U.S. patent application number 15/406733 was filed with the patent office on 2017-06-08 for method for producing and vaccine composition of neisseria meningitidis serogroups a, c, y, and w-135 oligosaccharides conjugated to glycan-free carrier protein.
The applicant listed for this patent is Jeeri R Reddy. Invention is credited to Jeeri R Reddy.
Application Number | 20170157235 15/406733 |
Document ID | / |
Family ID | 38957435 |
Filed Date | 2017-06-08 |
United States Patent
Application |
20170157235 |
Kind Code |
A1 |
Reddy; Jeeri R |
June 8, 2017 |
METHOD FOR PRODUCING AND VACCINE COMPOSITION OF NEISSERIA
MENINGITIDIS SEROGROUPS A, C, Y, AND W-135 OLIGOSACCHARIDES
CONJUGATED TO GLYCAN-FREE CARRIER PROTEIN
Abstract
A multivalent vaccine composition, a method of producing the
multivalent vaccine composition, and an apparatus containing the
multivalent vaccine composition. The multivalent vaccine
composition may include a mixture. The mixture may include
Neisseria meningitides serogroups A, C, Y, and W-135
oligosaccharides conjugated to glycan-free carrier proteins. When
administered, the multivalent vaccine composition may provide
long-lasting immunity to humans of all age groups, including
infants.
Inventors: |
Reddy; Jeeri R; (Waterloo,
NE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Reddy; Jeeri R |
Waterloo |
NE |
US |
|
|
Family ID: |
38957435 |
Appl. No.: |
15/406733 |
Filed: |
January 15, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11680471 |
Feb 28, 2007 |
7491517 |
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15406733 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 31/04 20180101;
A61K 39/385 20130101; C07K 19/00 20130101; A61K 2039/6037 20130101;
A61K 2039/70 20130101; A61K 39/095 20130101; A61K 2039/55
20130101 |
International
Class: |
A61K 39/095 20060101
A61K039/095; A61K 39/385 20060101 A61K039/385 |
Claims
1. A multivalent vaccine composition, comprising: a mixture
comprising: conjugated Neisseria meningitides serogroup A
oligosaccharides, each of the conjugated Neisseria meningitides
serogroup A oligosaccharides being conjugated to a first particular
glycan-free carrier protein; conjugated Neisseria meningitides
serogroup C oligosaccharides, each of the conjugated Neisseria
meningitides serogroup C oligosaccharides being conjugated to a
second particular glycan-free carrier protein; conjugated Neisseria
meningitides serogroup Y oligosaccharides, each of the conjugated
Neisseria meningitides serogroup Y oligosaccharides being
conjugated to a third particular glycan-free carrier protein; and
conjugated Neisseria meningitides serogroup W-135 oligosaccharides,
each of the conjugated Neisseria meningitides serogroup W-135
oligosaccharides being conjugated to a fourth particular
glycan-free carrier protein.
2. The multivalent vaccine composition of claim 1, wherein at least
one of the first, second, third and fourth particular glycan-free
carrier proteins is a glycan-free diphtheria toxoid.
3. The multivalent vaccine composition of claim 2, wherein the
conjugated Neisseria meningitides serogroup A, C, Y, and W-135
oligosaccharides of the mixture have sizes approximately between
5100 and 9900 Daltons.
4. The multivalent vaccine composition of claim 3, wherein the
first, second, third, and fourth particular glycan-free carrier
proteins are glycan-free diphtheria toxoid, wherein, with respect
to at least one of the conjugated Neisseria meningitides serogroup
A, C, Y, and W-135 oligosaccharides of the multivalent vaccine
composition, a ratio of a mass of unconjugated Neisseria
meningitides serogroup oligosaccharides to a mass of glycan-free
diphtheria toxoid is approximately between 1:1 and 1:2.
5. The multivalent vaccine composition of claim 3, wherein the
multivalent vaccine composition does not include an adjuvant.
6. The multivalent vaccine composition of claim 5, wherein the
glycan-free diphtheria toxoid is not in a detoxified state.
7. The multivalent vaccine composition of claim 6, wherein the
glycan-free diphtheria toxoid is glycan-free and formalin-free
diphtheriatoxoid.
8. The multivalent vaccine composition of claim 6, wherein the
glycan-free diphtheria toxoid is hydrazide-activated glycan-free
diphtheriatoxoid.
9. The multivalent vaccine composition of claim 1, wherein each of
the first, second, third, and fourth particular glycan-free carrier
protein is of a single glycan-free diphtheria toxoid species.
10. The multivalent vaccine composition of claim 1, wherein the
multivalent vaccine composition is formulated as an aseptic liquid
and is sterilized after formulation.
11. The multivalent vaccine composition of claim 10, wherein the
multivalent vaccine composition contains no preservative.
12. The multivalent vaccine composition of claim 1, wherein the
multivalent vaccine composition is produced and processed by
utilizing animal-component-free reagents and chemicals.
13. The multivalent vaccine composition of claim 1, wherein at
least one of the first, second, third, and fourth particular
glycan-free carrier proteins is glycan-free and formalin-free
diphtheria toxoid.
14. The multivalent vaccine composition of claim 1, wherein the
multivalent vaccine composition is a tetravalent vaccine
composition.
15. The multivalent vaccine composition of claim 1, wherein the
multivalent vaccine composition is a tetravalent vaccine
composition and each of the first, second, third, and fourth
particular glycan-free carrier proteins is glycan-free and
formalin-free diphtheria toxoid.
16. The multivalent vaccine composition of claim 1, wherein the
mixture comprises four distinct and collective protein-capsular
oligosaccharide monoconjugates of serogroups A, C, Y, and W-135 of
Neisseria meningitides.
17. The multivalent vaccine composition of claim 1, wherein the
conjugated Neisseria meningitides serogroup A, C, Y, and W-135
oligosaccharides of the mixture have sizes approximately between
5100 and 9900 Daltons.
18. The multivalent vaccine composition of claim 17, wherein the
multivalent vaccine composition is configured to provide immunity
to humans of all age groups for at least three years.
19. The multivalent vaccine composition of claim 17, wherein the
multivalent vaccine composition is configured to provide immunity
to infants.
20. The multivalent vaccine composition of claim 17, wherein the
multivalent vaccine composition is configured to provide immunity
to children less than 2 years of age for at least three years.
21. The multivalent vaccine composition of claim 1, wherein the
multivalent vaccine composition is configured to provide immunity
to humans of all age groups for at least three years.
22. The multivalent vaccine composition of claim 1, wherein the
multivalent vaccine composition is configured to provide immunity
to infants.
23. The multivalent vaccine composition of claim 1, wherein the
multivalent vaccine composition is configured to provide immunity
to children less than 2 years of age for at least three years.
24. A method for producing a multivalent vaccine composition
comprising Neisseria meningitides serogroups A, C, Y, and W-135
oligosaccharides conjugated to glycan-free carrier proteins, the
method comprising: non-chemically adjusting sizes of Neisseria
meningitides serogroups A, C, Y, and W-135 oligosaccharides;
activating the Neisseria meningitides serogroups A, C, Y, and W-135
oligosaccharides upon non-chemically adjusting the sizes of the
Neisseria meningitides serogroups A, C, Y, and W-135
oligosaccharides; activating glycan-free carrier proteins;
conjugating at least some of the activated Neisseria meningitides
serogroups A, C, Y, and W-135 oligosaccharides to at least some of
the activated glycan-free carrier proteins; and producing a
multivalent vaccine composition comprising a mixture, the mixture
comprising: conjugated Neisseria meningitides serogroup A
oligosaccharides, each of the conjugated Neisseria meningitides
serogroup A oligosaccharides being conjugated to a first particular
glycan-free carrier protein of the activated glycan-free carrier
proteins; conjugated Neisseria meningitides serogroup C
oligosaccharides, each of the conjugated Neisseria meningitides
serogroup C oligosaccharides being conjugated to a second
particular glycan-free carrier protein of the activated glycan-free
carrier proteins; conjugated Neisseria meningitides serogroup Y
oligosaccharides, each of the conjugated Neisseria meningitides
serogroup Y oligosaccharides being conjugated to a third particular
glycan-free carrier protein of the activated glycan-free carrier
proteins; and conjugated Neisseria meningitides serogroup W-135
oligosaccharides, each of the conjugated Neisseria meningitides
serogroup W-135 oligosaccharides being conjugated to a fourth
particular glycan-free carrier protein of the activated glycan-free
carrier proteins.
