CA2489940C - Relay vaccine - Google Patents

Abstract

The present invention provides a method and composition for raising an immune response in an animal. The method comprising administering to the animal a composition comprising a carrier and an antigen bound to a targeting moiety. The targeting moiety binds to at least one receptor that is upregulated on lymphocytes that home to MAdCAM+ mucosal lymphoid tissues.

Description

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RELAY VACCINE
FIELD OF THE INVENTION
The present invention relates to a targeted vaccine strategy. In particular the present invention relates to a vaccination strategy where the antigen is targeted to lymphocytes which carry the antigen to MAdCAM+ mucosal lymphoid tissues.
BACKGROUND OF THE INVENTION
DCs are the centre-piece of the immune system. They orchestrate the type and intensity of the immune mechanisms that prevent disease. Given the heterogeneity of these immune mechanisms, it is not surprising that the DC subsets that control them are equally diverse.
From the first description of DCs as antigen presenting cells [1], there has been a concerted effort to categorise the phenotypically distinct subsets and ascribe functional differences.
Such studies unveiled that DCs could be divided into subsets by their expression of COB
into CD84, CD8th (intermediated expression) and CDS- populations [2-4]. The CDS' populations can be further divided into C04-CD8' (double negative DN) and CD4*CD8-populations [5, 61. Segregation of DCs by these markers has uncovered significant differences in anatomical localization [7-9], antigen uptake and processing [10, 11] as well as cytokine production [12-15]. Although maturation/activation states of the subset, as well as the nature and amount of the antigen encountered can play a large role in dictating the functional outcome, some functional ascriptions have been made. For example, it is believed that CDS' DCs (found mainly in the T-cell areas) are preferentially involved in cross-priming CTL responses [16-18], whereas CDS' DCs (mainly found in non T-cell areas) may be more associated with potentiating T helper type 2(171 and B-cell responses [7,9].
Along with phenotypic distinctions associated with the expression of CD8, the origin of the DCs can have significant impact on the induction of cytokine as well as T and B-cell responses. This is particularly evident when comparing DCs isolated from mucosal versus peripheral tissues. For example, DCs isolated from Peyer's Patches stimulate the production of more 1L-4 and IL-10 but less ITN-y [19]. In fact these IL-4 and IL-10 inducing cells are far more potent in stimulating allogenic T-cell proliferation compared with splenic DCs [19]. CD11b+ CD8- DCs isolated from the Peyer's Patches also preferentially secrete 1L-6 and induce the secretion of IgA from naive B-cells [20]. DCs from the mesenteric lymph nodes preferentially enhance T-cell expression of the irmcosal homing receptor LPAM-1 as well as chemokine receptor CCR9 [21, 22].

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2 Targeting DCs via non-lineage specific cell surface markers such as, MHC class II [23], CD11c [24], CD80/CD86 [25], toll-like receptors [26], DEC205 [27] and irrmaunoglobulin Fc-receptors [28, 291, can enhance the systemic response to antigen. These strategies however, fail to deliver antigen at the levels required for the induction of mucosal immunity. One approach to mucosal vaccination attempted to target M-cells (in gut lining) that shuttle antigen from the mucosal surface to underlying lymphoid tissue [30, 31].
Although somewhat promising, these techniques have so far failed to overcome the problems faced in effectively delivering antigens for the efficient induction of mucosal immune responses.
To combat the constant threat of infection, the mucosa is littered with a diverse assortment of specialised lymphocyte populations. Positioning of these lymphocytes throughout the mucosa is critical for proper effector function. Thus, lymphocyte positioning is tightly regulated by the coordination of a number of unique horning receptors. These include the specific expression of cellular adhesion molecules VCAM-1, ICAM-1, MAdCAM-1 and E-cadherin. Lymphocytes use heterodimeric complexes of the integrin family such as cc407 (LPAM-1) [32-34], a4f3-, (LPAM-2) [35], and ccEP7 [36, 37] to bind to these adhesins and move through the tissue in search of antigen presenting DCs. Once lymphocytes encounter DC-s, they form close interactions that are required for antigen recognition.
Indeed, these interactions are so close that the DC can acquire membrane components (including bound antigens) from the lymphocyte [38-40). It is therefore possible that lymphocyte populations programmed with unique and well-characterised homing potentials could be used as carriers to deliver antigen to particular DC populations. Such a mechanism may overcome the lack of suitable markers for the efficient delivery of vaccine antigens to mucosal DCs and may overcome the normal barriers to the induction mucosal IgA responses.
Given that DCs are constantly bombarded with lymphocytes that do express unique homing receptors, the present inventors have proposed a 2-step targeting model whereby lymphocytes could "relay" antigen to sites of immune induction. The mucosal homing receptor lymphocyte Peyer's Patch adhesion molecule-1 (LPAM.) was investigated. LPAM
is upregulated on mucosally targeted lymphocytes where it facilitates the interaction with the mucosal addressin cellular adhesion molecule-1 (MAdCAM) [32, 411 The present inventors provide a model for lymphocyte mediated delivery of antigen to mucosal lymphoid tissues of the gut as well as to peripheral lymphoid tissues.
SUMMARY OF THE INVENTION
In a first aspect, the present invention provides a method of raising an immune response in an animal, the method comprising administering to the animal a composition comprising a CA 02489940 2004-12-08 = = .=
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3 carrier and an antigen bound to a targeting moiety, wherein the targeting moiety binds to at least one receptor, the receptor being characterised in that it is upregulated on lymphocytes that home to MAdCAW mucosal lymphoid tissues.
In a second aspect, the present invention provides a targeted antigen comprising an antigen bound to a targeting moiety wherein the targeting moiety binds to at least one receptor, the receptor being characterised in that it is upregulated on lymphocytes that home to MAdCAM* mucosal lymphoid tissues.
In a third aspect, the present invention provides an antigenic composition, the composition comprising a carrier and an antigen bound to a targeting moiety wherein the targeting moiety binds to at least one receptor, the receptor being characterised in that it is upregulated on lymphocytes that home to MAdCAM+ mucosal lymphoid tissues.
In a fourth aspect, the present invention provides a method of raising an immune response in an animal, the method comprising ad.ministering to the animal a composition comprising a carrier and an isolated nucleic acid molecule, the nucleic acid molecule encoding an antigen and a targeting moiety which binds to at least one receptor, the receptor being characterised in that it is upregulated on lymphocytes that home to MAdCAM+ mucosal lymphoid tissues.
In a fifth aspect, the present invention provides an isolated nucleic add molecule, the nucleic acid molecule encoding an antigen and a targeting moiety which binds to at least one receptor, the receptor being characterised in that it is upregulated on lymphocytes that home to MAdCAM+ mucosal lymphoid tissues.
In a sixth aspect, the present invention provides an antigenic composition, the composition comprising a carrier and an isolated nucleic acid molecule, the nucleic add molecule encoding an antigen and a targeting moiety which binds to at least one receptor, the receptor being characterised in that it is upregulated on lymphocytes that home to MAdCA/v1+ mucosal lymphoid tissues.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Targeting LPAM enhances mucosai and systemic antibody responses in a dose dependent manner. Mice were immunized intravenously with 10 us of either anti-LPAM
mAb DATK32 or the rat IgG2a isotype control GL117 in 0.2ml of saline. Rat IgG2a specific Ab responses for fecal IgA (a) and serum IgG (b) were measured by ELISA at 0,2,4 and 6 weeks. 2 weeks after immunization IgM, IgE and lgA as well as IgG subclass (IgGl, IgG2a = CA 02489940 2004-12-08 _ .

