Link:  Pharm/Biotech Resources

Title:  Plant lectins as mucosal adjuvants

United States Patent:  6,863,896

Issued:  March 8, 2005

Inventors:  O'Hagan; Derek (Berkeley, CA); Lavelle; Edward C. (Dublin, IE)

Assignee:  Chiron Corporation (Emeryville, CA)

Appl. No.:  696194

Filed:  October 26, 2000

The invention provides a method of increasing an immune response in a mammal. The method involves administering to the mammal an admixture comprising an immunogen and a plant lectin. The plant lectin acts as an adjuvant to increase an immune response against the immunogen. The method is especially well-suited for mucosal administration to humans and other mammals.

Description of the Invention

TECHNICAL FIELD OF THE INVENTION

The invention relates to the enhancement of an immune response in a mammal. More particularly, the invention relates to the use of plant lectins as adjuvants.

BACKGROUND OF THE INVENTION

Because most pathogens colonize and invade the host at mucosal surfaces, the induction of immunity at these sites is a rational and attractive approach to prevent infection (1). Mucosal routes for vaccine delivery are non-invasive, so administration is relatively simple and inexpensive. Furthermore, the potential to induce a range of mucosal and systemic immune responses after mucosal vaccine delivery allows the possibility of effective immunization against many diseases. For example, specific IgA alone can protect mice against intranasal infection with influenza (2) and intestinal infection with Vibrio cholerae (3). However, mucosal delivery of nonreplicating immunogens typically does not stimulate strong immune responses. Where responses are induced, the delivery of multiple high doses is often necessary (4). In addition, mucosal delivery of immunogens frequently results in systemic unresponsiveness (1).

A number of strategies may be used to enhance responses to mucosally delivered vaccines. Live bacterial and viral vectors which colonize the mucosae can be used to deliver immunogens (5). Imparting particulate characteristics to immunogens by association with biodegradable microparticles (6) or liposomes (7) can also enhance mucosal immunogenicity.

Another approach is the use of lectin-like molecules with adjuvant properties. The most powerful mucosal adjuvants identified to date are cholera toxin produced by Vibrio cholerae (CT) and heat-labile enterotoxin (LT) from enterotoxigenic strains of Escherichia coli (8, 9). CT and LT are well-characterized mucosal immunogens and adjuvants for bystander proteins. These toxins contain separate A and B subunits (referred to as CTA and CTB, respectively). The B subunits mediate binding to cell surface receptors (20). GM1 ganglioside is considered to the principal receptor for CT (21), but CTB may bind to cell surface receptors other than GM1 (22). After binding of the B subunit, the A subunit reaches the cytosol and activates adenyl cyclase leading to a large increase in [cAMP]_i (10, 11). LT is structurally and functionally similar to CT and is comparable to CT as a systemic or mucosal adjuvant (23, 24). In mice, CT strongly stimulates humoral and cell-mediated immune responses, including mucosal IgA production and cytotoxic T cell effector functions (10). Stimulation of toxin-specific local and systemic responses and responses to co-administered immunogens distinguish these molecules from most soluble proteins which are poorly immunogenic when administered mucosally (10, 11). The toxicity of these molecules, however, prevents clinical application.

Certain plant lectins have been investigated as agents for specific targeting of molecules to a mucosal epithelium. Plant lectins are proteins containing at least one non-catalytic domain, which binds specifically and reversibly to a monosaccharide or oligosaccharide (13). For example, Giannasca et al. (14) discloses that intranasal immunization with a lectin-immunogen conjugate stimulated induction of specific IgG antibodies, while immunogen alone or admixed with lectin did not. U.S. Pat. No. 4,470,967 discloses that a complex of a glycoprotein immunogen with a lectin can act as an adjuvant to increase the immune response against the immunogen. Similarly, WO 86/06635 discloses a chemically modified immunogen-lectin complex which can be used to elicit an immune response in vertebrates, including mammals. In each of these cases, however, the lectin was physically coupled to the immunogen. This requires at least one extra preparation step and may actually alter an epitope of the immunogen against which an immune response is desired, such as an epitope against which a neutralizing immune could be directed.

Thus, there is a need in the art for simple, effective, and non-toxic methods of increasing immune responses in a mammal, particularly after mucosal administration, without the need to complex the immunogen with another molecule and potentially mask or alter desirable epitopes.

