Note: Descriptions are shown in the official language in which they were submitted.
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0-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.
Field of the Invention
[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. It is
caused by Salmonella typhi (S. typhi). It is estimated that 16-33 mon new
typhoid fever cases and 500,000 - 600,000 deaths occur annually.
Contaminated food and water 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
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dose and an oral live attenuated Ty2la 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,
[0006] 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 ([alpha]1-4), 2-deoxy-2-N-acetyl
galacturonic
acid that is variably 0-acetylated at C3 (60-90%; Szu and Bystricky, Enzymoi,
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.
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[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/IVB/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 0-acetylation and molecular weight are the critical
determinants of the immunogenicity of Vi polysaccharides. Studies have shown
that removal of the 0-acetyl group at C3 reduces its immunogenicity. Jarvis et
al., J. Bacteriol, 94:1406-1410 (1967); Szewczyk and Taylor, Infect. Irnmun.
29:539-544 (1980); Szu et al., Infect, immun. 59;4555-44561 (1991); Rijpkema
et al., Biologicals. 32:11-6 (2004). Structural modeling showed that the bulky
nonpolar 0-acetyl groups at C3 make up most of the surface of the
polysaccharide molecule by protruding in rows on both sides, whereas the
carboxyl and N-acetyl (at C2) groups are mostly embedded or located close to
the axis. Szu et al., Infect. Immun. 59:4555-4561 (1991). This is consistent
with the 0-acetyl group being the dominant immunogenicity determinant. The
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amount of 0-acetylation is expressed as the degree of 0-acetylation (D0Ac,
ratio of 0-acetyl group/Gal UA [mole/mole]) or as the acetyl group content
(pmole) 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 at,,
õI, Bacterial. 94:1411-1416 (1967); Szu et at., Infect. Irnmun, 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 at., Vaccine, 26:292-300 (2008).
[0012] The 0-acetyl 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
polysaccharide 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 polygalacturonic acid (PGA) that is
variably methylated. Those from apple 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 methoxyl (LM) pectin,
whereas those with a DM of >50% are high methoxyl (HM) pectins. LM pectins
with a DM below 10% are considered as PGA. As a final product, the PGA is
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often produced in the sodium salt form or sodium polygalacturonate. Thus, the
term "polygalacturonic acid" is used herein interchangeably with
"polygalacturonate." LM pectins and PGAs are commonly obtained by
dernethyiation 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 0-acetylated in
efforts to analyze the antigenicity of Vi polysaccharides and production of a
synthetic Vi polysaccharide vaccine. The resulting acetylated product was
found to share the same antigenicity with native Vi polysaccharide. Szewczyk
and Taylor, Infect. Immun. 29:539-544 (1980); Sal et al., Infect. Immun.
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 105 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 irnmunogenicity on molecular weight has been
demonstrated with other polysaccharide antigens. Gonzalez-Fernandez et al.,
Vaccine. 26:292-300 (2008). However, the 0-acetylated LM pectin was
immunogenic once conjugated to a protein carrier, although its antibody
response level was much lower than that of Vi polysaccharide-protein
conjugate. Szu et al., Infect. Immun. 62:5545-5549 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.
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surtwRy
[0015]The instant disclosure provides 0-acetylated high molecular
weight polygalacturonic acids (0Ac-HPGA) or pharmaceutically acceptable
salts thereof, having at least one of: (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,
useful as a synthetic immunogenic Vi antigen. The instant disclosure further
provides methods of preparing an 0-acetylated 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.
[0016] Pectins from different plant sources have different chemical and
physical characteristics. A high molecular weight polygalacturonic 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 7,691,986, all 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 NaCl), Like a LM
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
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chemical and physical properties distinguish HPGA from all other existing
pectins or PGAs.
I
talilei.,. Physical and chemical proporties of HPGA¨:::::!:'¨:::!:::-----g¨
: Molecular weight : > 1 x 106 Da (SEC; Pextran Standards)
Pol dissersit. MW/Mn), ___________ 5.1.8 ............................. --- -
,i
Anhydro-Gal UA content (w I wrlo 80-90% ......
