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Número de publicaciónWO2015172077 A1
Tipo de publicaciónSolicitud
Número de solicitudPCT/US2015/029979
Fecha de publicación12 Nov 2015
Fecha de presentación8 May 2015
Fecha de prioridad8 May 2014
También publicado comoCA2946900A1, CN106459225A, EP3140324A1, WO2015172077A8
Número de publicaciónPCT/2015/29979, PCT/US/15/029979, PCT/US/15/29979, PCT/US/2015/029979, PCT/US/2015/29979, PCT/US15/029979, PCT/US15/29979, PCT/US15029979, PCT/US1529979, PCT/US2015/029979, PCT/US2015/29979, PCT/US2015029979, PCT/US201529979, WO 2015/172077 A1, WO 2015172077 A1, WO 2015172077A1, WO-A1-2015172077, WO2015/172077A1, WO2015172077 A1, WO2015172077A1
InventoresYawei Ni, Michael Springer
SolicitanteYawei Ni, Michael Springer
Exportar citaBiBTeX, EndNote, RefMan
Enlaces externos:  Patentscope, Espacenet
O-acetylated high molecular weight polygalacturonic acids and their use as vi polysaccharide vaccine
WO 2015172077 A1
Resumen
The instant disclosure provides O-acetylated high molecular weight polygalacturonic acids or pharmaceutically acceptable salts thereof, having at least one of: (a) a molecular weight greater than 1 x 106 Da; (b) a degree of mefhylation less than about 10% per mole; and (c) an intervening rhamnose content ranging from about 2% to about 15% per mole, useful as a synthetic immunogenic Vi antigen. The instant disclosure further provides methods of preparing an O-acetylated high molecular weight polygalacturonic acid or pharmaceutically acceptable salt thereof of, pharmaceutical compositions and/or vaccine compositions comprising the same, and methods of immunization using any of the foregoing.
Reclamaciones  (El texto procesado por OCR puede contener errores)
WHAT IS CLAIMED IS:
1. An O-acetylated high molecular weight polygalacturonic acid (OAc- HPGA) or pharmaceutically acceptable salt thereof, having at least one of the following:
(a) a molecular weight greater than 1 x 106 Da,
(b) a degree of methylation less than about 10% per mole, and
(c) an intervening rhamnose content ranging from about 2% to about 15% per mole.
2. The OAc-HPGA or pharmaceutically acceptable salt thereof according to claim 1 having a molecular weight greater than 1 x 106 Da.
3. The OAc-HPGA or pharmaceutically acceptable salt thereof according to any one of claims 1 or 2, wherein the degree of O-acetylation is greater than 100% or is greater than 50% at either the C2 or C3 position.
4. The OAc-HPGA or pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein the OAc-HPGA is a synthetic immunogenic Vi antigen.
5. The OAc-HPGA or pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein the OAc-HPGA or pharmaceutically
acceptable salt thereof has substantially the same antigenicity as Vi
polysaccharide vaccine and is immunogenic.
8. The OAc-HPGA or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein the OAc-HPGA or pharmaceutically
acceptable salt thereof induces a 2-fold or greater rise in antibody titers upon second immunization.
7. The OAc-HPGA or pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, wherein the OAc-HPGA or pharmaceutically
acceptable salt thereof is effective as a synthetic immunogenic Vi antigen in people under 2 years of age without conjugation to a protein carrier.
8. The OAc-HPGA or pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein the OAc-HPGA or pharmaceutically
acceptable salt thereof is effective as a vaccine against typhoid fever.
9. TheOAc-HPGA or pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, having a molecular weight greater than 1 x 10s Da, a degree of methylation less than about 10% per mole, and a degree of O- acetylafion greater than 100%, or a degree of O-acetylation greater than 50% at either the C2 or C3 position.
10. An O-acetylated high molecular weight polygalacturonic acid (OAc- HPGA) or a pharmaceutically acceptable salt thereof, formed by a process comprising reacting a high molecular weight polygalacturonic acid (HPGA) or a salt thereof with a mixture of acetic acid and acetic anhydride, wherein the HPGA has at least one of the following:
(a) a molecular weight greater than 1 x 106 Da,
(b) a degree of methylat on less than about 10% per mole, and
(c) an intervening rhamnose content ranging from about 2% to about 15% per mole.
11. The OAc-HPGA or pharmaceutically acceptable salt thereof according to claim 10, wherein the process further comprises formation of an acid gel with the HPGA or a salt thereof prior to reaction with the mixture of acetic acid and acetic anhydride. 2, The OAc-HPGA or pharmaceutically acceptable salt thereof according to any one of ciaims 10 or 11 , wherein the process further comprises reacting the HPGA or a salt thereof with perchloric acid,
13, The OAc-HPGA or pharmaceutically acceptable salt thereof according to any one of claims 10 to 12, wherein the HPGA is derived from Aloe vera.
14. The OAc-HPGA or pharmaceutically acceptable salt thereof according to any one of claims 10 to 13, wherein the degree of O-acetylation of the OAc- HPGA is greater than 100% or is greater than 50% at either the C2 or C3 position.
15. The OAc-HPGA or pharmaceuiically acceptable salt thereof according to any one of claims 10 to 14, wherein the OAc-HPGA or pharmaceutscally acceptable salt thereof is a synthetic immunogenic VI antigen.
18. The OAc-HPGA or pharmaceutically acceptable salt thereof according to any one of claims 10 to 15, wherein the OAc-HPGA has substantially the same antigenicity as Vi polysaccharide vaccine and is immunogenic.
17. The OAc-HPGA or pharmaceutically acceptable salt thereof according to any one of claims 10 to 16, wherein the OAc-HPGA or pharmaceutically acceptable salt thereof is effective as a synthetic immunogenic Vi antigen in people under 2 years of age without conjugation to a protein carrier.
18. The OAc-HPGA or pharmaceuticaiiy acceptable salt thereof according to any one of claims 10 to 17, wherein the OAc-HPGA or pharmaceutically acceptable salt thereof is effective as a vaccine against typhoid fever.
19. The OAc-HPGA or pharmaceutscally acceptable salt thereof according to any one of claisTis 10 to 18. wherein the OAc-HPGA induces a 2-fold or greater rise in antibody titers upon second immunization.
20. A process for preparing an O-acefylated high molecular weight polygalacturonlc acid (OAc-HPGA) or a pharmaceutically acceptable salt thereof, comprising reacting a high molecular weight polygalacturonlc acid
38 (HPGA) or a salt thereof, with a mixture of acetic acid and acetic anhydride, wherein the HPGA has at !east one of the following:
(a) a molecular weight greater than 1 x 106 Da,
(b) a degree of methy!ation less than about 10% per mole, and
(c) an intervening rhamnose content ranging from about 2% to about 15% per mole.
21 . The process according to claim 20, further comprising forming an acid gel with the HPGA or a salt thereof prior to reacting with the mixture of acetic acid and acetic anhydride.
22. The process according to any one of claims 20 or 21 , further comprising reacting the HPGA or a salt thereof with perchloric acid.
23. The process according to any one of claims 20 to 22. wherein the HPGA is derived from Aloe vera,
24. An O-acetylated high molecular weight polygalacturonic acid (OAc- HPGA) or a pharmaceutically acceptable salt thereof, formed by the process according to any one of claims 20 to 23.
25. The OAc-HPGA or pharmaceutically acceptable salt thereof according to claim 24, wherein the degree of O-acetylafion of the OAc-HPGA is greater than 100%, or is greater than 50% at either the C2 or C3 position.
28. The OAc-HPGA or pharmaceutically acceptable salt thereof according to any one of claims 24 or 25, wherein the OAc-HPGA or pharmaceutically acceptable salt thereof is a synthetic immunogenic Vi antigen having
substantially the same antigenicity as Vi polysaccharide vaccine and is immunogenic.
27. The OAc-HPGA or pharmaceutically acceptable salt thereof according to any one of claims 24 to 28, wherein the OAc-HPGA or pharmaceutically acceptable salt thereof is effective as a synthetic immunogenic Vi antigen in people under 2 years of age without conjugation to a protein carrier.
28. The OAc-HPGA or pharmaceutically acceptable salt thereof according to any one of claims 24 to 27, wherein the OAc-HPGA or pharmaceutically acceptable salt thereof is effective as a vaccine against typhoid fever.
29. The OAc-HPGA or pharmaceutically acceptable salt thereof according to any one of claims 24 to 28, wherein the OAc-HPGA induces a 2~fold or greater rise in antibody titers upon second immunization.
30. A pharmaceutical composition comprising the OAc-HPGA or
pharmaceutically acceptabte salt thereof according to any one of claims 1 to 19 or 24 to 29 and at least one pharmaceutically acceptable excipient.
31. The pharmaceutical composition according to claim 30, further comprising a protein carrier.
32. The pharmaceutical composition according to claim 31 , wherein the protein carrier is conjugated to the OAc-HPGA.
33. A method of immunizing a subject against typhoid fever, comprising administering to the subject in need thereof an effective amount of the OAc- HPGA or pharmaceutically acceptable salt thereof according to any one of claims 1 to 19 or 24 to 29, or the pharmaceutical composition according to any one of claims 30 to 32.
34. The method according to claim 33, wherein administration of the pharmaceutical composition induces an antibody response.
35. The method according to any one of claims 33 or 34, wherein administration of the pharmaceutical composition induces a 2-fold or greater rise in antibody titers upon second immunization.
36. Use of an O-acetylated high molecular weight polygalacturonic acid (OAc-HPGA) or pharmaceutically acceptable salt thereof according to any one of claims 1 to 19 or 24 to 29, or a pharmaceutical composition according to any one of claims 30 to 32, as a synthetic immunogenic Vi antigen, wherein the Vi antigen has substantially the same antigenicity as Vi polysaccharide vaccine and is immunogenic.
37. Use of an O-aceiylaied high molecular weight polygafacturonic acid (OAc-HPGA) or pharmaceutically acceptable salt thereof according to any one of claims 1 to 9 or 24 to 29, or a pharmaceutical composition according to any one of claims 30 to 32, for the manufacture of a medicament for vaccination against typhoid fever.
Descripción  (El texto procesado por OCR puede contener errores)