SUMMARY OF THE INVENTION

The invention provides a method of increasing an immune response in a mammal by administering to the mammal an admixture comprising an immunogen and a plant lectin. The mammal thereby produces an immune response which is increased relative to an immune response produced in the absence of the plant lectin.

The invention thus provides a simple and effective method of increasing an immune response in a mammal.

DETAILED DESCRIPTION OF THE INVENTION

It is an aspect of the present invention that certain plant lectins act as mucosal adjuvants to increase immune responses, including an increased antibody titer, against a variety of immunogens, thus permitting simple, non-toxic, and cost-effective vaccine or immunogenic compositions to be prepared. Vaccine or immunogenic compositions of the invention are admixtures comprising a plant lectin and an immunogen. Such admixtures are especially suitable for mucosal delivery to mammals, including humans, and are thus useful for veterinary as well as human medical purposes.

Admixtures of the invention comprise a plant lectin and an immunogen. The immunogen and the lectin are not coupled together chemically, but are simply mixed together in an appropriate liquid medium, such as phosphate buffered saline or other isotonic saline solution. Optionally, an admixture can comprise stabilizing agents, including antimicrobial agents, preservatives, and the like. The proportions of immunogen and lectin in the admixture can be varied, such as at least about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1, depending on the particular immunogen and lectin combination selected. If desired, at least 2, 3, 4, or more different immunogens and/or lectins in varying proportions can be included in an admixture.

Lectins useful in the invention include plant lectins such as mistletoe lectin I (ML-I), mistletoe lectin II (ML-II), mistletoe lectin III (ML-III), wheat germ agglutinin (WGA), and Ulex europaeus (UEA-1). Other lectins which may be useful include lentil bean lectin, jack bean lectin (concanavalin A), and asparagus pea, broad bean, camel's foot tree, castor bean, fava bean, hairy vetch, horse gram, Japanese wisteria, Jequirity, Scotch laburnum, lima beam, lotus, mung bean, Osage orange, Pagoda tree, garden pea, potato, red kidney bean, Siberian pea tree, spindle tree, sweet pea, tomato, and winged pea lectins.

Type 2 ribosome inactivating proteins (RIP), such as nigrin b, basic nigrin b, ebulin l, ebulin r, ebulin f, nigrin f, SNA1, SNA1, SNAV, SNAVI, Sambucus nigra SNLRP1, SNLRP2, ricin, Ricinus lectin, Polygonatum RIP, Sieboldin-6, abrin, abrin 11, modeccin, volkensin, SSA, Cinnamonin, porrectin, gelorin, Evanthis hyemalis, RIP, Iris agglutinin, ML-I, ML-II, and ML-III, are especially useful as adjuvants. Such lectins contain an N-glycosidase A subunit responsible for the ribosome-inactivating activity and a galactose-specific carbohydrate-binding B subunit (29). ML-I, ML-II, and ML-III are strong mucosal adjuvants, which can stimulate high antibody titers in sera and mucosal secretions. Type 2 RIPs which do not show in vivo toxicity, such as ebulin-1 (32), nigrin b (33) and basic nigrin b (34), are particularly useful. Alternatively, lectins can be genetically "detoxified," for example by modifying one or more amino acids by site-directed mutagenesis such that the lectins retain their adjuvant properties but are non-toxic to the mammalian recipient (see 35-39; EP 0880361; EP 620850; EP 95/903889.4).

Lectins in an admixture are preferably in an unbound, water-soluble form. Suitable lectins for use in admixtures of the invention can be purchased from commercial suppliers, such as Sigma. Alternatively, lectins can be purified using protein purification protocols well known in the art, including size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, crystallization, electrofocusing, and preparative gel electrophoresis.

Immunogens against which a cellular and/or humoral response can be increased using a plant lectin adjuvant include proteins of infectious agents, such as viruses, bacteria, mycoplasmas, prions, and yeast, as well as hormones, allergens such as grass, weed, tree, and plant pollens, epithelia of animals such as cats, dogs, rats, and pigs, house dust, and wheat chaff. Means of obtaining such immunogens are well known in the art. An immunogen need not be able to raise a cellular and/or humoral response in the absence of the plant lectin.