4 and 1gG3) anti-rat 1gG2a responses in scrum were measured by ELISA (a). Means SE are shown.
Figure 2. LFAM-targeting localizes antigen to penpheral and mucosa' tLcsucw to enhance systemic and mucosa] IgA and I,KG production. (a) Mice were immunized intravenously with 100 ug of either anti-LPAM mAb DATK32 or the isotype control GL117. 1 hr after, blood, ILN, and MLN were harvested and lymphocytes stained for antigen with PE-conjugated anti-rat Ig and analysed by FACS. (b) Mice were immunized intravenously with 10 ug of either anti-LPAM mAb DATK32 or the isotype control GL117. 11 d after, spleen, 1LN, MLN and PP were harvested and assayed for rat IgG2a specific IgA
and IgG
Ab secreting cells (ASC) by ELISPOT; Means SE (spots/106 cells) are shown.
(c) Mice were immunized intravenously with 10 pg of either anti-LPAM mAb DATK32 or the isotype control GL117. 11 d after, spleen, ILN, MLN and PP cells were harvested cultured for 5 days and supernatant assayed for rat IgG2a specific IgA and IgG Ab by ELISA. Means SE of OD 450 run are shown.
Figure 3. Immunizing doses of anti-LPAM mAbs do not deplete target populations, inhibit homing or alter antigen specific proliferation of CO4+ T-cells in-vivo. (a) Mice were immunized intravenously with either 10 jig of anti-LPAM mAb DATK32 in 0.2 ml of PBS
or a PBS only control. 1 day after, white blood cell (WBC) percentages and total cell numbers were analysed from the blood using a coulter-counter. CD4+, CDS+ and B220' lymphocyte proportions were analysed by FACS (inlay of bottom panel in a). (b) Mice received an intravenous injection of 5 x 106 CFSE labelled MLN cells mixed with 10 jig of either anti-LPAM mAb DATK32 or isotype control mAb GL117 in 0.2 ml of saline.
Three hr after, MLN and 1LN were harvested and homing of CFSE labelled cells analysed by FACS.
(c) Mice received an intravenous injection of 5 x 106 CFSE labelled OT-II
(CD4+ class II
restricted OVA specific TCR transgenic). 1 d after, mice were immunized 50 fig of OVA
only or mixed with 10 jig of anti-LPAM mAb DATK32 0.2 ml of saline. Three days after, MLN and 1LN were harvested and proliferation CFSE labelled OVA specific CD4+ T-cells cells analysed by FACS.
Figure 4. Enhanced responses induced by anti-LFAM require targeting but not intravenous delivery. (a) Mice were immunized intravenously with 10 us of either anti-LPAM
mAb DATK32 or isotype control mAb GL117 mixed with 50 jig of ovalbumin (OVA) in 0.3 ml of saline. OVA and rat IgG2a specific Ab responses for fecal IgA and serum TgG
were measured by ELISA at 2 wk. (b) Mice were immunized intravenously with 10 jig of either - - , -rt=
anti-0.437 (LFAM) mAb DATK32, isotype control mAb GL117, anti-intraepithelial lymphocyte (IEL) mAb M290, anti-a4 integrin mAb MFR4.B or the anti-n, integrin mAb FIB27. Rat IgG2a specific Ab responses for fecal IgA and serum IgG were measured by ELISA at 2 wk. (c) Mice were immunized orally, intravenously, subcutaneously or

5 intramuscularly with 10 lig of either anti-LPAM mAb DATK32 or the isotypc control mAb GL117. Rat IgG2a specific Ab responses for fecal IgA and serum IgG were measured by EL1SA at 2 wk.
Figure 5. An LPAM-1 targeted DNA vaccine azzgrnents systemic Ab responses.
Mice (5 per group) were immunised intramuscularly with 200 ug of plasmid encoding either targeted MAdCA/v1-1-hlg, or non-targeted CD5 leader (CD5L)-Mg (expresses the human Ig only) at 0 and 6 weeks. Human IgG specific faecal IgA, serum IgA and serum IgG
Ab responses were measured by EL1SA at 8 weeks. Means + SD of O.D. 450 nut are shown for faecal IgA (1:10 dilution of faecal sample) and serum IgA (1:50 dilution of serum). Means +
SD log Ab titres are shown for serum IgG response.
DETAILED DESCRIPTION OF THE INVENTION
The present inventors have identified a unique relay approach to antigen targeting that substantially improves mucosal and systemic antibody responses.
In a first aspect, the present invention provides a method of raising an immune response in an animal, the method comprising administering to the animal a composition comprising a carrier and an antigen bound to a targeting moiety, wherein the targeting moiety binds to at least one receptor, the receptor being characterised in that it is upregulated on lymphocytes that home to MAdCAM+ mucosal lymphoid tissues.
In a second aspect, the present invention provides a targeted antigen comprising an antigen bound to a targeting moiety wherein the targeting moiety binds to at least one receptor, the receptor being characterised in that it is upregulated on lymphocytes that home to MAdCAM+ mucosal lymphoid tissues.
In a preferred embodiment of the present invention, the targeting moiety binds to a receptor which is a member of the integrin family. More preferably, the receptor is the mucosal homing receptor lymphocyte Peyer's Patch adhesion molecule (LPAM).
Still more preferably, the receptor is a4137 integrin (LPAM-1).
LPAM facilities the interaction with the mucosal addressin cellular adhesion molecule-1 (MAdCAM) [32, 41} present in mucosal lymphoid tissue. LPAM-1' lymphocytes are = CA 02489940 2004-12-08 -r!

