PEPTIDE MIMOTOPE THAT INDUCES AN IMMUNE RESPONSE AGAINST MYCOBACTERIUM TUBERCULOSIS LIPOARABINOMANNAN (LAM)

Abstract

The present invention concerns methods and compositions for treating or preventing infection or dissemination of the bacterium Mycobacterium tuberculosis and for stimulating an immune response against the bacteria. In certain embodiments, the methods and compositions involve anti-LAM peptides or mimotopes. In some embodiments, the methods and compositions involve vaccine compositions. In additional embodiments, the present invention concerns peptide sequences and their use in the development of therapeutics, detection assays or vaccines against M. tuberculosis.

Claims

1-83. (canceled)

84. A method of eliciting an immune response against Mycobacterium tuberculosis or Mycobaterium bovis in a subject comprising administering to the subject an effective amount of a composition comprising a peptide of SEQ ID NO:1.

85. The method of claim 84, wherein the composition is formulated as a vaccine.

86. An isolated peptide having at least about 80% sequence identity or similarity to SEQ ID NO.:1.

87. The isolated peptide of claim 86, wherein the peptide is conjugated to a moiety, a carrier protein, or a vector.

88. The isolated peptide of claim 86, wherein the peptide is conjugated to a recombinant baculovirus.

89. The isolated peptide of claim 88, wherein the recombinant baculovirus is Autographa californica multinuclear polyhedrosis virus (AcMNPV).

90. The isolated peptide of claim 4, wherein the carrier protein is keyhole limpet hemocyanin (KLH) protein.

91. A vaccine composition comprising the isolated peptide of claim 86.

92. The vaccine composition of claim 46, further comprising an additional M. tuberculosis vaccine.

93. The vaccine composition of claim 47, wherein the additional M. tuberculosis vaccine is comprising a Bacillus Calmette Guerin (BCG) vaccine.

94. A pharmaceutical composition comprising the isolated peptide of claim 86.

95. A kit comprising a composition comprising the isolated peptide of claim 86.

96. A method of preparing an immunoglobulin to prevent or treat M. tuberculosis or M. bovis comprising the steps of immunizing a recipient with a composition comprising SEQ ID NO:1 and isolating the immunoglobulin from the recipient.

97. A method of detecting the presence of M. tuberculosis or M. bovis in a subject, the method comprising detecting anti-LAM antibodies in a sample obtained from the subject using a composition comprising SEQ ID NO:1 to determine the presence or absence of M. tuberculosis.

98. A lipoarabinomannan (LAM) peptide mimotope comprising SEQ ID NO:1, wherein the peptide mimotope is conjugated to a carrier or a vector.

99. The LAM peptide mimotope of claim 98, wherein the peptide is an anti-Mycobacterium tuberculosis LAM peptide mimotope.

100. The LAM peptide mimotope of claim 98, wherein the peptide is an anti-Mycobacterium bovis LAM peptide mimotope.

101. The peptide mimotop of claim 98, wherein the vector is a recombinant baculovirus.

102. The peptide mimotope of claim 101, wherein the recombinant baculovirus is Autographa californica multinuclear polyhedrosis virus (AcMNPV).

103. The peptide mimotope of claim 98, wherein the carrier is keyhole limpet hemocyanin (KLH) protein.

Description

DESCRIPTION OF THE DRAWINGS

[0069] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. It is to be noted, however, that the appended drawings illustrate certain embodiments of the invention and therefore are not to be considered limiting in their scope.

[0070] FIGS. 1A-M Analysis of previously published LAM mimotopes. (A) Testing a polyclonal rabbit and two mouse monoclonal antibodies in a LAM ELISA. (B-E) Mimotope peptides or their scramble controls were conjugated to KLH and mimotope behavior determined by ELISA using the rabbit polyclonal anti-LAM antibody. (F-J) Mimotope peptides or their scramble controls were conjugated to KLH and mimotope behavior determined by ELISA using the mouse monoclonal CS-35 anti-LAM antibody. K-M Mice (n=5 per peptide) were vaccinated with PBS, KLH alone, or mimotope peptides or scramble controls conjugated to KLH and sera collected after 4 vaccinations. Mouse sera (1:100 dilution) was tested by ELISA against LAM (K), KLH (L) or specific peptides (M) with CS-35 as a positive control and preimmune 413 sera or mouse IgG as negative controls. One of two representative experiments is shown.

[0071] FIGS. 2A-C Analysis of novel anti-LAM mimotopes by ELISA. (A) Plates were coated with either phage alone, LAM, HS peptide-expressing phage, or SG peptide-expressing phage (10 g/ml for each coating molecule) and binding by rabbit anti-LAM polyclonal antibody determined by ELISA. (B) Plates were coated with BSA, LAM, HS peptide-expressing phage, HS peptide alone, HS peptide conjugated to KLH, HS scramble peptide alone (HSSR) or HSSR conjugated to KLH (10 g/ml for each coating molecule) and binding by rabbit anti-LAM polyclonal antibody determined by ELISA. (C) Plates were coated with BSA, LAM, SG peptide-expressing phage, SG peptide alone, SG peptide conjugated to KLH, SG scramble peptide alone (SGSR) or SGSR conjugated to KLH (10 g/ml for each coating molecule) and binding by rabbit anti-LAM polyclonal antibody determined by ELISA. One of two representative experiments is shown

[0072] FIGS. 3A-B Vaccination with HS-peptide conjugated to KLH induces anti-LAM antibodies. Mice (n=5) per group were vaccinated with HS peptide conjugated to KLH (A) or HSSR peptide conjugated to KLH (B) and sera was collected after 4 vaccinations. Serum was collected from individual mice and tested for binding (1:100 dilution) to BSA, LAM, HS peptide conjugated to KLH, HSSR conjugated to KLH or KLH alone (10 g/ml for each coating molecule) by ELISA. Shown is a scatter plot from one experiment of two. (STATS).

[0073] FIGS. 4A-D Baculovirus conjugated HS peptide is protective in a low dose aerosol tuberculosis infection model in mice. (A) Test peptide (P1 biotin) was conjugated to baculovirus and detected by anti-biotin Western blot. Lane 1 is baculovirus conjugated to P1 biotin, lane 2 is peptide alone, and lane 3 is baculovirus alone. (B) Plates were coated with BSA, LAM, HS peptide435 expressing phage, HS peptide alone, HSSR peptide alone, baculovirus alone, HS peptide conjugated to baculovirus or HSSR conjugated to baculovirus (10 g/ml for each coating molecule) and binding by rabbit anti-LAM polyclonal antibody determined by ELISA. (C) Mice (n=5) per group were vaccinated intranasally with HS peptide conjugated to baculovirus or HSSR peptide conjugated to baculovirus and sera was collected after 4 vaccinations. Serum was collected from individual mice and tested for binding (1:100 dilution) to BSA, LAM, or baculovirus (10 g/ml for each coating molecule) by ELISA. Shown is a scatter plot from one experiment of two. (STATS). (D) Mice (n=10 per group) vaccinated as in C with baculovirus alone, HS peptide conjugated to baculovirus, HSSR peptide conjugated to baculovirus or M. bovis var BCG were infected with 200 CFU M. tuberculosis Erdman strain and monitored for survival. *p<0.05, **<p<0.01 by Gehan-Breslow-Wilcoxon test compared to baculovirus vaccination.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0074] Described herein is the use of anti-LAM monoclonal antibodies to pan a phage display library and uncover novel mimotopes that can serve, for example, in the generation of vaccines against M. tuberculosis. A phage display library was panned using two monoclonal antibodies against M. tuberculosis lipoarabinomannan (LAM) and identified two peptide sequences with high antibody affinity after multiple rounds of panning. Recombinant M13 phages displaying the peptides HSFKWLDSPRLR (SEQ ID NO:1) or SGVYKVAYDWQH (SEQ ID NO:2) on the g3p minor coat protein showed strong binding affinity with both mAbs. Both peptides were able to induce an anti-LAM response when conjugated to either keyhole limpet hemocyanin (KLH) or to the baculovirus Autographa californica multicapsid nucleopolyherovirus (AcMNPV) and introduced into mice by injection or via intranasal infection, respectively. Vaccination with AcMNPV conjugated HSFKWLDSPRLR peptide delayed mortality in a mouse model of tuberculosis.

