U.S. patent application number 16/284910 was filed with the patent office on 2019-06-20 for single dose non-adjuvanted cutaneous tetanus vaccine and uses thereof.
The applicant listed for this patent is Sporos Therapeutics, LLC. Invention is credited to Richard Compans, E. Stein Esser, Shaguna Seth, Ioanna Skountzou.
Application Number | 20190183790 16/284910 |
Document ID | / |
Family ID | 56887117 |
Filed Date | 2019-06-20 |
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United States Patent
Application |
20190183790 |
Kind Code |
A1 |
Skountzou; Ioanna ; et
al. |
June 20, 2019 |
SINGLE DOSE NON-ADJUVANTED CUTANEOUS TETANUS VACCINE AND USES
THEREOF
Abstract
A method for preventing or treating tetanus infection in a
subject, comprising administering a non-adjuvanted tetanus toxoid
vaccine to the skin. The vaccine can be delivered by intradermal
injection or microneedle patch and does not require cold
storage.
Inventors: |
Skountzou; Ioanna; (Atlanta,
GA) ; Esser; E. Stein; (Atlanta, GA) ;
Compans; Richard; (Atlanta, GA) ; Seth; Shaguna;
(Bothell, WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Sporos Therapeutics, LLC |
Bothell |
WA |
US |
|
|
Family ID: |
56887117 |
Appl. No.: |
16/284910 |
Filed: |
February 25, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15066961 |
Mar 10, 2016 |
10213378 |
|
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16284910 |
|
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62130744 |
Mar 10, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/54 20130101;
A61K 2039/55 20130101; A61K 39/08 20130101; A61K 9/0021 20130101;
A61K 2039/575 20130101 |
International
Class: |
A61K 9/00 20060101
A61K009/00; A61K 39/08 20060101 A61K039/08 |
Claims
1. A microneedle patch containing a non-adjuvanted tetanus toxoid
vaccine.
2. The microneedle patch of claim 1, wherein the patch is loaded
with commercially-available non-adjuvanted tetanus toxoid monobulk
that is concentrated at least five times its original
concentration.
3. The microneedle patch of claim 2, wherein the non-adjuvanted
tetanus toxoid comprises 3% polyvinyl alcohol and 10% sucrose as
excipients.
4. A method for treating tetanus infection in a subject, comprising
administering a non-adjuvanted tetanus toxoid vaccine to the skin
by applying the patch of claim 1.
5. The method of claim 4, wherein the vaccine does not require cold
storage or cold transport.
6. The method of claim 4, wherein the subject is pregnant.
7. The method of claim 4, further comprising: administering a
non-adjuvanted tetanus toxoid vaccine by placing a microneedle
patch containing the vaccine on the skin of the subject; holding
the patch in place to allow the vaccine to dissolve into the
skin.
8. The method of claim 4, wherein the microneedles of the patch
penetrate the skin and the patch is held in place for at least 10
minutes.
9. The method of claim 4, wherein at least five-fold higher level
of tetanus toxoid-specific antibodies are induced in non-pregnant
subjects than for intramuscular administration in non-pregnant
subjects.
10. The method of claim 4, wherein at least two-fold higher level
of tetanus toxoid-specific antibodies are induced in pregnant
subjects than for intramuscular administration in pregnant
subjects.
11. The method of claim 4, wherein at least a six-fold higher level
of tetanus toxoid-specific antibodies IgG2a are induced in a
pregnant subject as compared to intramuscular administration in a
non-pregnant subject.
12. The method of claim 4, wherein at least a ten-fold increase in
antibody levels is induced at week 3 as compared to intramuscular
administration.
13. The method of claim 4, wherein the induced ratio IgG1/IgG2a is
lower as compared to intramuscular administration.
14. The method of claim 4, wherein the patch is loaded with
commercially-available non-adjuvanted tetanus toxoid monobulk that
is concentrated at least five times its original concentration.
15. The method of claim 4, wherein the non-adjuvanted tetanus
toxoid comprises 3% polyvinyl alcohol and 10% sucrose as
excipients.