O-ACETYLATED HIGH MOLECULAR WEIGHT POLYGALACTURONIC ACIDS AND THEIR USE AS VI POLYSACCHARIDE VACCINE

[0001] This application claims the benefit of U.S. Provisional Application No. 61/990,493, filed May 8, 2014, which Is incorporated herein by reference in its entirety.

[0002] The present disclosure relates generally to the field of medicine, and specifically to microbiology, immunology, and vaccines, and more specifically, typhoid vaccines,

Background

[0003] Typhoid fever is an acute and life-threatening febrile illness, If is caused by Saimoneiia typhi (8, typhi). It is estimated that 16-33 million new typhoid fever cases and 500,000 - 600,000 deaths occur annually. Contaminated food and wafer are the main sources of infection. The risk of infection is the highest in developing countries with poor sanitation, e.g., Asia, Africa, and Latin America.

[0004] The bacteria penetrate the mucosal barrier in the small intestine after being ingested. They then reach the liver and spleen via blood circulation and cause the disease. Several different antibiotic therapies have been used to treat typhoid fever. However, multi-drug resistant strains of S. typhi have emerged and rendered these costly treatments ineffective. Thus, the widespread use of low-cost efficacious vaccines is still the most effective way to reduce the impact of typhoid fever.

[0005] Currently, there are two types of vaccines available for controlling typhoid fever, a Vi polysaccharide vaccine administered parenterally in a single dose and an oral live attenuated Ty21 a vaccine. The Vi polysaccharide vaccine is licensed for use in persons≥2 years old and provides about 70% protection that lasts for three years. The oral liquid live vaccine is licensed for use in persons over 2 years old and confers about 53-78% protection after three or four doses. Another oral live vaccine in capsule form is approved for persons over 5 years old and provides a similar level of protection after four doses,

[0008] The continued high incidence of typhoid fever in many regions, along with the rise and spread of drug-resistant strains, led the WHO in 2000 to recommend immunizing school-age children with these vaccines in areas where typhoid fever is a substantial public health problem. In the US, the vaccines are costly and mainly given to travelers and military personnel.

[0007] Vi polysaccharide vaccine is based on the Vi capsular polysaccharide (Fig. 1). It is an ([a!pha]1~4), 2-deoxy-2-A/-acetyl galacturonic acid that is variably O-acetylated at C3 (60-90%; S∑u and Bystricky, Enzymo!, 363:552-567 (2003)) and forms a capsule that protects the bacteria against complement-mediated lysis and phagocytosis. The VI vaccine was developed after many years of research that eventually demonstrated that the Vi polysaccharide is the protective antigen and can be produced without denaturation with improved purification methods. Currently, two Vi polysaccharide vaccines are available in the US and Europe, one (Typhim Vi®) made by Sanofi-Pasteur and the other (Typherix®) made by GSK. Each vaccine dose is formulated with 25 pg Vi polysaccharide and phenol as the preservative and is administered by intramuscular or subcutaneous injection. Revaccination is recommended every two years in the US. The Vi polysaccharide vaccine is also being made in other countries, including India and China. [0008] These vaccines are produced by large-scale fermentation of the wild-type S. typhi strain Ty2 and precipitation of the polysaccharide from the culture supernatant, followed by further downstream purification steps (Product inserts, Typhim Vi® and Typherix®), The production technology was developed in the 1970s and 1980s. Since S. typhi is a gram-negative bacteria, contamination of the Vi vaccine by endotoxin or lipopolysaccharide, a cell wall component of the gram-negative bacteria, is always a potential safety risk.

[0009] The Vi vaccine, similar to most polysaccharide-based vaccines, is a T~independent antigen and does not elicit a boosting or memory response upon revaccination (WHO/!VB/12,02). It is not effective in infants or toddlers under 2 years old. Thus, a Vi polysaccharide-protein conjugate vaccine is being developed by covalent linking of Vi polysaccharide to a protein carrier. The conjugate vaccine is a T-dependent antigen and has a boosting effect upon revaccination.

[0010] The O-acetyiation and molecular weight are the critical determinants of the immunogenicify of Vi polysaccharides. Studies have shown that removal of the O-acetyl group at C3 reduces its immunogenicity, Jarvis et al., J. Bacterial. 94:1408-1410 (1967); Szewczyk and Taylor, Infect immun. 29:539-544 (1980); Szu et al., infect immun, 59:4555-4581 (1991); Rijpkema et al., Bioiogicais. 32:1 1-6 (2004). Structural modeling showed that the bulky nonpolar O-acetyl groups at C3 make up most of the surface of the polysaccharide molecule by protruding in rows on both sides, whereas the carboxy! and N-acetyl (at C2) groups are mostly embedded or located close to the axis, Szu et al., infect, imrnun. 59:4555-4561 (1991 ). This is consistent with the O-acetyl group being the dominant immunogenicity determinant. The amount of O-acetylation is expressed as the degree of O-acetylation (DOAc, ratio of O-acetyl group/Gal UA [moie/moie]) or as the acetyl group content (μιηοίθ) per mg polysaccharide. N-acetyl groups at C2 also play an important role in the immunogenicity,

[0011] Studies have also shown that the immunogenicity of the Vi polysaccharide decreases when its molecular weight is reduced. Martin et a!., J, Bacteriol. 94:141 1-1416 (1967); Szu et a!., Infect Immun. 59:4555-4561 (1991 ), This is consistent with findings made with a model polysaccharide antigen Dextran B512 (dx), indicating that the immunogenicity of this polysaccharide antigen decreases with reduction in molecular weight. Gonzalez-Fernandez et al. , Vaccine, 26:292-300 (2008).