Admixtures of the invention can be administered to a recipient mammal in a variety of formulations. For example, admixtures can be entrapped in or adsorbed to the surface of microparticles, such as poly(lactide-co-glycolides) (PLG) (35; U.S. Pat. Nos. 5,804,212, 6,876,761, and 5,603,960; PCT/US99/17308). Admixtures can also be administered in conjunction with bioadhesive polymers, such as those described in PCT/US99/12105, PCT/US99/11906, and U.S. Pat. Nos. 5,955,097, 5,800,832, 5,744,155, and 5,814,329. Alternatively, enteric formulations of admixtures can be used for oral administration (see U.S. Pat. No. 5,968,554).

An admixture of the invention can be administered to a mammal by injection, i.e., subcutaneous, intramuscular, or other parenteral injection, such as transdermal or transcutaneous injection, by oral ingestion, or by intranasal administration. Admixtures can be administered to any mammal in which it is desired to increase an immune response, including but not limited to rats, cats, dogs, rabbits, horses, cows, mice, guinea pigs, chimpanzees, baboons, and humans.

Mucosal administration, particularly intranasal administration into either one or both nostrils, is preferred. Doses can be delivered, for example, in one or more drops or using a spray, such as an aerosol or non-aerosol spray. If desired, multiple administrations of an admixture can be used to increase antibody titers against a particular immunogen. Intervals between multiple administrations can be at least 1, 2, 3, 4, 5, 6, or 7 or more days, or at least 2, 3, or 4 or more weeks, depending on the particular immunogen and/or lectin in the admixture. The volume of admixture to be administered will vary according to the mode of administration and size of the mammal. Typical volumes for intranasal administration vary from at least 5, 10, 15, 25, 50, 75, 100, 200, or 250 .mu.l, to at least 500 .mu.l or more per intranasal dose.

The concentration of immunogen in an admixture also will vary according to the particular immunogen and route of administration selected. For intranasal administration, for example, the concentration of an immunogen in an admixture varies from at least 0.033, 0.67, 0.1, 0.2, 0.33, 0.5, 0.67, 0.75, 1, 2, 2.5, 5, 7.1, 10, 12.5, 15, 17.5, 20, or 25 .mu.g/.mu.l.

Admixtures of the invention preferably increase antibody production as well as T cell responses, including cytokine production, target-cell killing, macrophage activation, B-cell activation, and lymphokine production. Admixtures of the invention preferably increase a T cell response or an antibody titer by at least 10, 15, 20, 25, 30, 40, 50, 75, or 100 percent or more relative to such responses to the immunogen alone in the absence of the plant lectin.

Methods of measuring T cell responses are well known in the art. (See Janeway et al., eds., 1997, IMMUNOBIOLOGY: THE IMMUNE SYSTEM IN HEALTH AND DISEASE, 3d ed., at pages 2:31-2-33; Abbas et al., 1997, CELLULAR AND MOLECULAR IMMUNOLOGY, 3d ed., at pages 250-277 and 290-293).

According to the invention, antibodies can be produced which are directed against the immunogen in the admixture. Antibodies which specifically bind to the immunogen typically provide a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in immunochemical assays, such as Western blots, ELISAs, radioimmunoassays, immunohistochemical assays, immunoprecipitations, or other immunochemical assays known in the art. Preferably, antibodies which specifically bind to a particular immunogen do not detect other proteins in immunochemical assays and can precipitate the immunogen from solution.

Antibody titer is preferably measured by ELISA, as described in Example 1, below. IgG, including IgG subtypes IgG1, IgG2a, IgG2b, and IgG3, as well as IgA antibodies directed against the immunogen can be measured in serum, in saliva, and in mucosal secretions, including vaginal, nasal, and gut washes (see Example 1).

The complete contents of all patents and patent applications cited in this disclosure are expressly incorporated herein.

The following examples are provided for exemplification purposes only and are not intended to limit the scope of the invention which has been described in broad terms above.

EXAMPLE 1

Materials and Methods

Immunogens and lectins. Cholera toxin (CT), ovalbumin (OVA, type V, hen egg) and WGA were obtained from Sigma (Poole, UK). PHA from kidney bean was prepared as described previously (15). UEA-I and LEA were obtained from Vector Laboratories. ML-I was isolated as described previously (16).

Animals. Eight week old female Balb/c mice (Harlan Olac, Bicester, UK) were given free access to commercial stock diet (Labsure, Manea, UK) and water.