[0075] The inventor began by first testing the known peptide sequences. Surprisingly, mimotope activity could not be detected by the inventor for the published peptide sequences, neither in vitro as antigenic substrates in ELISA nor in vivo after vaccinating experimental animals.

[0076] It was thus found that a peptide mimotope against the mycobacterial cell wall component LAM was identified by panning a phage display library, this mimotope can induce an anti-LAM antibodies after vaccination either when conjugated to KLH or baculovirus, and a baculovirus-mimotope anti-LAM vaccination strategy can afford partial protection against mortality from M. tuberculosis infection.

A. LIPOARABINOMANNAN (LAM)

[0077] A critical contributor to the ability of M. tuberculosis to evade the host immune system is its hydrophobic and complex cell wall, which is composed of peptidoglycan, arabinogalactan, mycolic acids, and glycolipids layered on top of the plasma membrane. Lipoarabinomannan (LAM), is a mannose-containing glycolipids that is an important component of the cell envelope. LAM is a polysaccharide chain containing -D-arabinofuranosyl (Araf) and mannopyranosyl residues. It spans the mycobacterial outer membrane and the covalently linked macromolecules of the cell envelope referred to as mycolyl-arabinogalactan-peptidoglycan complex (mAGP), terminating in a phosphatidylinositol-diacylglycerol moiety that is embedded within the cytoplasmic membrane. In addition to serving as a major cell wall component, it is thought that LAM serves as a modulin by controlling the host immune and inflammatory responses. This ensures the survival of the bacterium by undermining host resistance and acquired immune responses. Immunoregulatory mechanisms include the inhibition of T-cell proliferation and of macrophage microbicidal activity via diminished IFN- response. Additional functions of lipoarabinomannan are thought to include the neutralization of cytotoxic oxygen free radicals produced by macrophages, inhibition of protein kinase C, and induction of early response genes.

B. MIMOTOPES AND PHAGE DISPLAY TECHNOLOGY

[0078] Mimotopes are peptides mimicking protein, carbohydrates, or lipid epitopes that are usually generated by phage display technology. When selected by antibodies, they represent exclusively B-cell epitopes and are devoid of antigen/allergen-specific T-cell epitopes. Coupled to carriers or presented in a multiple antigenic peptide forms, mimotopes achieve immunogenicity and induce epitope-specific antibody responses upon vaccination.

[0079] Phage display technology is an advanced tool to define peptide mimotopes that mimic natural epitopes including both conformational and linear epitopes. It is one of the most powerful and widely used laboratory technique for the study of protein-protein, protein-peptide, and protein-DNA interactions. The technology is mainly based on displaying the interest protein (peptides, antibodies, scaffolds or others) on the surface of employing phage so as to be used to interrogate the constructed libraries containing millions or even billions of displayed phages. The strength of the phage technology lies in this display of up to 10.sup.9 different peptides in a library form enabling the selection of mimotopes in a repetitive screening procedure. Theoretically, phage display is an exogenous gene expression method which the gene encoding the interest protein is inserted into bacteriophage coat protein gene then displaying the interest protein on the phage surfaces, resulting in a connection between genotype and phenotype. A large number of protein-antibody, virus-antibody and ligand-receptor interfaces have already been mapped by the use of phage display technology.

[0080] For the creation of a peptide phage display library random oligonucleotides are inserted into the genome of the filamentous bacteriophage M13 using either the minor coat protein pIII (display of 3-5 copies/phage) or the major coat protein pVIII (display of up to 2700 copies/phage) as display system on the surface of the phages. The peptides can be presented in either linear or circular form. To construct circular peptides the sequence has to be flanked by two cysteine residues. By building a disulfide bond a constrained cycle is formed and presented on the phage surface. Beside the different structural presentations, the length of the peptides can vary from 6 to 38 amino acids, Thus, within this phage system only the presentation of relatively short peptide sequences is possible, whereas larger gene insertions of proteins or antibody fragments appear to prevent pIII and pVIII functions necessary for phage reproduction, To overcome this problem phagemid systems have been developed. The phagemid itself carries only the phage gene gill or gVIII, containing the foreign sequence, and needs a helper phage with all the necessary genes for phage production including also a copy of the wild-type gene used for display. Therefore, both recombinant and wild type proteins will be produced and incorporated into the phage particle. For pill one of five copies, and for pVIII from 1% to 30% of the 2700 copies will display the foreign protein. For the production of combinatorial antibody libraries primarily phagemid systems are used resulting in the display of giant mimotopes, most often in association with pIII. Antibody display can be performed: i) by antibody fragment (Fab) systems, including two light chain domains (variable and constant) and the variable and first constant domain of the heavy chain; ii) by single-chain variable fragment (scFv) systems where only the variable domains of each chain are presented; or iii) by single-chain Fab (scFab).

[0081] In biopanning allergen- or antigen-specific antibodies of interest, monoclonal or polyclonal, are adsorbed on microtiter plates and incubated with a phage display library containing the complete repertoire of the respective library. Phages displaying peptide or protein domains which bind to the antibodies are caught whereas unbound phages are removed by washing steps. Bound phages are then eluted from the complexes by acidic solutions (such as HCl or glycine buffer) or by competition with the original antigen, if available. Amplified phage particles of the preceding round are used as starting material for the next panning round. Thereby, specifically interacting ligands can be amplified with great efficacy. Eluents from the biopanning rounds are-tested by ELISA or other immunological methods. An increase in the titre of phage particles specifically binding to the selection antibody during the panning rounds is a first indicator of successful selection. Subsequently, the colony screening method is used to identify specifically interacting phage clones for further analysis. Generally, a strong signal in these tests predicts good mimicry of the original antigen, but this has to be additionally proven by competition assays with the original antigen or allergen. After sequencing the most promising clones, computational matching studies can be performed using a software program rendering visualization of the location and the structural features of the epitope of interest Subsequent immunization studies with the mimotopes must prove molecular mimicry and should lead to antibodies recognizing the original allergen/antigen.

C. VACCINES

[0082] The methods of the disclosure also include the administration of vaccines. As used herein, the term in vitro administration refers to manipulations performed on cells removed from or outside of a subject, including, but not limited to cells in culture. The term ex vivo administration refers to cells that have been manipulated in vitro, and are subsequently administered to a subject. The term in vivo administration includes all manipulations performed within a subject, including administrations.

[0083] In certain aspects of the present disclosure, the compositions may be administered either in vitro, ex vivo, or in vivo. In certain in vitro embodiments, autologous T cells are incubated with compositions of this disclosure. The cells can then be used for in vitro analysis, or alternatively for ex vivo administration.

[0084] Method aspects of the disclosure include vaccinating a subject with a variety of different immunotherapeutic compositions. In some embodiments, the methods further comprise administration of SEQ ID NO: 1 to a subject. The route of administration of the immune cell may be, for example, intratumoral, intracutaneous, subcutaneous, intravenous, intralymphatic, and intraperitoneal administrations. In some embodiments, the administration is intratumoral or intralymphatic.