25. The method of claim 24, wherein the conjugated A, C, Y, and
W-135 oligosaccharides have sizes approximately between 5100 and
9900 Daltons.
26. The method of claim 24, further comprising: removing
unconjugated oligosaccharides from a mixture including the
activated A, C, Y, and W-135 oligosaccharides conjugated to the
glycan-free carrier proteins.
27. The method of claim 24, further comprising: sterilizing a
mixture including the activated A, C, Y, and W-135 oligosaccharides
conjugated to the glycan-free carrier proteins.
28. The method of claim 24, further comprising: vialing at least a
portion of the multivalent vaccine composition comprising Neisseria
meningitides serogroups A, C, Y, and W-135 oligosaccharides
conjugated to the glycan-free carrier proteins.
29. The method of claim 24, wherein the glycan-free carrier
proteins are glycan-free and formalin-free diphtheria toxoid.
30. The method of claim 24, wherein the multivalent vaccine
composition is a tetravalent vaccine composition.
31. The method of claim 24, wherein the glycan-free carrier
proteins are glycan-free diphtheria toxoid.
32. An apparatus, comprising: a vessel containing a multivalent
vaccine composition, the multivalent vaccine composition
comprising: a mixture comprising: conjugated Neisseria meningitides
serogroup A oligosaccharides, each of the conjugated Neisseria
meningitides serogroup A oligosaccharides being conjugated to a
first particular glycan-free carrier protein; conjugated Neisseria
meningitides serogroup C oligosaccharides, each of the conjugated
Neisseria meningitides serogroup C oligosaccharides being
conjugated to a second particular glycan-free carrier protein;
conjugated Neisseria meningitides serogroup Y oligosaccharides,
each of the conjugated Neisseria meningitides serogroup Y
oligosaccharides being conjugated to a third particular glycan-free
carrier protein; and conjugated Neisseria meningitides serogroup
W-135 oligosaccharides, each of the conjugated Neisseria
meningitides serogroup W-135 oligosaccharides being conjugated to a
fourth particular glycan-free carrier protein.
33. The apparatus of claim 32, wherein the vessel is a vial.
34. A multivalent vaccine composition, comprising: a mixture
comprising: a plurality of conjugated Neisseria meningitides
serogroup oligosaccharides, the plurality of conjugated Neisseria
meningitides serogroup oligosaccharides including at least two
serogroups of conjugated Neisseria meningitides serogroup A, C, Y,
and W-135 oligosaccharides, each of the plurality of conjugated
Neisseria meningitides serogroup oligosaccharides being conjugated
to a particular glycan-free carrier protein.
35. The multivalent vaccine composition of claim 34, wherein the
plurality of conjugated Neisseria meningitides serogroup
oligosaccharides including at least three serogroups of conjugated
Neisseria meningitides serogroup A, C, Y, and W-135
oligosaccharides.
36. A vaccine composition, comprising: a mixture comprising:
conjugated Neisseria meningitides serogroup oligosaccharides, each
of the conjugated Neisseria meningitides serogroup oligosaccharides
being conjugated to a particular glycan-free carrier protein.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is related to U.S. patent
application Ser. No. 11/680,471, filed on Feb. 28, 2007, now issued
as U.S. Pat. No. 7,491,517, which is herein incorporated by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The present disclosure relates generally to the fields of
medical microbiology, immunology, vaccines and the prevention of
infection by a bacterial pathogen by immunization.
BACKGROUND
[0003] Meningococcal meningitis is an infection of the meninges,
which is a thin lining that surrounds the brain and the spinal
cord. The causative agent, Neisseria meningitidis (also referred to
as meningococcus), was identified in 1887. Meningococcal disease
was first reported in 1805 when an outbreak swept through Geneva,
Switzerland.
[0004] Thirteen subtypes or serogroups of N. meningitidis have been
identified and six (N. meningitidis A, B, C, X, Y and W-135) are
known to cause epidemics. Pathogenicity, immunogenicity, and
epidemic capabilities differ according to the serogroup.
Identification of the serogroup responsible for a sporadic case is
important for epidemic containment. The most common symptoms are a
stiff neck, high fever, and sensitivity to light, confusion,
headaches, and vomiting. Even when the disease is diagnosed early
and adequate therapy instituted, 5% to 10% of patients die,
typically within 24-48 hours of the onset of symptoms. Bacterial
meningitis may result in brain damage, hearing loss, or learning
disability in 10 to 20% of survivors. A less common but more severe
(and often fatal) form of meningococcal disease is meningococcal
septicemia which is characterized by a hemorrhagic rash and rapid
circulatory collapse.
[0005] Major African epidemics are associated with N. meningitidis
serogroups A, W-135 and C, and serogroup A is usually the cause of
meningococcal disease in Asia. Outside of Africa, Mongolia reported
a large epidemic in 1994 to 1995. There is increasing evidence of
serogroup W-135 being associated with outbreaks of considerable
size. In 2000 and 2001 several hundred pilgrims attending the Hajj
in Saudi Arabia were infected with N. meningitidis W-135. Then in
2002, W-135 emerged in Burkina Faso, striking 13,000 people and
killing 1,500.
[0006] The highest burden of meningococcal disease occurs in
sub-Saharan Africa, which is sometimes referred to as the
"Meningitis Belt", an area that stretches from Senegal in the west
to Ethiopia in the east, with an estimated total population of 300
million people. This hyper-endemic area is characterized by a
particular climate and social habits. During the dry season,
between December and June, because of dust winds and upper
respiratory tract infections due to cold nights, local immunity is
diminished, increasing the risk of meningitis. At the same time,
the transmission of N. meningitidis is favored by the overcrowded
housing at the family level and by large population displacements
due to pilgrimages and traditional markets at the regional level.
This conjunction of factors contributes to the large epidemics
which occur during this season in the meningitis belt area. Partly
due to herd immunity (whereby transmission is blocked when a
threshold percentage of the population has been vaccinated, which
extends some protection to the unvaccinated), these epidemics tend
to occur in a cyclic mode. N. meningitidis A, C, and W-135 are now
the main serogroups involved in the meningococcal meningitis
activity in Africa.
[0007] In 1996, Africa experienced the largest recorded outbreak of
epidemic meningitis in history, with over 250,000 cases and 25,000
deaths registered. Between that crisis and 2002, 223,000 new cases
of meningococcal meningitis were reported to the World Health
Organization. The countries most affected have been Burkina Faso,
Chad, Ethiopia, and Niger. In 2002, the outbreaks occurring in
Burkina Faso, Ethiopia, and Niger accounted for about 65% of the
total cases reported on the African continent. Furthermore, the
meningitis belt appears to be extending further south. In 2002, the
Great Lakes region was affected by outbreaks in villages and
refugee camps which caused more than 2,200 cases, including 200
deaths.
[0008] In 2006 and 2007, outbreaks of the disease occurred in the
North of Ivory Coast and the southern region of Burkina Faso,
Southern Sudan, and Uganda, killing several children and adults.
Meningococcal meningitis impacts not only Africa but also the rest
of the world. Meningococcal meningitis impacts not only sub-Saharan
Africa but also North America, the United Kingdom, Ireland, Europe,
South East Asia, the Middle East, and New Zealand.
[0009] Currently, the capsular polysaccharides of Neisseria
meningitidis have been considered as having highly conserved and
highly exposed bacterial surface antigens. Currently, capsular
polysaccharides have been used as immunoprophylactic agents against
human disease caused by encapsulated bacteria. Currently, the
capsular polysaccharides of the meningococcus are negatively
charged and are obtained in a high-molecular-weight immunogenic
form by precipitation. Currently, meningococcal polysaccharide
vaccines are relatively efficacious for protection from meningitis
disease in adults; however, the duration of protection elicited by
existing meningococcal polysaccharide vaccines is not long lasting
and has been estimated to be 18 months in adults and children above
four years of age. Currently, for children from one to four years
old, the duration of protection is less than three years.