Neutral sugar content (w/w)% <5%
Degree of methylationgo mole/mole) <6%
Sodium content (w/w) !1012% ___
Protein content (w/w) ........... <0.5% ............................ ,
[0017] As defined herein, HPGA is a high molecular weight
polygalacturonic 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 0-acylated high molecular
weight polygalacturonic 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 rharnnose content ranging
from about 2% to about 15% per mole.
[0019] In some embodiments of this disclosure, the HPGA is GelSite .
GelSite is manufactured under cGMP from the Aloe vera L. plant. The
manufacturing process includes mincing of plant materials, extraction,
clarification and 0.2 pm filtration followed by the purification steps as
described
in detail in U.S. Patent 7,691,986, The final product is a dried substance.
Commercially, the resulting HPGA is also known as GelSite polymer. A Drug
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Master File (DMF) 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 0-acetylated high molecular weight
polygalacturonic acid (flAc-HPGA, e.g., GelSite-OAcTm) or pharmaceutically
acceptable salt thereof is generated by 0-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 0-acetylation.
[0021] OAc-HPGA, for example, GelSite.-OAcTM, is produced by a
chemical process at a very high yield (weight yield >100%). It has a high
degree of 0-acetylation (DOAc) as the ratio of 0-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 (>4.6 pmole/mg), and a
high molecular weight (>1 x 106 Da), thus exceeding the potency specifications
of the current Vi vaccines. GelSiteOAcTM 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.
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[0022] Accordingly, it is an objective of the instant disclosure to provide a
0-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 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.
[0023] In some embodiments, the OAc-HPGA or pharmaceutically
acceptable salt thereof has a molecular weight greater than 1 x 106 Da.
[0024] In some embodiments, the degree of 0-acetylation (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 0-acetylation greater than 100%, or a degree of 0-acetylation
greater than 50% at either the C2 or C3 position,
[0026] In some embodiments, the 0Ao-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.
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[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., GelSite..OAcTM) can be produced in large quantities from the
plant-based starting material (e.g., GelSitee).
[0032] Accordingly, it is another objective of the instant disclosure to
provide an 0-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:
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(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.
[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.
[0036] 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
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subject in need thereof an effective amount of the OAc-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 0-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] In some embodiments, administration of the pharmaceutical
composition induces an antibody response,
[0044] In some embodiments, administration of the pharmaceutical
composition induces a 24o1d 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] It 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
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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 plgidose. It is plant-based and therefore does not
contain
endotoxin (lipopolysaccharide, 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 DE5_CRIPTION OF THE DRAWINGS
[0049] Fig. '1 illustrates structures of Vi polysaccharide, HPGA (e.g,,
GelSite ), and OAc-HPGA (GelSite-OAcTm). The basic galacturonic acid (Gal
UA) residue is shown for Vi polysaccharide, HPGA and OAc-HPGA.
[0050] Fig. 2 illustrates the 0-acetylation process for production of OAc-
HPGA (GelSite-OAcTm), The asterisk indicates the optional use of a small
amount of perch lone acid as the catalyst.
[0051] Fig. 3 illustrates size exclusion chromatograms of GelSite and
GelSite-OAcTM, together with Dextran Standards. The Dextran Standards used
were 1.597, 214.8, and 39.9 kDa.
[0052] Fig. 4 illustrates the antigenicity of 0-acetylated polygalacturonic
acid (GelSite-OAcTm), as tested in irnmunodiffusion assay. Each well received
20 pl polysaccharide (200 pgiml) or reference serum (center well). The agarose
plate was kept in a wet chamber overnight. The wells were charged as follows;
1) GelSite ; 2) 0-acetylated polygalacturonic acid lx; 3) 0-acetylated
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polygalacturonic acid 2x; 4) O-acetylated polygalacturonic acid 3x; and C) Vi
polysaccharide.
[0053] Fig. 5 illustrates any effect of DOAc on immune responses to
GelSite-OAcTm. Balb/c mice were immunized twice with Vi vaccine or GelSite-
OAcTm having different DOAc at 2.5 pg/mouse , 4 weeks apart. Specific IgG
was measured using the Vi polysaccharide (A) or GelSiteOAcTM (B) as the
antigen.