[0085] The present invention includes methods for preventing or ameliorating M. tuberculosis and/or M. bovis infections. As such, the invention contemplates vaccines for use in both active and passive immunization embodiments. Immunogenic compositions, proposed to be suitable for use as a vaccine, may be prepared from immunogenic peptides comprising SEQ ID NO:1 or variants thereof as described herein. In certain aspects, antigenic material is extensively dialyzed to remove undesired small molecular weight molecules and/or lyophilized for more ready formulation into a desired vehicle.

[0086] The present invention includes peptides and compositions for preventing, eliciting an immune response against, diagnosing and detecting M. tuberculosis and/or M. tuberculosis LAM. As such, the invention contemplates vaccines for use in both active and passive immunization embodiments. Immunogenic compositions, proposed to be suitable for use as a vaccine, may be prepared from peptides of SEQ ID NO:1.

[0087] Embodiments provided include methods of preventing, vaccinating or treating Mycobacterium bovis infections in animals comprising a step of administering to an animal an effective amount of a pharmaceutical composition comprising SEQ ID NO:1 or variants thereof, such as a vaccine. In some embodiments the animals are livestock animals including but not limited to cows, bulls, pigs, sheep, goats, yak, bison, buffalo, oxen and horses.

[0088] Other options for a protein/peptide-based vaccine involve introducing nucleic acids encoding the antigen(s) as DNA vaccines. In this regard, reports described construction of recombinant vaccinia viruses expressing either 10 contiguous minimal CTL epitopes (Thomson, 1996) or a combination of B cell, cytotoxic T-lymphocyte (CTL), and T-helper (Th) epitopes from several microbes, and successful use of such constructs to immunize mice for priming protective immune responses. Thus, there is ample evidence in the literature for successful utilization of peptides, peptide-pulsed antigen presenting cells (APCs), and peptide-encoding constructs for efficient in vivo priming of protective immune responses. The use of nucleic acid sequences as vaccines is exemplified in U.S. Pat. Nos. 5,958,895 and 5,620,896.

[0089] The preparation of vaccines that contain polypeptide or peptide sequence(s) as active ingredients is generally well understood in the art, as exemplified by U.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792; and 4,578,770, all of which are incorporated herein by reference. Typically, such vaccines are prepared as injectables either as liquid solutions or suspensions: solid forms suitable for solution in or suspension in liquid prior to injection may also be prepared. The preparation may also be emulsified. The active immunogenic ingredient is often mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the vaccine may contain amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants that enhance the effectiveness of the vaccines. In specific embodiments, vaccines are formulated with a combination of substances, as described in U.S. Pat. Nos. 6,793,923 and 6,733,754, which are incorporated herein by reference.

[0090] Vaccines may be conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly. Additional formulations which are suitable for other modes of administration include suppositories and; in some cases, oral formulations. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70%.

[0091] The peptides and peptide-encoding DNA constructs may be formulated into a vaccine as neutral or salt forms. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the peptide) and those that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.

[0092] Typically, vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective and immunogenic. The quantity to be administered depends on the subject to be treated, including the capacity of the individual's immune system to synthesize antibodies and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner. However, suitable dosage ranges are of the order of several hundred micrograms of active ingredient per vaccination. Suitable regimes for initial administration and booster shots are also variable; but are typified by an initial administration followed by subsequent inoculations or other administrations.

[0093] The manner of application may be varied widely. Any of the conventional methods for administration of a vaccine are applicable. These are believed to include oral application within a solid physiologically acceptable base or in a physiologically acceptable dispersion, parenterally, by injection and the like. The dosage of the vaccine will depend on the route of administration and will vary according to the size and health of the subject.

[0094] In certain instances, it will be desirable to have multiple administrations of the vaccine, e.g., 2, 3, 4, 5, 6 or more administrations. The vaccinations can be at 1, 2, 3, 4, 5, 6, 7, 8, to 5, 6, 7, 8, 9, 10, 11, 12 twelve week intervals, including all ranges there between. Periodic boosters at intervals of 1-5 years will be desirable to maintain protective levels of the antibodies. The course of the immunization may be followed by assays for antibodies against the antigens

[0095] 1. Carriers

[0096] A given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin, or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimyde, and bis-biazotized benzidine.

[0097] KLH (Keyhole Limpet Hemocyanin) is used extensively as a carrier protein in research and therapeutic vaccine applications. Peptides, small proteins and drug molecules of low molecular weight are not usually immunogenic. They require the aid of a carrier protein to stimulate antibody production. KLH is a very effective and popular carrier protein due to its large size, numerous epitopes, plentiful sites for antigen conjugation, and strong safety history. KLH is also well known as a safe, potent stimulator of humoral and cellular immune responses. It used in research and clinical studies as an antigen for assessing immune function and in immunotoxicology studies such as monitoring the immunosuppressive effects of drug candidates.

[0098] 2. Adjuvants

[0099] The immunogenicity of peptide compositions can be enhanced by the use of nonspecific stimulators of the immune response, known as adjuvants. Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins, or synthetic compositions. A number of adjuvants can be used to enhance an antibody response against a variant of SEQ ID NO:1 or combination contemplated herein. Adjuvants can (1) trap the antigen in the body to cause a slow release; (2) attract cells involved in the immune response to the site of administration; (3) induce proliferation or activation of immune system cells; or (4) improve the spread of the antigen throughout the subject's body.

[0100] Adjuvants include, but are not limited to, oil-in-water emulsions, water-in-oil emulsions, mineral salts, polynucleotides, and natural substances. Specific adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12, -interferon, GMCSP, BCG, aluminum salts, such as aluminum hydroxide or other aluminum compound, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM), and cell wall skeleton (CWS) in a 2% squalene/Tween 80 emulsion. MHC antigens may even be used. Others adjuvants or methods are exemplified in U.S. Pat. Nos. 6,814,971, 5,084,269, 6,656,462, each of which is incorporated herein by reference.

[0101] Various methods of achieving adjuvant affect for the vaccine includes use of agents such as aluminum hydroxide or phosphate (alum), commonly used as about 0.05 to about 0.1% solution in phosphate buffered saline, admixture with synthetic polymers of sugars (Carbopol) used as an about 0.25% solution, aggregation of the protein in the vaccine by heat treatment with temperatures ranging between about 70 to about 101 C. for a 30-second to 2-minute period, respectively. Aggregation by reactivating with pepsin-treated (Fab) antibodies to albumin; mixture with bacterial cells (e.g., C. parvum), endotoxins or lipopolysaccharide components of Gram-negative bacteria; emulsion in physiologically acceptable oil vehicles (e.g., mannide mono-oleate (Aracel A)); or emulsion with a 20% solution of a perfluorocarbon (Fluosol-DA) used as a block substitute may also be employed to produce an adjuvant effect.

[0102] Examples of and often preferred adjuvants include complete Freund's adjuvant (a nonspecific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants, and aluminum hydroxide.

[0103] In some aspects, it is preferred that the adjuvant be selected to be a preferential inducer of either a Th1 or a Th2 type of response. High levels of Th1-type cytokines tend to favor the induction of cell mediated immune responses to a given antigen, while high levels of Th2-type cytokines tend to favor the induction of humoral immune responses to the antigen.

The distinction of Th1 and Th2-type immune response is not absolute. In reality an individual will support an immune response, which is described as being predominantly Th1 or predominantly Th2. However, it is often convenient to consider the families of cytokines in terms of that described in murine CD4+ T cell clones by Mosmann and Coffman (Mosmann, and Coffman, 1989). Traditionally, Th1-type responses are associated with the production of the INF- and IL-2 cytokines by T-lymphocytes. Other cytokines often directly associated with the induction of Th1-type immune responses are not produced by T-cells, such as IL-12. In contrast, Th2-type responses are associated with the secretion of IL-4, IL-5, IL-6, IL-10.