6 targeted to mucosal lymphoid tissue through their specific interaction with the mucosal homing receptor MAdCAM-1. Accordingly, such lymphocytes provide a mechanism for "relaying" antigen to sites of immune induction.
In a third aspect, the present invention provides an antigenic composition, the composition comprising a carrier and an antigen bound to a targeting moiety wherein the targeting moiety binds to a receptor, the receptor being characterised in that it is upregulated on lymphocytes that home to MAdCAM+ mucosal lymphoid tissues.
In a preferred embodiment of the third aspect of the invention, the composition is administered to the animal parenterally. Various routes of administration may be employed including intravenous (IV), intramuscular (TM), intraperitorteal (IP), subcutaneous (SC) and intradermal (TD). It is preferred that the administration is by a haematogenous route.
In a fourth aspect, the present invention provides a method of raising an immune response in an animal, the method comprising administering to the animal a composition comprising a carrier and an isolated nucleic acid molecule, the nucleic acid molecule encoding an antigen and a targeting moiety which binds to a receptor, the receptor being characterised in that it is upregulated on lymphocytes that home to MAdCAM
mucosal lymphoid tissues.
In a fifth aspect, the present invention provides an isolated nucleic acid molecule, the nucleic acid molecule encoding an antigen and a targeting moiety which binds to a receptor, the receptor being characterised in that it is upregulated on lymphocytes that home to MAdCAlvr mucosal lymphoid tissues.
In a sixth aspect, the present invention provides an antigenic composition, the composition comprising a carrier and an isolated nucleic add molecule, the nucleic add molecule encoding an antigen and a targeting moiety which binds to a receptor, the receptor being characterised in that it is upregulated on lymphocytes that home to MAdCAlvr mucosal lymphoid tissues.
Preferably, the nucleic add molecule according to the fourth, fifth and sixth aspects is a DNA molecule. More preferably, it is a cDNA.
Trt a preferred embodiment of the fourth, fifth and sixth aspect, the targeting moiety binds a receptor which is a member of the 'integrin family. More preferably, the receptor is the =

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7 mucosal homing receptor lymphocyte Peyer's Patch adhesion molecule (LPAM).
Still more preferably, the receptor is o.4137 integrin (LPAM-1).
Molecules which target LPAM are known in the art, for example the LPAM
specific rat Ig,G2a monoclonal antibody DATK32 which is commercially available. It is presently preferred that the targeting moiety is an antibody, an antibody fragment or an antibody binding domain. Further information regarding antibody fragments such as single chain Fvs can be found in, for example, Hudson PJ & Kortt AA. "I-ligh avidity scFv multimers;
diabodies and triabodies". J Immunol. Meth. 231 (1999) 177-189; Adams GP &
Schier R.
"Generating improved single-chain Fv molecules for tumor targeting". J.
Trnrrturiol. Meth.
231 (1999) 249-260; Raag R & Whitlow M. "Single-chain Fvs" FASEB J. 9 (1995) 73-80;
Owens RJ & Young RJ. -The genetic engineering of monoclonal antibodies". J.
Immunol.
Meth. 168 (1994) 149-165.
Additional ligands that target LPAM4 and adhesins may be generated by using peptide display libraries such as those made in phage display technology (Burton DR.
"Phage display. irnmunotedmology." 1995 1:87-94; Cwirla SE, Peters EA, Barrett RW, Dower WJ.
Peptides on phage: a vast library of peptides for identifying ligands. Proc Natl Acad Sci U S
A. 1990 87:6378-82; Scott J.K, Smith GP. "Searching for peptide ligands with an eryitope library." Science. 1990 249:386-90; Dubree et al, J Med Chem, 45:3451) as well as peptide libraries displayed on other surface components e.g. on flagella molecules (Westerlund-Wikstrom 13. "Peptide display on bacterial flagella: principles and applications." Int J Med Microbiol. 2000 290:223-30) or on yeast (Boder ET, Wittrup KD. "Yeast surface display for screening combinatorial polypeptide libraries." Nat 13iotechnol. 1997 15:553-7).
As will be recognised by those skilled in the field of protein chemistry there are numerous methods by which the antigen may be bound to the targeting moiety. Examples of such methods include:
1) affinity conjugation such as antigen-ligand fusions where the ligand has an affinity for the targeting antibody (examples of such ligands would be streptococcal protein G, staphylococcal protein A, peptostreptococcal protein L) or bispecific antibody to cross-link antigen to targeting moiety.
2) chemical cross-linking. There are a host of well known cross-linking methods including periodate-borohydride, carbodiimide, glutaraldehyde, photoaffinity labelling, oxirane and various suceinimide esters such as maleimiclobenzoyl-succinimide ester. Many of these are readily available =

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8 commercially e.g. from Pierce, Rockford, IL, USA- There are many references to cross-linking techniques including T-Iermartson GT "Bioconjugate Techniques"
Academic Press, San Diego 1996; Lee 'IC, Lee RT. Conjugation of glycopeptities to proteins. Methods Enzyrnol. 1989;179:253-7; Wong SS "Chemistry of Protein Conjugation and Cross-linking" CRC Press 1991; Harlow E & Lane D "Antibodies:
A Laboratory Manual" Cold Spring Harbor Laboratory, 1988; Marriott G, Ottl J.
Synthesis and applications of heterobifunctional photocleavable cross-linking reagents. Methods Enzymol. 1998;291:155-75.
3) genetic fusions. These can be made as recombinant antibody-antigen fusion proteins (in bacteria, yeast, insect or mammalian systems) Or used for DNA
immunization with or without spacers between the antibody and antigen. There are many publications of immunoglobulin fusions to other molecules. Fusions to antigens like influenza hemagglutinin are known in the art see, for example, Deliyartnis G, Boyle JS, Brady JL, Brown LE, Lew AM. "A fusion DNA vaccine that targets antigen-presenting cells increases protection from viral challenge."
Proc Nati Acad SO. U S A. 2000 97:6676-80. Short sequences can also be inserted into the immunoglobulin molecule itself ILun.d.e E, Western KU, Rasmussen 1.13, Sandlie Bogen B. "Efficient delivery of T cell epitopes to APC by use of MHC class II-specific Troybodies." J Immunol. 2002 168:2154-621. Shortened versions of antibody molecules (e.g. Fv fragments) may also be used to make genetic fusions (Reiter Y, Pastan I. "Antibody engineering of recombinant Fv immunotoxins for improved targeting of cancer: disulfide-stabilized Pv immunotoxins." Clin Cancer Res. 1996 2:245-521 As will be understood by persons skilled in the art, whatever the method of targeting moiety-antigen fusion used, such fusions need to be able to target the receptor, such as LPAM-1, in vivo. It is therefore highly preferable that binding of the targeting moiety-antigen fusion to cells other than mucosal homing lymphocytes is rninimised.
Furthermore, antigens which have a high propensity for binding to cells or tissues other than mucosal homing lymphocytes and/or cells or tissues on route to mucosal lymphoid tissues should also be avoided. This can be tested in vitro for example by determining whether the targeting moiety-antigen fusion non-specifically bind to cryostat sections of the mucosal tissue by irnmunohistology.
The antigen used in the present invention can be any antigen against which it is desired to raise an immune response. It is preferred that the antigen is selected such that an immune response is generated against any pathogen whose main portal of entry is the gut and those . .
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9 that colonise mucosal surface. This would include .5//monena, Cholera, Helicobacter pylori, rectally introduced HIV, Candida, F gingivalls, gut parasites or gut associated toxins. Moreover, the present invention may be used to induce an immune response to gut hormones (e.g. gastrin) or their receptors for gut associated cancers [Watson SA, Clarke PA, Morris TM, Caplin ME. "Antiserum raised against an epitope of the cholecystokirtin B/gastrin receptor inhibits hepatic invasion of a human colon tumor." Cancer Res. 2000 60:5902-7; Smith AM, Justin T, Michaeli ID, Watson SA. "Phase I/II study of G17-DT, an anti-gastrin irnmunogen, in advanced colorectal cancer." Clin Cancer Res. 2000 6:4719-241_ Information regarding HIV antigens such as gp120 and other candidates can be found in Stott J, Hu SL, Almond N. "Candidate vaccines protect macaques against primate inumuriodeficiency viruses." AIDS Res Hum Retroviruses. 1998 Oct;14 Suppl 3:S265-70.
Information regarding Helicobacterpylori antigens such as urease of Helicobacter pylori and other candidates can be found in Lee CK. "Vaccination against Helicohacter pyloriin non-human primate models and humans." Sca rid J Imm-unol. 2001 May53(5):437-42.
In addition, the antigen may be derived from intimin which is the ligand by which enteropathogenic Escherichia colicelLs adhere to gut epithelial cells causing haemorrhagic enteritis.
Further information regarding antigens in which mucosal immunity is important may be found in van Ginkel FW, Nguyen HH, McGhee JR. "Vaccines for mucosal immunity to combat emerging infectious diseases." Emerg Infect Dis. 2000 Mar-Apr;6(2):123-32; and Neutra MR, Pringault E, Kraehenbuhl JP. "Antigen sampling across epithelial barriers and =
induction of mucosal immune responses." Arum Rev Imrnunol. 1996;14:275-300.
As will be recognised by those skilled in the art, the fourth to sixth aspects of the present invention relate to DNA vaccination.
The ability of direct injection of non-replicating plasmid DNA coding for viral proteins to elicit protective immune responses in laboratory and preclinical models has created increasing interest in DNA immunisation. A useful review of DNA vaccination is Provided in Donnelly et al, Journal of Immunological Methods 176(1994) 145-152.
DNA vaccination involves the direct in vivo introduction of DNA encoding an antigen into tissues of a subject for expression of the antigen by the cells of the subject's tissue. DNA
vaccines are described in US 5,939,400, US 6,110,898, WO 95/20660 and WO