[0054] Fig. 6 illustrates any dose-dependent effect of GelSite-OAcTM.
Balbic mice were immunized twice with GelSite-OAcTM (DOAc, 1.75) at the
different indicated doses, 6 weeks apart, Specific IgG was measured by ELISA
using the Vi polysaccharide (A) or GelSite-OAc (B) as the antigen.
[0055] Fig. 7 illustrates any cross reactivity. Pooled serum samples from
different study groups were reacted with GelSite (A), G&SiteOAcTM (B) or Vi
polysaccharide (C).
[0056] Fig. 8 illustrates the cross-boosting effect. Specific IgG antibodies
were measured with Vi polysaccharide (A) or GelSite-OAcTm(B).
[0057] Fig. 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-OAcTm(B) as the antigen.
[0058] Fig. 10 illustrates protection against lethal challenge with live S.
Typhi. Balbic mice immunized twice with Vi vaccine or GelSite-OAcTm 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.
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[0059] Fig. 11 illustrates the correlation of DOAc with immunogenicity of
GeISiteOAcTM. Balbic mice (n=6) were immunized with GelSiteOAcTM having
different DOAc at 2.5 pgimouse 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 Ge]SiteOAcTM
(B) as the antigen,
[0060] Fig, 12 illustrates the correlation of DOAc with protection of
GelSiteOAcTM. Balbic mice (n=6) were immunized with GelSite-OAcTM having
different DOAc at 2.5 pgimouse twice, 4 weeks apart. Animals were challenged
with 100 L.D50 of S. Typhi at week 3 following the second immunization. A, %
survival; B, Mean body weight.
DESCRIPTION
[0061] The 0-acetylation 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
OAc-
HPGA beads were solubilized by increasing the acidic pH to neutral pH. The
acetylation process is highly efficient with >100% yield due to the simplicity
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 Maclay. J. Am. Chem. Soc.
68:1015-1017 (1946).
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[0063] The term "0-acetylation" or "0-acetylated" refers to addition of
acetyl groups at the C2 and C3 positions of a sugar residue. The maximum
degree of 0-acetylation (200%) is reached when both the C2 and C3 positions
of each sugar residue are acetylated.
[0064] The resulting flAc-HPGA (e.g., GelSfte-OACTM) has a degree of
0-acetylation (DOAc) of at least 130% or 100%, or at least 65%, or 50% at
either the C2 or C3 position, and a high molecular weight (>1.0 x 106 Da),
thus
closely resembling the native Vi polysaccharide (60-90% 0-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, flAc-HPGA also
has
an increased molecular weight.
[0065] The potency indicators for the current licensed Vi vaccines are the
0-acetyl group content pmoie 0-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 Biological Standardization, 1993). Thus, OAc-
HPGA readily meets and exceeds both potency indicators for current Vi
vaccines (>2.5 urnol 0-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., GelSite-OAcTM)
no longer forms a gel with calcium. Also unlike HPGA, OAc-HPGA can directly
dissolve in saline or buffered saline (150 mM NaCl). These results indicate
that
the 0-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). It shared the same antigenicity as the Vi
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polysaccharide. Importantly, no reaction with HPGA was obtained, indicating
that the 0Ac-HPGA does not cross-react with its unacetylated parental
molecule.
[0068] 0Ac-HPGA, such as GelSite-OAcT", is immunogenic in mice (Fig.
5). Specific antibodies can be detected by ELISA with Vi polysaccharide or
0Ac-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. 62:5545-5549 (1994), 0Ac-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 0Ac-HPGA is directed toward
the 0-acetyl group, the most critical immunogenicity determinant of Vi
polysaccharide.
[0069] Remarkably, 0Ac-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, 0Ac-
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-OAcTM, but also
Vi
vaccine as the second dose. This further confirms the structural similarity
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between the OAc-HPGA and Vi polysaccharide. This boosting effect potentially
makes it effective in people 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 ug 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.