[0012] The O-acety! group content and molecular weight are the potency indicators for the current Vi vaccines (Product insert, Typhim Vi®). Production of potent Vi polysaccharide vaccines is dependent on the preservation of the Vi poiysaccharide structure. Many early attempts to produce potent Vi polysaccharides failed because the polysaccharide was degraded during the purification process.

[0013] Plant pectins share the same backbone with the Vi polysaccharide, They are alpha 1-4 linked polygalacturonsc acid (PGA) that is variably methylated. Those from appie and citrus fruits are most widely used in the food industry. Methylation of pectins occurs naturally through esterification of the carboxyl groups in galacturonic acid residues by methanol. Pectins with a degree of methylation (DM) <50% are defined as a low methoxy! (LM) pectin, whereas those with a DM of >50% are high methoxyi (HM) pectins. LM pectins with a DM below 10% are considered as PGA. As a final product, the PGA is often produced in the sodium salt form or sodium pofygalacturonate, Thus, the term "polygalacturonic acid" is used herein interchangeably with "polygafacturonate." LM pectins and PGAs are commonly obtained by demeihylation of HM pectins under alkaline pH conditions. While these conditions remove the methyl groups, they invariably break the polymer backbone, which results in a decreased molecular weight.

[0014] The commercial LM pectins or PGAs have been O-acetyiated in efforts to analyze the antigenicity of Vi polysaccharides and production of a synthetic VI polysaccharide vaccine. The resu ting acetylated product was found to share the same antigenicity with native Vi polysaccharide. Szewczyk and Taylor, Infect. Immun. 29:539-544 (1980); Szu et al. , Infect !mmun. 62;5545™5549 (1994). However, it was not immunogenic in animals. Szu et al., Infect Immun. 62:5545-5549 (1994). This was attributed to the low molecular weight (-4 x 10D Da) of the commercial LM pectin used as compared to the 1-2 x 106 Da of the native Vi polysaccharide. Id. The dependence of the polysaccharide antigen's immunogenicity on molecular weight has been demonstrated with other polysaccharide antigens. Gonzalez-Fernandez et al., Vaccine. 28:292-300 (2008). However, the O-acetylated LM pectin was immunogenic once conjugated to a protein carrier, although its antibody response level was much lower than that of Vi polysaccharide-profein conjugate, Szu et al., Infect. Immun. 82:5545-5549 1994); Kossaczka et al., Infect Immun. 67:5806-5810 (1997), Therefore, there exists a need to develop novel synthetic Vi polysaccharide vaccines against typhoid fever that is immunogenic, even without conjugation to a protein carrier. [0015] The instant disclosure provides O-acetyiated high moiecular weight poiygalacturonic acids (GAc-HPGA) or pharmaceuiscally acceptable salts thereof, having at least one of: (a) a molecular weight greater than 1 x 106 Da; (ta) a degree of methylation iess than about 10% per mole; and (c) an intervening rhamnose content ranging from about 2% to about 15% per mole, useful as a synthetic immunogenic Vi antigen. The instant disclosure further provides methods of preparing an O-acetylated poiygalacturonic acid or pharmaceutically acceptable salt thereof of, pharmaceutical compositions and/or vaccine compositions comprising the same, and methods of immunization using any of the foregoing.

[0016] Pectins from different plant sources have different chemical and physical characteristics. A high moiecular weight poiygalacturonic acid (HPGA) from Aloe vera L, (GelSite®, including pharmaceutically acceptable salts thereof) has been identified and manufactured as described in U.S. Patent Nos. 5,929,051 , 7,705,135, and 7,691 ,988, ali of which are incorporated by reference. Aloe vera is a plant widely cultivated in tropical and subtropical regions of the world. HPGA possesses distinctive features. It has a naturally low methoxyl content (!!DM"<10%), a very high molecular weight (>1 x 106 Da), and a high Gal UA (sodium salt) content (>90%). HPGA's chemical and physical properties are summarized in Table 1. HPGA is soluble in water, but poorly soluble directly in saline or buffered saline (150 mM NaCi). Like a L pectin, it can form a gel with calcium ions. The gelation occurs immediately when mixed with calcium. HPGA typically has a purity of >99% and contains a minimal amount of neutral sugars and proteins (< 0.5%). These superior chemical and physical properties distinguish HPGA from all other existing pectins or PGAs.

[0017] As defined herein, HPGA is a high molecular weight polyga!acturonic acid having at least one of the following characteristics: (1) a molecular weight greater than 1 x 106 Da, (2) a degree of methylation less than about 10% per mole, and (3) an intervening rhamnose content ranging from about 2% to about 15% per mole.

[0018] As defined herein, OAc-HPGA is an O-acylated high molecular weight polyga!acturonic acid having at least one of the following characteristics: (1) a molecular weight greater than 1 x 106 Da, (2) a degree of methylation less than about 10% per mole, and (3) an intervening rhamnose content ranging from about 2% to about 15% per mole.

[0019] In some embodiments of this disclosure, the HPGA is GelSite® GeiSite® is manufactured under cGMP from the Abe vera L. plant. The manufacturing process includes mincing of plant materials, extraction, clarification and 0.2 prn filtration followed by the purification steps as described in detail in U.S. Patent 7,691 ,988. The final product is a dried substance. Commercially, the resulting HPGA is also known as GelSite® polymer. A Drug Master File (D F) on the GelSite® was filed with the FDA in 2005 and is updated annually with new product information.

[0020] The present disclosure provides a synthetic and immunogenic Vi antigen which can be used as a new typhoid vaccine with significant advantages over the current Vi vaccines with respect to safety, effectiveness, and cost. The synthetic Vi antigen, an O-acetylated high molecular weight polygalacturonic acid (OAc-HPGA, e.g., GelSife-OAc™) or pharmaceutically acceptable salt thereof is generated by O-acetylation of a novel high molecular weight polygalacturonic acid (HPGA, e.g., GelSite®). HPGA novel properties discussed above make it an ideal substrate for a synthetic Vi polysaccharide analog by O-acetylation.

[0021] OAc-HPGA, for example, GelSite-OAc™, is produced by a chemical process at a very high yield (weight yield >100%). It has a high degree of O-acetylation (DOAc) as the ratio of O-acetyl group/Gal UA [mole/mole] (>100% or >50% (mole/mole) at either the C2 or C3 position) or as the acetyl group content (pmole) per mg polysaccharide (>4.8 pmole/mg), and a high molecular weight (>1 x 106 Da), thus exceeding the potency specifications of the current Vi vaccines. GelSite-OAc™ has similar or substantially the same antigenicity to Vi polysaccharide and is highly immunogenic on its own. More importantly, this synthetic antigen was fully protective in animals challenged with a lethal dose of live S. typhi. Furthermore, OAc-HPGA possesses a boosting effect or memory immune response, exhibiting more than 2-fold rise in antibody titers upon second immunization. This is very unique among polysaccharide antigens and potentially makes it effective in people under 2 years of age without conjugation to a protein carrier. [0022] Accordingly, it is an objective of the instant disclosure to provide a O-acetylated high molecular weight polygaiacturonic acid (OAc-HPGA) or pharmaceutically acceptable salt thereof, having at least one of the following:

(a) a molecular weight greater than 1 x 10¾ Da,

(b) a degree of methylation less than about 10% per mole, and

(c) an intervening rhamnose content ranging from about 2% to about 15% per mole.