The ability of directly injected DNA that encodes an antigen to elicit a protective immune response has been demonstrated in numerous experimental systems (see, for example, Conry eta]., Cancer Res 54:1164-1168, 1994; Cardoso eta]., Immuniz Virol 225:293-299, 1996;
Cox eta]., J
5 Virol 67:56645667, 1993; Davis et al,Hum Mol Genet 2:1847-1851, 1993;
Sedegah eta]., Proc Natl Acad Sci USA 91:9866-9870, 1994; Montgomery eta]., DNA Cell Biol 12:777-783, 1993; Ulmer eta]., Science 259:1745-1749, 1993; Wang eta!, Proc Nat! Acad Sc!
USA
90:4156-4160, 1993; Xiang et ELL, Virology 199:132-140, 1994; Yang et Vaccine 15:888-891, 1997; Ulmer eta/Science 259:1745, 1993; Wolff et al Biotechniques 11:474, 1991).

10 To date, most DNA vaccines in mammalian systems have relied upon viral promoters derived from cytomegalovirus (civiv). These have had good efficiency in both muscle and skin inoculation in a number of mammalian species. A factor known to affect the immune response elicited by DNA immunization is the method of DNA delivery, for example, parenteral routes can yield low rates of gene transfer and produce considerable variability of gene expression (Montgomery eta]., DNA Cell Biol 12:777-783, 1993)_ High-velocity inoculation of plasmids, using a gene-gun, enhanced the immune responses of mice (Fynart etal., Proc Nall Acad Sci USA 90:11478-11482, 1993; Eisenbraun etal., DNA Cell Bi01 12:791-797, 1993), presumably because of a greater efficiency of DNA
transfection and more effective antigen presentation by dendritic cells. Vectors containing the nucleic acid-based vaccine of the invention may also be introduced into the desired host by other methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE d.extran, calcium phosphate precipitation, lipofection (lysosome fusion), or a DNA vector transporter.
Additionally, other modes of delivery of DNA vaccines may be contemplated including the use of viral vectors via retrovirus or adenovirus mediated transduction of target cells (such as reviewed in Alexander Pfeifer and in.der M Verma. Annual Review of Genornics and Human Genetics. V012:177-211 "Gene Therapy: Promises and Problems" (2001).
Alternatively, bacteriophage mediated DNA transfer may be employed. Suitable bacteriophage vectors include M13 bacteriophage, fl bacteriophage, lambda bacteriophage, .P1 bacteriophage, SP6 bacteriophage, T3 bacteriophage, 17 bacteriophage and bacteriophage.
Suitable carriers for use in the present invention will be familiar to those skilled in the art, such as for example phosphate buffered saline.

11 As used herein the term "animal" encompasses both human and non-human animals.
As used herein the term "mucosal lymphoid tissue" encompasses tissue that is associated with mucosal surfaces and is referred to as mucosa associated lymphoid tissue (MALT) of the gut, respiratory tract and genital tract. It has commonly been called GALT
or gut-associated lymphoid tissue when associated with the alimentary tract.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.
In order that the nature of the present invention may be more clearly understood preferred forms thereof will be described with reference to the following Examples.
MATERIALS AND METHODS
/mm wit:5-fifions The rat IgG2a mAb immunogens used were an anti-LPAM mAb DATK32, a rat IgG2a isotype control (GL117 that recognizes bacteria113-galactosidase), an anti-intraepithelial lymphocyte (IEL) mAb M290, an anti-a., integrin mAb MER4.B and an anti-I17 integrin mAb FIB27. These mAbs were purchased from Pharmingen (San Diego, CA, USA) or isolated from hybrid oma supernatants and purified on immobilized protein G (Amersham Pharmacia Biotech, Little Chalfont, UK). CBA mice (6-8 wk old female; 5 per group unless otherwise stated) were used for all experiments. Oral immunizations were performed by gavage in 3% wt/vol sodium bicarbonate buffer after light anaesthesia with methoxyflurane (Medical developments, Spring-vale, Australia). For co-immunization and OT-II proliferation experiments ovalburnin (OVA) grade V (Sigma. St Louis, MO, USA) was dissolved in PBS and mixed with targeting or control mAbs. All irrununogens

