Synthetic fragment of mycobacterium tuberculosis methylglucose lipopolysaccharide with the capacity to induce protection against tuberculosis

Abstract

The present disclosure is directed to the generation and use of a synthetic tetrasaccharide designed based on a portion of a mycobacterial polar glycolipid as a vaccine antigen to limit or prevent tuberculosis infections.

Description

[0001] DESCRIPTION SYNTHETIC FRAGMENT OF MYCOBACTERIUM TUBERCULOSIS METHYLGLUCOSE LIPOPOLYSACCHARIDE WITH THE CAPACITY TO INDUCE PROTECTION AGAINST TUBERCULOSIS STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT This invention was made with government support under R01AI048391 awarded by National Institutes of Health. The government has certain rights in the invention. PRIORITY CLAIM This application claims benefit of priority to U.S. Provisional Application Serial No. 63 / 704,270, filed October 7, 2024, the entire contents of which are hereby incorporated by reference. BACKGROUND 1. Field of the Disclosure The present disclosure relates generally to the fields of immunology, infectious disease and medicine. More particularly, the disclosure relates to a synthetic tetrasaccharide fragment matching the nonreducing end of 6-O-methyl-glucose lipopolysaccharide (mGLP). 2. Background Tuberculosis (TB), also known colloquially as the "white death", or historically as consumption, is an infectious disease usually caused by Mycobacterium tuberculosis (Mtb) bacteria. Tuberculosis generally affects the lungs, but it can also affect other parts of the body. Most infections show no symptoms, in which case it is known as latent tuberculosis. Around 10% of latent infections progress to active disease which, if left untreated, kill about half of those affected. Typical symptoms of active TB are chronic cough with blood-containing mucus, fever, night sweats, and weight loss. Infection of other organs can cause a wide range of symptoms. Treatment requires the use of multiple antibiotics over a long period of time. Antibiotic resistance is a growing problem, with increasing rates of multiple drug- resistant tuberculosis (MDR-TB). Tuberculosis is spread from one person to the next through the air when people who have active TB in their lungs cough, spit, speak, or sneeze. People with latent TB do not spread the disease. Active infection occurs more often in people with HIV / AIDS and in those who smoke. Diagnosis of active TB is based on chest X-rays, as well as microscopic examination and culture of body fluids. Diagnosis of latent TB relies on the tuberculin skin test (TST) or blood tests. Prevention of TB involves screening those at high risk, early detection and treatment of cases, and vaccination with the Bacillus Calmette-Guérin (BCG) vaccine. Those at high risk include household, workplace, and social contacts of people with active TB. Tuberculosis prevention and control efforts rely primarily on the vaccination of infants and the detection and appropriate treatment of active cases. The World Health Organization (WHO) has achieved some success with improved treatment regimens, and a small decrease in case numbers. Some countries have legislation to involuntarily detain or examine those suspected of having tuberculosis, or involuntarily treat them if infected. The only available vaccine as of 2024 is BCG. In children it decreases the risk of getting the infection by 20% and the risk of infection turning into active disease by nearly 60%. It is the most widely used vaccine worldwide, with more than 90% of all children being vaccinated. The immunity it induces decreases within 5-10 years. As tuberculosis is uncommon in most of Canada, Western Europe, and the United States, BCG is administered to only those people at high risk. Part of the reasoning against the use of the vaccine is that it makes the tuberculin skin test falsely positive, reducing the test's usefulness as a screening tool. As such, improved vaccines with higher effectiveness, lower cost and longer duration are needed. The inventors’ lab reported in 1998 the first evidence that human γ9δ2 T cells could develop a memory immune response which made them potential vaccine targets (1). More importantly they reported that mycobacteria-specific identified TB-specific human γ9δ2 T cells could recognize human monocytes infected with Mtb or BCG and inhibit the intracellular replication of these mycobacteria (2, 3). Then in collaboration with Drs. Dobos and Chatterjee at Colorado State University, they found specific polar lipid Mtb antigens contained within the cell wall, methylglucose polysaccharides (mGLP), that could induce activation and expansion of γ9δ2T cells that could potently inhibit intracellular mycobacterial replication (4, 5). Nonetheless, further research is needed to determine which components in this compound are not only necessary but sufficient for stimulating an effective immune response.