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EXAMPLES
Example 1: 0-Acetylation of GelSite Polymer
[0072] Two different methods were evaluated for 0-acetylation of
GelSite: one was described by Carson and Maclay (1946) and the other by
Schweiger (1964), Both methods were capable of 0-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 0-acetylated 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 hi 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 pH, 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 0-acetylation of GelSite . After acetylation, the
gel beads or strands are washed in deionized water to remove the acetylation
reagents without any loss as acetylated GelSite in acid form is insoluble in
water. After the wash, the acetyiated GelSite is then solubilized by
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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 GelSite solution in water (200ml at 5 mg/ml, 1 gram total)
was dropped into 1 liter of 0,1 M HCI to produce GelSite gel beads. The gel
beads were recovered and washed in 100 ml 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 perchioric 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 M
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 gel beads are at least 2 mm in
diameter
and can be readily recovered during the acetylation process.
2) Degree of 0-Acetylation (DOAc). The DOAc is a key parameter for
the GelSite-OAcTM. The acetylation process was highly efficient, yielding a
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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 1Jmoleimg 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 GelSiteOAcTM 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-OAcTm.
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
low
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. It was found that 20% perchloric
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
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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.
6) Drying, At the end of the process, the GelSite-OAcTM could be readily
dried by lyophilization, thus eliminating the use of ethanol precipitation.
T:.tible;i2074iitehtititiplil(aGetSite:IMPGAt'141(iii!Oditi..:00!::A0.1
18)Veitjtitithethdd
Parametersii8W04.0iiV:
____________________________
130101070910:AS:WW071009:::143.0t01::i0701,025i:!::.
Starting material GP 060701; Mw 216.1 kDa (Dextran Std)
I¨Amount of the starting_HPGA 0.359 .. 1.15 g 1.04 g
Rounds of acetylation reaction 1 _1 1
Yield of acetylated HPGA 0.39 g (111%) 1.21 9(105%) 1.04 g
(99.8%)
Mw (Dextran Std) ___________ 2996.8 kDa 2136.1 kDa 4554.1 kDa
Polydispersjy 1.624 1.522 1.546
Degree of 0-Acetylation, 147% 135% 134%
mole/mole (pmole/mg) (6.31) (5.97) (5.75)
Cross reaction with reference Yes Yes Yes
serum and antigen (NICHD)
Soluble in saline Yes Yes Yes __
I-Gelation with calcium L!o No .................... No
Example 2; Characterization of Acetylated GelSiteq.4.) Polymer
1. Molecular weight
[0077] The HPLC SEC (size exclusion chromatography) with Dextran
standards was used to determine the molecular weight of GelSite.OAcTM. The
molecular weight of polymers such as polysaccharides 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
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measurement, thus aliowing the results to be directly comparable to those
reported previously. Briefly, the Shodex OH pak SB-806HQ (300 x 8 mm),
Shodex 0Hpak SB-805HQ (300 x 8 mm), and Shodex 0Hpak SB-804HQ (300
x 8 mm) columns were used in tandem with 0.1 M ammonium acetate and 200
ppm sodium azide as the mobile phase at a flow rate of 0,5 ml/min. Molecular
weight values of the peak apex were reported as compared to the Dextran
Standards (Phenomenex broad MWD, 8 standards, 10-200 kDa) (Fig, 3). The
molecular weight of GelSite-0AG TM 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 0-acetylation (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, hydroxyiamine
hydrochloride and iron chloride. The assay was conducted in 96-well plates,
Samples were tested at -0,2 mg/m1 (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 pmole/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-
OAcTM 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 also be
expressed based on umole/mg (Table 2). The DOAc could be further increased
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by extending the acetylation 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 TM to gel in the presence of
calcium was determined by mixing a GelSite or GelSiteOAcTM solution with a
calcium solution. Thus, a - 0.2% (wiv) 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-OAcTM. Gel formation was indicated by the whole sample solution
changing into a gel and which could no longer flow freely. The GelSite-OAcTm
solution remained as a free flowing solution after mixing with the calcium
chloride.
[0081] Also, unlike GelSite , GelSite-OAcTM could be directly dissolved in
saline or buffered saline (150 mM NaCI). These results indicate that the 0-
acetyiation has altered the basic chemical and functional properties of the
GelSite and that the resulting product, GelSite-OAcTM, is a new chemical
entity.