Description
BACKGROUND OF THE INVENTION
[0001] Tetanus is an acute disease caused by a neurotoxin produced
by the anaerobe Clostridium tetani with motor and autonomous
nervous system manifestations such as rigidity and convulsive
spasms. Neonatal tetanus (NT) is around 10% in these countries
where antenatal care is also well below 50%. According to the
latest WHO estimates in 2010, NT claimed 58,000 newborn lives and
is still a major public health concern. Mothers and newborns are at
high risk when delivery and umbilical cord stump are performed
under unsanitary conditions leading to tissue infection. The
Centers of Disease Control and Prevention (CDC) and World Health
Organization (WHO) recommend vaccinations of pregnant women at 20
weeks of gestation against tetanus and other preventable diseases
due to increased rate of complications in mothers and their
fetuses. These immunizations not only confer protective immunity to
the mother, but also provide a passive immune response to the
infant prior to the development of their own antibodies. Maternal
antibodies are transferred through the umbilical cord blood during
fetal development and breast milk during infant nursing. Thus, two
doses of tetanus toxoid can reduce mortality from neonatal tetanus
by 94%.
[0002] Although tetanus is a vaccine preventable disease, it has
not been fully eradicated despite the efforts of Maternal and
Neonatal Tetanus (MNT) Elimination Initiative launched by UNICEF,
WHO and UNFPA in 1999. The problem of inadequate vaccine coverage
is more critical in developing countries where many women do not
receive vaccinations before pregnancy due to limited access to
health care providers. For the same reason, pregnant women deliver
their babies in non-sanitary conditions resulting in high mortality
rates from tetanus infections. A critical bottleneck for effective
vaccination is the absence of electricity in rural areas of
developing countries, which is required for vaccine transportation
and storage at low temperatures.
[0003] Tetanus toxoid is a small protein with low immunogenicity,
so the vaccine has low potency and requires the presence of an
adjuvant (Alum) and multiple immunization doses to induce robust
antibody titers and affinity maturation.
[0004] To this day tetanus infections claim at least 60,000
maternal and neonatal lives worldwide. Two doses of adjuvanted
tetanus vaccine are recommended to protect pregnant women during
labor and their newborns, but in many areas vaccination is still
unavailable or insufficient.
[0005] It has been demonstrated that pregnancy hormones modulate
maternal immunity to tolerate the developing fetus, but a downside
of tolerance is the increased rate and severity of infections due
to compromised immune responses. Pregnant women infected with
influenza virus are at higher risk than healthy non-pregnant
controls. Influenza infection-induced inflammation may compromise
the well-being of their offspring by stillbirth, preterm labor,
small for gestation age newborns, fetal abnormalities and even
death. The Centers of Disease Control and Prevention (CDC) and
World Health Organization (WHO) recommend vaccinations of pregnant
women at 20 weeks of gestation against preventable diseases
including tetanus, due to higher risk of complications in mothers
and their fetuses. These immunizations not only confer protective
immunity to the mother, but also provide a passive immune response
to the infant prior to the development of their own antibodies.
Maternal antibodies are transferred through the umbilical cord
blood during fetal development and breast milk during infant
nursing.
[0006] The problem of inadequate vaccine coverage is more critical
in developing countries where many women do not receive these
vaccines before pregnancy and frequently they do not have access to
health care providers delivering their babies in non-sanitary
conditions resulting in high mortality rates from tetanus
infections. Moreover, intramuscular delivery of tetanus toxoid
requires the presence of an adjuvant (Alum) due to low vaccine
potency, and multiple immunization doses to induce robust antibody
titers and affinity maturation.
[0007] Vaccine delivery in the skin has been shown to utilize
antigen presenting cells (APCs) residing in the epidermis and the
dermis, which leads to an increased immune response compared to the
traditional route of intramuscular delivery. Skin vaccination
during pregnancy via intradermal injection can be more immunogenic
than intramuscular vaccine delivery.
[0008] What is needed are methods, compositions and devices for
tetanus immunization.
[0009] There is a continuing need for methods, compositions and
devices for tetanus immunization.
BRIEF SUMMARY
[0010] This invention relates to the fields of vaccines for
tetanus.
[0011] More particularly, this invention relates to methods and
devices for cutaneous delivery of tetanus vaccine.