[0002] 2 {01122399}4928-7409-1886, v. 1 SUMMARY Thus, in accordance with the present disclosure, there is provided a tetrasaccharide having the following formula: (I) C1-C8 substituted acyl; R2 is C1-C8 alkyl, C1-C8 substituted alkyl, C1-C8 acyl, C1-C8 substituted acyl, or ; R3is alkyl, C1-C8 substituted alkyl, C1-C8 acyl, C1-C8 substituted acyl; R4is C1-C8 alkyl, C1-C8 substituted alkyl, C1-C8 acyl, C1-C8 substituted acyl, or ; R5 is wherein Y1 is C1-C12 alkanediyl or C1-C12 substituted alkanediyl and R′ and R′′ are each independently hydrogen, C1-C8 alkyl, C1- C8 substituted alkyl, C1-C8 acyl, or C1-C8 substituted acyl; or

[0003] 3 {01122399}4928-7409-1886, v. 1 a monosaccharide or disaccharide, optionally comprising one or more methyl groups. R1 may be –C(O)CH(CH3)2, and / or R2 may be –C(O)CH3. R3 may be hydrogen, R4 may be – C(O)CH3, and / or R5 may be –(CH2)8NHAc. The polysaccharide may be further defined as: polysaccharide as defined herein, dispersed in a pharmacologically acceptable buffer, diluent or excipient. The pharmaceutical composition of claim 8 may be formulated for injection, and / or further comprise an adjuvant. Such a pharmaceutical composition may comprise a polysaccharide further defined as:

[0004] 4 {01122399}4928-7409-1886, v. 1

[0005] In another embodiment, there is provided a method of inducing an immune response in a subject comprising administering to said subject the polysaccharide or the pharmaceutical formulation as defined herein. The subject may be at increased risk of developing tuberculosis as compared to populational average and / or the subject may have previously received a tuberculosis vaccine or not previously received a tuberculosis vaccine. The method may further comprise administering said polysaccharide or pharmaceutical composition a second time. The administering may comprise intramuscular, subcutaneous, intradermal, aerosol, intranasal or oral delivery. Administering may be repeated at least once. The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and / or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The word “about” means plus or minus 5% of the stated number. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein. Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0006] 5 {01122399}4928-7409-1886, v. 1 BRIEF DESCRIPTION OF THE DRAWINGS The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. FIG.1. Chemical structure of mGLP from Mtb (4, 5). Sugars are color-coded. Black = terminal 3-O-Me Glc; Green = variably acylated Glc; Red = 6-O-me-Glc; Purple = Glc. All main chain linkages are α-(1->4). Treatment with α-amylase hydrolyzes α-linked glucans, but mGLP is a substrate only when the first Glc on the terminal 6-O-me-Glc is not aceylated (R = H); the 6-O-methylation of Glc residues prevent cleavage. Native mGLP has both acetylated (R = Ac) and non-acylated Glc (R = H). The reducing end is composed of diglucosylglycerate (DGG) and the second Glc of DGG is α-(1->4) linked to the mGLP main chain, which contains two additional branching β-(1->3)-linked Glc (in blue) to which succinate esters are attached (6). Upon treatment with mild alkali (deacylation) all esters are removed but the sugar backbone is unaffected. The location of the octanoate ester has been debated (6;7;5) but it is currently thought to be located as shown. FIGS.2A-C. A synthetic tetrasaccharide matching the non-reducing end of native mGLP expands a subset of anti-mycobacterial Vgamma9-Vdelta2 T cells. (FIG. 1A) PBMC from 2 PPD+volunteers were cultured for 7 days with medium alone, live BCG, or native mGLP versus synthetic mGLP (smGLP) before flow cytometry to identify expanded gamma delta T cells. (FIG. 1B) Gamma delta T cell expansion indices were compared to medium plus IL-2 alone. (FIG. 1C) Expanded gamma delta T cells were purified by immunomagnetic negative selection and co-cultured with BCG-infected syngeneic monocytes for 3 days in intracellular Mycobacterial Growth Inhibition Assays (MGIA) after which the estimated persistently viable BCG were assessed by tritiated uridine incorporation. FIG.3. Synthesis of mGLP derivative 1f. A key feature of the strategy is to minimize the number of glycosylation reactions needed, by using commercially available D-maltose, which already has one α-(1→4)-glucosyl-linkage, as one of the starting materials. Thus, D- maltose and D-glucose (also commercial) were converted to appropriate building blocks (4–6),