4. Antigenicity
[0082]The immunodiffusion assay was performed in 1% agarose as
described by Szu et al Infect, immun. 62:5545-5549 (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-OAcTm formed positive
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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
Maclay method (Fig. 4). GetSite-OAcTM shared the same or substantially the
same antigenicity as the Vi polysaccharide as evidenced by the merging of the
precipitation lines from GelSiteOAcTM and Vi polysaccharide; no reaction was
observed with the un-acetylated GelSite (Fig. 4). As used herein, the terms
the same or substantially the same antigenicity as the Vi polysaccharide"
means that the GelSiteOAcTM is reactive with the anti-Vi polysaccharide serum
or antibodies. For example, as seen in Fig. 4 and discussed_above, GelSite-
OAcTM formed positive precipitation lines with the reference serum in the
immunodiffusion assay of Szu et at. which merged with the immunodiffusion
assay precipitation lines of the Vi polysaccharide.
5. Comparison to Vi polysaccharide and vaccine
[0083] The GelSite-OAc TM has a degree of 0-acetylation of >130% (5.58
pm/mg), or >65% (2.79 pm/mg) at either the C2 or C3 position, assuming an
even distribution between these two 0-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 0-acetylation at the C3 position of 60-90%
(Szu
and Bystricky, 2003). Thus, the GelSite-OAcTm 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
0-acetylation content (?.2 pmole /mg [at C3]) and molecular weight (50% of the
polysaccharide .zz 2.5 x 104 Da) of the Vi polysaccharide (WHO Expert
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Committee on Biological Standardization, 1993; Keitel et al., 1994). With a
DOAc of readily >2.5 pmol /mg (> 65%) at either the C2 or C3 position and a
molecular weight of M x 106 Da for ?.70% of the polysaccharide (Table 2 and
Fig 3), GelSiteOAcTM exceeds both potency specifications.
Example 3: Immunogenicity of O-Acetylated GelSite (GelSite-
OAc TM )
[0085] The immunogenicity of GelSiteOAcTM was examined in Balt*
mice in comparison with a licensed Vi vaccine (Typhim Sanofi-
Pasteur).
Groups of mice (n=10) were immunized with the GelSiteOAcTM 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 ELISA in 96-wel1 plates as
described previously. Szu et al., Enzyma 363:552-567 (2003). The Vi
polysaccharides from NICHD and International Vaccine Institute (IV]), 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 ELISA units/ml obtained from NICDH. The
antibody titers were determined using the CDC ELISA program
(http://www.cdc.govincidod/dbmdibimbielisa.htm) with the reference serum as
the standard.
1. Effects of DOAc and antigen dose
[0087] Effect of DOAc: Three GelSite-OAcTM samples with different
DOAc (136%, 155%, or 175% mole/mole, or 5.6, 6,5, or 7.5 pmole/mg) were
tested. The results showed that levels of specific IgG responses were found to
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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
GelSite-OAcTm (Fig. 6B), The GelSite-OAcTM with the lowest DOAc (136%)
consistently yielded the lowest titer at most time points. The GelSiteOAcTM
samples with 155% or 175% DOAc exhibited very similar response levels,
suggesting that the GelSite-OAcTM with 155% or 175% DOAc could be equally
effective. This DOAc-dependent effect on immune responses were further
demonstrated by evaluating GelSite-OAcTM with a DOAc as low as 80%
(Example 5).
[0088] Effect of antigen dose: The effect of GelSite-OAcTm antigen dose
was also tested at four different dose levels (1, 2.5, 5, and 10 pg) using the
GelSite-OAcTm with 175% DOAc. The antibody responses measured with
either Vi or GelSiteOAcTM were antigen dose-dependent, especially after the
second immunization (Fig. 6).
[0089] Comparison with the Vi vaccine: GelSite-OAcTM with 155% or
175% DOAc consistently induced comparable 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. 5 and 6). When measured with
the GelSite-OAcTM antigen, the specific antibody titers induced by GelSite-
OAcTm 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-OAcTM. Together, these results indicate that the GelSite-OAcT" is
highly immunogenic when compared to the Vi vaccine.