[0104] In addition to adjuvants, it may be desirable to co-administer biologic response modifiers (BRM) to enhance immune responses. BRMs have been shown to upregulate T cell immunity or downregulate suppresser cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA); or low-dose Cyclophosphamide (CYP; 300 mg/m.sup.2) (Johnson/Mead, NJ) and cytokines such as -interferon, IL-2, or IL-12 or genes encoding proteins involved in immune helper functions, such as B-7.

D. FORMULATIONS AND ROUTES OF ADMINISTRATION

[0105] In some embodiments, pharmaceutical compositions are administered to a subject. Different aspects of the present invention involve administering an effective amount of a composition to a subject. In some embodiments of the present invention, anti-LAM peptide mimotopes or antigens, may be administered to a recipient to protect against infection by M. tuberculosis. Alternatively, an expression vector encoding one or more such peptides may be given to a patient as a preventative treatment. Additionally, such compounds can be administered in combination with an antibiotic or an antibacterial. Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.

In addition to the compounds formulated for parenteral administration, such as those for intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration, time release capsules; and any other form currently used, including inhalants and the like.

[0106] The active compounds of the present invention can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes. The preparation of an aqueous composition that contains a compound or compounds that increase the expression of an MHC class I molecule will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and, the preparations can also be emulsified.

[0107] Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0108] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil, or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that it may be easily injected. It also should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.

[0109] The compositions may be formulated into a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

[0110] The carrier also can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0111] Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques, which yield a powder of the active ingredient, plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0112] Administration of the compositions according to the present invention will typically be via any common route. This includes, but is not limited to oral, nasal, or buccal administration. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intranasal, or intravenous injection. In certain embodiments, a vaccine composition may be inhaled (e.g., U.S. Pat. No. 6,651,655, which is specifically incorporated by reference). Such compositions would normally be administered as pharmaceutically acceptable compositions that include physiologically acceptable carriers, buffers or other excipients. As used herein, the term pharmaceutically acceptable refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem complications commensurate with a reasonable benefit/risk ratio. The term pharmaceutically acceptable carrier, means a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a chemical agent.

[0113] For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in isotonic NaCl solution and either added to hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, Remington's Pharmaceutical Sciences, 1990). Some variation in dosage will necessarily occur depending on the condition of the subject. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

[0114] An effective amount of therapeutic or prophylactic composition is determined based on the intended goal. The term unit dose or dosage refers to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of the composition calculated to produce the desired responses discussed above in association with its administration, i.e., the appropriate route and regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the protection desired.

[0115] Precise amounts of the composition also depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dose include physical and clinical state of the subject, route of administration, intended goal of treatment (alleviation of symptoms versus cure), and potency, stability, and toxicity of the particular composition.

[0116] Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically or prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above.

E. IMMUNOGLOBULINS, ANTIBODIES AND PASSIVE IMMUNIZATION

[0117] Another aspect of the invention is a method of preparing an immunoglobulin for use in prevention or treatment of staphylococcal infection comprising the steps of immunizing a recipient or donor with the vaccine of the invention and isolating immunoglobulin from the recipient or donor. An immunoglobulin prepared by this method is a further aspect of the invention. A pharmaceutical composition comprising the immunoglobulin of the invention and a pharmaceutically acceptable carrier is a further aspect of the invention that could be used in the manufacture of a medicament for the treatment or prevention of M. tuberculosis disease. A method for treatment or prevention of M. tuberculosis infection comprising a step of administering to a patient an effective amount of the pharmaceutical preparation of the invention is a further aspect of the invention.

[0118] Inocula for polyclonal antibody production are typically prepared by dispersing the antigenic composition in a physiologically tolerable diluent such as saline or other adjuvants suitable for human use to form an aqueous composition. An immunostimulatory amount of inoculum is administered to a mammal and the inoculated mammal is then maintained for a time sufficient for the antigenic composition to induce protective antibodies.

[0119] The antibodies can be isolated to the extent desired by well-known techniques such as affinity chromatography (Harlow and Lane, 1988). Antibodies can include antiserum preparations from a variety of commonly used animals, e.g. goats, primates, donkeys, swine, horses, guinea pigs, rats or man.

[0120] An immunoglobulin produced in accordance with the present invention can include whole antibodies, antibody fragments or subfragments. Antibodies can be whole immunoglobulins of any class (e.g., IgG, IgM, IgA, IgD or IgE), chimeric antibodies or hybrid antibodies with dual specificity to two or more antigens of the invention. They may also be fragments (e.g., F(ab)2, Fab, Fab, Fv and the like) including hybrid fragments. An immunoglobulin also includes natural, synthetic, or genetically engineered proteins that act like an antibody by binding to specific antigens to form a complex.

[0121] A vaccine of the present invention can be administered to a recipient who then acts as a source of immunoglobulin, produced in response to challenge from the specific vaccine. A subject thus treated would donate plasma from which hyperimmune globulin would be obtained via conventional plasma fractionation methodology. The hyperimmune globulin would be administered to another subject in order to impart resistance against or treat staphylococcal infection. Hyperimmune globulins of the invention are particularly useful for treatment or prevention of staphylococcal disease in infants, immune compromised individuals, or where treatment is required and there is no time for the individual to produce antibodies in response to vaccination.

[0122] An additional aspect of the invention is a pharmaceutical composition antibodies (or fragments thereof; preferably human or humanized) reactive against at least two constituents of the immunogenic composition of the invention, which could be used to treat or prevent infection M. tuberculosis. Such pharmaceutical compositions comprise monoclonal antibodies that can be whole immunoglobulins of any class, chimeric antibodies, or hybrid antibodies with specificity to two or more antigens of the invention. They may also be fragments (e.g., F(ab)2, Fab, Fab, Fv and the like) including hybrid fragments.

[0123] Methods of making monoclonal and polyclonal antibodies are well known in the art and can include the fusion of spleenocytes with myeloma cells (Kohler and Milstein, 1975; Harlow and Lane, 1988). Alternatively, monoclonal Fv fragments can be obtained by screening a suitable phage display library (Vaughan et al., 1998). Monoclonal antibodies may be humanized or part humanized by known methods.

F. IMMUNOASSAYS

[0124] Embodiments of the current invention include methods of detection and immunoassays related to peptides of SEQ ID NO:1 or immunoglobulins against said peptides. Immunoassays generally include immunoblotting (e.g., Western blotting), RIA, and ELISA. More specific types of immunoassays include antigen capture/antigen competition, antibody capture/antigen competition, two-antibody sandwiches, antibody capture/antibody excess, and antibody capture/antigen excess. Methods of making antibodies are described herein and in Harlow and Lane, Antibodies: A Laboratory Manual, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA. Phospho-specific antibodies can be made de novo or obtained from commercial or noncommercial sources. Phosphorylation levels and/or status can also be determined by metabolically labeling cells with radioactive phosphate in the form of [.gamma.-.sup.32P]ATP or [.gamma.-.sup.33P]ATP. Phosphorylated proteins become radioactive and hence traceable and quantifiable through scintillation counting, radiography, and the like (see, e.g., Wang et al., J. Biol. Chem., 253:7605-7608 (1978)).