[0012] Embodiments of this invention provide a single dose,
non-adjuvanted tetanus toxoid vaccine that can be delivered
cutaneously, either by intradermal injection, or by a microneedle
patch device.
[0013] Embodiments of this invention include:
[0014] A method for preventing or treating tetanus infection in a
subject, comprising administering a non-adjuvanted tetanus toxoid
vaccine to the skin.
[0015] The method above, wherein the tetanus toxoid vaccine is
delivered by intradermal injection or microneedle patch.
[0016] The method above, wherein the subject is pregnant.
[0017] The method above, further comprising:
[0018] administering a non-adjuvanted tetanus toxoid vaccine by
placing a microneedle patch containing the vaccine on the skin of
the subject;
[0019] holding the patch in place to allow the vaccine to dissolve
into the skin.
[0020] The method above, wherein the microneedles of the patch
penetrate the skin and the patch is held in place for at least 10
minutes.
[0021] The method above, wherein at least five-fold higher level of
tetanus toxoid-specific antibodies are induced in non-pregnant
subjects than for intramuscular administration in non-pregnant
subjects.
[0022] The method above, wherein at least two-fold higher level of
tetanus toxoid-specific antibodies are induced in pregnant subjects
than for intramuscular administration in pregnant subjects.
[0023] The method above, wherein at least a six-fold higher level
of tetanus toxoid-specific antibodies IgG2a are induced in a
pregnant subject as compared to intramuscular administration in a
non-pregnant subject.
[0024] The method above, wherein at least a ten-fold increase in
antibody levels is induced at week 3 as compared to intramuscular
administration.
[0025] The method above, wherein the induced ratio IgG1/IgG2a is
lower as compared to intramuscular administration.
[0026] The method above, wherein the patch is loaded with
commercially-available non-adjuvanted tetanus toxoid monobulk that
is concentrated at least five times its original concentration.
[0027] The method above, wherein the non-adjuvanted tetanus toxoid
comprises 3% polyvinyl alcohol and 10% sucrose as excipients.
[0028] A microneedle patch containing a non-adjuvanted tetanus
toxoid vaccine.
[0029] The microneedle patch above, wherein the patch is loaded
with commercially-available non-adjuvanted tetanus toxoid monobulk
that is concentrated at least five times its original
concentration.
[0030] The microneedle patch above, wherein the non-adjuvanted
tetanus toxoid comprises 3% polyvinyl alcohol and 10% sucrose as
excipients.
BRIEF DESCRIPTION OF THE DRAWINGS
[0031] This patent or application file contains at least one
drawing executed in color. Copies of this patent or patent
application publication with color drawings will be provided by the
US Patent Office upon request and payment of the necessary fee.
[0032] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present disclosure, the inventions of which can be
better understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0033] FIG. 1: FIG. 1 shows SDS-page analysis of tetanus toxoid. As
shown in FIG. 1 (left), proteins were separated on 10% gel under
denaturing conditions, transferred onto nitrocellulose membrane and
reversibly stained with PONSEAU S protein stain. Tetanus toxoid
(Serum Institute of India), 7.5 .mu.g total protein. As shown in
FIG. 1 (right), the same membrane developed with anti-tetanus
toxoid HRP-labeled antibodies (#TTX-314, Alpha Diagnostics).
Tetanus toxoid (positive control, Alpha Diagnostics kit), 7.5 .mu.g
total protein.
[0034] FIG. 2A: FIG. 2A shows an array of microneedles loaded with
TT (red color to facilitate imaging).
[0035] FIG. 2B: FIG. 2B shows an array of microneedles loaded with
sulforhodamine (red color to facilitate imaging).
[0036] FIG. 3A: FIG. 3A shows second-generation microneedles before
insertion into mouse skin in vivo.
[0037] FIG. 3B: FIG. 3B shows second-generation microneedles 20 min
after insertion into mouse skin in vivo.
[0038] FIG. 3C: FIG. 3C shows mouse skin with dissolved
nicroneedles.
[0039] FIG. 4A: FIG. 4A shows Anti-TT IgG antibodies in adult
non-pregnant and pregnant population.