[0010] Polysaccharides, themselves, have been found to be at least
typically poor at stimulating an effective antibody response in the
highest risk age groups (e.g., infants). Children less than two
years of age are more susceptible to diseases caused by microbes
that have polysaccharide capsules, such as Neisseria meningitidis.
Current vaccines are expensive and have short duration of
protection.
[0011] Discovery of a low-cost, more easily manufactured, and i m
proved meningitis vaccine would be desirable for providing
affordable vaccines to third world countries and would reduce
mortality of infants, children, and adults. Additionally, there is
a need for a method of producing a meningococcal meningitis vaccine
at least substantially without chemical impurities or residues to
improve depolymerization and conjugation by chemical means and
capsular polysaccharide size. Also, there is a need for a medium
that produces a higher yield of polysaccharides and a lower yield
of cellular biomass to facilitate the production and purification
processes for vaccine production.
SUMMARY
[0012] In one aspect, embodiments of the inventive concepts
disclosed herein are directed to a multivalent vaccine composition.
The multivalent vaccine composition may include a mixture. The
mixture may include conjugated Neisseria meningitides serogroup A
oligosaccharides, where each of the conjugated Neisseria
meningitides serogroup A oligosaccharides are conjugated to a first
glycan-free carrier protein. The mixture may also include
conjugated Neisseria meningitides serogroup C oligosaccharides,
each of the conjugated Neisseria meningitides serogroup
[0013] C oligosaccharides being conjugated to a second particular
glycan-free carrier protein. The mixture may further include
conjugated Neisseria meningitides serogroup Y oligosaccharides,
each of the conjugated
[0014] Neisseria meningitides serogroup Y oligosaccharides being
conjugated to a third particular glycan-free carrier protein. The
mixture may additionally include conjugated Neisseria meningitides
serogroup W-135 oligosaccharides, each of the conjugated Neisseria
meningitides serogroup W-135 oligosaccharides being conjugated to a
fourth particular glycan-free carrier protein. [0013] In a further
aspect, embodiments of the inventive concepts disclosed herein are
directed to method for producing a multivalent vaccine composition
comprising Neisseria meningitides serogroups A, C, Y, and W-135
oligosaccharides conjugated to glycan-free carrier proteins. The
method may include non-chemically adjusting sizes of
[0015] Neisseria meningitides serogroups A, C, Y, and W-135
oligosaccharides. The method may include activating the Neisseria
meningitides serogroups A, C, Y, and W-135 oligosaccharides upon
non-chemically adjusting the sizes of the Neisseria meningitides
serogroups A, C, Y, and W-135 oligosaccharides. The method may
include activating glycan-free carrier proteins. The method may
include conjugating at least some of the activated Neisseria
meningitides serogroups A, C, Y, and W-135 oligosaccharides to at
least some of the activated glycan-free carrier proteins. The
method may include producing a multivalent vaccine composition
comprising a mixture. The mixture may include conjugated Neisseria
meningitides serogroup A oligosaccharides, each of the conjugated
Neisseria meningitides serogroup A oligosaccharides being
conjugated to a first particular glycan-free carrier protein of the
activated glycan-free carrier proteins; conjugated Neisseria
meningitides serogroup C oligosaccharides, each of the conjugated
Neisseria meningitides serogroup C oligosaccharides being
conjugated to a second particular glycan-free carrier protein of
the activated glycan-free carrier proteins; conjugated Neisseria
meningitides serogroup Y oligosaccharides, each of the conjugated
Neisseria meningitides serogroup Y oligosaccharides being
conjugated to a third particular glycan-free carrier protein of the
activated glycan-free carrier proteins; and conjugated Neisseria
meningitides serogroup W-135 oligosaccharides, each of the
conjugated Neisseria meningitides serogroup W-135 oligosaccharides
being conjugated to a fourth particular glycan-free carrier protein
of the activated glycan-free carrier proteins
[0016] In a further aspect, embodiments of the inventive concepts
disclosed herein are directed to an apparatus. The apparatus may
include a vessel. The vessel may contain a multivalent vaccine
composition. The multivalent vaccine composition may include a
mixture. The mixture may include conjugated Neisseria meningitides
serogroup A oligosaccharides, where each of the conjugated
Neisseria meningitides serogroup A oligosaccharides being
conjugated to a first glycan-free carrier protein. The mixture may
also include conjugated Neisseria meningitides serogroup C
oligosaccharides, each of the conjugated Neisseria meningitides
serogroup C oligosaccharides being conjugated to a second
particular glycan-free carrier protein. The mixture may further
include conjugated Neisseria meningitides serogroup Y
oligosaccharides, each of the conjugated Neisseria meningitides
serogroup Y oligosaccharides being conjugated to a third particular
glycan-free carrier protein. The mixture may additionally include
conjugated Neisseria meningitides serogroup W-135 oligosaccharides,
each of the conjugated Neisseria meningitides serogroup W-135
oligosaccharides being conjugated to a fourth particular
glycan-free carrier protein.
[0017] In one aspect, embodiments of the inventive concepts
disclosed herein are directed to a multivalent vaccine composition.
The multivalent vaccine composition may include a mixture. The
mixture may include a plurality of conjugated Neisseria
meningitides serogroup oligosaccharides. The plurality of
conjugated Neisseria meningitides serogroup oligosaccharides may
include at least two serogroups of conjugated Neisseria
meningitides serogroup A, C, Y, and W-135 oligosaccharides. Each of
the plurality of conjugated Neisseria meningitides serogroup
oligosaccharides being conjugated to a glycan-free
carrierprotein.
[0018] In one aspect, embodiments of the inventive concepts
disclosed herein are directed to a vaccine composition. The
multivalent vaccine composition may include a mixture. The mixture
may include conjugated Neisseria meningitides serogroup
oligosaccharides, each of the conjugated Neisseria meningitides
serogroup oligosaccharides being conjugated to a particular
glycan-free carrier protein.
DETAILED DESCRIPTION
[0019] Before explaining at least one embodiment of the inventive
concepts disclosed herein in detail, it is to be understood that
the inventive concepts are not limited in their application to the
details of construction and the arrangement of the components or
steps or methodologies set forth in the following description or
illustrated in the drawings. In the following detailed description
of embodiments of the instant inventive concepts, numerous specific
details are set forth in order to provide a more thorough
understanding of the inventive concepts. However, it will be
apparent to one of ordinary skill in the art having the benefit of
the instant disclosure that the inventive concepts disclosed herein
may be practiced without these specific details. In other
instances, well-known features may not be described in detail to
avoid unnecessarily complicating the instant disclosure. The
inventive concepts disclosed herein are capable of other
embodiments or of being practiced or carried out in various ways.
Also, it is to be understood that the phraseology and terminology
employed herein is for the purpose of description and should not be
regarded as limiting.
[0020] As used herein a letter following a reference numeral is
intended to reference an embodiment of the feature or element that
may be similar, but not necessarily identical, to a previously
described element or feature bearing the same reference numeral
(e.g., 1, 1 a, 1b). Such shorthand notations are used for purposes
of convenience only, and should not be construed to limit the
inventive concepts disclosed herein in any way unless expressly
stated to the contrary.
[0021] Further, unless expressly stated to the contrary, "or"
refers to an inclusive or and not to an exclusive or. For example,
a condition A or B is satisfied by anyone of the following: A is
true (or present) and B is false (or not present), A is false (or
not present) and B is true (or present), and both A and B are true
(or present).
[0022] In addition, use of the "a" or "an" are employed to describe
elements and components of embodiments of the instant inventive
concepts. This is done merely for convenience and to give a general
sense of the inventive concepts, and "a" and "an" are intended to
include one or at least one and the singular also includes the
plural unless it is obvious that it is meant otherwise.