[0125] The present invention includes the implementation of serological assays to evaluate whether and to what extent an immune response is induced or evoked by compositions of the invention. There are many types of immunoassays that can be implemented. Immunoassays encompassed by the present invention include, but are not limited to, the Immunoassays described in U.S. Pat. No. 4,367,110 (double monoclonal antibody sandwich assay) and U.S. Pat. No. 4,452,901 (western blot). Other assays include immunoprecipitation of labeled ligands and immunocytochemistry, both in vitro and in vivo. Immunoassays generally are binding assays. Certain preferred immunoassays are the various types of enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays (RIA) known in the art. For example, peptide mimotopes comprising SEQ ID NO:1 can be used as antigens in ELISA detection assays which are assays well known to a person of skill in the art.

[0126] Immunohistochemical detection using tissue sections is also particularly useful. In one example, antibodies or antigens are immobilized on a selected surface, such as a well in a polystyrene microtiter plate, dipstick, or column support. Then, a test composition suspected of containing the desired antigen or antibody, such as a clinical sample, is added to the wells. After binding and washing to remove non-specifically bound immune complexes, the bound antigen or antibody may be detected. Detection is generally achieved by the addition of another antibody, specific for the desired antigen or antibody that is linked to a detectable label. This type of ELISA is known as a sandwich ELISA. Detection also may be achieved by the addition of a second antibody specific for the desired antigen, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label.

[0127] Competition ELISAs are also possible implementations in which test samples compete for binding with known amounts of labeled antigens or antibodies. The amount of reactive species in the unknown sample is determined by mixing the sample with the known labeled species before or during incubation with coated wells. The presence of reactive species in the sample acts to reduce the amount of labeled species available for binding to the well and thus reduces the ultimate signal. Irrespective of the format employed, ELISAs have certain features in common, such as coating, incubating or binding, washing to remove non-specifically bound species, and detecting the bound immune complexes.

[0128] Antigen or antibodies may also be linked to a solid support, such as in the form of plate, beads, dipstick, membrane, or column matrix, and the sample to be analyzed is applied to the immobilized antigen or antibody. In coating a plate with either antigen or antibody, one will generally incubate the wells of the plate with a solution of the antigen or antibody, either overnight or for a specified period. The wells of the plate will then be washed to remove incompletely-adsorbed material. Any remaining available surfaces of the wells are then coated with a nonspecific protein that is antigenically neutral with regard to the test antisera. These include bovine serum albumin (BSA), casein, and solutions of milk powder. The coating allows for blocking of nonspecific adsorption sites on the immobilizing surface and thus reduces the background caused by nonspecific binding of antisera onto the surface.

G. DIAGNOSIS OF M. TUBERCULOSIS INFECTION

[0129] In addition to the use of peptides, as well as antibodies binding these M. tuberculosis antigens or peptide mimotopes comprising SEQ ID NO: 1 or variants described herein, to prevent an infection as described above, the present invention contemplates the use of these peptides, and/or antibodies in a variety of ways, including the detection of the presence of M. tuberculosis to diagnose an infection, whether in a patient or a subject at risk of developing an infection.

[0130] Current methods to detect exposure to M. tuberculosis rely on either skin test (PPD) or blood test (IGRA), both of which require 2-3 days to complete. For diagnostic purposes, detection of anti-LAM antibodies against a readily produced peptide could be significantly more rapid. In addition, the novel methods of diagnosis provided by the current invention are more rapid and direct for detecting exposure to tuberculosis as the current tuberculosis vaccine, BCG has limited efficacy.

[0131] In accordance with the invention, a preferred method of detecting the presence of infections involves the steps of obtaining a sample suspected of being infected by one or more M. tuberculosis or strains thereof, such as a sample taken from an individual, for example, from one's blood, saliva, tissues, fluids, lungs, for example. Following isolation of the sample, diagnostic assays utilizing the peptides, and/or antibodies of the present invention may be carried out to detect the presence of M. tuberculosis, and such assay techniques for determining such presence in a sample are well known to those skilled in the art and include methods such as radioimmunoassay, western blot analysis and ELISA assays. In general, in accordance with the invention, a method of diagnosing an infection is contemplated wherein a sample suspected of being infected with M. tuberculosis has added to it the polypeptide, protein, peptide, antibody, or monoclonal antibody in accordance with the present invention, and M. tuberculosis are indicated by antibody binding to the polypeptides, proteins, and/or peptides, or polypeptides, proteins, and/or peptides binding to the antibodies in the sample.

[0132] Accordingly, antibodies in accordance with the invention may be used for the prevention of infection from M. tuberculosis (i.e., passive immunization), for the treatment of an ongoing infection, or for use as research tools. The term antibodies as used herein includes monoclonal, polyclonal, chimeric, single chain, bispecific, simianized, and humanized or primatized antibodies as well as Fab fragments, such as those fragments which maintain the binding specificity of the antibodies, including the products of an Fab immunoglobulin expression library. Accordingly, the invention contemplates the use of single chains such as the variable heavy and light chains of the antibodies. Generation of any of these types of antibodies or antibody fragments is well known to those skilled in the art. Specific examples of the generation of an antibody to a bacterial protein can be found in U.S. Patent Application Pub. No. 20030153022, which is incorporated herein by reference in its entirety.

[0133] Any of the above described polypeptides, proteins, peptides, mimotopes and/or antibodies may be labeled directly with a detectable label for identification and quantification of staphylococcal bacteria. Labels for use in immunoassays are generally known to those skilled in the art and include enzymes, radioisotopes, and fluorescent, luminescent and chromogenic substances, including colored particles such as colloidal gold or latex beads. Suitable immunoassays include enzyme-linked immunosorbent assays (ELISA).

H. DELIVERY SYSTEMS

[0134] In one insect expression system that may be used to produce the chimeric proteins of the invention, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express the foreign genes. The virus grows in Spodoptera frugiperda cells. A coding sequence may be cloned into non-essential regions (for example, the polyhedron gene) of the virus and placed under control of an AcNPV promoter (for example, the polyhedron promoter). Successful insertion of a coding sequence will result in inactivation of the polyhedron gene and production of non-occluded recombinant virus (i.e. virus lacking the proteinaceous coat coded for by the polyhedron gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (see, e.g., Smith et al. 1983, J. Virol. 46:584; U.S. Pat. No. 4,215,051). Further examples of this expression system may be found in Ausubel et al., eds. 1989, Current Protocols in Molecular Biology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience.

I. COMBINATION THERAPIES

[0135] The compositions and related methods, particularly the administration of an anti-LAM peptide mimotope of SEQ ID NO: 1 can be also used in combination with the administration of conventional therapies and vaccinations against M. tuberculosis infections. The compositions and related methods of the present invention, particularly administration of vaccine or immunoglobulin prepared in connection with SEQ ID NO:1, including a variant peptide, can also be used in combination with the administration of traditional therapies such as antibiotics or other vaccines such as a Bacillus Calmette Guerin (BCG) vaccine. The Bacillus Calmette-Guerin (BCG) vaccine has existed for decades and is one of the most widely used of all current vaccines, reading >80% of neonates and infants in countries where it is part of the national childhood immunization programs. BCG vaccine has a documented protective effect against meningitis and disseminated TB in children. It does not prevent primary infection and, more importantly, does not prevent reactivation of latent pulmonary infection, the principal source of bacillary spread in the community. Combination with other vaccine such as the vaccines contemplated in the current disclosure could provide a more effective measure against M. tuberculosis infections. To that end, identification of anti-LAM mimotopes such as provided by the current invention, might provide even greater protection when combined with other vaccines or therapies.

[0136] In one aspect, it is contemplated that a polypeptide vaccine and/or therapy is used in conjunction with an antibacterial treatment. Alternatively, the therapy may precede or follow the other agent treatment by intervals ranging from minutes to weeks. In embodiments where the other agents and/or a proteins or polynucleotides are administered separately, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and antigenic composition would still be able to exert an advantageously combined effect on the subject. In such instances, it is contemplated that one may administer both modalities within about 12-24 h of each other or within about 6-12 h of each other. In some situations, it may be desirable to extend the time period for administration significantly, where several days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.