[0023] In some embodiments, the OAc-HPGA or pharmaceutically acceptable salt thereof has a molecular weight greater than 1 x 10a Da.

[0024] in some embodiments, the degree of O-acetyiation (DOAc) of the OAc-HPGA or pharmaceutically acceptable salt thereof is greater than 100%, or is greater than 50% at either the C2 or C3 position.

[0025] In some embodiments, the OAc-HPGA has a molecular weight greater than 1 x 106 Da, a degree of methylation less than about 10% per mole, and a degree of O-acefylation greater than 100%, or a degree of O-acetyiation greater than 50% at either the C2 or C3 position.

[0028] In some embodiments, the OAc-HPGA is a synthetic immunogenic Vi antigen.

[0027] In some embodiments, the OAc-HPGA or pharmaceutically acceptable salt thereof has substantially the same antigenicity as Vi polysaccharide vaccine and is immunogenic.

[0028] In some embodiments, the OAc-HPGA or pharmaceutically acceptable salt thereof induces a 2-fold or greater rise in antibody titers upon second immunization. [0029] In some embodiments, the OAc-HPGA or pharmaceutically acceptable salt thereof is effective as a synthetic immunogenic Vi antigen in people under 2 years of age without conjugation to a protein carrier.

[0030] In some embodiments, the OAc-HPGA or pharmaceutically acceptable salt thereof is effective as a vaccine against typhoid fever.

[0031] The present disclosure further provides a method of producing OAc-HPGA, which incorporates the unique properties of HPGAs of the disclosure to simplify the process and ensure a high recovery or yield. OAc- HPGAs (e.g., GelSsfe-OAc™) can be produced in large quantities from the plant-based starting material (e.g.. GelSite^).

[0032] Accordingly, it is another objective of the instant disclosure to provide an G-acetylated high molecular weight polygalacturonic acid (OAc- HPGA) or a pharmaceutically acceptable salt thereof, formed by a process comprising reacting a high molecular weight polygalacturonic acid (HPGA) or salt thereof with a mixture of acetic acid and acetic anhydride, wherein the HPGA has at least one of the following:

(a) a molecular weight greater than 1 x 106 Da,

(b) a degree of methylation less than about 10% per mole, and

(c) an intervening rhamnose content ranging from about 2% to about 15% per mole.

[0033] It is a further objective of the instant disclosure to provide a process for preparing an OAc-HPGA or a pharmaceutically acceptable salt thereof, comprising reacting a high molecular weight polygalacturonic acid (HPGA) or a salt thereof, with a mixture of acetic acid and acetic anhydride, wherein the HPGA has at least one of the following: (a) a molecular weight greater than 1 x 10 Da,

(b) a degree of methylaison less than about 10% per mole, and

(c) an intervening rhamnose content ranging from about 2% to about 15% per mole,

[0034] In some embodiments, the process further comprises forming an acid gel with HPGA or a salt thereof prior to reacting with the mixture of acetic acid and acetic anhydride,

[0035] in some embodiments, the process further comprises reacting the HPGA or a salt thereof with perchloric acid.

[0038] In some embodiments, the HPGA used in the process is derived from Aloe vera.

[0037] It is also an objective of this disclosure to provide an OAc-HPGA or a pharmaceutically acceptable salt thereof formed by the process according to any of the processes described.

[0038] An additional objective of the disclosure is to provide pharmaceutical compositions comprising the OAc-HPGA or pharmaceutically acceptable salts thereof of the disclosure, optionally comprising at least one pharmaceutically acceptable excipient.

[0039] In some embodiments, the pharmaceutical composition further comprises a protein carrier.

[0040] In some embodiments, the protein carrier is conjugated to the OAc-HPGA.

[0041 ] It is another objective of this disclosure to provide a method of immunizing a subject against typhoid fever, comprising administering to the subject in need thereof an effective amount of the QAc~HPGA or pharmaceutically acceptable salt,

[0042] In some embodiments, the present disclosure also provides a method of inducing a protective immune response against Salmonella typhi by administering O~acetylated polygalacturonic acid (OAc-HPGA) to an animal or human. For example, the disclosure provides methods of immunizing a subject against Salmonella typhi and/or typhoid fever, comprising administering to the subject in need thereof an effective amount of an OAc-HPGA or pharmaceutically acceptable salt thereof or pharmaceutical compositions of any of the foregoing according to the disclosure.

[0043] !n some embodiments, administration of the pharmaceutical composition induces an antibody response,

[0044] In some embodiments, administration of the pharmaceutical composition induces a 2-fold or greater rise in antibody titers upon second immunization.

[0045] It is an objective of this disclosure to provide use of an OAc-HPGA or pharmaceutically acceptable salt thereof, or a pharmaceutical composition, as a synthetic immunogenic Vi antigen that has substantially the same antigenicity as Vi polysaccharide vaccine and is immunogenic.

[0046] !t is a further objective of this disclosure to provide use of an OAc- HPGA or pharmaceutically acceptable salt thereof, or a pharmaceutical composition, for the manufacture of a medicament for vaccination against typhoid fever.

[0047] The present disclosure further provides a typhoid vaccine formulated with OAc-HPGA optionally comprising at least one pharmaceutically acceptable excipient. OAc-HPGA may optionally be conjugated to a protein carrier to potentially further enhance its immunogenicity.

[0048] One kg of OAc-HPGA can potentially make 20-40 million doses of the vaccine at 25-50 ^xg/dose. It is plant-based and therefore does not contain endotoxin (lipopolysacchande, LPS) which is the most dangerous contaminant in vaccines purified from gram negative bacteria such as S. typhi. Compared to the existing Vi polysaccharide vaccine, OAc-HPGA could be a much safer, less expensive and more effective vaccine. The economic advantage also makes it easier and more affordable to expand production and use of the typhoid vaccine worldwide, especially in endemic areas of developing countries.

BRIEF .DESCRIPTION OF THE DRAWINGS

[0049] Fig. 1 illustrates structures of Vi polysaccharide, HPGA (e.g., GelSite®), and OAc-HPGA (GelSite-OAc™). The basic galacfuronic acid (Gal UA) residue is shown for Vi polysaccharide, HPGA and OAc-HPGA.

[0050] Fig. 2 illustrates the O-acetylation process for production of OAc- HPGA (GelSite-OAc™). The asterisk indicates the optional use of a small amount of perchloric acid as the catalyst.

[0051] Fig, 3 illustrates size exclusion chromatograms of GeiSite® and GelSite-OAc™, together with Dextran Standards. The Dextran Standards used were 1.597, 214.8, and 39.9 kDa.

[0052] Fig. 4 illustrates the antigenicity of O-acetylated poiygalacturonic acid (Ge!Site-OAc™), as tested in immunodiffusion assay. Each well received 20 μ! polysaccharide (200 Mg/ml) or reference serum (center well). The agarose plate was kept in a wet chamber overnight. The wells were charged as follows: 1 ) GelSite®; 2) O-acetylated poiygalacturonic acid 1x; 3) O-acetylated polygalacturonic acid 2x; 4) 0-acetylated polygalacturonic acid 3x; and C) Vi polysaccharide.