Mucosal immunization schedule. Groups of mice (n=10) were bled one week prior to the first immunization. On days 1, 14, 28, and 42, mice were immunized intranasally with PBS, OVA (10 .mu.g) alone, or OVA (10 .mu.g) mixed with CT (1 .mu.g), ML-I (1 .mu.g), LEA (10 .mu.g), PHA (10 .mu.g), WGA (10 .mu.g), or UEA-I (10 .mu.g). In other examples, mice were immunized intranasally with 5 .mu.g glycoprotein D2 (gD2) from Herpes simplex virus type 2 on days 1, 14, 28, and 49 alone or in an admixture with 1 .mu.g of either CT, ML-I, ML-II, or ML-III. Mice were dosed with 30 .mu.l of each preparation (15 .mu.l per nostril) through fine tips attached to a pipette.

Collection of blood and mucosal secretions. Blood samples were collected one day prior to each immunization by bleeding from the tail vein following a 10 minute incubation at 37^oC. Two weeks after the final immunization, animals were terminally anesthetized (hypnorin plus diazepam) to allow collection of salivary and vaginal secretions. Mice were then killed by anesthetic overdose followed by exsanguination. Blood was immediately collected and centrifuged, and the serum was stored at -20^oC.

Absorbent cellulose wicks (Whatman International, UK) were used for collection of saliva and vaginal fluid as described previously (17). Wash fluid (ice-cold 0.01 M PBS, 50 mM EDTA, 5 mM PMSF, 5 .mu.g/ml Aprotinin) was used for elution of antibody from wicks and for nasal and intestinal washes. Saliva was collected by the insertion of a wick tip into the mouth for 2 minutes (17). Antibody was extracted from wicks into 400 .mu.l mucosal wash fluid. Vaginal fluid was collected by repeated flushing and aspiration of 50 .mu.l wash fluid and insertion of a wick for 2 minutes. Antibody was extracted from wicks into 400 .mu.l wash fluid. Nasotracheal washes were collected from decapitated animals by backflushing 0.5 ml of mucosal wash fluid from the trachea. Intestinal washes were obtained by flushing the small intestine with 10 ml of ice-cold wash fluid. All secretions were stored at -20^oC. until required for analysis.

Detection of specific antibodies by ELISA. ELISAs were set up to enable measurement of specific IgG, IgA, and IgG subclasses to OVA, CT, and plant lectins. Sera (from 1:100) and mucosal secretions (from 1:2) were titrated in the appropriate dilution buffer. Microtiter plates (Immunolon 4, Dynatech) were coated with 75 .mu.l of immunogen per well (1 .mu.g/ml for CT/lectins, 50 .mu.g/ml when measuring responses to OVA and 2 .mu.g/ml when measuring responses to gD2) in carbonate-bicarbonate buffer, pH 9.6, and incubated at 4^oC. overnight. After washing, plates were blocked with 2% gelatin/dilution buffer and incubated at 37^oC. for 1 hour. Plates were washed, and samples were added, serially diluted, and incubated at 37^oC. for 1 hour.

Biotinylated antiserum in dilution buffer was added and incubated at 37^oC. for 1 hour. After further washes, ExtrAvidin.RTM. peroxidase (Sigma) diluted 1:750 in dilution buffer was added and incubated at 37^oC. for 30 minutes. Plates were washed, and 50 .mu.l/well of developing solution (TMB microwell peroxidase substrate (1-C), Kirkegaard and Perry Laboratories, Gaithersburg, Md.) was added. Plates were incubated in the dark with shaking at 37^oC. for 30 minutes. The reaction was stopped by addition of 1M H₂ SO₄, and the absorbance was read at 450 nm.

ELISA dilution buffers were as follows: CT (PBS+0.1% Tween (PBST)), OVA (PBST), WGA (100 mM N-acetylglucosamine/PBST), PHA (0.1% Fetuin/PBST), UEA-I (30 mM L-fucose/PBST), LEA (Chitin hydrolysate (1:200) (Vector)/PBST), ML-I (100 mM D-galactose/PBST). Working dilutions of anti-IgG (1:8000) and IgA (1:2600) biotinylated capture antisera (Sigma) were determined after preliminary assays with pre-immune and pooled positive sera. Working dilutions of IgG subclass antisera (Serotect) were as recommended by the manufacturers (IgG1 (1:4000), IgG2a (1:4000), IgG2b (1:2000), IgG3 (1:2000)). Endpoint titers were determined as the dilution of a serum or mucosal sample giving an OD value of 0.1 units greater than the mean of control samples at the same dilution.