[0137] Various combinations may be employed, for example antibiotic therapy is A and the immunogenic molecule given as part of an immune therapy regime, such as an antigen, is B: [0138] A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B [0139] B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A [0140] B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

[0141] Administration of the immunogenic compositions of the present invention to a patient/subject will follow general protocols for the administration of such compounds, taking into account the toxicity, if any, of the current composition, or other compositions described herein. It is expected that the treatment cycles would be repeated as necessary. It also is contemplated that various standard therapies, such as hydration, may be applied in combination with the described therapy.

J. KITS

[0142] Certain aspects concern kits containing compositions described herein or compositions to implement methods described herein.

In some embodiments, kits may be provided to evaluate the presence of M. tuberculosis or LAM or antibodies against LAM. Such kits can be prepared from readily available materials and reagents. For example, such kits can comprise any one or more of the following materials: enzymes, reaction tubes, buffers, detergent, primers and probes, ELISA reagents, antibodies, enzymes. In a particular embodiment, these kits allow a practitioner to obtain various biological samples.

[0143] The kit may further include an instruction sheet that outlines the procedural steps of the methods set forth herein, and will follow substantially the same procedures as described herein or are known to those of ordinary skill. The instruction information may be in a computer readable media containing machine-readable instructions that, when executed using a computer, cause the display of a real or virtual procedure of delivering a pharmaceutically effective amount of a therapeutic agent.

[0144] Kits may comprise components, which may be individually packaged or placed in a container, such as a tube, bottle, vial, syringe, or other suitable container means. The components may include probes, primers, antibodies, arrays, negative and/or positive controls. Individual components may also be provided in a kit in concentrated amounts; in some embodiments, a component is provided individually in the same concentration as it would be in a solution with other components. Concentrations of components may be provided as 1, 2, 5, 10, or 20 or more.

[0145] The components of the kits may be packaged either in aqueous media or in lyophilized form. The container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquotted. Where there is more than one component in the kit (labeling reagent and label may be packaged together), the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial. The kits may also include a means for containing the nucleic acids, antibodies or any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow molded plastic containers into which the desired vials are retained.

[0146] When the components of the kit are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.

[0147] Alternatively, the components of the kit may be provided as dried powder(s). When reagents and/or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means. In some embodiments, labeling dyes are provided as a dried power. It is contemplated that 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 g or at least or at most those amounts of dried dye are provided in kits in certain aspects. The dye may then be resuspended in any suitable solvent, such as DMSO.

[0148] The container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the nucleic acid formulations are placed, preferably, suitably allocated. The kits may also comprise a second container means for containing a sterile, pharmaceutically acceptable buffer and/or other diluent.

[0149] The kits may include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained.

[0150] A kit may also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions may include variations that can be implemented.

K. EXAMPLES

[0151] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. One skilled in the art will appreciate readily that the present invention is well adapted to carry out the objects and obtain the ends and advantages mentioned, as well as those objects, ends and advantages inherent herein. The present examples, along with the methods described herein are presently representative of particular embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

Example 1: Testing Existing Mimotopes In Vitro and in Mice

[0152] To test the ability of peptide mimotopes to induce an anti-LAM response and protect mice in vivo, either a rabbit polyclonal anti-LAM antibody or two mouse monoclonal anti-LAM antibodies (CS-35 and CS-40) were tested for their ability to bind LAM by ELISA (FIG. 1A). All three antibodies were able to bind LAM, though the rabbit polyclonal could bind at a lower titer compared to the two mouse monoclonal antibodies tested (FIG. 1A). Next 4 previously published peptides and their scrambled controls were synthesized (Table 1) (Gevorkian et al., 2005; Sharma et al., 2006; and Barenholz et al., 2007), conjugated to KLH and tested the ability of either the rabbit anti-LAM polyclonal antibody or the mouse monoclonal antibody CS-35 to bind the KLH-conjugated peptides in vitro by ELISA (FIG. 1B-J). Of the previously published peptides, only P8 could be specifically detected by the rabbit anti-LAM polyclonal antibody above control (FIG. 1E), and only at the 1:1000 dilution. Likewise, none of the peptides conjugated to KLH were bound by the CS-35 mAb (FIG. 1F-J). Next, the peptides were tested as to whether they could induce an anti-LAM immune response in mice. Outbred Swiss-Webster mice were injected 4 times with PBS, KLH alone, KLH conjugated to peptide or KLH conjugated to control peptide along with an alum adjuvant, and sera from the mice after the 4.sup.th injection were collected. Using a LAM ELISA, it was found that as with the in vitro assay, none of the vaccinations with KLH-conjugated mimotope peptides induced anti-LAM antibodies at an antibody dilution of 1:100, while the CS-35 mAb showed a strong response (FIG. 1K). As expected, immunization induced a robust anti-KLH (FIG. 1L) and anti-peptide (FIG. 1M) response. Taken together, the data indicate that none of the previously identified anti-LAM mimotope peptides could be bound by anti-LAM antibodies in vitro or induce an anti-LAM mimotope response during experimental vaccination.

TABLE-US-00001 Peptide Sequence P1 CQEPLMGTVPIRAGGGS P1scramble CQLGAIEMPGSPGTVGR P1biotin CQEPLMGTVPIRAGGGSR-Biotin P4 CMSPRATI P4scramble CIPARMTS P6 CSHRLLQTYWSSA P6scramble CQSTSHLYASWLR P8 CISLTEWSMWYRH P8scramble CRSWEWHSTLMYI HS CHSFKWLDSPRLR HSscramble CPHDFRLWSLSRK SG CSGVYKVAYDWQH SGscramble CGSYVVAYWDKHQ

Example 2: Screening Phage Display Libraries with Anti-LAM Monoclonal Antibodies CS-35 and CS-40

[0153] Since monoclonal antibodies have defined epitope specificity compared to polyclonal antibodies, biopanning with the monoclonal antibodies rather than the polyclonal antibody was pursued. Five rounds of bio-panning of a 12-mer phage display library (NEB) were conducted independently, with either CS-35 or CS-40. As expected, the number of high-affinity phages recovered increased significantly over the course of the biopanning. After the fifth panning cycle, 10 randomly selected phages per antibody were sequenced. When biopanning with CS-40 mAb, 2 peptide motifs predominated: HSFKWLDSPRLR (hereafter called 232 HS peptide after the two N-terminal amino acids OR SEQ ID NO:1) accounted for 70% ( 7/10) of the sequenced phages, while SGVYKVAYDWQH (hereafter called SG peptide or SEQ ID NO:2) accounted for 30% ( 3/10). Biopanning with CS-35 mAb revealed the predominance of only 1 peptide motif, SGVYKVAYDWQH, which was identical to the SG peptide obtained from biopanning with CS-40. To determine if the two identified peptide motifs represented false-positive target-unrelated peptides (TUP) (Bakhshinejad et al., Amino Acids. 2016; 48:2699-716), the SAROTUP (Huang et al., J Biomed Biotechnol. 2010; 2010:101932) and Mimo-DB (Huang et al. Nucleic Acids Res. 2012; 40:D271-7) databases were searched with each peptide. While neither peptide was identified as having previously been found to be a TUP using the TUPScan software, both peptides had previously been identified during other target searches. In particular, the SG peptide has been identified 5 times previously (Chen et al., Mol Pharm. 2015; 12:2180-8; Ren et al., Biochem Pharmacol. 2016; 107:91-100; and Diaz-Perlas et al., Biopolymers. 2016), while the HS peptide has only been identified once (Chen et al., 2015). Interestingly, both the SG and HS peptide were identified in the same screen for peptides that bind the calcium-independent mannose-6-phosphate receptor (Chen et al., 2015). Thus, since LAM is composed of a number of mannose residues and the calcium-independent mannose-6-phosphate receptor binds a modified mannose motif, it is feasible that both peptide mimotopes create a three-dimensional structure similar to one or more mannose residues.