[0007] 6 {01122399}4928-7409-1886, v. 1 which were then coupled to give protected tetrasaccharide 7; subsequent N-acetylation 7 and deprotection yielded 1f. In the glycosylation reactions, we relied on powerful methodology reported by Codée and coworkers (Wang et al., J Am Chem Soc., 2018, PMID: 29553729).

[0008] 7 {01122399}4928-7409-1886, v. 1 DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS As discussed above, there remains a need for improved vaccine formulations that can reduce, limit or prevent development of tuberculosis. The inventors’ prior research on mGLP led them to develop a synthetic fragment of mGLP matching the tetrasaccharide on the non- reducing end of the native mGLP molecule. The mGLP purified from Mtb cell walls was shown in a small, to date unpublished study, to induce apparent protective γ9δ2 T cells in nonhuman primates which were associated with reduced pathogen loads and pathology after Mtb challenge (unpublished). Then, Dr. Hoft recruited Dr. Lowary, an additional glycochemist whose lab synthesized a tetrasaccharide matching the non-reducing end of native mGLP based on their hypothesis that this fragment of mGLP contained the biological activity capable of inducing protective γ9δ2T cells (unpublished but shown below in Figure 1). Being a synthetic molecule not purified from native virulent Mtb and potentially amenable to GMP scaled-up manufacturer, this synthetic molecule can be used as a novel TB vaccine with potential to be protective in humans. As noted above, the inventors have demonstrated that this synthetic tetrasaccharide was able to activate human gamma / delta T cells capable of recognizing and inhibiting intracellular mycobacteria. Normally, targeting alpha / beta T cells is problematic because these cells respond to antigens presented to human T cells by highly polymorphic HLA molecules, in contrast to the ability of the potential for an antigen like mGLP to induce immunity in everyone expressing a highly conserved nonpolymorphic restriction element, so the ability of the synthetic tetrasaccharide is a key aspect of its immunobiology. The synthetic mGLP fragment can activate gamma / delta T cells in the context of highly conserved butyrophylin molecules that are the same in all humans. Therefore, a synthetic mGLP fragment vaccine could induce the same protective gamma / delta T cell responses in all humans (universally relevant for all people). Furthermore, since Mtb has a relatively stable genome and Mtb genes involved in drug resistance are not known to be involved in T cell immunity, a synthetic mGLP fragment vaccine should work universally against all Mtb drug sensitive and resistant strains of the pathogen. These and other aspects of the disclosure are described in detail below. I. Mycobacterium tuberculosis Mycobacterium tuberculosis (Mtb), also known as Koch's bacillus, is a species of pathogenic bacteria in the family Mycobacteriaceae and the causative agent of tuberculosis. Mtb has an unusual, waxy coating on its cell surface primarily due to the presence of mycolic