[0053] Fig. S illustrates any effect of DOAc on immune responses to GeiSite-OAc™. Balb/c mice were immunized twice with Vi vaccine or GelSite- OAc™ having different DOAc at 2.5 pg/mouse , 4 weeks apart. Specific IgG was measured using the Vi polysaccharide (A) or GelSite-OAc™ (B) as the antigen.

[0054] Fig. S illustrates any dose-dependent effect of GeiSite-OAc™. Baib/c mice were immunized twice with GeiSite-OAc™ (DOAc, 1.75) at the different indicated doses, 6 weeks apart. Specific IgG was measured by ELISA using the Vi polysaccharide (A) or GeiSite-OAc (B) as the antigen.

[0055] Fig, 7 illustrates any cross reactivity. Pooled serum samples from different study groups were reacted with GelSite® (A), GeiSite-OAc™ (B) or Vi polysaccharide (C).

[0056] Fig. 8 illustrates the cross-boosting effect. Specific IgG antibodies were measured with Vi polysaccharide (A) or GelSite~OAc™(B).

[0057] Fsg. 9 shows IgG subclass distribution. Specific IgG antibodies of different subclasses were measured by ELISA. Titers were determined by the end point (2-fold higher than the background, > 0.2 OD). Antibodies were measured with Vi polysaccharide (A) or GelSite~OAc™(B) as the antigen.

[0058] Fig, 10 illustrates protection against lethal challenge with live S. Typhi. Balb/c mice immunized twice with Vi vaccine or GelSite-OAc™ having different DOAc at 2.5 pg/mouse , 4 weeks apart. Animals were challenged with 100 LD50 of S, Typhi at week 2 following the second immunization. A, % survival; B, Mean body weight. [0059] Fig. 11 illustrates the correlation of DOAc with immunogenicity of GelSite-OAc™. Baib/c mice (n=6) were immunized with GelSite-OAc™ having different DOAc at 2.5 Mg/mouse twice, 4 weeks apart. Specific IgG in individual serum samples collected at week 2 (w2) following the first and second immunization was measured using the Vi polysaccharide (A) or GelSite-OAc™ (B) as the antigen.

[0060] Fig, 12 illustrates the correlation of DOAc with protection of GelSite-OAc™. Balb/c mice (n~6) were immunized with GelSite-OAc™ having different DOAc at 2.5 g/mouse twice, 4 weeks apart. Animals were challenged with 100 LD50 of S. Typhi at week 3 following the second immunization. A; % survival; B, Mean body weight.

DESCRIPTION

[0081] The O-acefy!ation process is based on the method described by Schweiger (1964). Due to its high molecular weight, HPGA (e.g., GelSite®) forms a gel at the low pH (~pH2.0), which eliminates the need to prepare a calcium precipitate as the initial step of the process described by Schweiger, J. Org, Chem. 29:2973 - 2975 (1964). This allows acetylation of the HPGA (e.g., GelSite®) in a solid or bead form which allows for easy handling and recovery during the acetylation process. At the end of the acetylation reaction, the QAc- HPGA beads were solubsHzed by increasing the acidic pH to neutral pH. The acetylation process is highly efficient with >100% yield due to the simplicit of the process and addition of acetyl groups.

[0062] The acetylation, however, can be achieved by other processes, including the one described by Carson and aclay, J. Am. Chem. Soc, 88:1015-1017 (1946). [0083] The term "O-acetylation" or "G-acetylated" refers to addition of acetyl groups at the C2 and C3 positions of a sugar residue. The maximum degree of O-acetylation (200%) is reached when both the C2 and C3 positions of each sugar residue are acetylated.

[0084] The resulting OAoHPGA (e.g., GelSite~OAc™) has a degree of O-acetylation (DOAc) of at least 130% or 100%, or at least 85%, or 50% at either the C2 or C3 position, and a high moiecular weight (>1.0 x 106 Da), thus closely resembling the native Vi polysaccharide (60-90% O-acetylation at C3 and 1-2 x 106 Da). The DOAc can be readily increased to≥175% by extending the reaction time. Due to the addition of the acetyl groups, OAc-HPGA also has an increased molecular weight.

[0065] The potency indicators for the current licensed Vi vaccines are the O-acetyl group content (≥2 pmole O-acetyl group [at C3]/mg) and molecular weight of the Vi polysaccharide (50% of the polysaccharide ≥ 2.5 x 104 Da) (WHO Expert Committee on Bioiogical Standardization, 1993). Thus, OAc- HPGA readily meets and exceeds both potency indicators for current Vi vaccines (>2.5 pmol O-acetyl group at either C2 or C3/mg and 70% of the polysaccharide≥1 x 106 Da, Table 2 and Fig. 3).

[0066] Unlike HPGA (e.g., GelSite®), OAc-HPGA (e.g., GeiSite-OAc™) no longer forms a gel with calcium. Also unlike HPGA, OAc-HPGA can directly dissolve in saline or buffered saline (150 mM NaCI). These results indicate that the O-acetylation has altered the basic chemical and functional properties of HPGA and that the resulting product, OAc-HPGA is a new chemical entity.

[0067] OAc-HPGA is antigenic as shown with the reference anti-Vi polysaccharide serum (Fig. 4). ^t shared the same antigenicity as the Vi polysaccharide. Importantly, no reaction with HPGA was obtained, indicating that the OAc~HPGA does not cross-react with its unacetyiated parental molecule.

[0088] OAc-HPGA, such as GelSite-OAc™, is immunogenic in mice (Fsg. 5). Specific antibodies can be detected by ELISA with Vi polysaccharide or OAc-HPGA as the antigen. Previously, it was found that the acetylated LM pectin was not immunogenic in mice, which was attributed to its low molecular weight (4 x 105 Da) in comparison to the native Vi polysaccharide. Szu et al. Infect, Immun. 82:5545-5549 (1994). OAc-HPGA has a much higher molecular weight (>1 x 106 Da), which may be in part responsible for its immunogenicity. Again, no cross reactivity with its parent molecule (HPGA) was observed, which indicates the specific antibodies induced by the OAc-HPGA is directed toward the O-acetyl group, the most critical immunogenicity determinant of Vi polysaccharide.

[0089] Remarkably. OAc-HPGA possesses the boosting effect or memory immune response, exhibiting more than a 2-fold rise in antibody titers upon the second immunization, No such boosting effect was observed with the Vi vaccine (Typhim Vi®) tested in parallel. The polysaccharide antigens are known to be T-independent and lack the immune memory or boosting effect. The memory or boosting immune response is only achieved with polysaccharide antigens by conjugating them with a protein carrier. Thus, OAc- HPGA is highly unique in possessing the boosting effect on its own without any conjugation, which is likely due to its novel chemical properties. Furthermore, the boosting effect can be induced not only with the GelSite-OAc™, but also Vi vaccine as the second dose. This further confirms the structural similarity between the OAc-HPGA and Vi polysaccharide. This boosting effect potentially makes si effective in peopie under 2 years of age without conjugation to a protein carrier,

[0070] The term "boosting effect" is used interchangeably with "memory immune response" and refers to an increase in immune response by about 2~ fold following the second immunization or revaccination.