[0040] FIG. 4B shows differences in antibody titers between
pregnant, non-pregnant cohorts and routes of immunization (Table
1).
[0041] FIG. 5A: FIG. 5A shows: Anti-TT IgG antibodies.
[0042] FIG. 5B: FIG. 5B shows: Anti-TT IgG1 antibodies.
[0043] FIG. 6A: FIG. 6A shows: Survival rates in healthy adult
non-pregnant population.
[0044] FIG. 6B: FIG. 6B shows: Survival rates in healthy adult
population vaccinated during pregnancy.
[0045] FIG. 6C: FIG. 6C shows: Anti-TT neutralizing antibodies.
[0046] FIG. 7A: FIG. 7A shows: Survival rates of 3-week old mice
after challenge with 8.33.times.LD.sub.50 tetanus toxin.
[0047] FIG. 7B: FIG. 7B shows: Survival rates of 6-week old mice
after challenge with 8.33.times.LD.sub.50 tetanus toxin.
[0048] FIG. 8A: FIG. 8A shows evaluation of humoral responses of
immunized adult mice. Serum samples from non-pregnant adult mice
vaccinated either intradermally (ID) or intramuscularly (IM) were
collected 7, 14, and 28 days after immunization. The samples were
analyzed against tetanus toxoid for the levels of total serum IgG
(FIG. 8A) antibody titers by ELISA. ELISA antibody data represent
the mean.+-.SEM. Statistics for the ID and IM group were performed
with two-tailed unpaired Student's t-test. *p<0.05.
[0049] FIG. 8B: FIG. 8B shows evaluation of humoral responses of
immunized adult mice. Serum samples from non-pregnant adult mice
vaccinated either intradermally (ID) or intramuscularly (IM) were
collected 7, 14, and 28 days after immunization. The samples were
analyzed against tetanus toxoid for the levels of total serum IgM
(FIG. 8B) antibody titers by ELISA. ELISA antibody data represent
the mean.+-.SEM. Statistics for the ID and IM group were performed
with two-tailed unpaired Student's t-test. *p<0.05.
[0050] FIG. 9A: FIG. 9A shows protective immune response in
immunized mice after lethal challenge with Tetanus Toxin. Eight
week old were immunized with 5 Lf of non-adjuvanted tetanus toxoid
by intradermal (ID) or intramuscular (IM) delivery. Twenty eight
days later, they were challenged with 10.times.LD.sub.50 tetanus
toxin and observed over a 4 day period. Survival rates (FIG. 9A)
during the observation period were recorded. All symptoms were
categorized according to the European Pharmacopeia's scale of
paralysis. T1: slight stiffness of toxin-injected hind leg, only
observed when the mouse is lifted by the tail. T2: paresis of the
toxin-injected hind leg, which can still function for walking. T3:
paralysis of the toxinOinjected hind leg, which does not function
for walking. Mice displaying symptoms of stages T2 and T3 were
considered unprotected and euthanized.
[0051] FIG. 9B: FIG. 9B shows protective immune response in
immunized mice after lethal challenge with Tetanus Toxin. Eight
week old were immunized with 5 Lf of non-adjuvanted tetanus toxoid
by intradermal (ID) or intramuscular (IM) delivery. Twenty eight
days later, they were challenged with 10.times.LD.sub.50 tetanus
toxin and observed over a 4 day period. Signs of symptoms (FIG. 9B)
during the observation period were recorded. All symptoms were
categorized according to the European Pharmacopeia's scale of
paralysis. T1: slight stiffness of toxin-injected hind leg, only
observed when the mouse is lifted by the tail. T2: paresis of the
toxin-injected hind leg, which can still function for walking. T3:
paralysis of the toxin0injected hind leg, which does not function
for walking. Mice displaying symptoms of stages T2 and T3 were
considered unprotected and euthanized.
DETAILED DESCRIPTION OF THE INVENTION
[0052] This invention provides methods for treating tetanus
infection, as well as a single dose, non-adjuvanted tetanus toxoid
vaccine.
[0053] A tetanus vaccine of this disclosure can be delivered by
intradermal injection, or via a microneedle patch device.