[0009] 8 {01122399}4928-7409-1886, v. 1 acid. This coating makes the cells susceptible to Gram staining more strongly than with most organisms for which acid washes remove the Gram stain, and as a result, Mtb can appear weakly Gram-positive. Acid-fast stains such as Ziehl–Neelsen, or fluorescent stains such as auramine are used instead to identify Mtb with a microscope. The physiology of Mtb is highly aerobic and requires high levels of oxygen. Primarily a pathogen of the mammalian respiratory system, it infects the lungs. The most frequently used diagnostic methods for tuberculosis are the tuberculin skin test, acid-fast stain, culture, and polymerase chain reaction. Mtb requires oxygen to grow and is nonmotile. It divides every 18–24 hours. This is extremely slow compared with other bacteria, which tend to have division times measured in minutes. It is a small bacillus that can withstand weak disinfectants and can survive in a dry state for weeks. Its unusual cell wall, rich in lipids such as mycolic acid and cord factor glycolipid, is likely responsible for its resistance to desiccation and is a key virulence factor. Humans are the only known reservoirs of Mtb. A misconception is that Mtb can be spread by shaking hands, making contact with toilet seats, sharing food or drink, or sharing toothbrushes. However, major spread is through the respiratory route via air droplets or aerosols originating from a person who has the disease either coughing, sneezing, speaking, or singing. When in the lungs, Mtb is phagocytosed by alveolar macrophages, but they are unable to kill and digest the bacterium. Its cell wall is made of cord factor glycolipids that inhibit the fusion of the phagosome with the lysosome, which contains a host of antibacterial factors. Specifically, Mtb blocks the bridging molecule, early endosomal autoantigen 1 (EEA1); however, this blockade does not prevent fusion of vesicles filled with nutrients. In addition, production of the diterpene isotuberculosinol prevents maturation of the phagosome. The bacteria also evade macrophage-killing by neutralizing reactive nitrogen intermediates. More recently, Mtb has been shown to secrete and cover itself in 1-tuberculosinyladenosine (1- TbAd), a special nucleoside that acts as an antacid, allowing it to neutralize pH and induce swelling in lysosomes. Granulomas, organized aggregates of immune cells, are a hallmark feature of tuberculosis infection. Granulomas play dual roles during infection: they regulate the immune response and minimize tissue damage, but also can aid in the expansion of infection. The ability to construct Mtb mutants and test individual gene products for specific functions has significantly advanced the understanding of its pathogenesis and virulence

[0010] 9 {01122399}4928-7409-1886, v. 1 factors. Many secreted and exported proteins are known to be important in pathogenesis. For example, one such virulence factor is cord factor (trehalose dimycolate), which serves to increase survival within its host. Resistant strains of Mtb have developed resistance to more than one TB drug, due to mutations in their genes. In addition, pre-existing first-line TB drugs such as rifampicin and streptomycin have decreased efficiency in clearing intracellular Mtb due to their inability to effectively penetrate the macrophage niche. Symptoms of Mtb include coughing that lasts for more than three weeks, hemoptysis, chest pain when breathing or coughing, weight loss, fatigue, fever, night sweats, chills, and loss of appetite. Mtb also has the potential of spreading to other parts of the body. This can cause blood in urine if the kidneys are affected, and back pain if the spine is affected. Mtb is a clonal organism and does not exchange DNA via horizontal gene transfer. Despite an additionally slow evolution rate, the emergence and spread of antibiotic resistance in Mtb poses an increasing threat to global public health. In 2019, the WHO reported the estimated incidence of antibiotic resistant (ABR) TB to be 3.4% in new cases, and 18% in previously treated cases. Geographical discrepancies exist in the incidence rates of drug- resistant TB. Countries facing the highest rates of ABR TB China, India, Russia, and South Africa. Recent trends reveal an increase in drug-resistant cases in a number of regions, with Papua New Guinea, Singapore, and Australia undergoing significant increases. Multidrug-resistant Tuberculosis (MDR-TB) is characterized by resistance to at least the two front-line drugs isoniazid and rifampin. MDR is associated with a relatively poor treatment success rate of 52%. Isoniazid and rifampin resistance are tightly linked, with 78% of the reported rifampin-resistant TB cases in 2019 being resistant to isoniazid as well. Rifampin-resistance is primarily due to resistance-conferring mutations in the rifampin- resistance determining region (RRDR) within the rpoB gene. The most frequently observed mutations of the codons in RRDR are 531, 526 and 516. However, alternative more elusive resistance-conferring mutations have been detected. Isoniazid function occurs through the inhibition of mycolic acid synthesis through the NADH-dependent enoyl-acyl carrier protein (ACP)-reductase. This is encoded by the inhA gene. As a result, isoniazid resistance is primarily due to mutations within inhA and the KatG gene or its promoter region - a catalase peroxidase which is required to activate Isoniazid. As MDR in Mtb becomes increasingly common, the emergence of pre-extensively drug resistant (pre-XDR) and extensively drug resistant (XDR-) TB threatens to exacerbate the public health crisis. XDR-TB is characterised by resistance to both rifampin and Isoniazid, as well second-line fluoroquinolones and at least