[0071] OAc-HPGA was fully protective in animals challenged with a lethal dose of live S. typhi, indicating the immune response induced by it is protective against S typhi. Together, these findings indicate that OAc-HPGA can potentially be developed as a new typhoid Vi vaccine which has distinct advantages over the current Vi vaccines with respect to manufacturing, cost, memory response and potential effectiveness in people under 2 years without conjugation to a protein carrier. OAc-HPGA can be produced in large quantities by a simple chemical process using the abundant, high quality HPGA from a plant source. Each kg of OAc-HPGA can potentially make 20-40 million doses of the vaccine at 25-50 pg polysaccharide per dose. Being of plant origin, OAc- HPGA does not contain endotoxin or LPS, which is a cell wall component of gram-negative bacteria such as S. typhi and the most dangerous contaminant in vaccines purified from these bacteria. Thus, this synthetic vaccine could be safer and less expensive than current licensed Vi vaccines. The economic advantage makes it easier and more affordable to expand production and use of typhoid vaccine worldwide, especially in endemic areas of developing countries. In addition, OAc-HPGA can be used for the development of a conjugate vaccine that may be even more immunogenic and protective for children under 2 years of age. Example 1 : O-Acetyfation of GelSite Polymer

[0072] Two different methods were evaluated for O-acetylation of GelSite®: one was described by Carson and aclay (1946) and the other by Schweiger (1964), Both methods were capable of O-acetylating the GelSite®. The Schweiger method was found to be more efficient and better suited for the unique property of GelSite® and for obtaining the Q-acety!ated product. The Schweiger method does not use formamide or pyridine, Instead it uses perchloric acid in a small amount as a catalyst. In addition, the whole process by the Schweiger method is conducted at room temperature, Thus, the Schweiger method was selected and modified for acetylation of GelSite ®.

[0073] The process. The process, modified from the Schweiger method, consists of six simple steps (Fig, 2). Compared to the original Schweiger method, it is much simplified by the two distinct properties of GelSite® - acid gelation and insolubility in water in its acid form. The first step is to convert the substrate or GelSite® to its acid form from the sodium salt form by rinsing in dilute HCI and then glacial acetic acid. One of the novel properties of GelSite® is that it gels efficiently at low H, forming strong acid gel beads or strands which can withstand the down-stream steps. The original Schweiger method first prepared calcium precipitates prior to acid wash steps. This is therefore not necessary and eliminated for O-acetylation of GelSite®. After acefylation, the gel beads or strands are washed in deionized water to remove the acetylation reagents without any loss as acetyiated GelSite® in acid form is insoluble in wafer. After the wash, the acetyiated GelSite® is then solubilized by neutralization with NaOH, which converts it from an acid to sodium salt, The whole process can be completed in one day except for the last drying step.

[0074] All reagents used were obtained from Sigma Chemical Co. (St, Louis, MO). The basic process, based on the 1 gram scale, is briefly described below.

[0075] The Ge!Site® solution in water (200ml at 5 mg/ml; 1 gram total) was dropped into 1 liter of 0.1 HCI to produce GelSite® gel beads. The gel beads were recovered and washed in 100 mi acetic acid three times for 15 minutes each time. The gel beads were suspended in 200 ml of an acetic acid/acetic anhydride (1 :1) mixture. While stirring, 2 ml perchloric acid (70%) was gradually added over 2 hrs with 0.5 ml at time 0, 30 min, 60 min, and 90 min in 0.1 ml portions. The gel beads were then recovered at 120 min with a stainless steel strainer and washed extensively with water. They were suspended in 300 ml water and dissolved by adjusting the pH to ~7 with 0.1 NaOH before being precipitated with ethanol and dried under vacuum.

[0076] Process evaluation. The process has been evaluated with respect to key parameters including yield and DOAc.

1) Yield. The weight yield had been consistently above 100%. Such a high yield assures efficient production and maximizes the use of starting materials and reagents. It is consistent with the fact that addition of the acetyl groups increases the molecular weight of the GelSite®, and the process can be carried out with minimal loss. The acid gei beads are at least 2 mm in diameter and can be readily recovered during the acetylation process.

2) Degree of O-Acetylation (DOAc). The DOAc is a key parameter for the GelSite-OAc™. The acetylation process was highly efficient, yielding a DOAc >130% after just one round of reaction. The DOAc could be controlled by the duration of the acetylation reaction as well as the concentration of the acetic anhydride used, It could be further increased, along with an increase in the reaction time, while maintaining the same 30 min interval for addition of perchloric acid. A DOAc as high as 175% or 7.5 pmole/mg has been obtained. The maximum DOAc is 200% when both the C2 and C3 sites are fully acetylated. On the other hand, lower DOAc, e.g., 80%, could also be obtained by lowering the concentration of acetic anhydride to as low as about 10% of the reaction mixture, thus allowing generation of GeiSite-OAc™ with an even wider range of DOAc for evaluation,

3) Molecular weight. The molecular weight was consistently increased following acetylation (Table 2 and Fig. 3). This is consistent with the addition of the acetyl groups to GelSite® and indicates that the process does not cause the degradation of the parent molecule (GelSite®), therefore assuring the retaining of the high molecular weight of the end product GelSite-OAc™.

4) Acid gelation. The formation of the strong acid gel in the dilute HCI is important to the efficiency of the acetylation process. It was observed that a Sow molecular weight commercial PGA could only form soft gels which disintegrated during the downstream processes. Thus, the high molecular weight of GelSite® is critical to the process efficiency.

5) Perchloric acid. The acetylation is initiated by addition of a small amount of perchloric acid as the catalyst. St was found that 20% perchioric acid was just as effective as 70% perchloric acid. Furthermore, a high DOAc (>100%) could be readily obtained with use of only 0.125 ml of 20% perchloric acid in the reaction mixture with 1 gram of GelSite® described above, In addition, the 1 % perchloric acid in acetic acid was also found to be equally effective when used at the same amount of perchloric acid. The 70% perchloric acid is a strong acid. Thus, use of 1 % or 20% perchloric acid minimizes the use of hazardous reagents, increasing process safety.

8) Drying. At the end of the process, the GeiSite-OAc™ could be readily dried by lyophiiization, thus eliminating the use of ethanol precipitation,

Gelation with calcium No No No

Exampte 2: Characterization of Acetyiated GeiSite® Polymer

1 , liytofecufar weight

[0077] The HPLC SEC (size exclusion chromatography) with Dextran standards was used to determine the molecular weight of GelSite-OAc™. The molecuiar weight of polymers such as poiysaccharides is now generally determined using the more advanced HPLC - MALLS (multiple-angle laser-light scattering) method. However, this HPLC SEC with dextran standard was adopted because it has been most widely used in studying the Vi polysaccharides in the published literature and for Vi vaccine potency measurement, thus allowing the results to be directly comparable to those reported previously. Briefly, the Shodex OH pak SB-806HQ (300 x 8 mm), Shodex OHpak SB-805HQ (300 x 8 mm), and Shodex OHpak SB-804HQ (300 x 8 mm) columns were used in tandem with 0.1 M ammonium acetate and 200 pprn sodium azide as the mobile phase at a flow rate of 0,5 m!/min. Molecular weight values of the peak apex were reported as compared to the Dextran Standards (Phenomenex broad WD, 8 standards, 10-200 kDa) (Fsg, 3). The molecular weight of GelSite-OAc™ was found to be the same as or higher (>2 x 106 Da) than that of the starting GelSite® in all batches examined (Table 2). This is consistent with the increase in the molecular weight by addition of the acetyl groups,

2. Degree of O-acetylatsort (DOAc)

[0078] The method described by Hestrin, J. Biol. Chem. 180:249-261 (1949) was used with modifications. All reagents were obtained from Sigma Chemical Co (St. Louis, MO), including acetylcholine, hydroxylamine hydrochloride and iron chloride. The assay was conducted in 96-well plates. Samples were tested at -0.2 mg/m! (w/v) in duplicate. Acetylcholine was used as the standard. The DOAc was expressed as a molar ratio or percent of acetyl groups over Gal UA residues, or mole/mg - the unit used for DOAc specification for the current VI vaccine. The Gal UA content was based on GelSite® product release testing results.