12 contained less than 0.06 ng of endotoxirt per mg of Ag, as determined by the Limulus Amebocyte Lysate assay (Kinetic-QCL, BioWhittaker, Walkersville, MD) Pµreparation of DIVA vacethe Large-scale isolation of DNA for immunization studies was performed using an alkaline lysis/triton X-114 method. Transformed DH5o:. E. calf were grown to saturation overnight in 100 ml of superbroth containing 100 ug/m1 of ampicillin at 37 C. 50 ml of starting cultures was then transferred to 600 ml of superbroth containing 100 ktg/m1 of arnpicillin and grown at 37 C for 24 hours. After centrifugation at 3000 g the pelleted bacteria were fully resuspended in a total of 40 ml of solution 1 (25mM pH=8, 10 mM EDTA
p118.0, 15% sucrose). 100 ml of fresh solution 11 (10 ml 2M NaOli, 85 H20, 5 ml 20% SDS) was added before incubation on ice for 10 min. 75 ml of cold solution III (5 M
potassium acetate pH5) was then mixed by inversion and left on ice for 30 min. After centrifugation at 10000 g, 30 min, 4 C, supernatant was filtered through 2 layers of ieunwipg (Kimberley-Clarke, NSW, Aust) and 0.6 volume of isopropanol added to precipitate DNA. DNA
was then resuspended in 5 ml of TE (10mMtris, 1mM EDTA pFI=8) with 20 mg/ml of DNAse-free RNAse (Prornega, Madison, WI, USA), and placed at 37 C for 30 min. Equal volume of polyethylene glycol (13% PEG 8000 in 1.6M NaC1) was added and left at 4 C
overnight before centrifugation at 4000 g for 20 min. DNA pellet was resuspended in 9.5 ml TE and 0.5 ml of 3 M sodium acetate p118.9 and phenol/chloroform extractions performed 3 times followed by a chloroform only extract-ion. DNA was precipitated by the addition of equal volumes of isopropanol and centrifugation at 4000 g. Pellet was then resuspended in 10 ml TM
of MTPBS and 100111 of tritonX-114 added to remove endotoxin. Solution was thoroughly mixed and placed on ice for 4 min until clear. Solution was then placed at 40 C for 5 min TM TM
then centrifuged at 3000 g for 5 min to pellet tritonX-114-endotoxin.
TritonX114 endotoxirt extractions were repeated 3 times in total. DNA was then precipitated with equal volume of isopropanol and stored at 4 C for after resuspension in normal saline Preparation of-fere-a/ samples Ja or analysis ofgrit .4721 responses Mucosa' Ab isolated from fecal samples was used as a measure of gastrointestinal immune responses [42J. Briefly, 1 ml of 0.1 mg/m1 soybean tr-ypsin inhibitor (Sigma Chemical Co, St Louis, MO, USA) in PBS was added per 0.1 g of faeces then vortexed in a mini-beadheater (Biospec Products, Bartlesville, OK, USA) for 10 s at 2500 rpm, debris removed by centrifugation 9000 g, 4 C, for 15 min, and supernatant assayed for Ab_ =
== CA 02489940 2004-12-08 =

13 ELISA
Rat IgG2a, and OVA specific Ab responses from serum, fecal and culture supernatant samples were determined by ELISA. Briefly, microtiter plates (Dynatech, Chantilly, VA, USA) coated with Ag (2 ig/m1 in PBS) were incubated with serially diluted sera, fecal extract, or culture supernatant (diluted in blocking buffer of 5% skim-milk powder in PBS) overnight at 4 C. Bound Ab was detected after 3 hr incubation at room temp with peroxidase-conjugated antibodies to mouse IgG (donkey anti-mouse, adsorbed against rat Ig; Chemicon, Temecula, CA), IgA, Ig.M, IgE (goat anti-mouse), IgG1, IgG2a, IgG2b, or Ig,G3 (rat anti-mouse) (Southern Biotechnology, Birmingham, AL) diluted in blocking buffer. The substrate used was tetramethyl-benzidine (TMB, Sigma Chemical Co, St Louis, MO, USA) in 0.1 M sodium acetate pH 6 and reactions stopped with 0.5 M
sulphuric acid.
IgG and IgA titres were defined as the reciprocal of the highest dilution to reach an Door=
of 0.1.
EL/SPOT
To determine the number of cells secreting Ab, ELISPOT assays were performed.
Briefly, 96 well sterile multiscreen filtration plates (Millipore S.A. Yvelines, Cedex, France) coated with rat IgG2a (GL117, 20 u.g/m1 in PBS) were incubated for 36 hr at 37 C 10%
CO2 with dilutions of single cell lymphocyte preparations isolated from mesenteric lymph nodes (MLN), Foyers Patch (PP), ing,uinal lymph nodes (ILN) or spleen. Bound Ab was detected after incubation with peroxidase-conjugated antibodies to mouse IgA (Southern Biotechnology, Birmingham, AL, USA) diluted in blocking buffer- Numbers of spots representing individual Ag specific ASC were counted under a stereo microscope after development with AEC substrate (Dako Co, Carpinteria, CA, USA).
Lymphocyte cell callarte Lymphocyte cell culture was used to reinforce ELISPOT data. Lymphocytes were prepared as described (see ELISPOT) and cultured for 5 days in 96 well plates in 0.2 ml of RPMI 10%
FCS, 37 C, 10% CO2. Supernatants were then removed and assayed for antigen specific IgG
and IgA responses by ELISA.
by-rtila 07-.1 pro/O.-rah:on assay Carboxyfluorescein diacetate, succinimidyl ester (CFSE) labelling of OVA
specific TCR
transgenic CD4+ T-cells (0T-11) cells was performed as previously described 1431 Briefly, OT-II T-cells were resuspended in PBS containing 0.1% BSA (Sigma, St. Louis, MO) at 107

14 cells/ml. For fluorescence labelling, 2 I of a CFSE (Molecular Probes, Eugene, OR) stock solution (5 mM in DMSO) was incubated with 107 cells for 10 min at 37C.
C57/B16 mice were primed with either OVA alone, or OVA mixed with various doses of anti-LPAM mAb DATK32. Three days after priming mice were sacrificed and single cell suspensions made from ILN and MLN. CD4+ T-cells were then stained in FACS buffer (PBS, 5% FCS, 2.5mM
EDTA) with anti-CD4-PE (Caltag, Burlingame, CA, USA) and proliferation of CFSE+ PE+
T-cells was measured by sequential loss of CFSE by flow cytometric analysis using a FACScan (Becton Dickinson, Mountain View, CA).
bi-vivo lymphocyte homing- assay CFSE labelling of MLN cells was performed as described above. Labelled cells were then mixed with various doses of anti-LPAM (DATK32) or isotype control (GL117) mAbs and immediately injected intravenously. 3 hr after, mice were sacrificed and single cell suspensions made from ILN and MLN and horning of CFSE+ cells from the blood analysed by flow cytometric analysis using a FACScan (Becton Dickinson, Mountain View, CA).
White blood eel/ aaalysi5 TM
Total white blood cells counts were performed using a Coulter-counter (Beckman Coulter, Miami, FL, USA). For analysis of lymphocyte subsets, red blood cells were first lysed from whole blood then remaining cells stained in FACS buffer with fluorescent labelled Ab against mouse CD4, CD8 and B220. Cells were then washed twice and analysed by flow cytometric analysis using a FACScan (Becton Dickinson, Mountain View, CA).
RESULTS
Jawlike-the mucosa/ lymphocyte ham ins- tear/or euhaaces gut mucosa/ /KA
re<spou-virs to a model alltiken To investigate the influence of LPAM (4:17 integrin) targeting on rnucosal Ab responses the rat IgG2a anti-mouse LPAM iriAb DATK32 was employed as a model targeted antigen.
Tsotypic differences between rat and mouse IgG renders rat IgC2a immunogenic in mice.
Thus, anti-rat IgG2a responses can be measured to this targeted antigen and compared to other non-targeted rat lgG2a inAbs (in this case the anti-bacterial fi-galactosidase mAb GL117 that has no known reactivity in the mouse). To analyze mucosa' responses in the gut, the fecal extract technique was used that allows for non-invasive monitoring of responses with minimum contamination from serum im.murioglobulins [441.
Remarkably it was found that gut mucosal responses could be significantly enhanced by targeting LPAM