[0054] The methods and apparatus of this invention can be used for
treating tetanus infection, especially in locations where cold
storage is not available.
[0055] Skin immunization during pregnancy can be an alternative and
efficient route to expand vaccination coverage and provide
protective immunity by administering only one vaccine dose.
[0056] In this invention, host immune responses to non-adjuvanted
tetanus toxoid (TT) induced by skin delivery were surprisingly
greater than for conventional intramuscular immunization.
[0057] In some embodiments, skin vaccination of BALB/c mice during
pregnancy via intradermal injection of non-adjuvanted tetanus
toxoid vaccine, employing microneedle patches fabricated from PVA
(polyvinyl alcohol) that encapsulated the non-adjuvanted vaccine,
was more immunogenic than intramuscular vaccine delivery.
[0058] In certain aspects, skin vaccination via microneedle patches
resulted in a greater passive immune response amongst offspring, as
well as significant protective efficacy against lethal infections
compared to the offspring of those vaccinated intramuscularly
during pregnancy.
[0059] In further embodiments, controlled vaccine delivery with
microneedle patches outperformed intramuscular or intradermal
delivery of non-adjuvanted TT with hypodermic needle. Robust
humoral immune responses were elicited after a single dose of 5 Lf
(flocculation units) and were sufficient to fully protect the
mothers and newborns against paralytic disease or death after
lethal challenge with tetanus toxin.
[0060] In further aspects, induction of TT-specific IgG titers with
microneedle patches was surprisingly higher than systemic
vaccination. This showed that the invention can improve affinity
maturation following isotype class switching. Protective immunity
can be a reliable parameter to compare the efficacy of vaccination
platforms.
[0061] In some embodiments, two types of effector CD4+T helper cell
responses can be induced by professional antigen presenting cells,
designated Th1 and Th2, each designed to eliminate different types
of pathogens. Although Th1 responses are more effective against
intracellular pathogens and viruses due to cell mediated immunity
triggered by IFN-.gamma. production and subsequent activation of
macrophages and B cell-secreted opsonizing and complement fixation
antibodies, Th2 responses are significant for humoral immunity
necessary for bacterial pathogens including tetanus due to
neutralizing antibody production by IL-4-activated B cells.
[0062] In some embodiments, skin immunization with microneedle
patches can induce both humoral and cellular immune responses.
Thus, skin immunization is a surprisingly effective vaccine
delivery platform in conferring protective immunity against lethal
challenge with tetanus toxin.
[0063] Embodiments of this invention can also provide a protocol
for tetanus toxin challenge in mice and establish an LD.sub.50.
[0064] This invention encompasses novel microneedle patches for
skin immunization to improve immunity of pregnant mothers against
tetanus infections and increase protection of offspring early after
birth. Skin can be a desirable vaccine target organ because it has
an increased number of professional antigen presenting cells
(APCs). Microneedles or micron-scale needles can be used to
administer vaccines, therapeutics, and other materials into cells
and tissues, including the skin. The vaccine can be targeted to
antigen-presenting cells in the skin, allowing administration of
lower doses and generating more potent, longer-lasting immune
responses. Microneedle patches of this disclosure can be
bio-degradable, and not leave any bio-hazardous sharps after
administration. Microneedle patches of this disclosure can provide
vaccine stability and storage at ambient temperatures, to
facilitate self-administration and expanded geographical
vaccination coverage.
[0065] In general, the skin is composed of three superimposed
layers: the epidermis, the dermis and the subcutaneous tissue. The
skin layers contain large numbers of specialized antigen presenting
cells (APCs), which can be targets for vaccine delivery. The
epidermis is rich in innate cell populations, namely keratinocytes,
melanocytes, Langerhans cells expressing CD207.sup.+ CD1a.sup.+ in
humans, and CD207.sup.+ CD205.sup.+ CD11c.sup.+Cd11b.sup.+ in mice.
The dermis is rich in Langerin positive
(CD207.sup.+CD11c.sup.+CD11b.sup.lowCD205.sup.-CD.sup.103+) and
Langerin negative (CD207.sup.-CD11c.sup.+CD11b.sup.+CD205.sup.+)
dermal dendritic cells (dDCs), macrophages and mast cells.