[0079] Based on the initial process conditions described above, GelSite- OAc™ with a DOAc of 134% - 153% was obtained, which corresponded to the 67% - 76% at either the C2 or C3 position (Table 2). The DOAc may aiso be expressed based on Mmole/mg (Table 2). The DOAc could be further increased by extending the acetyiaiion reaction time to as high as 175% or 87.5% at either the C2 or C3 position, approaching the theoretical maximum of 200%. On the other hand, by adjusting the reaction conditions or lowering the concentration of acetic anhydride in the reaction mixture, lower DOAc such as 80% (Example 5) could also be obtained.

3. Calcium gelation

[0080] The ability of GelSite® and GelSite-OAc™ to gel in the presence of calcium was determined by mixing a GelSite® or GelSite-OAc™ solution with a calcium solution. Thus, a - 0.2% (w/v) sample solution (2-3 ml) was mixed directly with 0.5 - 1 ml of a 1 % calcium chloride solution. The GelSite® solution immediately formed the gel, whereas no gel formation occurred with the GelSite-OAc™. Gel formation was indicated by the whole sample solution changing into a ge! and which could no longer flow freely. The GelSite-OAc™ solution remained as a free flowing solution after mixing with the calcium chloride.

[0081] Also, unlike GelSite®, GelSite-OAc™ could be directly dissolved in saline or buffered saline (150 mM NaCI). These results indicate that the O- acetylation has altered the basic chemical and functional properties of the GelSite® and that the resulting product, GelSite-OAc™, is a new chemical entity.

4, Antigenicity

[0082] The immunodiffusion assay was performed in 1 % agarose as described by Szu et al. Infect, Immun. 82:5545-5549 (1994). The reference Vi polysaccharide antigen from Citrobacter freundii and reference burro serum against S. typhi were obtained from the National Institute of Child Health and Human Development (NICHD). The GelSite-OAc™ formed positive precipitation lines with the reference serum (Fig. 4). The strength of precipitating lines increased along with the increase in the DOAc, as shown with samples obtained after 1 , 2, and 3 rounds of acetylation with the Carson and aclay method (Fig, 4). Ge!Site~OAc™ shared the same or substantially the same antigenicity as the VI polysaccharide as evidenced by the merging of the precipitation lines from GelSite-QAc™ and Vi polysaccharide; no reaction was observed with the un-acetylated GelSite® (Fsg, 4). As used herein, the terms "the same or substantially the same antigenicity as the Vi polysaccharide" means that the GelSite~OAc™ is reactive with the anti-Vi polysaccharide serum or antibodies. For example, as seen in Fig. 4 and discussedjabove, GelSite- OAc™ formed positive precipitation lines with the reference serum in the immunodiffusion assay of Szu et a!. which merged with the immunodiffusion assay precipitation lines of the Vi polysaccharide.

5, Comparison to VI polysaccharide and vaccine

[0083] The Ge!Site-OAc™ has a degree of O-acetylation of >130% (5.58 μηι/mg), or >85% (2.79 μηη/mg) at either the C2 or C3 position, assuming an even distribution between these two O-acetylation sites, and a molecular weight of >1 x 106 Da (Table 2). The native Vi polysaccharide has a molecular weight of 1 -2 x 106 Da and a degree of O-acetylation at the C3 position of 80-90% (Szu and Bystricky, 2003). Thus, the GelSite-OAc™ is very similar to Vi polysaccharide, based on the DOAc and molecular weight, the two critical determinants of immunogenicity.

[0084] The potency indicators for the current licensed Vi vaccines are the O-acetylafion content (>2 pmole /mg [at C3]) and molecular weight (50% of the polysaccharide > 2.5 x 104 Da) of the Vi polysaccharide (WHO Expert Committee on Biological Standardization, 1993; Keitel et al., 1994), With a DOAc of readily >2.5 pmoi /mg {> 85%) at either the C2 or C3 position and a molecular weight of >1 x 106 Da for >70% of the polysaccharide (Table 2 and Fig.3), GelSite-OAc™ exceeds both potency specifications.

Example 3: Immunogen city of O-Acetylatad GelSste® fGelSste-

OAc™)

[0085] The immunogenicity of GelSite-OAc™ was examined in Balb/c mice in comparison with a licensed Vi vaccine (Typhim Vi®; Sanofi-Pasteur), Groups of mice (n= 0) were immunized with the GelSite-OAc™ or Typhim Vi vaccine in 50 pi PBS at the indicated doses by intramuscular injection in the right hind leg. Animals were immunized 2 times 4 weeks apart and serum samples were collected every two weeks.

[0086] Specific antibodies were measured by EUSA in 96-weiI plates as described previously, Szu et al., Enzynioi. 383:552-587 (2003). The Vi polysaccharides from N CHD and International Vaccine Institute (IVi), both of which are well known institutions in typhoid fever research, were used as the antigens for coating the ELISA plates. The reference serum was a hyperimmune mouse serum with 40 EL!SA units/ml obtained from NICDH. The antibody titers were determined using the CDC ELISA program (http://www.cdc.gov/ncidod/dbmd/bimb/elisa.htm) with the reference serum as the standard.

1. Effects of DOAc and antigen dose

ΙΌ0871 Effect of DOAc: Three GelSite-OAc™ samples with different DOAc (138%, 155%, or 175% mole/mole, or 5.8, 8.5, or 7.5 pmole/mg) were tested. The results showed that levels of specific IgG responses were found to

28 correlate with DOAc - the higher the DOAc, the higher the immune response (Fig, 5). This was especially true after the 1st immunization, no matter whether the specific IgG was measured using Vi antigen (Fig, 5A) or synthetic antigen Ge!Site~OAc™ (Fig. SB), The GelSite-OAc™ with the lowest DOAc (138%) consistently yielded the lowest titer at most time points. The GelSite-OAc™ samples with 155% or 175% DOAc exhibited very similar response levels, suggesting that the GelSite-OAc™ with 155% or 175% DOAc could be equally effective. This DOAc-dependent effect on immune responses were further demonstrated by evaluating Ge!Site-OAc™ with a DOAc as low as 80% (Example 5).

[0088] Effect of antigen dose: The effect of Ge!Site-OAc™ antigen dose was also tested at four different dose levels (1 , 2.5, 5, and 10 pg) using the GelSite-OAc™ with 175% DOAc. The antibody responses measured with either Vi or GelSite-OAc™ were antigen dose-dependent, especially after the second immunization (Fig. β).