[0023] Finally, as used herein any reference to "one embodiment,"
or "some embodiments" means that a particular element, feature,
structure, or characteristic described in connection with the
embodiment is included in at least one embodiment of the inventive
concepts disclosed herein. The appearances of the phrase "in some
embodiments" in various places in the specification are not
necessarily all referring to the same embodiment, and embodiments
of the inventive concepts disclosed may include one or more of the
features expressly described or inherently present herein, or any
combination of sub-combination of two or more such features, along
with any other features which may not necessarily be expressly
described or inherently present in the instant disclosure.
[0024] Broadly, embodiments of the inventive concepts disclosed
herein are directed to a vaccine composition, a method for
producing the vaccine composition, and an apparatus containing the
vaccine composition.
[0025] Embodiments may include coupling T-cell independent
saccharides to a T-cell dependent protein that allows an infant
immune system to provide T-cell help to B-cells to produce IgG
antibody of high affinity to the polysaccharide antigen. For
example, T-Independent (TI) antigens may improve vaccines.
Molecules, such as polysaccharides, that have numerous identical
evenly spaced epitopes characterize one type of TI antigen. In one
embodiment, as clusters of B-cell receptors bind the antigen
simultaneously or approximately simultaneously, it causes B-cell
activation without the help of T-helper cells. Such antigens may be
particularly important in young children who typically respond
poorly to these antigens.
[0026] In one embodiment, Neisseria meningitidis serogroup A, C,
W-135 & Y Polysaccharide may be extracted from cultures grown
in NMFM2 media in a fermenter vessel. For example, the cultures may
be incubated at 36+1.degree. C. up to a maximum of 24 hours while
controlling temperature, pH, aeration, agitation, pressure, and
dissolved oxygen content and antifoam additions whenever required.
After the incubation period, a batch may be terminated when the
glucose concentration reaches a threshold concentration (e.g.,
below 1 g/L), pH, and when dissolved oxygen content increases. The
culture may be killed by the addition of formaldehyde; for example,
0.5% volume/volume concentration of formaldehyde may kill the
culture. For example, adding about 420 mL of formaldehyde may kill
80L of broth. Polysaccharide size adjustment may be achieved by a
non-chemical method (e.g., sonication) to produce low molecular
weight meningococcal oligosaccharides (e.g., with a minimum range
of 5000 to 10,000 Daltons size). Sizes of oligosaccharides before
and after sonication were compared by high-performance liquid
chromatography-size exclusion chromatography (HPLC-SEC). In one
embodiment, the carrier protein used in the conjugation with
oligosaccharide is glycan-free diphtheria toxoid (GFDT), though
other carrier proteins are contemplated. Glycan may be removed by
Octyl-sepharose hydrophobic interaction chromatography and
confirmed by high-performance anion exchange chromatography-pulsed
amperometric detection analysis. In one embodiment, potluck
oligosaccharide activation may be performed on multiple (e.g., 4)
serogroup oligosaccharides that may be together activated by sodium
periodate reaction to generate aldehyde active groups on them. For
example, potluck oligosaccharide activation may be performed on 4
serogroup oligosaccharides that are together activated by 6mM
sodium periodate reaction at 22.+-.2.degree. C. for 4 hours to
generate aldehyde active groups on them. After activation, the
activated oligosaccharides may be diafiltered against HEPES using
filters (e.g., membrane filters, such as MWCO membrane filters) to
remove traces of sodium periodate. For example, after activation,
the activated oligosaccharides are diafiltered against 10 mM HEPES
pH7.5 at 2-8.degree. C. for 15 h using 1K MWCO membrane filters to
remove traces of sodium periodate. Oligosaccharide activation may
be confirmed by colorimetric estimation of aldehyde groups using
purpald assay. Acidic activation of diphtheria toxoid (DT) in the
presence of EDC (3-ethyl-1-[3-dimethylamino) propyl] carbodiimide
hydrochloride) and hydrazine may be used to create hydrazide groups
on the DT. For example, acidic activation of the DT in the presence
of EDC (3-ethyl-1-[3-dimethylamino) propyl] carbodiimide
hydrochloride) hydrazine may be converted to hydrazide groups.
Generated hydrazide groups on the toxoid may be quantified using
colorimetric assay using trinitrobenzene sulfonic acid (TNBS)
reagent where adipic acid dihydrazide (ADH) may be used as a
standard in this assay. Protein content may be estimated using BCA
assay kit following manufacturer's instructions. A degree of DT
activation may be arrived at by measuring hydrazide groups per mole
of DT by TNBS assay. For determination of DT degree of hydrazide
activation measurement, an average molecular weight of DT may be
taken as 63,000 Da. Embodiments may include conjugation of
activated oligosaccharide to activated GFDT. For example, potluck
activated oligosaccharides may be combined at suitable estimated
concentration of each activated oligosaccharide (e.g., 20 mg/ml)
and activated GFDT (e.g., 20 mg/ml) in a suitable weight ratio
(e.g., 1:1), and mixture may be continuously mixed by rotating in
suitable sealed containers, for example at 22.+-.2.degree. C. for
18-24 hours. The mixture may be treated with sodium borohydride,
for example for 6 h at 22.+-.2.degree. C., to perform reductive
amination and capping of unused aldehyde groups in a single step.
Conjugate ultra-purification may be performed by filtration (e.g.,
10K MWCO membrane filtration) to remove unconjugated low molecular
weight oligosaccharide. For example, the conjugated and capped
mixture may be diafiltered to remove unconjugated low molecular
weight oligosaccharide using 10K MWCO membrane filters. Each
oligosaccharide in the pure potluck conjugate may be quantified by
HPAEC PAD analysis. With respect to the above potluck tetravalent
conjugate, for example, the ultrapure mixture may be formulated in
isotonic 1.times.PBS to contain a final concentration of 8 .mu.g of
each serogroup oligosaccharide conjugated to a total 32-64 .mu.g
glycan-free diphtheria Toxoid per each milliliter of formulated
vaccine. For example, above formulated vaccine may be passed
through 0.2.mu. membrane filters to obtain sterile final vaccine to
consider for vialing.
[0027] An exemplary vaccine product was tested for safety and
efficacy in 560 human volunteers comprising male and females of the
ages 18 to 55. A safety study on the human subjects was observed
for a total of 26 weeks for any safety concerns. No SAEs were
observed in the phase 1 trial. Immunization with 4 .mu.g of each
serogroup oligosaccharide conjugated with 4-6 .mu.g Glycan-free
Diphtheria toxoid (per serogroup) produced seroconversion rates of
46-71% of subjects at 4 weeks: Serogroup A 46%, C 71%, W-135 63%
and Y 67%. Higher seroconversion rates (71-92%) were observed at 8
weeks: A 71%, C 92%, W-135 67% and Y 75%.
[0028] Embodiments include a method of producing meningococcal
meningitis vaccine comprising N. meningitidis serotypes A, C, Y and
W-135 that have a long lasting effect and provide broad spectrum
immunity to humans of all age groups.
[0029] Embodiments include a method wherein trace chemical
impurities currently present in the available meningococcal
meningitis vaccine are reduced (e.g., eliminated) by a mechanical
method, such as sonication.
[0030] Embodiments include a composition of a medium that yields a
higher percentage of polysaccharides in comparison to known media
employed for producing meningococcal meningitis vaccine.
[0031] Embodiments include a composition of a medium that yields a
lower percentage of cellular biomass in comparison with known media
employed for producing meningococcal meningitis vaccine.
[0032] Embodiments include method of producing a vaccine
composition and a vaccine composition having improved (e.g.,
optimum) molecular size of N. meningitidis polysaccharides of
serogroups A, C, Y and W-135 that confers broad spectrum
immunogenic protection against meningitis.