Langerhans cells and dermal dendritic cells are characterized by
differential chemokine receptor expression and cytokine
production.
[0066] In this invention, cutaneous immunization can be used to
provide antigen capture and processing by APCs, which can be
activated to migrate towards the proximal lymph nodes. Cutaneous
immunization can be achieved by intradermal injection of a tetanus
vaccine of this invention, or though the use of a microneedle patch
of this invention.
[0067] In general, migration of APCs depends on the cutaneous
environment, and the balance between pro- and anti-inflammatory
signals. The presentation of processed antigen to immature CD4 or
CD8 T cells or naive follicular B cells can result in clonal
expansion of T cells to effector and memory populations or
activation of B cells to plasma cells or plasmablasts.
[0068] Embodiments of this invention can provide surprisingly
increased effectiveness of tetanus vaccine, as compared to systemic
immunization delivered to the muscle.
Example 1: Vaccines
[0069] Non-adjuvanted tetanus toxoid was provided by the Serum
Institute of India. Commercially available alum-adjuvanted tetanus
toxoid vaccine was purchased from IBSS Biomed S.A. (Krakow,
Poland). Vaccine stability was tested with ELISA (Alpha Diagnostics
International Inc., San Antonio, Tex.) and SDS-page analysis of
tetanus toxoid. Tetanus toxoid and positive control were separated
on 10% gel under denaturing conditions, transferred onto
nitrocellulose membrane which was either reversibly stained with
PONSEAU S protein stain or developed with anti-tetanus toxoid
HRP-labeled antibodies (Alpha Diagnostics).
[0070] Animals.
[0071] Six to 8-week-old female BALB/c mice (Harlan Laboratories,
Dublin, Va.) were bred and housed in a biosafety level 1 facility
for immunizations, and tetanus challenges were performed in a
biosafety level 2 facility (Emory University Whitehead animal
facility).
[0072] Determination of Lethal Dose 50 (LD.sub.50) in BALB/c
Mice.
[0073] Healthy adult BALB/c mice 6-weeks old were divided in groups
of five and challenged with tetanus toxin (Sigma-Aldrich, St.
Louis, Mo.) at concentrations of 100, 50, 10, 5, 2, 1, and 0.1
ng/50 ul PBS via subcutaneous injection in the dorsal surface of
their right hind leg. The mice were examined twice a day, once in
the morning and once in the evening for four days post-challenge.
The experiment was repeated twice. The effect of tetanus toxin on
mice was measured based on European Pharmacopeia's scale of
paralysis and symptoms were recorded twice per day up to 4 days.
The stages of tetanus are: T0=Normal; T1=Slight Stiffness of toxin
injected hind leg, only observed when mouse is lifted by the tail;
T2=paresis of the toxin injected hind leg, which can still function
for walking; T3=paralysis of the toxin-injected hind leg, which
does not function for walking; T4=the toxin-injected hind leg is
completely paralyzed.
[0074] Adult Immunizations, Lethal Challenge and Sample
Collection.
[0075] Groups of ten pregnant or non-pregnant mice received a
single dose of non-adjuvanted tetanus toxoid containing 5 Lf for
intramuscular (IM), intradermal (ID) and microneedle (MN)
immunization. Pregnant mice were vaccinated on day 12 of gestation.
For cutaneous immunization, the dorsal hair was removed. Dissolving
microneedle patches encapsulating the antigen were placed on the
skin, and held in place for 20 minutes to allow the vaccine to
dissolve in the skin. For intramuscular vaccinations, the
non-adjuvanted TT was diluted in 50 .mu.l of PBS and injected into
the upper quadrant of the hind leg.
[0076] Blood samples were collected from adult pregnant and
non-pregnant mice via submandibular bleeds at days 7, 14, and 28
post-immunization. The offspring of pregnant mice were bled at
weeks 3, 4, 5, 6, 8, 10, and 12 from birth to assess humoral
responses. Following blood collection, the serum was separated via
centrifugation and stored at -20.degree. C.