Γ00891 Comparison with the Vi vaccine; GelSite-OAc™ with 155% or 175% DOAc consistently induced comparabUe or higher antibody titers compared to Vi vaccine, except for the time points after the first immunization and when measured with the Vi antigen (Figs. § and 6). When measured with the GeiSite~OAc™ antigen, the specific antibody titers induced by GelSite- OAc™ were higher than those by the Vi vaccine, by as much as 10-fold. This is likely due to the higher DOAc or presence of more acetyl groups with the GelSite-OAc™. Together, these results indicate that the GelSite-OAc™ is highly immunogenic when compared to the Vi vaccine. 2. Cross reactivity

[0090] The specificity of the antibodies induced by GelSite-OAc™ was evaluated by reaction with its parental molecule (GelSite®) in comparison with Vi polysaccharide and GelSite-OAc™. The results with the pooled serum samples collected at week 8 post the second immunization from the antigen dose- dependency experiment (Fig, 6) are shown in Fig. 7. The serum samples were tested at a tow starting dilution of 1 :40 and the reference serum obtained from N!CDH was also included in the test (at the 1 :2000 starting dilution). No reaction with the GelSite® was observed at any dose levels of GelSite-OAci M (Fsg, ?A), while high saturating levels of reactions were obtained against the GelSite-OAc™ (Fig. 7B), These results are consistent with the results obtained by the immunodiffusion test described above, They indicate that the response generated against the Ge!Site-OAc™ is directed toward the 0~acetyl groups on the GelSite-OAc™, This has been further confirmed by using GelSite-OAc™ with different DOAc (134% - 175%) as the antigens in EUSA, which showed that the antibody reaction levels were correlated with the DOAc.

3. Boosting effect

[0091] A unique boosting effect or memory immune response was consistently observed with GelSite-OAc™ upon the second immunization in all animal experiments conducted. As shown in Figs, 5 and 6, the titers of all three GelSite-OAc™ groups, as measured against the Vi or Ge!Site-OAc™ antigen, increased after the second immunization by at least 2-fold and up to > 4-fold compared to the highest titer after the first immunization, and became much higher than those by the Vi vaccine. No such boosting effect was observed with the Vi vaccine. Further evaluation with GelSite-OAc™ with a wide range of DOAc showed that the boosting effect and full protection could be obtained at a DOAc as low as 100% (Example 5),

[0092] To further confirm this novel boosting effect, a cross-boosting experiment was conducted, in which animals were first immunized with one antigen and followed by two boosting immunizations with the same or a different antigen (Fig. S). The results showed that all animals primed with GelSite- OAc™ exhibited the boosting effect upon the second immunization with Ge!Site-OAc™ or Vi vaccine (Fig. 8). The boosting level obtained with VI vaccine was higher than that with GelSite-OAc™ when measured with Vi polysaccharide (Fsg, 8A). This may reflect the additive effect of boosting based on the O-acetyl groups and induction of antibodies against other parts of the Vi polysaccharide.

[0093] Animals primed and boosted only with Vi vaccine did not show a boosting effect, Those primed with Vi vaccine and boosted with GelSite-OAc™ did show a boosting effect, although to a lesser extent and only detectable when antibodies were measured against GelSite-OAc™ (Fsg. 8B). No further boosting effect was observed at the third immunization. These results together showed that cross-boosting with Vi polysaccharide can occur, especially when GelSite-OAc™ is used as the priming immunization, and therefore further confirming the novel boosting effect of the GelSite-OAc™ and its structural similarity to Vi polysaccharide.

4, HgG subclasses

[0094] The unique boosting effect of GelSite-OAc™ suggests that it might be a T-dependent antigen. One of the features for the T- dependent immune response is the subclass change or switching following the boosting immunization or in comparison with the pure polysaccharide antigen in the case of the polysaccharide conjugate vaccines. Pooled serum samples collected at week 2 after the first and second immunizations were therefore used to examine the distribution of IgG subclasses (lgG1 , lgG2a, gG2b, lgG3) and IgM by ELISA. Animals injected with Ge!Site-OAc™ exhibited high levels of lgG1 after either one or two immunizations (Fig, 9). The titers for all IgGs increased by >2-fold following the second immunization. A greater increase in the titers for SgG1 ( igG2a, and lgG2b was observed after the second immunization with the lgG2a exhibiting the highest magnitude of titer increase (Fig. 9), This IgG subclass profile suggests that aTh2-biased T cell activation might be involved in the response against the GelSite-OAc™. The response is similar to the one obtained with the Vi-conjugate vaccine (An et ai.s 2012) and Streptococcus pneumonia polysaccharide conjugate vaccine (Safari et a!., 201 1). Unlike GelSite-OAc™, the Vi vaccine exhibited the same or decreased titers for all IgG subclasses following the second immunization, with the exception of gG2a, which showed a 2-fold increase. No apparent change in IgM levels were observed with either vaccine.

Example 4; Protective effect of 0»acetylated Ge!Site® polymer

[0095] The protective effect of the specific immune responses induced by GelSite-OAc™ was evaluated in the challenge experiment with live S. typhi as described previously (Park et aL, 2002). Groups of 15 six-to eight-week-old female Balb/c mice were immunized with GelSite-OAc™ with different DOAc (138% or 155%) or Vi vaccine at 2.5 pg/mouse by intramuscular injection twice, four weeks part. The control received the buffer solution. At week 2 after the second immunization, 10 mice from each group were challenged intraperitoneal!y with 100 LD50 (1 ,000 CPU/mouse) of the bacteria in 0.5 ml 5% porcine gastric mucin. The bacteria used were Salmonella enterica subsp. The Enterica serovar typhi (S. typhi) was obtained from ATCC (item number 19430),

[0098] The results showed that all immunized animals were protected, including the two GelSite-OAc™ groups, and the Vi vaccine group (Fig. 10). All unimmunized control mice died within three days. There were no differences among the protected groups (Fig.10), indicating that GelSite-OAc™ was just as protective as the Vi vaccine. In addition, the two GelSite-OAc™ groups with a DOAc of 138% or 155% showed no difference in protection, indicating the GelSite-OAc™ with 136% DOAc could be just as protective. All protected mice experienced a minor transient loss of body weight (<10%) at days 1-3 post challenge, but recovered to normal level afterwards (Fig.10B). The specific antibodies were also measured with Vi or Ge!Site-OAc™ antigen as described above and similar results to those shown in Fig, 5 were obtained.

[0097] The DOAc is a critical specification for GelSite-OAc™. Thus, it is important to further demonstrate the correlation between DOAc and protection. A series of additional GelSite-OAc™ samples with a wider range of DOAc (80% -153%) were therefore generated and screened in mice for immunogenicity and protection. The results showed that the levels of antibody titers and protection increased in direct correlation with DOAc (Fig 1 1 and 12). The unacetylated GelSite® with a 0% DOAc did not induce any detectable antibodies against either Vi polysaccharide or GelSite-OAc™, nor provided any protection, further indicating that the antibodies induced by GelSite-OAc™ are directed at the O- acetyl group. Importantly, GelSite-OAc™ with an 80% DOAc exhibited the lowest antibody titer and no boosting effect after the second immunization, and provided only a partial protection, while others with a higher DOAc (>100%) generated much higher antibody titers (>10 fold) with boosting effect and provided the full protection. On the other hand, even with a low antibody titer (0.239 U/mi against the Vi after the second immunization; Fig. 11 A), the GelSite-OAc™ with the 80% DOAc still provided a 83% protection (Fig. 12A), suggesting that the antibodies induced by GeiSite-OAc™ are highly protective. Together, these results suggest that a GelSite-OAc™ with DOAc of 100% or higher couid be fully protective along with the boosting effect, thus forming a solid foundation to establish a DOAc specification for the vaccine.

[0098] Other embodiments of the disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein. St is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the disclosure being indicated by the following claims.

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20WHO EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION, 1993
Clasificaciones
Clasificación internacionalC08B37/00, A61P31/04, A61K31/732
Clasificación cooperativaC08B37/0045
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