[0033] Embodiments include a multivalent (e.g., tetravalent)
vaccine and methods of producing such a vaccine. Embodiments may
include a meningococcal meningitis serogroups A, C, Y & W-135
vaccine made by using a fastidious culture medium (NMFM2). The
downstream process may be animal component-free. Polysaccharide
Size Adjustment may be performed by a non-chemical method (e.g.,
sonication) to produce low molecular weight polysaccharides and to
produce conjugated meningococcal polysaccharides, for example, with
a minimum range of 5000 to 10,000 Daltons size. A carrier protein
used in the conjugation with oligosaccharide may be glycan-free
diphtheria toxoid (GFDT). In an exemplary tetravalent embodiment,
potluck oligosaccharide activation may be performed on at least
some (e.g., all) of 4 serogroup oligosaccharides that may be
together activated by (e.g., 6 mM) sodium periodate reaction.
Activated oligosaccharides may be combined at estimated
concentration of each activated oligosaccharide (e.g., 20 mg/ml)
and activated GFDT (e.g., 20 mg/ml) in an exemplary weight ratio of
approximately 1:1. For such a potluck tetravalent conjugate, a
mixture (e.g., an ultrapure mixture) may be formulated in isotonic
1.times.PBS to contain a final concentration of 8 .mu.g, for
example, of each serogroup oligosaccharide conjugated to a total
32-64 .mu.g, for example, glycan-free diphtheria Toxoid per each
milliliter of formulated vaccine. Formulated vaccine may be
sterilized at the stage of final fill.
[0034] Some embodiments may include a vaccine composition which
does not include an adjuvant, but may include Glycan purified
Diphtheria Toxoid. Further, in some embodiments, the Diphtheria
Toxoid is not in a detoxified state. In some embodiments, the
Glycan free Diphtheria Toxoid is free from traces of Formaldehyde.
Some embodiments include a hydrazide activated Glycan free
Diphtheria Toxoid. In some embodiments, the carrier protein of the
vaccine composition is or includes a single Glycan free Diphtheria
Toxoid carrier protein species.
[0035] In some embodiments, the vaccine composition is formulated
as an aseptic liquid. In some embodiments, the vaccine composition
may be sterilized after formulation. In some embodiments, the
vaccine composition is preservative-free. In some embodiments, the
vaccine composition is a tetravalent conjugate vaccine comprising a
mixture of four distinct and collective protein-capsular
oligosaccharide conjugates of Serogroups A, C, Y and W-135 of
Neisseria meningitidis.
[0036] In some embodiments, the capsular oligosaccharides have a
minimum range of 5000 to 10,000 Daltons size of each of the
serogroups depolymerized by non-chemical methods.
[0037] In some embodiments, the capsular oligosaccharide and
Glycan-free Diphtheria Toxoid is produced and processed with
animal-component-free reagents and/or chemicals.
[0038] Trials were performed using ELISA bioassays because of
transportation problems of live bacteria from the United States to
Africa for performing SBA bioassays.
[0039] Meningococcal serogroup A, C, W-135, and Y polysaccharides
and DT or CRM197-based conjugates may be prepared using suitable
processes and conjugation chemistry. The polysaccharide content of
serogroups C, W-135, and Y conjugates may be quantified by sialic
acid determination. The Serogroup A conjugate may be quantified by
mannosamine-1-phosphate chromatographic determination.
[0040] The protein content may be measured by a micro-bicinchoninic
acid assay. For example, the polysaccharide-to-protein ratio of
conjugates may have any suitable range, such as between 0.3 and
1.5, which may be similar to that of cross-reacting material DT and
CRM-based conjugates.
[0041] A lymphocyte proliferation assay may be performed.
[0042] In addition, antigenic variation and human complement
sensitivity of Neisseria meningitidis may be a barrier to relying
on SBA bioassays.
[0043] Embodiments may include conjugation of bacterial
polysaccharides to immunogenic carrier proteins that results in
conjugates that induce strong anti-polysaccharide T-helper-cell
dependent immune responses in young infants.
[0044] Neisseria meningitidis are gram negative diplococci with
polysaccharide capsules that have led to the identification of at
least 13 different serogroups based on immunochemical studies.
Epidemics caused by N. meningitidis of Group A, C, Y, and W are
usually characterized by a predominance of a single meningococcal
genotype and a shift of cases towards older age groups. These four
serogroups express structurally different capsular polysaccharides
(shown below) that determine their distinct serological properties.
O-acetylation of sugar units at distinct locations is seen in all
these serogroups although at different levels.
##STR00001##
[0045] With respect to the above polysaccharide structures, the
structural characterization and verification of vaccine grade
polysaccharides may be performed by chemical and biophysical
methods. NMR spectroscopy may be used for characterizing the
meningococcal purified polysaccharides, and 1H NMR may be utilized
for the estimation of the percentage O-acetylation in each
serogroup. The O-acetyl transferases occurs in the serogroup A
Manacc O-acetylation.
[0046] Group A isolates are now principally responsible for
recurrent epidemics in the so called "meningitis belt" countries in
sub-Saharan Africa. Existing Polysaccharide vaccines offer
protection against groups A, C, Y, and W-135; however, such
existing vaccines produce poor immune responses in young children
and do not produce long-lasting immunity. Such existing vaccines
utilize polysaccharides that are T-cell independent antigens such
that they are poorly or not at all immunogenic in children aged
less than 2 years, and such existing vaccines do not induce vaccine
memory. Such existing vaccines use may predispose to blunting of
the immune response to subsequent doses.
[0047] Embodiments may utilize chemical conjugation of the capsular
polysaccharides to a protein carrier capable of eliciting T-cell
dependent immune responses.
[0048] In an exemplary embodiment, a method of producing (e.g.,
preparing) Meningococcal meningitis vaccine may include some or all
of the following steps:
[0049] A, C, W-135 & Y Polysaccharide preparation;
[0050] Mechanical Size Adjustment of Polysaccharide;
[0051] Glycan- & Formalin-free Diphtheria Toxoid (GFDT);
[0052] Potluck Oligosaccharide Activation;
[0053] GFDT Activation;
[0054] Conjugation of Activated oligosaccharide to Activated GFDT
to make individual Monoconjugate;
[0055] Conjugate ultra-purification by 10K MWCO membrane
filtration;
[0056] Formulation of Tetravalent conjugates vaccine in
1.times.PBS;
[0057] Sterilization of final vaccine by 0.2-micron filtration;
and
[0058] Vialing.
[0059] Such steps are exemplarily described, below.
[0060] A, C, W-135 & Y Polysaccharide Preparation:
[0061] In some embodiments, Neisseria meningitidis serogroup A, C,
W135, and Y frozen seed cultures may be thawed and streaked onto
NMFM2 Yeast extract meat free agar media. For example, such plates
may be incubated at 36.+-.1.0.degree. C. with CO.sub.2: 4.+-.2%
.sub.in the CO.sub.2 incubator for 12 to 18 hours. Some embodiments
may include inoculating 500 mL flasks containing 100 mL media with
2-3 lapful's of culture grown on the NMFM2 Yeast extract agar
plates. Some embodiments may include incubating flasks at
36.+-.1.0.degree. C. on an incubator shaker at 170.+-.30 RPM for
12.+-.4 hours until OD.sub.600 nm reaches 2.5.+-.1.0.
[0062] In some embodiments, once the flasks attain desired OD and
pass Gram stain test, some embodiments may include pipetting 70 mL
of inoculum into 630 mL media in 2 or 2.8 L flasks and incubating
at 36.+-.1.degree. C. with 170.+-.30 RPM on an incubator shaker for
about 12.+-.4 hours until the OD.sub.600 nm reaches in the range of
2.5.+-.1.0.
[0063] The contents of the flasks may be transferred to a fermenter
vessel containing sterile NMFM2 yeast extract medium. For example,
the fermenter vessel may be incubated at 36+1.degree. C. for up to
a maximum of 24 hours with controlling temperature, pH, aeration,
agitation, pressure, and dissolved oxygen content and antifoam
additions whenever required. After the incubation period, the batch
may be terminated when the glucose concentration reaches below 1
g/L, pH, and dissolved oxygen content increases. The culture may be
killed by the addition of 0.5% v/v. Formaldehyde (e.g., add about
420 mL of Formaldehyde to kill 80L broth).