µs.
CA 02489940 2004-12-08 .
in this model (Fig I a). Gut mucosal response could be induced after parenteral immunization with as little as 10 ng of LPAM-targeted antigen (Fig la).
Moreover, these could be induced in the absence of any additional adjuvants normally required to stimulate mucosal responses (Fig la). Mucosal IgA responses peaked 2 weeks after immunization 5 were still present 6 weeks after a single immunization (Fig la). In contrast to this, immunization with the non-targeted isotype control failed to induce a detectable response (Fig la).
LFAM-larse enhances ssle in Ic Ab re..4ponses With significant enhancements in gastrointestinal IgA responses after LPAM-targeting, the 10 inventors then investigated the influence of targeting on the systemic Ab response.
Following the finding in the gut mucosal compartment, LPAM-targeting lead to significant improvements in the systemic Ab response (Fig lb). Parenteral targeting of LPAM with as little as 10 ug of mAb, stimulated a significant anti-rat IgG2a IgG response in the serum (Fig lb). In fact, the systemic IgG response was approximately 10,000 fold higher than that

15 of the non-targeted control (Fig lb). Like the mucosal response, serum IgC responses appeared rapidly, being detected as early as 2 weeks after a single inununization.
I Iowever, unlike mucosal responses, serum igG response remained around their elevated peak long after 2 weeks (Fig lb).
With the different inununoglobulin isotypes possessing different protective effects, it was important to consider the influence of LPAM-targeting on the Ab subclass response.
Despite a remarkable enhancement in total systemic IgG responses, no difference was found in early IgM responses (Fig lc). The IgM response was not influenced by antigen targeting or antigen dose (Fig 1c). Similarly, the IgE responses were not influenced by antigen targeting or dose, with no detectable responses induced (Fig 1c).
However, serum IgA anti-rat IgG2a responses were influenced by antigen targeting and dose (Fig lc).
Immunization with 10 lig of LPAM-targeted antigen led to enhancements in both the IgG1 and Ig-G2a subclass response (Fig lc). Serum IgG3 responses could not be detected in any animals (Fig 1c).
LPAM-laryelliw localizes atri9keff to mucosa/ am/ pen,itheral lymph o tr:cstie hi -vivo To investigate the in-vivo localization of intravenously delivered anti-LPAM
mAb, both FACS and immunohistological based techniques were employed. The anti-LPAM mAb DATK32 does not recognize its ligand in immunohistology (Pharmingen, unpublished results). These findings were confirmed due to a failure to detect binding to LPAM on = CA 02489940 2004-12-08 . , =
=

16 lymph node sections stained via intravenous injection or ex-vivo incubation (data not shown). As a result it was decided to move to the FACS based assay that measured mAb binding to surface LPAM.. Consistent with the reported expression of Ll'AM
1451, it was found that intravenous injection of this mAb stained the majority of circulating blood lymphocytes (Fig 2a). More importantly paren.teral LPAM-targeting lead to preferential localization of antigen within both the peripheral (ILN) and mucosal (MLN) compartments (Fig 2a).
Enhanced Ah responses irlditced by ZFAM-targetzirf is the result a/ improved ./ey,4 and .18-C praa'action from /Jag mucosal and per4theral lymphoid capipartmea&
Given the substantial increase in Ab responses induced by LPAM-targeting the inventors wanted to investigate the origin of these enhancements. To achieve this ELISPOT and lymphocyte culture assays were performed to evaluate IgG and IgA responses within the mucosal and peripheral lymphoid compartments. Consistent with the analysis of serum and fecal Ab responses, LPAM-targeting enhanced the IgG and IgA response in both mucosal and systemic tissues (Fig 2 b&c). Elevated IgA responses could be detected from cells in the MLN and PP (Fig 2 b&c), characteristic of local gut mucosal responses.
Although TgA responses could not be detected in the TLN, the centrepiece of peripheral lymphoid machinery, the spleen, contributed remarkably to the IgA response (Fig 2 b&c).
Taken together these findings are consistent with the enhanced mucosal and systemic IgA
responses induced by LPAM-targeting. Similarly, IgG response were enhanced in both the mucosal and systemic compartments (Fig 2 b&c). Targeting significantly elevated IgG
responses in both the spleen and ILN (Fig 2 b&c). Although we failed to detect improvements in IgG responses from PP cells, the MLN contributed significantly to these responses (Fig 2 b&c). Overall, ELISPOT and lymphocyte culture assays were consistent with our previous findings revealing significant improvements in IgG and IgA
responses in peripheral and mucosal responses.
liamaffizing doses of anti-LPAM gtAbs do not a'eplek larget pointlations, hour /17K aril/It-rapt/I:fen .s.,aczyjefi:wroliftratiati of CD44- T-ceIls Anti-LPAM inAbs such as DATK32 can have a number of effects on LPAM-populations.
For example, Abs that bind the a437 integrin complex at high doses can block lymphocyte horning. It was also possible that this mAb depletes particular target cells in-vivo, influencing the Ab response. To segregate such bystander effects from that of antigen targeting, the influence of immunizing doses of anti-LPAM mAb DATK32 on normal blood cell populations, homing and T-cell activation within both the mucosal and systemic -.,,= .
CA 02489940 2004-12-08'