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2. Cross reactivity
[0090] The specificity of the antibodies induced by GelSite-OAcTm was
evaluated by reaction with its parental molecule (GelSite ) in comparison with
Vi
polysaccharide and GelSite-OAcTM. 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 low starting dilution of 1:40 and the reference serum obtained
from
NICDH was also included in the test (at the 1:2000 starting dilution). No
reaction with the GelSite was observed at any dose levels of GelSiteOAcTM
(Fig, 7A), while high saturating levels of reactions were obtained against the
GelSite-flAc TM (Fig. 7B). These results are consistent with the results
obtained
by the immunodiffusion test described above. They indicate that the response
generated against the GetSite-OAcTM is directed toward the 0-acetyl groups on
the GelSite-OAcTM, This has been further confirmed by using GelSite-OAcTM
with different DOAc (134% - 175%) as the antigens in ELISA, 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 GelSiteOAcTM upon the second immunization in all
animal experiments conducted. As shown in Figs. 5 and 6, the titers of all
three GelSiteOAcTM groups, as measured against the Vi or GelSiteOAcTM
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-OAcTm with a
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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. 8). The results showed that all animals primed with GelSite-
OAcTM exhibited the boosting effect upon the second immunization with
G&Site-OAcTM or Vi vaccine (Fig. 8). The boosting level obtained with Vi
vaccine was higher than that with GelSiteOAcTM when measured with Vi
polysaccharide (Fig. 8A), This may reflect the additive effect of boosting
based
on the 0-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-OAcTM
did show a boosting effect, although to a lesser extent and only detectable
when antibodies were measured against GelSite-OAcTM (Fig. 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
GelSiteOAcTM is used as the priming immunization, and therefore further
confirming the novel boosting effect of the GelSite-OAcTm and its structural
similarity to Vi polysaccharide.
4. IgG subclasses
[0094] The unique boosting effect of GelSite-OAcTM 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
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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 (IgG1 IgG2a, IgG2b, IgG3) and Igrvi
by ELISA. Animals injected with GeISite-OAcTM exhibited high levels of IgG1
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 IgG1 IgG2a, and IgG2b was observed after the second immunization with
the IgG2a 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 GelSiteOAcTM. The response is similar to the one
obtained with the Vi-conjugate vaccine (An et al., 2012) and Streptococcus
pneumonia polysaccharide conjugate vaccine (Safari et al., 2011). Unlike
GelSite-OAcTM, the Vi vaccine exhibited the same or decreased titers for all
IgG
subclasses following the second immunization, with the exception of IgG2a,
which showed a 2-fold increase. No apparent change in IgM levels were
observed with either vaccine.
Example 4: Protective effect of 0-acetylated GelSite polymer
[0095] The protective effect of the specific immune responses induced by
GelSite-OAcTM 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 Balbic mice were immunized with GelSite-OAcTM with different DOAc
(136% or 155%) or Vi vaccine at 2.5 pgimouse 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
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intraperitoneally 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),
[0096] The results showed that all immunized animals were protected,
including the two GelSite-OAcTM 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-OAcTM was just as
protective as the Vi vaccine. In addition, the two GelSite-OAcTm groups with a
DOAc of 136% or 155% showed no difference in protection, indicating the
GelSite-OAcTM 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 GelSiteOAcTM antigen as described
above and similar results to those shown in Fig. 5 were obtained.
Example 5: Further correlation of DOAc with immunogenicity and
protection
[0097] The DOAc is a critical specification for GelSite-OAcTM. Thus, it is
important to further demonstrate the correlation between DOAc and protection.
A series of additional GelSite-OAcTM 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 11 and 12). The unacetylated
GelSite with a 0% DOAc did not induce any detectable antibodies against
either Vi polysaccharide or GelSite-OAcT", nor provided any protection,
further
indicating that the antibodies induced by GelSite-OAcTM are directed at the 0-
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acetyl group. Importantly, GelSiteOAcTM 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/m1 against the Vi after the second immunization; Fig. 11A), the
GelSite-OAcTm with the 80% DOAc still provided a 83% protection (Fig. 12A),
suggesting that the antibodies induced by GelSite-OAcTM are highly protective.
Together, these results suggest that a GelSiteOAcTM with DOAc of 100% or
higher could 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. It 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.
32