[0064] Some embodiments may include holding the broth for about 1
hour to ensure that the culture was properly inactivated.
Additionally, some embodiments may include setting the fermenter
temperature between 10 to 20.degree. C. before centrifuging the
broth.
[0065] Some embodiments may include centrifuging the broth at
around 15,000 to 20,000 g force with temperature of about 4.degree.
C. Some embodiments may include discarding the cell pellet and
retaining the supernatant. Cetrimonium Bromide (CTAB) may be added
to the supernatant to a final concentration of 0.1% to the 80 L
supernatant (e.g., by adding 800 mL of 10% CTAB to make CTAB final
concentration to 0.1%), and the material may be mixed for about 5
minutes and the contents may be left overnight (8-24 hours) for
precipitation at room temperature (20-29.degree. C.).
[0066] After overnight precipitation, some embodiments may include
collecting the sediment portion and discarding the supernatant. The
precipitated CTAB portion may be centrifuged at around 15,000 to
20,000 g force with a temperature of about 4.degree. C. (CTAB
pellet can be kept in -80.degree. C. until further processing).
[0067] Some embodiments may include suspending the CTAB pellet in 4
L of WFI or process water and stirring the contents for a minimum
of 30 to 60 minutes at room temperature (20-29.degree. C.) by using
a magnetic stir bar and stirrer.
[0068] Some embodiments may include adding 4 L of 2M CaCl.sub.2 to
the CTAB pellet in WFI or process water, stirring the contents for
about 30 to 60 minutes at 2-8.degree. C., and ensuring that the
final concentration of the mixture will be 1M CaCl.sub.2.
[0069] Some embodiments may include, after the completion of
stirring, centrifuging the above contents for 30 to 60 minutes at
around 15,000 to 20,000 g force with a temperature of 4.degree. C.
Some embodiments may include retaining supernatant and discarding
the pellet. Some embodiments may include adding 3200 mL of 200
proof ethanol to the above 8 L solution to make a final
concentration of around 25% ethanol. (E.g., now, the volume will be
11.2 L).
[0070] Some embodiments may include mixing the calcium chloride
wash with 25% ethanol and gently mixing for few minutes using a
clean spatula. Some embodiments may include holding for 30-60
minutes at 2-8.degree. C. and centrifuging the contents for about
30-60 minutes at around 15,000 to 20,000 g force with a temperature
of 4.degree. C. After centrifugation, some embodiments may include
retaining supernatant and discarding the pellet. Some embodiments
may include collecting the above supernatant and adding 3.4 to 4.0
(approximately 42 L to 50 L) volumes of 200 proof ethanol into
clean 20L glass bottles to make the contents reach around 80%
ethanol concentration (+1-5% variation in ethanol concentration is
acceptable). Some embodiments may include mixing the contents for
about 5 minutes with a portable stirrer and then storing the
contents 12-24 hours in a cold room at 2-8.degree. C.
[0071] Some embodiments may include, after such 80% ethanol
precipitation step, siphoning-off the top clear layer and
discarding. Some embodiments may include collecting the
precipitated portion and centrifuging the contents for about 30-60
minutes at around 15,000 to 20,000 g force with a temperature of
4.degree. C. Some embodiments may include retaining the 80% ethanol
pellet. The ethanol pellet can be stored in a -80.degree. C.
freezer until further processing. If the pellet is not stored in a
-80.degree. C. freezer, then some embodiments may include
proceeding for pellet dissolution in water. For example, the 80%
ethanol pellet can be processed in 1 or more parts.
[0072] Some embodiments may include, to the crude polysaccharide
solution, adding enough Tris (e.g., pH 7.5 stock solution) and 1 M
MgCl.sub.2 stock solution to obtain a concentration of 20 mM Tris
and 1 mM MgCl.sub.2 and adding Benzonase at 1-54 per 100 mL of
crude polysaccharide solution. Some embodiments may include
incubating the contents in an incubator water bath shaker and
shaking at 100 rpm at a temperature of 36.+-.1.degree. C. for about
12-16 hours. Some embodiments may include, to the above reaction
mix, adding enough freshly prepared 5 mg/mL Proteinase K to obtain
a final concentration of 50.mu.g/mL. Some embodiments may include
continuing to mix at 100 RPM in the incubator-shaker at
36.+-.1.degree. C. for about 6-8 hours. Some embodiments may
include, to the polysaccharide obtained in the above step, adding 1
M CaCl.sub.2 of approximately 100 mL to 1L of polysaccharide
solution to obtain a final concentration of 0.1 M CaCl.sub.2and
placing in an ultracentrifuge (e.g., at 100,000 g force) for 2
hours at 4.degree. C. Some embodiments may include collecting the
supernatant and discarding the pellet. Some embodiments may include
proceeding with an ultrafiltration having a molecular weight
cut-off of 100 membrane filters. If the above sample indicates
non-permissive levels (>100 EU/.mu.g of Polysaccharide) of
endotoxin, some embodiments may include performing endotoxin
removal using sartobind Q membranes to remove further endotoxin
levels below 100 EU/.mu.g of polysaccharide to ensure removal of
endotoxin to a permissive level. Quality control passed material
will be freeze dried under a vacuum and stored in a -80 degree
Celsius freezer until further use.
[0073] Polysaccharide Size Adjustment:
[0074] Embodiments may include a non-chemical method (e.g.,
sonication) to make low molecular weight polysaccharides and to
produce conjugated meningococcal polysaccharides with a minimum
range of 5100 to 9900 Daltons size. Sizes of oligosaccharides
before and after sonication sizes may be compared by HPLC-SEC
(high-performance liquid chromatography-size exclusion
chromatography).
[0075] Potluck Polysaccharide Activation:
[0076] In some embodiments, suitable amounts of four serogroup
polysaccharides may be together activated by 6mM sodium periodate
reaction at 22.+-.2.degree. C. for 4 hours to generate aldehyde
active groups on them. After the activation, the activated
polysaccharides are diafiltered against 10 mM HEPES pH7.5 at
2-8.degree. C. for 15 hours using 1K MWCO membrane filters to
remove traces of sodium periodate. Oligosaccharide activation is
confirmed by colorimetric estimation of aldehyde groups using
purpald assay.
[0077] GFDT Activation:
[0078] During acidic activation of DT in the presence of EDC
(3-ethyl-1-[3-dimethylamino) propyl] carbodiimide hydrochloride),
hydrazine gets converted to hydrazide groups. In some embodiments,
generated hydrazide groups on the toxoid are quantified using a
colorimetric assay by using (TNBS) trinitrobenzene sulfonic acid
reagent where adipic acid dihydrazide (ADH) is used as a standard
in this assay.
[0079] In some embodiments, protein content is estimated using BCA
assay kit following manufacturer's instructions.
[0080] In some embodiments, the degree of DT activation is arrived
at by measuring hydrazide groups per mole of DT by TNBS assay. For
determination of DT degree of hydrazide activation measurement,
some embodiments may include an average molecular weight of DT
taken as 63,000 Da (0.4 mM). For example, the below Table 1 shows a
specification of 0.2-0.5 mN of hydrazide per mole of DT, i.e. with
a 50-80% degree of activation.
TABLE-US-00001 TABLE 1 Experminent Hydrazide content Degree of No.
mN activation % 1 0.23 57.5 2 0.29 72.5 3 0.31 77.5 4 0.30 75.0
[0081] Conjugation of Activated Oligosaccharide to Activated GFDT
Potluck-Conjugates:
[0082] For example, potluck activated oligosaccharides are combined
at estimated concentration of each activated oligosaccharide (20
mg/ml) and activated GFDT (20 mg/ml) in the weight ratio of 1:1,
and such mixture is continuously mixed by rotating in suitable
sealed containers at 22.+-.2.degree. C. for 18-24 hours. The
resulting mixture may then be treated with sodium borohydride for
6h at 22.+-.2.degree. C. to perform reductive amination and capping
of unused aldehyde groups in a single step.
