to 2 % by weight, 0.1 to 2 % by weight, 0.5 to 2 % by weight 0.5 to 1 % by weight of active.

In one embodiment of the disclosure the phosphoantigen is employed in a prime boost regime, as the priming and/or boosting dose. Priming in this context refers to priming the immune system for a response, such as Vy9V2 T cell proliferation and/activation. Boosting : . * refer to increasing or sustaining the immune response of said priming. *

* Whilst not wishing to be bound by theory if the appropriate number of doses is exceeded in a relatively short period of time then it is possible induce aiiergy in the immune system, which is undesirable and should be avoided.

in the one embodiment the dose is in the range lpg to i000pg per Kg, such as Ing to 10 pg per Kg.

in one embodiment the disclosure provides use of a phosphoantigen for enhancing protective host immune responses specifically those mediated by Vy9S2 T cells to enable the host to fight infection with intracellular pathogens such as Fran.cisel/a lu/ct rens is.

In one embodiment the disclosure provides a method of stimulating an immune response to fight Francisella ía larensis infect ion.

In one embodiment the bioburden is lowered in t'it.'o after administration of the

phosphoantigen of the disclosure.

EXAMPLES

Materials and methods Preparation of Peripheral Blood Moiioii.itclecir Cells (PBMCs) Human blood (60m1) was collected into BD Vacutainer CPT� sodium citrate tubes and centrifuged (25mm; 1500g (no brake)). Buffy coats were removed, placed in Falcon tubes and centrifuged (300g; 15mm). The cell pellet was resuspended in lOmI medium (RPMI +10% Foetal calf serum (FCS), 200U/ml penicillin, 200tg/nil streptomycin and L-glutamine (10mM)). Cell numbers were counted with trypan blue and centrifuged (300g: 15mm).

isolation of y T ce//s front PBMC /,repara/ions yS T cells were isolated from PBMC preparations using human Anti-TCRyTh Microhead Kit (Miltenyi Biotech. UK) according to the method described below. The PBMC pellet was resuspended in 40pl medium and lOpI anti-TCR yö Hapten-Antibody per lxlO7 cells, mixed well and refrigerated for 10mm. Medium (30 p1) and MACS anti-hapten microbeads-FITC (20 il) pei lx IO cells were added to cells and mixed well and refrigerated for 15mm. Medium was added to cells and centrifuged (300g:I5min). The cell pellet was resuspended in 500 pl medium. Media (500 p1) was added to MACS column (MS 130-042-201: Miltenyi Biotec, UK). A BD Falcon cell strainer (70 pm) was placed on top of the column and the cell suspension added. The cell effluent was collected. The column was washed 3 times with medium (500 p1). The column was removed from the magnetic 0.

separator and place iii a Bijoux. Medium (I ml) was added to the column and immediately flushed *::35 through using plunger from column kit. This is positive/enriched fraction contained the yö T cells.

The number of cells in positive fraction was counted.

* Culture of yô Tcells *.* * The T cell population was cultured in a 24-well plate in medium (RPMI + 10% Foetal calf serum (FCS), 200U/nil peiiicillin, 200.tg/ml streptomycin and L-glutamine (10mM)) supplemented with : .. * I OOU/nil human recombinant intcrleukin-2 (lL-2) (Sigma, UK) and isopentcnyl pyrophosphonale (IPP) 3pg/ml (Sigma, UK). Additional 1L-2 (100 U/mI) was added to cells every 3 days. Cells were * removed from wells by gently pipetling and scraping the bottom of the well with the tip to mix and break up cell clumps. Cells were counted and resuspended in I nil fresh medium without antibiotics to the correct cell concentration (Ix l0 cells/mI).

THP-/ human In000c)'te cell line The non-adherent human monocylic cell line THP-l (ATCC�) (ECACC, UK) was crown in RPMI.

10% Foetal calf serum (FCS). 200U/ml penicillin, with or without 200ig/ml strcptomycin and L-glutamine (I 0mM) all obtained from Sigma, UK in cell culture flasks under sterile conditions. Prior to infection. THP-I cells were removed from cell culture flasks and centrifuged (300g; I 5mm). Cells were resuspended in medium without antibiotics, counted and readjusted to requ irecl cell concentration (2x I O6cells/ml). THP-I cells (I mI/well) were plated into 24 well plates and activated with PMA (phorbol l2-nlyristate 13-acetate: Sigma. UK) at a final concentration of lOng/mi and were incubated overnight (37C; 5%C02) to produce an adherent monolayer. Media was removed from wells prior to infection and replaced with fresh media (900sl) without antibiotics.

Growth of F. tularensis LVS A viable frozen culture of R lularetisis LVS was thawed and 40u1 drops were spread oii dried BCGA plates at ACDP containment level 2. Plates were incubated overnight (37°C; 5%C02). Loops of bacterial lawn were taken and placed in PBS to make a cloudy suspension with an OD450 reading of between 0.2-0.3 (l-2x 106 cfu/ml). A 1:10 dilution of the suspension was performed prior to infection of THP-1 to provide an approximate infection dose of lx l0 cfu/ml.