17 compartments was investigated. It was found that mAb DATIC32 did not influence the normal numbers or proportions of white blood cells (Fig 3a). Lymphocyte, monocyte, neutrophil, eosinophil and basophil numbers were all equivalent to animals injected with controls, suggesting that this mAb does not deplete these cells (Fig 3a). This was extended further to show that animals immunized with anti-LPAM mAb had normal proportions of CD4+ and CD8 T-cells as well as B220+ B-cells in the blood (Fig 3a inset of bottom panel). It was also found that immunizing doses of anti-LPAM mAb did not affect the homing of cells from the blood into the MLN or ILN (Fig 3b). No significant differences in the proportion of CFSE labelled MLN cells homing back to the MLN or to the peripheral ILN
was observed during administration of immunizing doses of anti-LPAM mAb (Fig 3b).
The LPAM complex has been Identified as a possible costimulatory interaction involved in T-cell activation. Natural ligartds as well as mAbs can bind to LPAM promoting intracellular signalling, aggregation and activation of T-cells [35, 46]. To explore the possible costimulatory effects of this mAb the inventors employed an in-vivo model widely used for studying CD4+ T-cell activation, the proliferation of CFSE labelled OVA specific class-H restricted T-cell transgenic OT-ll cells. This model enabled the study of T-cell proliferation in-vivo within both the mucosal and systemic compartments. It was found that immunizing doses of anti-LPAM mAb had no influence on the proliferative response of OT-II cells to OVA (Fig 3c). The percentage of divided and undivided cells was not significantly different if mice were immunized with OVA alone or in combination with 10 I.A.g of the anti-LPAM mAb (Fig 3c).
Tatske.firtg LPAM required' for enhanced mucosa/ 1;f:e1 responses As well as T-cells, LPAM ligands such as mAbs could directly influence B-cell responses.
To further dissect possible bystander effects from that of antigen targeting, co-immunization experiments were performed. Animals received the anti-LPAM mAb DATK32 or the non-targeted isotype control GL117 mixed with 501.t.g of OVA.
Two weeks after immunization, fecal and serum samples were collected and IgG and IgA
responses were measured against rat IgG2a as well as OVA. It was found that targeting with anti-LPAM mAb DATIC32, had no effect on the anti OVA response (Fig 4a). Anti-OVA
serum IgG was equivalent in both the LPAM-targeted and non-targeted groups (Fig 4a).
Furthermore, co-immunization of OVA with anti-LPAM mAb did not result in the induction of anti-OVA fecal IgA responses (Fig 4a). Co-immunization of OVA
with the mAb had no effect on the anti-rat IgG responses in the mucosal or systemic compartments (Fig 4a). Taken together these data argue against the involvement of bystander effects of , =

. _

18 anti-LPAM mAbs in the induction of improved systemic and mucosal Ab responses.

Moreover, it shows that targeting of antigen to LPAM is required for the observed effects.
The inventors wanted to further analyse the requirement for antigen targeting in the induction of mucosal and systemic responses within their model. To achieve this, mice were immunized with other rat IgG2a mAb antigens including those recognizing mucosal intraepithelial lymphocytes (TEL) as well as the individual components of the LPAM
complex (cf.4 and 137 integrins). As ot.4 and 137 pair with other molecules (e.g. 13 and ccE
respectively), it would be expected that mAb to these components would not be as specific as the anti-LPAM mAb (that only recognizes the er.4137 complex. It was found that only targeting to LPAM with DAT1C32 induced a fecal IgA response (Fig 4b).
Targeting IEL as well as the sub-components of LPAM failed to induce a fecal IgA response (Fig 4b). The same effect was observed in animals immunized with up to 10 fold higher doses of anti-IEL, anti-4 and anti-7 (data not shown). However, targeting with anti-TEL, anti-a4 or anti-137 mAbs all enhanced the systemic response (Fig 4b), pointing to possible avenues for future application of these targets.
ZP/1/14-frzwettitg enhances respowses gfterparenteral hut no/ iwzecosal of lawified amtrikert Parenteral vaccines are usually ,Oven via subcutaneous or intramuscular routes. To investigate whether such routes could mirror the responses induced intravenously, mice were immunized intramuscularly and subcutaneously with either anti-LPAM mAb (DATK32) or non-targeted control (GL117). Importantly, these routes were found to be equally efficient in improving responses by LPAM-targeting (Fig. 4c). LPAM-targeting led to mucosal responses through the intramuscular and subcutaneous routes at similar levels to those through the intravenous route (Fig. 4c). Similarly, serum IgG
responses remained greatly elevated and were not significantly different to intravenous immunizations (Fig 4c).
As expected, oral immunization in the absence of mucosal adjuvants failed to induce a mucosal response to either the targeted or non-targeted antigen (Fig. 4c).
Targeting did however improve the systemic IgG response to orally administered antigen, however this enhancement was only modest and remained 1(X) fold less than responses induced by parenteral antigen targeting.
LPAM-2 targeted DNA immunisalion A DNA immunisation strategy was employed to investigate the ability of LPAM-1 targeted antigen to enhance mucosal and systemic immune responses. Mice were immunised :
=' CA 02489940 2004-12-08 .=== =

19 intramuscularly with either a non-targeted or LPAM-1 targeted DNA vaccine.

targeting was achieved through fusion of human Ig antigen to the extraceLlular region of MAdCAM-1. It was found that LPAM-1 targeted DNA vaccination enhanced the subsequent Ab response (Figure 5). Immunisation with either targeted or non-targeted DNA vaccine was unable to induce a significant faecal or serum IgA response (Figure 5); it is not known whether the lack of IgA response was because not enough protein was expressed in the DNA vaccine to achieve targeting or because the affinity of MAtiCAM-hig was insufficient compared with anti-LPAM antibody. In contrast, both targeted and non-targeted DNA vaccines were able to induce significant serum IgG responses (Figure 5).
Moreover, the systemic Ab induced by LPAM-1 targeting was 10-100 fold higher than the non-targeted control immunisation (Figure 5).
LPAM is used by naive, activated and memory lymphocyte populations to home to MAdCAM+ mucosal lymphoid tissues [32,34, 45, 471. Blocking this interaction with either anti-MAdCAM or anti-LPAM mAb inhibits the homing of mucosal lymphocytes [33, 48].
Given the high levels of expression of LPAM along with its unique role in directing lymphocytes to mucosal tissues, it was proposed that it may be an ideal target for systemic delivery of rnucosally targeted vaccines. Targeting antigen to this specialized lymphocyte population may direct antigen to mucosal inductive tissue in a 2 step or "relay" fashion.
Therefore a protein immunization model using a tat IgG2a anti-mouse LPAM inAb DATK32 was chosen, whereby the mAb serves as both immunog,en and targeting moiety.
Remarkably, it was found that targeting LPAM with DATK32 induced a potent mucosal and systemic response. Systemic responses were greatly enhanced, and importantly, targeted protein immunization enabled sufficient localization of antigen to the mucosal associated lymphoid tissue for induction of mucosal Ab responses.
Closer investigation of the Ab response to LPAM-targeting immunizations revealed several interesting findings. Although targeting did not improve the 1gM or lgE
response, IgA, IgG1 and IgG2a responses were significantly enhanced. Elevated IgA, IgG1 and IgG2a responses are consistent with enhancements in both Thl and Th2 type responses.
ELISPOT
and lymphocyte culture assays showed that elevated serum IgG responses were the result of improved production from both mucosal and systemic tissues. These responses are consistent with the localisation of targeted antigen to these sites.
Interestingly, the same pattern emerged when the origin of IgA responses were investigated. Elevated serum and fecal TgA responses were associated with IgA responses in peripheral and mucosal tissues.
In fact the IgA responses in the spleen were remarkable and equally as potent as those induced in mucosal tissues that normally dominate the IgA response. It remains to be = .
=