[0083] Conjugate Ultra-Purification by 10K MWCO Membrane
Filtration:
[0084] For example, the above conjugated and capped mixture is
diafiltered to remove unconjugated low molecular weight
oligosaccharide using 10K MWCO membrane filters. Quantification of
each Oligosaccharide in the pure potluck conjugate may be performed
by HPAEC PAD analysis.
[0085] Formulation of Tetravalent Conjugate Vaccine in
1.times.PBS:
[0086] In some embodiments, the above potluck tetravalent conjugate
ultrapure mixture is formulated in isotonic 1.times.PBS to contain
a final concentration of 8 .mu.g of each serogroup oligosaccharide
conjugated to a total 32-64 .mu.g diphtheria Toxoid per each
milliliter of formulated vaccine.
[0087] Sterilization of final vaccine by 0.2-micron filtration: For
example, the above formulated vaccine may be passed through 0.2.mu.
membrane filters to obtain a sterile final vaccine to consider for
vialing.
[0088] Phase 1 Study: A Double-Blind, Randomized, Controlled, Two
Arm Phase 1 Clinical Trial of the Safety and Immunogenicity of
Group A, C, Y & W-135 Meningococcal Polysaccharide DT Conjugate
Vaccine: NmVac4-A/C/Y/W-135-DT.TM..
[0089] Clinical Trail Study Location: Hanford, Calif., USA
[0090] Our previous pre-clinical experience and the experience of
others with similar vaccines which are already in the market,
suggests that one dose of Meningococcal meningitis A, C, Y &
W-135 polysaccharide diphtheria conjugate vaccine is likely to show
satisfactory safety and induce a protective immune response in
naive individuals.
[0091] Safety Results:
[0092] In our Phase, 1 Safety Study, 60 age 18-50-year-old subjects
(30 test vaccine, 30 active control vaccine) were observed for a
total of 26 weeks for any safety concerns. No SAEs were observed in
the phase 1 trial. Adverse event severity and frequency were
similar to those reported for published meningococcal vaccine
studies. Treatment-related adverse events observed in our phase 1
study are shown in Table 1. Most were mild or moderate. Two Grade 3
AEs (a headache and anorexia) were possibly associated with the
vaccine. The subjects were fully recovered within 1 week of
vaccination.
TABLE-US-00002 TABLE 2 Summary of Treatment-Related Adverse Events
Subject Count (%) by Strata System Organ Class Control Control
MedDRA Preferred Total NmVac4- NmVac4- vaccine/ vaccine/ Term
(sorted by Subject DT/Female DT/Male Female Male frequency) Count
(%) (N = 15) (N = 15) (N = 15) (N = 15) Any adverse event 32 (53%)
7 (47%) 9 (60%) 9 (60%) 7 (47%) General Disorders: 27 (45%) 6 (40%)
8 (60%) 7(40%) 6 (40%) Administration site conditions General
disorders: 9 (15%) 0 (0%) 3 (20%) 3 (20%) 3 (20%) Chills, Fatigue,
Malaise Gastrointestinal 9 (15%) 3 (20%) 3 (20%) 3 (20%) 0 (0%)
disorders: nausea, diarrhea Musculoskeletal and 8 (13%) 2 (13%) 3
(20%) 2 (13%) 1 (7%) connective tissue disorders Nervous system 8
(13%) 1 (7%) 3 (20%) 2 (13%) 2 (13%) disorders: Headache Metabolism
and 2 (3.3%) 0 (0%) 1 (7%) 1 (7%) 0 (0%) nutrition disorders:
Anorexia Vital Signs 1 (1.7%) 0 (0%) 0 (0%) 0 (0%) 1 (7%)
[0093] Immunogenicity Results:
[0094] Immunization with 4 .mu.g of each serogroup oligosaccharide
conjugated with 4-6 .mu.g GF Diphtheria toxoid (per serogroup)
produced seroconversion rates (by rSBA) of 46-71% of subjects at 4
weeks: Serogroup A 46%, C 71%, W-135 63% and Y 67%. Higher
seroconversion rates (71-92%) were observed at 8 weeks: A 71%, C
92%, W-135 67% and Y 75%. Immunogenicity results were similar for
control and test vaccines (Table 2). These preliminary results
indicate that a single dose may be sufficient to protect adult
subjects from Meningococcal disease and may be useful for endemic
seasonal immunization as an effective response to the global need
for vaccination against this pandemic and endemicdisease.
TABLE-US-00003 TABLE 3 Seroconversion Rate and GMT in the Per
Protocol Population by Treatment. Sero- NmVac4-DT Control vaccine
Variable group Visit (N = 24) (N = 25) Seroconversion A Week 4 46%
(26%, 67%) 68% (46%, 85%) % (95% CI) GMT (95% CI) A Week 4 12.7
(10.4, 33.0) 12.8 (11.3, 31.7) Seroconversion A Week 8 71% (49%,
87%) 80% (59%, 93%) % (95% CI) GMT (95% CI) A Week 8 13.0 (12.0,
33.8) 15.8 (20.8, 68.7) Seroconversion C Week 4 71% (49%, 87%) 80%
(59%, 93%) % (95% CI) GMT (95% CI) C Week 4 18.5 (10.4, 33.0) 18.9
(11.3, 31.7) Seroconversion C Week 8 92% (73%, 99%) 68% (46%, 85%)
% (95% CI) GMT (95% CI) C Week 8 22.6 (14.6, 35.1) 19.4 (12.5,
30.2) Seroconversion W-135 Week 4 63% (41%, 81%) 76% (55%, 91%) %
(95% CI) GMT (95% CI) W-135 Week 4 18.0 (9.1, 35.6) 29.4 (14.4,
60.0) Seroconversion W-135 Week 8 67% (45%, 84%) 68% (46%, 85%) %
(95% CI) GMT (95% CI) W-135 Week 8 19.6 (11.3, 33.9) 24.3 (12.9,
45.5) Seroconversion Y Week 4 67% (45%, 84%) 72% (51%, 88%) % (95%
CI) GMT (95% CI) Y Week 4 22.0 (12.6, 38.5) 31.1 (16.7, 57.9)
Seroconversion Y Week 8 75% (53%, 90%) 60% (39%, 79%) % (95% CI)
GMT (95% CI) Y Week 8 19.0 (11.3, 32.0) 21.7 (11.9, 39.6)
[0095] Some embodiments may include at least one processor
configured to run various software applications or computer code
stored in a non-transitory computer-readable medium and configured
to execute various instructions or operations, including but not
limited to instructions or operations associated with processes,
systems, equipment, functions, and methods disclosed throughout.
The at least one processor may be configured to run various
software applications or computer code stored in a non-transitory
computer-readable medium and configured to execute various
instructions or operations as disclosed throughout and configured
to perform any suitable functions. The at least one processor may
be implemented in any number (e.g., at least one, two, or more) of
computing devices that may or may not be interconnected over a
network (e.g., a local area network (LAN), wireless area network
(WAN), the Internet, or a combination thereof) and/or
communicatively coupled (e.g., via a network) with other computing
devices. Such computing devices may include at least one processor,
memory, storage, or the like, and such computing devices may
include or be communicatively coupled to any of various suitable
sensors configured to measure properties (e.g., pressure, gas
composition, spectrometer, etc.) associated with systems and
processes disclosed throughout.
[0096] Referring now to FIG. 1A, an exemplary embodiment of a
vessel 100A containing a multivalent vaccine composition 102
according to the inventive concepts disclosed herein is depicted.
The vessel 100A may be or may be implemented as any vessel (e.g., a
container, a vial (e.g., vial 100B, as shown in FIG. 1B), or a
syringe chamber) suitable for containing the multivalent vaccine
composition 102.