[0011] 10 {01122399}4928-7409-1886, v. 1 one additional front-line drug. Thus, the development of alternative therapeutic and / or prophylactic measures are of utmost priority. II. mGLP Through a multi-step route starting from glucose and maltose, a tetrasaccharide matching the non-reducing end of native mGLP complete with native mGLP acetylation, isobutylation and methylation has been generated: were confirmed by a combination of one- and two-dimensional1H and13C Nuclear Magnetic Resonance (NMR) spectroscopy, and high-resolution mass spectrometry (HRMS). The Hoft lab has shown that this tetrasaccharide activates γδ T cells with the ability to inhibit intracellular replication of mycobacteria. FIG. 3 summarizes our synthesis of the mGLP nonreducing end tetrasaccharide. The main synthetic challenge is the stereoselective α-glycosylation reactions (8). Therefore, a key feature of the inventors’ strategy is to minimize the number of glycosylation reactions needed, by using commercially available D-maltose, which already has one α-(1→4)-glucosyl-linkage, as one of the starting materials. Thus, D-maltose and D-glucose (also commercial) were converted to appropriate building blocks (4–6), which were then coupled to give protected tetrasaccharide 7; subsequent N-acetylation 7 and deprotection yielded 1f. In the glycosylation reactions, the inventors relied on powerful methodology reported by Codée and coworkers (Wang et al., 2018).

[0012] 11 {01122399}4928-7409-1886, v. 1 For the chemical groups and compound classes, the number of carbon atoms in the group or class is as indicated as follows: “Cn” or “C=n” defines the exact number (n) of carbon atoms in the group / class. “C≤n” defines the maximum number (n) of carbon atoms that can be in the group / class, with the minimum number as small as possible for the group / class in question. For example, it is understood that the minimum number of carbon atoms in the groups “alkyl(C≤8)”, “alkanediyl(C≤8)”, “heteroaryl(C≤8)”, and “acyl(C≤8)” is one, the minimum number of carbon atoms in the groups “alkenyl(C≤8)”, “alkynyl(C≤8)”, and “heterocycloalkyl(C≤8)” is two, the minimum number of carbon atoms in the group “cycloalkyl(C≤8)” is three, and the minimum number of carbon atoms in the groups “aryl(C≤8)” and “arenediyl(C≤8)” is six. “Cn-n′” defines both the minimum (n) and maximum number (n′) of carbon atoms in the group. Thus, “alkyl(C2-10)” designates those alkyl groups having from 2 to 10 carbon atoms. These carbon number indicators may precede or follow the chemical groups or class it modifies and it may or may not be enclosed in parenthesis, without signifying any change in meaning. Thus, the terms “C1-4-alkyl”, “C1-4-alkyl”, “alkyl(C1-4)”, and “alkyl(C≤4)” are all synonymous. Except as noted below, every carbon atom is counted to determine whether the group or compound falls with the specified number of carbon atoms. For example, the group dihexylamino is an example of a dialkylamino(C12) group; however, it is not an example of a dialkylamino(C6) group. Likewise, phenylethyl is an example of an aralkyl(C=8)group. When any of the chemical groups or compound classes defined herein is modified by the term “substituted”, any carbon atom in the moiety replacing the hydrogen atom is not counted. Thus methoxyhexyl, which has a total of seven carbon atoms, is an example of a substituted alkyl(C1-6). Unless specified otherwise, any chemical group or compound class listed in a claim set without a carbon atom limit has a carbon atom limit of less than or equal to twelve. The term “alkyl” refers to a monovalent saturated aliphatic group with a carbon atom as the point of attachment, a linear or branched acyclic structure, and no atoms other than carbon and hydrogen. The groups −CH3 (Me), −CH2CH3 (Et), −CH2CH2CH3 (n-Pr or propyl), −CH(CH3)2(i-Pr,iPr or isopropyl), −CH2CH2CH2CH3(n-Bu), −CH(CH3)CH2CH3(sec-butyl), −CH2CH(CH3)2 (isobutyl), −C(CH3)3 (tert-butyl, t-butyl, t-Bu ortBu), and −CH2C(CH3)3 (neo- pentyl) are non-limiting examples of alkyl groups. The term “alkanediyl” refers to a divalent saturated aliphatic group, with one or two saturated carbon atom(s) as the point(s) of attachment, a linear or branched acyclic structure, no carbon-carbon double or triple bonds, and no atoms other than carbon and hydrogen. The groups −CH2− (methylene), −CH2CH2−, −CH2C(CH3)2CH2−, and −CH2CH2CH2− are non-limiting examples of alkanediyl groups. The