elucidated whether splenic IgA responses were dependent on the traffic of DCs from mucosal tissues or whether IgA responses were primed at this site by a presentation mechanism skewed towards IgA production. Given the potency of this response, it is suggested that the later may be more likely. Overall, it is clear that LPAM-targeting results 5 in enhanced Ab responses by stimulating production across a number of lymphoid sites.
The inventors found no differences in the total numbers or proportions of white blood cells in mice treated with anti-LPAM. In fact, lymphocyte homing, T-cell activation and the induction of Ab response against a co-injected antigen in either the mucosal and systemic compartments were not different in animals that received anti-LPAM rnAb. From these 10 results it was concluded that bystander effects are unlikely to contribute. Importantly, these studies also revealed that IgA and IgG responses induced by anti-LPAM
mAb are not the result of aberrant homing, T-cell proliferative or B-cell responses and are mostly likely resultant of antigen localization to LPAM. This was reinforced by the finding that targeting intraepithelial lymphocytes or the separate components of LPAM (04 and 137 integrin 15 individually; these molecules can pair with other molecules found in other cells) failed to induce a mucosal response. Although the enhanced systemic IgG responses that were induced warrant further investigation, this data further illustrates that LPAM-targeting is required for mucosal IgA responses_ Route of administration is an important consideration in the development of novel vaccine

20 strategies. This is particularly salient for vaccines aimed at inducing mucosal responses as such responses are rarely induced to parenterally delivery vaccines. Direct mucosal administration provides a better chance of reaching the specialized lymphoid tissues that governs protection of these sites; however, adjuvants are almost always required to overcome the biases that maintain tolerance or non-responsiveness to the heavy burden of harmless antigens. Mucosal IgA responses could however, be induced by delivery of LPAM-targeted antigen by traditional parenteral routes (subcutaneous and intramuscular).
This reinforced the findings using the intravenous route and highlights a layer of flexibility that may be important in the future clinical application of this technology.
Although the highest expression of LPAM can be found in mucosal lymphocytes, a high proportion of peripheral lymphocytes also express low to intermediate levels [33, 49]. For example, 30% of the total lymphocyte population in the spleen express LPAM
[331. This was illustrated in the localisation studies by a large proportion of LPAM' cells staining for antigen in the blood, ILN as well as the MLN. It is likely that the localisation of antigen to both the systemic and mucosal compartments underlies the improved Ab responses.
However, it is not clear which population of lymphocytes is responsible for this effect.
=

21 Interestingly, this is not the first report of enhanced Ab responses induced by targeting antigen to specialized populations of lymphocytes. An early study of the immunotargeting approach by Skca, ctn.], (1993), reported that Ab responses could be enhanced by targeting T cells [501. They found that targeting antigen to CDT and CD4+ lymphocytes via specific mAb, enhanced the subsequent Ab response [50]. They failed to elucidate any of the underlying mechanisms of this enhancement but proposed that B cell binding to antigen coated T cells may focus T cell help and promote antigen specific B cell responses. A
similar mechanism may also function in the LPAM.-targeting model. This group also reported enhance Ab responses by targeting CD45RA that is predominantly expressed on B
cells [501. Other groups have also reported similar effects [51, 521 which have been attributed to the antigen presenting function of targeted B cells [53]. As B
cells express LPAM through various stages of maturation [45, 47], B cell targeting may also play a role in enhanced responses to LPAM-targeted antigen_ To add further complexity, LPAM
expression is not limited to lymphocyte populations. For instance, although blood monocytes are LPAM", they dramatically upreg,ulate LPAM after activation with IFN-y [54].
The importance of targeting various cell populations for enhanced responses remains to be elucidated and highlights an important avenue for future experiments.
In conclusion, the present study provides a model and proof of principle that targeting lymphocytes in an antigen relay approach to vaccination can improve antigen specific responses. Most importantly this approach facilitated the induction of mucosa/
IgA
responses not normally induced to parenteral vaccines. Furthermore, induction of mucosal IgA was associated with a marked elevation of systemic responses. Further characterization of the LPAM-targeting model, for example dissecting the mechanisms of antigen transfer, may lead to further improvements in the induction of mucosa' and systemic immune responses.

22 DNA inummisation with the LPAM-1 targeted antigen resulted in greatly enhanced systemic Ab responses. However, this strategy was unable to significantly enhance the faecal or serum IgA response. Assuming that localisation of LPAM-1 targeted antigen follows its predominant expression on mucosal lymphocytes, the dichotomy noted 30 between the mucosal and systemic Ab responses to DNA immunisation may reflect a lower antigenic threshold required for induction of systemic immune responses.

= CA 02489940 2004-12-08 '2. - =

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Claims (10)

1. Use of a composition comprising a carrier and an antigen bound to a targeting moiety, wherein the targeting moiety is antibody DATK32, a binding fragment thereof or a binding domain thereof; and wherein the targeting moiety binds lymphocyte Peyer's Patch adhesion molecule (LPAM) on lymphocytes that home to MAdCAM+ mucosal lymphoid tissues in the preparation of a medicament for raising an IgG and IgA immune response in an animal following parenteral administration, and wherein the antigen is from Salmonella, Cholera, Helicobacter pylori, HIV, Candida, P. gingivalis, enteropathogenic Escherichia coli, gut parasites, gut associated toxins, gut hormones, gut hormone receptors or gut associated cancers.

2. The use according to claim 1 wherein the medicament is to be administered by haematogenous route.

3. The use according to claim 1 or claim 2 wherein the antigen is bound to DATK32 by affinity conjugation, chemical cross-linking or genetic fusions.

4. A targeted antigen comprising an antigen bound to a targeting moiety wherein the targeting moiety is antibody DATK32, a binding fragment thereof or a binding domain thereof; and wherein the targeting moiety binds lymphocyte Peyer's Patch adhesion molecule (LPAM) on lymphocytes that home to MAdCAM+ mucosal lymphoid tissues, and wherein the antigen is from Salmonella, Cholera, Helicobacter pylori, HIV, Candida, P. gingivalis, enteropathogenic Escherichia coli, gut parasites, gut associated toxins, gut hormones, gut hormone receptors or gut associated cancers.

5. The targeted antigen according to claim 4 wherein the antigen is bound to DATK32 by affinity conjugation, chemical cross-linking or genetic fusions.

6. An antigenic composition comprising a carrier and the targeted antigen according to claim 4 or claim 5.

7. An isolated nucleic acid molecule encoding the targeted antigen according to claim 4 or 5.

8. The isolated nucleic acid molecule according to claim 7 which is a DNA
molecule.

9. An antigenic composition comprising a carrier and the isolated nucleic acid molecule according to claim 7 or 8.

10. Use of the composition according to claim 9 in the preparation of a medicament for raising an IgG and IgA immune response in an animal following parenteral administration.