[0097] In some embodiments, the multivalent vaccine composition 102
may comprise a vaccine composition including two or more Neisseria
meningitides serogroup oligosaccharides conjugated to glycan-free
carrier proteins. For example, the multivalent vaccine composition
102 may include a liquid mixture including Neisseria meningitides
serogroups A, C, Y, and W-135 oligosaccharides conjugated to
glycan-free carrier proteins. For example, the multivalent vaccine
composition 102 may include a mixture of conjugated Neisseria
meningitides serogroup A, C, Y, and W-135 oligosaccharides, where
each of the conjugated Neisseria meningitides serogroup A, C, Y,
and W-135 oligosaccharides is conjugated to a glycan-free carrier
protein. For example, the multivalent vaccine composition may
include a mixture of the following: a) conjugated Neisseria
meningitides serogroup A oligosaccharides, each of the conjugated
Neisseria meningitides serogroup A oligosaccharides being
conjugated to a first particular glycan-free carrier protein; b)
conjugated Neisseria meningitides serogroup C oligosaccharides,
each of the conjugated Neisseria meningitides serogroup C
oligosaccharides being conjugated to a second particular
glycan-free carrier protein; c) conjugated Neisseria meningitides
serogroup Y oligosaccharides, each of the conjugated Neisseria
meningitides serogroup Y oligosaccharides being conjugated to a
third particular glycan-free carrier protein; and d) conjugated
Neisseria meningitides serogroup W-135 oligosaccharides, each of
the conjugated Neisseria meningitides serogroup W-135
oligosaccharides being conjugated to a fourth particular
glycan-free carrier protein.
[0098] When administered, the multivalent vaccine composition 102
may provide long-lasting immunity against two or more serogroups
(e.g., four, such as serogroups A, C, Y, and W-135) of N.
meningitidis to humans of all age groups, including infants.
[0099] While the vessel 100A exemplarily includes elements as
shown, in some embodiments, the vessel 100A may include
otherelements.
[0100] Referring now to FIG. 1B, an exemplary embodiment of a vial
100B containing the multivalent vaccine composition 102 according
to the inventive concepts disclosed herein is depicted. The vial
100B containing the multivalent vaccine composition 102 may be
implemented similarly to the vessel 100A containing a multivalent
vaccine composition 102.
[0101] Referring now to FIG. 2, an exemplary embodiment of a method
200 for method for producing a multivalent vaccine composition
according to the inventive concepts disclosed herein may include
one or more of the following steps, which, for example, may be
controlled and/or performed by any suitable components disclosed
throughout. Additionally, for example, some embodiments may include
performing and/or controlling one more instances of the method 200
iteratively, concurrently, and/or sequentially.
[0102] A step 202 may include non-chemically adjusting sizes of
Neisseria meningitides serogroups A, C, Y, and W-135
oligosaccharides.
[0103] A step 204 may include activating the Neisseria meningitides
serogroups A, C, Y, and W-135 oligosaccharides upon non-chemically
adjusting the sizes of the Neisseria meningitides serogroups A, C,
Y, and W-135 oligosaccharides.
[0104] A step 206 may include activating glycan-free carrier
proteins.
[0105] A step 208 may include conjugating at least some of the
activated Neisseria meningitides serogroups A, C, Y, and W-135
oligosaccharides to at least some of the activated glycan-free
carrier proteins.
[0106] A step 210 may include producing a multivalent vaccine
composition including a mixture. The mixture may include one or
more (e.g., one, two, three, or four) of the following: a)
conjugated Neisseria meningitides serogroup A oligosaccharides,
each of the conjugated Neisseria meningitides serogroup A
oligosaccharides being conjugated to a first particular glycan-free
carrier protein of the activated glycan-free carrier proteins; b)
conjugated Neisseria meningitides serogroup C oligosaccharides,
each of the conjugated Neisseria meningitides serogroup C
oligosaccharides being conjugated to a second particular
glycan-free carrier protein of the activated glycan-free carrier
proteins; c) conjugated Neisseria meningitides serogroup Y
oligosaccharides, each of the conjugated Neisseria meningitides
serogroup Y oligosaccharides being conjugated to a third particular
glycan-free carrier protein of the activated glycan-free carrier
proteins; and d) conjugated Neisseria meningitides serogroup W-135
oligosaccharides, each of the conjugated Neisseria meningitides
serogroup W-135 oligosaccharides being conjugated to a fourth
particular glycan-free carrier protein of the activated glycan-free
carrier proteins.
[0107] Further, the method 200 may include any of the operations,
steps, and/or actions disclosed throughout. For example, the method
200 may include removing unconjugated oligosaccharides from a
mixture including the activated A, C, Y, and W-135 oligosaccharides
conjugated to the glycan-free carrier protein. Further, for
example, the method 200 may include sterilizing a mixture including
the activated A, C, Y, and W-135 oligosaccharides conjugated to the
glycan-free carrier protein. Also, for example, the method 200 may
include vialing at least a portion of the multivalent vaccine
composition comprising Neisseria meningitides serogroups A, C, Y,
and W-135 oligosaccharides conjugated to a glycan-free carrier
protein in a vessel.
[0108] As will be appreciated from the above, embodiments of the
inventive concepts disclosed herein may be directed to a vaccine
composition, a method for producing the vaccine composition, and an
apparatus containing the vaccine composition.
[0109] As used throughout and as would be appreciated by those
skilled in the art, "at least one non-transitory computer-readable
medium" may refer to as at least one non-transitory
computer-readable medium (e.g., memory, storage, or a combination
thereof; e.g., at least one computer-readable medium implemented as
hardware; e.g., at least one non-transitory processor-readable
medium, at least one memory (e.g., at least one nonvolatile memory,
at least one volatile memory, or a combination thereof; e.g., at
least one random-access memory, at least one flash memory, at least
one read-only memory (ROM) (e.g., at least one electrically
erasable programmable ROM (EEPROM), at least one on-processor
memory (e.g., at least one on-processor cache, at least one
on-processor buffer, at least one on-processor flash memory, at
least one on-processor EEPROM, or a combination thereof), or a
combination thereof), at least one storage device (e.g., at least
one hard-disk drive, at least one tape drive, at least one
solid-state drive, at least one flash drive, at least one readable
and/or writable disk of at least one optical drive configured to
read from and/or write to the at least one readable and/or writable
disk, or a combination thereof), or a combination thereof.
[0110] As used throughout, "at least one" means one or a plurality
of; for example, "at least one" may comprise one, two, three, . . .
, one hundred, or more. Similarly, as used throughout, "one or
more" means one or a plurality of; for example, "one or more" may
comprise one, two, three, . . . , one hundred, or more. Further, as
used throughout, "zero or more" means zero, one, or a plurality of;
for example, "zero or more" may comprise zero, one, two, three, . .
. , one hundred, or more.
[0111] In the present disclosure, the methods, operations, and/or
functionality disclosed may be implemented as sets of instructions
or software readable by a device (e.g., a processor of a computing
device or a controller). Further, it is understood that the
specific order or hierarchy of steps in the methods, operations,
and/or functionality disclosed are examples of exemplary
approaches. Based upon design preferences, it is understood that
the specific order or hierarchy of steps in the methods,
operations, and/or functionality can be rearranged while remaining
within the scope of the inventive concepts disclosed herein. The
accompanying claims may present elements of the various steps in a
sample order, and are not necessarily meant to be limited to the
specific order or hierarchy presented.
[0112] It is to be understood that embodiments of the methods
according to the inventive concepts disclosed herein may include
one or more of the steps described herein. Further, such steps may
be carried out in any desired order and two or more of the steps
may be carried out simultaneously with one another. Two or more of
the steps disclosed herein may be combined in a single step, and in
some embodiments, one or more of the steps may be carried out as
two or more sub-steps. Further, other steps or sub-steps may be
carried in addition to, or as substitutes to one or more of the
steps disclosed herein.
[0113] From the above description, it is clear that the inventive
concepts disclosed herein are well adapted to carry out the objects
and to attain the advantages mentioned herein as well as those
inherent in the inventive concepts disclosed herein. While
presently preferred embodiments of the inventive concepts disclosed
herein have been described for purposes of this disclosure, it will
be understood that numerous changes may be made which will readily
suggest themselves to those skilled in the art and which are
accomplished within the broad scope and coverage of the inventive
concepts disclosed and claimed herein.
* * * * *