[0013] 12 {01122399}4928-7409-1886, v. 1 term “alkylidene” refers to the divalent group =CRR′ in which R and R′ are independently hydrogen or alkyl. Non-limiting examples of alkylidene groups include: =CH2, =CH(CH2CH3), and =C(CH3)2. An “alkane” refers to the class of compounds having the formula H−R, wherein R is alkyl as this term is defined above. The term “acyl” refers to the group −C(O)R, in which R is a hydrogen, alkyl, cycloalkyl, or aryl as those terms are defined above. The groups, −CHO, −C(O)CH3(acetyl, Ac), −C(O)CH2CH3, −C(O)CH(CH3)2, −C(O)CH(CH2)2, −C(O)C6H5, and −C(O)C6H4CH3 are non- limiting examples of acyl groups. A “thioacyl” is defined in an analogous manner, except that the oxygen atom of the group −C(O)R has been replaced with a sulfur atom, −C(S)R. The term “aldehyde” corresponds to an alkyl group, as defined above, attached to a −CHO group. When a chemical group is used with the “substituted” modifier, one or more hydrogen atom has been replaced, independently at each instance, by −OH, −F, −Cl, −Br, −I, −NH2, −NO2, −CO2H, −CO2CH3, −CO2CH2CH3, −CN, −SH, −OCH3, −OCH2CH3, −C(O)CH3, −NHCH3, −NHCH2CH3, −N(CH3)2, −C(O)NH2, −C(O)NHCH3, −C(O)N(CH3)2, −OC(O)CH3, −NHC(O)CH3,−S(O)2OH, or−S(O)2NH2. For example, the following groups are non-limiting examples of substituted alkyl groups: −CH2OH, −CH2Cl, −CF3, −CH2CN, −CH2C(O)OH, −CH2C(O)OCH3, −CH2C(O)NH2, −CH2C(O)CH3, −CH2OCH3, −CH2OC(O)CH3, −CH2NH2, −CH2N(CH3)2, and −CH2CH2Cl. The term “haloalkyl” is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to halo (i.e. −F, −Cl, −Br, or −I) such that no other atoms aside from carbon, hydrogen and halogen are present. The group, −CH2Cl is a non- limiting example of a haloalkyl. The term “fluoroalkyl” is a subset of substituted alkyl, in which the hydrogen atom replacement is limited to fluoro such that no other atoms aside from carbon, hydrogen and fluorine are present. The groups −CH2F, −CF3, and −CH2CF3are non- limiting examples of fluoroalkyl groups. Non-limiting examples of substituted aralkyls are: (3-chlorophenyl)-methyl, and 2-chloro-2-phenyl-eth-1-yl. The groups, −C(O)CH2CF3, −CO2H (carboxyl),−CO2CH3 (methylcarboxyl),−CO2CH2CH3,−C(O)NH2 (carbamoyl), and −CON(CH3)2, are non-limiting examples of substituted acyl groups. The groups −NHC(O)OCH3and −NHC(O)NHCH3are non-limiting examples of substituted amido groups. III. Mtb Vaccines A. General Formulation and Administration The present disclosure provides pharmaceutical compositions containing the synthetic tetrasaccharide of interest. Such compositions comprise a prophylactically or therapeutically effective amount of an agent, and a pharmaceutically acceptable carrier. In a specific

[0014] 13 {01122399}4928-7409-1886, v. 1 embodiment, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term “carrier” refers to a diluent, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a particular carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical agents are described in “Remington's Pharmaceutical Sciences.” Such compositions will contain a prophylactically or therapeutically effective amount of the agent, preferably in purified form, together with a suitable amount of carrier to provide the form for proper administration to the patient. The formulation should suit the mode of administration, which can be oral, intravenous, intraarterial, intrabuccal, intranasal, nebulized, bronchial inhalation, intra-rectal, vaginal, topical or delivered by mechanical ventilation. Pharmaceutically acceptable salts include the acid salts and those 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 may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. Generally, the compositions of the disclosure are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.