Example 3: Characterization of Mimotope Behavior of Identified Peptides

[0154] To determine if the putative mimotope peptides identified by biopanning using the mouse monoclonal antibodies can also be detected by LAM antibodies from another species, the ability of the rabbit polyclonal anti-LAM antibody to bind the putative mimotope peptides was tested. Thus, plates were coated with LAM alone as a positive control, HS-conjugated phage, SG-conjugated phage or phage alone and the magnitude of binding of the rabbit anti-LAM antibody was determined. Coating the plate with LAM yielded the greatest OD value (3.23), which was similar to coating with the HS phage (2.82) (FIG. 2A). Coating wells with an equivalent amount of SG-conjugated phage yielded the lowest response (OD 1.56).

[0155] The ability of the rabbit anti-LAM polyclonal antibody to recognize the mimotope peptides under a variety of conditions was compared. Thus, plates were coated with LAM as a positive control or HS peptide alone, HS peptide displayed on bacteriophage, HS peptide conjugated to KLH, the scrambled version of the HS peptide (HSSR) or the HSSR peptide conjugated to KLH and the magnitude of binding of rabbit polyclonal anti-LAM antibody to each hapten was determined by ELISA. The OD values were 2.8, 0.45, 1.81, and 2.56 for LAM, HS peptide alone, HS phage, and HS-KLH respectively (FIG. 2B). No binding was detected for the scramble peptide alone or when conjugated to KLH. When the experiment was repeated for the SG peptide, the OD values were 2.8, 0.32, 1.36, and 0.57 for LAM, SG peptide alone, SG phage, and SG-KLH respectively (FIG. 2C). For both peptides, coating with synthetic peptide alone did not result in significant antibody binding. In contrast, while the anti-LAM polyclonal antibody bound both HS and SG peptides displayed on bacteriophage, only the HS-peptide could be bound when conjugated to KLH. Thus, it can be concluded that both peptides require conjugation to a larger protein to be successfully recognized by anti-LAM antibody, and that the HS peptide is more consistently detected by the anti-LAM polyclonal antibody. Finally, to determine if mice can generate anti-LAM antibodies after vaccination with LAM, and if such antibodies can detect the HS-peptide, Swiss-Webster mice were vaccinated with LAM combined with alum and tested the serum against various antigens by ELISA (FIG. 2D). As expected, serum from LAM vaccinated mice robustly bound LAM. Mouse anti-LAM antibodies also bound the HS-peptide conjugated to KLH, but not the HSSR control peptide conjugated to KLH or KLH alone (FIG. 2D). Thus, it can be concluded that anti-LAM antibodies from either rabbit (polyclonal) or mouse (monoclonal or polyclonal) bind the HS-peptide when it is conjugated to a carrier molecule.

Example 4: Mimotope Activity of KLH-Conjugated HS-Peptide in Vivo

[0156] To confirm that the HS-peptide had mimotope activity in vivo, Swiss-Webster mice were vaccinated with KLH-conjugated HS284 peptide or KLH-conjugated HSSR peptide with alum adjuvant and their antibody responses were measured after 4 injections with KLH-conjugated mimotope peptides. All mice vaccinated with the HS peptide conjugated 286 to KLH generated antibodies against HS peptide and KLH but not the HSSR peptide or BSA, as expected (FIG. 3A). Likewise, all mice vaccinated with the HSSR peptide conjugated to KLH generated antibodies against HSSR peptide and KLH but not HS peptide or BSA (FIG. 3B) However, only mice vaccinated with the HS peptide conjugated to KLH developed antibodies against LAM, though there was significant variation in the response of individual mice (FIG. 3A). No HSSR-vaccinated mice generated antibodies reactive with LAM by ELISA (FIG. 3B). Taken together, the data show that the HS peptide can function as a mimotope in vivo when conjugated to the hapten KLH in the presence of an adjuvant.

Example 5: Peptide Conjugation to Baculovirus

[0157] While in the process of biopanning for novel mimotopes, the ability to conjugate peptides successfully to baculovirus as has been previously reported was tested (Wilson et al., 2008). A biotinylated version of Peptide 1 (Table 1) was synthesized, conjugated to baculovirus and then the conjugation was tested by Western blot and ELISA. Both peptide conjugated baculovirus (FIG. 4A, lane 1) and free peptide (FIG. 4A, lane 2) could be detected with an anti-biotin antibody, but baculovirus alone (FIG. 4A, lane 3) could not.

Example 6: Immunogenicity of Mimotope Conjugated Baculovirus

[0158] To test the ability of HS-peptide to be recognized as a LAM mimotope when conjugated to baculovirus, ELISA plates were coated with HS-peptide conjugated to baculovirus, the HSSR-peptide conjugated to baculovirus as well as controls and measured binding by anti-LAM ELISA. The anti-LAM antibody bound LAM, the HS-peptide conjugated to phage and also to baculovirus (FIG. 4B). Neither baculovirus alone nor HSSR-peptide conjugated baculovirus were detected by the anti-LAM antibody, demonstrating the specificity of the HS-peptide.

[0159] To further test the immunogenicity of the baculovirus conjugates as LAM mimotopes, mice were vaccinated intranasally with either HS-conjugated baculovirus or HSSR-conjugated baculovirus and evaluated the serum IgG response after 4 vaccinations. While both vaccinations yielded anti-baculovirus antibodies (FIG. 4C), only vaccination with the HS-peptide conjugated baculovirus induced an anti-LAM mimotope response (FIG. 4C). This mimotope activity was lower than the activity induced by KLH-conjugated HS peptide (compare FIG. 4C to FIG. 3A). In addition, no anti-LAM IgA antibodies could be detected in broncheoalveolar lavage of vaccinated mice (data not shown).

Example 7: Effect of Vaccination on Mouse Survival

[0160] To test the ability of baculovirus conjugated HS-peptide to protect mice from tuberculosis, Swiss-Webster mice were vaccinated with either baculovirus alone, HS-conjugated baculovirus, HSSR-conjugated baculovirus or BCG as a positive control and then all of the mice were infected with 200 CFU of M. tuberculosis Erdman strain via aerosol. Vaccination with the HS-conjugated baculovirus partially protected mice from M. tuberculosis mortality 321 compared to baculovirus alone, though the protection was not as robust as vaccination with BCG (FIG. 4D). Despite the relatively modest mimotope response, vaccination with the HS-baculovirus did protect mice against M. tuberculosis mortality, though not as effectively as the established BCG vaccine. To that end, identification of mimotopes against other important cell wall antigens might provide even greater protection when combined with anti-LAM mimotopes.

Example 8: Experimental Procedures

[0161] A. LAM Antibodies and Screening

[0162] Anti-LAM mouse monoclonal antibodies specific either for non-mannose-capped LAM, CS35 (Antibody class IgG3; NR-13811), or mannose-capped LAM (ManLAM), CS40 (Antibody class IgG1; NR-13812) and rabbit polyclonal rabbit anti-Mtb LAM antiserum (NR-13821) were generously provided by BEI Resources NIAID, NIH. To compare binding reaction of these antibodies with LAM, a LAM coating ELISA was performed. Five g of LAM (NR-14848) provided by BEI Resources NIAID, NIH was used as a coating antigen following classic ELISA methods. Secondary anti-mouse and anti-rabbit antibodies conjugated to HRP were from Jackson ImmunoResearch Laboratories Inc. (catalogue #715-035-151 and 111-035-003, respectively). Detection of bound antibody complexes was with the 1-Step Ultra TMB-ELISA substrate solution (Thermo Scientific Co, Ltd; catalogue #34028).