Growth ofF. tularensis SC/-lU S4 A viable frozen culture of F. tularensis SCHU-S4 was thawed and 40u1 drops were spread on dried BCGA plates. All work with this organism was performed at ACDP containment level 3. Loops of bacterial lawn were taken and placed in PBS to make a cloudy suspension with an OD4SQrni reading of between 0.2-0.3 ( l-2x 106 cfu/ml). A 1:10 dilution of the suspension was performed prior to infection of THP-1 to provide an approxinlae infection dose of lxl0 cfu/mi.

infection of THP-I inonolaver F. tularensis suspension (lOOpI /well) was added to the THP-l monolayer and incubated (30-45niin; 37°C; C01). Media was then removed and fresh media without antibiotics (500d) was added. Isolated yö T cells or media only were added to the infected THP-l monolayer and incubated in a sealed container (with CO2 packet) in 37°C incubator for 24 h. Supernatants were removed from wells following incubation and stored at -20°C for further bacteriological analysis. Cells were lysed using sterile distilled water (liiil), pipetting up and down several times to disrupt cells. The lysed cell suspension (lOOpi) was added to PBS (900pi) to prevent further cell lysis. Droplets of the lysed cell Suspension stabilised in PBS or thawed cell culture supernatants (3x201.d) were placed onto duplicate ::35 BCGA plates and incubated for 5 days (37°C; 5%C02). Viable colonies were then counted. ** *

* * * Results * ** * Human yr5 T cells and growth of F. ruk,rensi.v The effect of human yö T cells treated with IPP+1L-2 on intracellular growth of F. rularensis LVS was investigated. In tile absence of yö T cells, the number of intracellular bacteria inlecting the human monocytic cell line (THP-l) was 1000-10000 F. tularensis LVS cfu/nil. In the presence of purified human y T cells (2x10 cells/nil) obtained from 6 different individLials. a significant decrease in the number of intracellular bacteria was observed (p<O.000I) IANOVAI in comparison with media oiily controls (Figure IA). No significant differences were observed between individuals in their ability to kill F. tuiaren.sis LVS (p=O.608). A reduction iii growth of F. tularensis SCHU S4 was also observed in the presence of human ö T cells obtained from 3 individuals in comparison with media only controls (Figure I B).

The number of bacteria released from THP-I during the infection assay was measured in supernatants from wells containing purified yö T cells or media alone. In the absence of y T cells, 1000-10000 cfu F. rularensis LVS /ml were identified. No bacterial growth was observed in supernatants retrieved from wells containing y T cells from 5 individuals.

Figure 1 shows human y6 T cell mediated killing of Fl lu/arensis LVS and [I iuiaren.vis SCHU-S4.

THP-I cells infected with F. rularensis (lx I ü cfu/ml: I xl 06 THP-I). were incubated for 24 hours in the presence of y8 T cells (*) or media alone (o). A) Intracellular F. iukirenci.s LVS retrieved following lysis of cells following infection. Data is shown as mean of triplicate wells for each individual (1-6) and any repeats performed on each individual are also displayed; B) Intracellular F. rularensis SCHU S4 retrieved following lysis of cells following infection from 3 diffferent individuals. Data is represented as mean of triplicate wells for each individual (1-3). Error bars represent SD of triplicate wells/individual.

Figure 2 shows human ö T cell mediated control of free F. rularensis LVS. Release of F. rukirensis LVS was investigated in supernatants from THP-l cells infected with F. rukirensis (lx lO cfu/ml: I xl 06 THP-I) incubated for 24 hours. Individual data showing mean of triplicate supernatants (n=5) in the presence of yS T cells (*) or media alone (n). Error bars represent SD of triplicate wells/individual It is envisaged that one or more embodiment described herein may he combined, as technically appropriate.

In the context of this specification "comprising" is to be interpreted as including.

Aspects of the disclosure comprising certain elements are also intended to extend 10 alternative embodiments consisting or consisting essentially" of the relevant elements. * I **..

S * * ** * * . . * I.

S

I * S * **S

*IIS*I * .

Claims (9)

1. Claims 1. A phosphoantigen for the prophylaxis and/or treatment of tularemia.
2. 2. A phosphoantigen according to claim I, wherein the phosphoantigen is [PP.
3. 3. A phosphoantigen according to claim I, where in the phosphoantigen is BrHPP or [HPP.
4. 4. A pharmaceutical composition comprising a phosphoantigen as recited in any one of claims 1 to 3.
5. 5. The pharmaceutical composition of claim 4. further comprising IL-2.
6. 6. The pharmaceutical composition of either claim 4 or 5 for use in the prophylaxis and/or treatment of tularemia.
7. 7. The pharmaceutical composition of claim 6 wherein the composition does not further comprise an antigen that is pharmaceutically active in the prophylaxis andlor treatment of tularem ia.
8. 8. A method of treatment for tularemia comprising administering a therapeutically effective amount of a phosphoantigen prophylactically or as treatment to a patient in need thereof.
9. 9. A method of treatment for tularemia comprising adrninisteriiig a therapeutically effective amount of the pharmaceutical composition of any one of claims 4 to 7 prophylactically or as treatment to a patient in need thereof.1 I. Use of the pharmaceutical composition of either claim 4 or 5 in the manufacture of a *111* medicament for the prophylaxis or treatment of tulareniia.I SS lS * . Il S * . S* 1.35 a..I S. * Sl*SS..... * I