[0015] 14 {01122399}4928-7409-1886, v. 1 Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration. B. Vaccines The present disclosure provides pharmaceutical compositions comprising vaccine components. Such compositions comprise a prophylactically or therapeutically effective amount of an immunogen, and a pharmaceutically acceptable carrier. Active vaccines produced an immune response against Mtb in vivo in a subject at risk of such as infection. Such vaccines can be formulated for parenteral administration, e.g., formulated for injection via the intradermal, intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Administration by intradermal and intramuscular routes are contemplated. The vaccine could alternatively be administered by a topical route directly to the mucosa, for example, by nasal drops, inhalation, by nebulizer, or via intrarectal or vaginal delivery. Pharmaceutically acceptable salts include acid salts and those 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 may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. The inventors have preliminary data showing that a synthetic monophosphoryl lipid A Toll-like Receptor 4 (TLR4) ligand, glucopyranosyl lipid adjuvant (GLA), can provide adjuvant activity for mGLP. IV. Subjects All living humans are at risk of infection with Mtb, with an estimated that one quarter of the world’s population is currently infected, and thus anyone can benefit from the disclosed vaccines. In addition, certain subjects may derive particular benefit from the disclosed vaccines, such as with increased risk of severe disease and death for individuals with immune compromised states. In addition, contact tracing to reveal exposed patients that remain asymptomatic for TB can also benefit from the detection of immune responses to these vaccine antigens. * * * * * * * * * * * * * * * * *

[0016] 15 {01122399}4928-7409-1886, v. 1 All of the compositions and 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 disclosure 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 compositions and 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 disclosure. 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 disclosure as defined by the appended claims.

[0017] 16 {01122399}4928-7409-1886, v. 1 V. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. 1. Hoft DF, Brown RM, Roodman ST. Bacille Calmette-Guérin vaccination enhances human γδ T cell responsiveness to mycobacteria suggestive of a memory-like phenotype. Journal of Immunology.1998;161(2):1045-54. PubMed PMID: 9670986. 2. Worku S, Hoft DF. In vitro measurement of protective mycobacterial immunity: antigen-specific expansion of T cells capable of inhibiting intracellular growth of bacille Calmette-Guerin. Clin Infect Dis. 2000;30 Suppl 3:S257-61. Epub 2000 / 06 / 30. doi: 10.1086 / 313887. PubMed PMID: 10875795. 3. Hoft DF, Worku S, Kampmann B, Whalen CC, Ellner JJ, Hirsch CS, Brown RB, Larkin R, Li Q, Yun H, Silver RF. Investigation of the relationships between immune-mediated inhibition of mycobacterial growth and other potential surrogate markers of protective Mycobacterium tuberculosis immunity. Journal of Infectious Diseases.2002;186(10):1448-57. 4. Xia M, Hesser DC, De P, Sakala IG, Spencer CT, Kirkwood JS, Abate G, Chatterjee D, Dobos KM, Hoft DF. A Subset of Protective γ9δ2 T Cells Is Activated by Novel Mycobacterial Glycolipid Components. Infect Immun.2016;84(9):2449-62. Epub 2016 / 06 / 15. doi: 10.1128 / iai.01322-15. PubMed PMID: 27297390; PMCID: PMC4995917. 5. De P, McNeil M, Xia M, Boot CM, Hesser DC, Denef K, Rithner C, Sours T, Dobos KM, Hoft D, Chatterjee D. Structural determinants in a glucose-containing lipopolysaccharide from Mycobacterium tuberculosis critical for inducing a subset of protective T cells. J Biol Chem. 2018;293(25):9706-17. Epub 2018 / 05 / 03. doi: 10.1074 / jbc.RA118.002582. PubMed PMID: 29716995; PMCID: PMC6016469. 6. Smith WL, Ballou CE. The 6-O-methylglucose-containing lipopolysaccharides of Mycobacterium phlei. Locations of the neutral and acidic acyl groups. J Biol Chem. 1973;248(20):7118-25. PubMed PMID: 4743516. 7. Maranha A, Moynihan PJ, Miranda V, Correia Lourenco E, Nunes-Costa D, Fraga JS, Jose Barbosa Pereira P, Macedo-Ribeiro S, Ventura MR, Clarke AJ, Empadinhas N. Octanoylation of early intermediates of mycobacterial methylglucose lipopolysaccharides. Sci Rep. 2015;5:13610. Epub 20150901. doi: 10.1038 / srep13610. PubMed PMID: 26324178; PMCID: PMC4555173.