Total IgA was quantified as specific IgA with the following modifications: plates were coated with goat anti-mouse IgA (1:8000; .alpha.-chain specific, Sigma), PBST was used as diluent, and 2% gelatin in PBST was used as blocking solution. Total IgA levels were calculated from the linear region of the IgA (IgA kappa, Sigma) standard curve. Total IgA endpoint titers were determined as the dilution of a sample giving an OD value of 0.1 units greater than buffer alone.

Statistics. Data are expressed as the mean+standard deviation. An unpaired two-tailed t-test was used to test for significance between groups. Where the standard deviations were significantly different between groups, a nonparametric test (Kruskal-Wallis test with Dunn's multiple comparison post test) was used to assess significance. Kruskal-Wallis nonparametric test with Dunn's multiple comparison post test was also used to assess significance of the total IgA data.

EXAMPLE 2

The Effect of Immunization on Total IgA Levels in Sera and Secretions

Mice were immunized by the intranasal route on days 1, 14, 28, and 42 with either PBS, OVA (10 .mu.g) alone, or OVA (10 .mu.g) together with CT (1 .mu.g), ML-I (1 .mu.g), LEA (10 .mu.g), PHA (10 .mu.g), WGA (10 .mu.g) or UEA-I (10 .mu.g). Samples were collected two weeks after the final immunization. The results are shown FIG. 1. Data represent the mean+SD.

After four intranasal immunizations with CT+OVA there was a significant increase in the concentration of total nasotracheal wash IgA (p<0.01) compared with all other groups. Co-administration of CT with OVA did not result in a significant rise in total IgA concentration in sera or the other mucosal secretions sampled. There was no significant effect of immunization with any of the plant lectins on total IgA levels in any of the secretions or in serum.

EXAMPLE 3

The Adjuvant Effect of Plant Lectins on OVA-specific Serum Antibody Responses

Mice were immunized intranasally on days 1, 14, 28, and 42 with either OVA (10 .mu.g), alone or OVA (10 .mu.g) together with CT (1 .mu.g), ML-I (1 .mu.g), LEA (10 .mu.g), PHA (10 .mu.g), WGA (10 .mu.g) or UEA-I (10 .mu.g). Sera were collected 1 day before each immunization and at the termination of the study. FIGS. 2A-D show the results of this experiment. Points refer to individual data, and the symbol (-) represents the mean titer.

Two weeks after a single immunization, OVA-specific serum IgG was detected in 5/10 mice immunized with CT+OVA and 1/10 mice immunized with ML-I+OVA but OVA-specific IgG was not detected in the other groups. After a second dose, higher responses were measured with detectable antibody in all mice immunized with CT+OVA (mean titer 40321) and in 9/10 mice immunized with ML-I+OVA (mean titer 11090). Of the other groups, specific IgG was only detected in mice immunized with UEA-I+OVA (mean titer 91).

After four doses, the highest mean IgG titers were in mice immunized with CT+OVA, being approximately 286-fold higher than in mice which received OVA alone. The mean titer in the group immunized with ML-I+OVA was approximately 118-fold higher than in mice which received OVA alone. Titers in mice immunized with PHA+OVA were similar to those in mice administered with OVA alone. Administration of LEA+OVA resulted in a small increase in mean titer compared with OVA alone (5-fold). Delivery of WGA and UEA-I with OVA respectively led to 41- and 51-fold increases in mean serum IgG anti-OVA titers compared with OVA alone.

In contrast to the groups which received CT+OVA and ML-I+OVA, responses in the groups immunized with WGA or UEA-I+OVA were highly variable. As a result, after the final dose only the CT+OVA and ML-I+OVA groups (difference not significant between groups) had mean OVA-specific IgG titers significantly higher (p<0.001) than the OVA only group. Titers in these groups were also significantly higher than in the PHA+OVA group (p<0.001).

In contrast to the high levels of specific IgG, very low titers of OVA-specific serum IgA were detected. In fact, after the final dose, significant levels of OVA-specific serum IgA were only detected in mice immunized with CT+OVA (mean titer, 220) and ML-I+OVA (mean titer, 80).

Claim 1 of 37 Claims

What is claimed is:

1. A method of producing an immune response in a mammal, comprising the step of:

administering mucosally to a mammal an admixture comprising an immunogen and a plant lectin selected from the group consisting of ML-I, ML-II, ML-III, and UEA-I, whereby the mammal produces an immune response to the immunogen which is greater relative to the immune response to the immunogen produced in the absence of the plant lectin.

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