[0163] B. Screening of Phage Displayed Peptide Library

[0164] Seven-mer (E8100s) and twelve-mer (E8110S) phage display peptide libraries (New England Biolabs) were panned with the selected mAb and titrated by plaque assay according to manufacturer's instructions. Briefly, a well of a 96-well microtiter plate was sensitized with 25 g of selected mAb and incubated at 4 C. overnight. Blocking was performed with 350 l of BSA (5 mg/ml) in 50 mM Tris-buffered saline (TBS) for 1 hour at room temperature followed by six washes with 50 mM TBS containing 0.1% Tween 20 (0.1% TBS-T) to remove excess antibody and blocking reagent. Approximately 21011 pfu recombinant phage were added to the well and incubated for 1 hour. Unbound phage were removed by repeat (10) washing with 0.1% TBS-T. Antibody-bound phage were eluted by treatment with 100 l of 0.2M Glycine-HCl (pH 2.2) followed by 15 l of 1M Tris-Cl (pH 9.1) containing BSA. The eluted phage were amplified in log phase E. coli ER2738 and harvested in the culture supernatant at 12,000g for 10 minutes 125 and concentrated by PEG/NaCl precipitation. Bio-panning was repeated 3 times on amplified phage eluate using a wash buffer containing 0.5% Tween 20 (0.5% TBS-T) to enrich the pool of phage with strong binding affinity to the mAb. Eluted and amplified phages were titrated after each round of panning.

[0165] C. Sequencing of mAb-Specific Phage

[0166] Following manufacturer's instructions, 10 phage colonies recovered after the fourth panning step were randomly selected and amplified. Each selected phage was excised from the bacterized agar and cultured in E. coli ER2738. Phage genomic DNA was extracted by treatment with Iodide buffer (pH 8.0) and purified by ethanol precipitation. The phage heptapeptide-gIII fusion gene was then sequenced and the corresponding amino acid sequence of the 7 or 12-mer peptide was deduced from the resulting nucleotide sequence using the reduced genetic code chart.

[0167] D. Peptide Synthesis

[0168] Peptides were synthesized and are summarized in Table 1. All peptides were synthesized with an N-terminal cysteine residue to permit chemical conjugation to KLH and baculovirus. Synthetic peptides were dissolved in distilled water and kept 20 C. until use.

[0169] E. Peptide ELISA Binding Assay

[0170] The ELISA plate wells were coated with by adding each peptide (2 g in 0.1M NaHCO.sub.3, pH 8.6) and incubating overnight at 4 C. After overnight incubation, plates were blocked with BSA (5 mg/ml) for 1 hour at 4 C. For controls, wells were coated with same amount of LAM or BSA. After washing with TBST, diluted 143 anti-LAM antibodies were added to the ELISA plate and incubated at room temperature for 1 hour. Plates were washed with TBST and HRP-conjugated secondary antibodies were added and incubated for 1 hour. After washing 3 times with TBST, 50 l of the 1-Step Ultra TMB-ELISA (Thermo Scientific Co, Ltd) substrates was added in the dark. After color development, 50 l of 2M sulfuric acid was added to stop the reaction and absorbance was measured at 450 nm.

[0171] F. KLH Conjugation

[0172] Peptide conjugation to KLH was performed using Imject Maleimide Activated Carrier Protein Kits (Thermo Scientific Co., Ltd; catalogue #77115) following the manufacturer's instructions.

[0173] G. Baculovirus Conjugation

[0174] Wild type AcMNPV (5108 PFU/ml) was obtained from Kinnakeet Biotechnology (USA). AcMNPV conjugation with target peptides was performed using the Sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate) reagent (Thermo Scientific catalogue #22322) following manufacturer's instructions as described (Wilson et al., 2008). Briefly, 4 mg/ml of Sulfo-SMCC crosslinking reagent was added to 10 mg AcMNPV in conjugation buffer and incubated for 30 minutes at room temperature. After desalting to remove unbound crosslinker, 10 g of peptide was added to the SMCC crosslinker-coated AcMNPV and incubated for 20 minutes at room temperature followed by a desalting step to remove unbound peptide. The AcMNPV-peptide conjugates were then stored at 80 C. until use.

[0175] H. Mouse Vaccination to Measure Antibody Response

[0176] Swiss Webster outbred mice (5 mice/group, Taconic Biosciences) were vaccinated with PBS or mimotopes-conjugated to KLH or baculovirus. For mice receiving PBS, KLH alone or peptide-conjugates to KLH, inoculations were via subcutaneous injection. For the baculovirus groups, inoculations were intranasal. On day 0, serum from each mouse was collected from the retroorbital plexus (pre-immune), and 10 g of peptide-conjugate or control was inoculated per animal. For testing mimotopes conjugated to KLH, vaccination also included alum adjuvant. Animals were boosted every two week interval for 3 additional times. Before each boost, serum was collected from each mouse and stored at 20 C. until use.

[0177] I. Mouse Serology

[0178] To perform ELISAS, 96-well ELISA plates (Greiner Bio-One Catalogue #655-001) were coated with 10 g of corresponding coating antigens: HS peptide, HSSR peptide, Baculovirus, SG peptide, SGSR peptide, or LAM resuspended in coating buffer (0.1M NaHCO.sub.3, pH 8.6). After overnight coating at 4 C., blocking (5% skim milk in PBS) was performed for 1 hour at room temperature. After washing 3 times with washing buffer (0.01% Tween-20 in PBS), mouse serum was added to the appropriate wells and binding proceeded at room temperature for 1 hour. Plates were washed 3 times with washing buffer (0.01% Tween-20 in PBS) and HRP177 conjugated anti-mouse IgG at 1:5,000 dilution (Jackson ImmunoResearch Laboratories Inc. catalogue #715-035-151) was added to each well. After incubating for 1 hour at room temperature, the plate was washed 3 times with TBST and 50 l 179 of the 1-Step Ultra TMB180 ELISA substrate (Thermo Scientific) was added. 50 ul of 2M sulfuric acid was then added to stop the reaction and absorbance was measured at 450 nm.

[0179] J. Mouse Aerosol Infection and Survival Study

[0180] To test the ability of the anti-LAM mimotope to protect mice from subsequent tuberculosis infection, outbred Swiss Webster mice were first vaccinated according to the protocol described above. Mice receiving BCG Pasteur were vaccinated once subcutaneously (s.c.) with 106 bacteria in 0.2 ml PBS 8 weeks prior to receiving a M. tuberculosis Erdman challenge. After 3 booster injections, mice were then infected with 200 CFU of M. tuberculosis Erdman strain in a Glas-Col aerosolization chamber as described (Nair et al., Cell reports. 2016; 16:1253-8; Collins et al., Cell host & microbe. 2015; 17:820-8; and Zacharia et al., MBio. 2013; 4:e00721-13). On day 0, 5 unvaccinated mice were sacrificed to determine the initial CFU. Afterwards, mice were monitored weekly for weight loss and were euthanized when they had lost >15% of their body weight.

[0181] K. Statistical Analysis

[0182] Statistical analysis was performed using GraphPad Prism version 6.01. Two tailed unpaired Student's t test was used for single comparisons. Analysis of Variance (ANOVA) was used for experiments with multiple comparisons. Gehan-Breslow-Wilcoxon test was used for mouse survival experiments.

[0183] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0184] The references recited in the application, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.