[0018] 17 {01122399}4928-7409-1886, v. 1 8. Ishiwata A, Tanaka K, Ito Y, Cai H, Ding F. Recent Progress in 1,2-cis glycosylation for Glucan Synthesis. Molecules. 2023;28(15). Epub 20230725. doi: 10.3390 / molecules28155644. PubMed PMID: 37570614; PMCID: PMC10420028. 9. Wang L, Overkleeft HS, van der Marel GA, Codee JDC. Reagent Controlled Stereoselective Synthesis of alpha-Glucans. J Am Chem Soc. 2018;140(13):4632-8. Epub 20180323. doi: 10.1021 / jacs.8b00669. PubMed PMID: 29553729; PMCID: PMC5890317.

[0019] 18 {01122399}4928-7409-1886, v. 1

Claims

WHAT IS CLAIMED IS:

1. A polysaccharide having the following formula: (I)R1 is hydrogen, C1-C8 alkyl, C1-C8 substituted alkyl, C1-C8 acyl, or C1-C8 substituted acyl; R2is C1-C8 alkyl, C1-C8 substituted alkyl, C1-C8 acyl, C1-C8 substituted acyl, or ; R3isalkyl, C1-C8 substituted alkyl, C1-C8 acyl, C1-C8 substituted acyl; R4 is C1-C8 alkyl, C1-C8 substituted alkyl, C1-C8 acyl, C1-C8 substituted acyl, or ; R5iswherein Y1is C1-C12 alkanediyl or C1-C12 substituted alkanediyl and R′ and R′′ are each independently hydrogen, C1-C8 alkyl, C1- C8 substituted alkyl, C1-C8 acyl, or C1-C8 substituted acyl; or19 {01122399}4928-7409-1886, v. 1a monosaccharide or disaccharide, optionally comprising one or more methyl groups.

2. The polysaccharide of claim 1, wherein R1is –C(O)CH(CH3)2.

3. The polysaccharide of either claim 1 or claim 2, wherein R2 is –C(O)CH3.

4. The polysaccharide according to any one of claims 1-3, wherein R3 is hydrogen.

5. The polysaccharide according to any one of claims 1-4, wherein R4is –C(O)CH3.

6. The polysaccharide according to any one of claims 1-5, wherein R5 is –(CH2)8NHAc.

7. The polysaccharide according to any one of claims 1-6, wherein the polysaccharide is further defined as:

8. A pharmaceutical composition comprising the polysaccharide according to any one of claims 1-7 dispersed in a pharmacologically acceptable buffer, diluent or excipient.

9. The pharmaceutical composition of claim 8, formulated for injection.20 {01122399}4928-7409-1886, v.

110. The pharmaceutical composition of claim 8 or claim 9, further comprising an adjuvant.

11. The pharmaceutical composition of any one of claims 8-10, wherein the polysaccharide is further defined as:

12. A method of inducing an immune response in a subject comprising administering to said subject the polysaccharide according to any one of claims 1-7 or the pharmaceutical formulation of any one of claims 8-10.

13. The method of claim 12, wherein the subject is at increased risk of developing tuberculosis as compared to populational average.

14. The method of claim 12 or claim 13, wherein the subject has previously received a tuberculosis vaccine.

15. The method of claim 12 or claim 13, wherein the subject has not previously received a tuberculosis vaccine.21 {01122399}4928-7409-1886, v.

116. The method of any one of claims 12-15, further comprising administering said polysaccharide or pharmaceutical composition a second time.

17. The method of any one of claims 12-16, wherein administering comprises intramuscular, subcutaneous, intradermal, aerosol, intranasal or oral delivery.

18. The method of any one of claims 12-17, wherein administering is repeated at least once.22 {01122399}4928-7409-1886, v. 1

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