[0077] At day 35 post-immunization, adult immunized mice were
challenged with 50.times.LD.sub.50 of tetanus toxin (Sigma-Aldrich,
St. Louis, Mo.) administered subcutaneously in the dorsal surface
of their right hind leg. Disease progression was monitored twice
daily for signs of paralysis (stage T1-T4), morbidity (decreased
mobility, and weight loss) and mortality for 4 days. Their
protective immunity was compared to unimmunized naive mice.
[0078] A separate cohort of 3 week-old pups were challenged with
8.33.times.LD.sub.50 of tetanus toxin and observed for signs of
paralysis for 4 days. All mice were humanely euthanized within 24
hours after exhibiting either paresis of the hind leg while still
maintaining the ability to walk (stage T2) or paralysis of the hind
leg and lacked the ability to function (stage T3). Their protective
immunity was compared to unimmunized naive mice.
[0079] All mice were euthanized according to IACUC guidelines when
they reached one of the following endpoints: Paralysis of the hind
leg in accordance with stage T3 in European Pharmacopeia's scale of
paralysis or weight loss exceeding 25% of the starting body
weight.
[0080] Evaluation of Humoral Immune Responses.
[0081] Anti-tetanus toxoid specific antibody levels were determined
quantitatively by ELISA according to the manufacturer's
instructions; the plates were coated with tetanus toxoid (Enzo Life
Sciences, Inc., Farmingdale, N.Y.) diluted in PBS, 100 ng total
protein per well. Neutralizing antibody titers were determined
using an in vivo tetanus toxin neutralization assay. For each
group, equal amounts of sera taken from all animals were pooled and
serially diluted in PBS. An equal volume of tetanus toxin (dosages:
5.times.LD.sub.50 for adults and 2.times.LD.sub.50 for young) was
mixed with the serum dilutions and incubated overnight at
+4.degree. C. The following day, each mixture was subcutaneously
injected into a single naive mouse and survival was monitored over
a 4 day period. Titers were established as the inverse serum
dilutions that demonstrated protection.
[0082] Fabrication of Dissolving Microneedle Patches for
Encapsulation of Tetanus Toxoid Vaccine.
[0083] PDMS molds were fabricated (GA Tech laboratory) and were
used to prepare microneedle patches (MN) carrying tetanus toxoid
(TT). 10.times.10 parallel rows of conical microcavities were
etched by precisely focused laser. Each individual cavity was 650
.mu.m deep and 250 .mu.m at base diameter.
[0084] Since the total volume of all those cavities was
approximately 1.6 .mu.L, the original non-adjuvanted tetanus toxoid
monobulk obtained from the manufacturer (Serum Institute of India)
was concentrated 5 and 10 times its original concentration by
ultrafiltration using 10 kDa MWCO spin filters (EMD Millipore,
Billerica, Mass.). The TT solution was modified by addition of 3%
polyvinyl alcohol (PVA) (Sigma-Aldrich St Louis, Mo.) and 10%
sucrose (Sigma-Aldrich) as excipients to increase protein stability
and improve geometrical shape of the microneedle. A 5 .mu.L mixture
of the vaccine and additives was loaded to fill in the cavities and
stay on a mold's surface. Vacuum was applied to force the TT
mixture to fill into the mold's cavities and excess of solution was
wiped off. The casting solution was air-dried in the cavities.
Twenty-four hours later, a second solution of 20% PVA and 20%
sucrose was loaded onto the mold to fill the remaining free space
in the cavities and the mold's surface. Vacuum was applied followed
by air-drying in order to form a hard backing. The resulting
microneedles had a conical shape with 650.times.250 .mu.m
dimension, and TT was localized at the tip of the microneedles.
Strength of microneedles was checked before and after short-term
skin insertion under the microscope, and were not bent or
brittle.
[0085] Statistics.
[0086] For ELISAs, the statistical significance of differences
between two groups was calculated by two-tailed unpaired Student's
t-test. For survival curves, statistics were calculated using a
Log-rank (Mantel-Cox) test. A p value less than 0.05 was considered
significant (on graphs: * p<0.05; ** p<0.01; ***
p<0.001).
Example 2: Vaccine Stability
[0087] We found that more than 95% TT was retained above the
membrane suggesting that the tetanus toxoid preparation was intact
and therefore suitable for microneedle fabrication (data not
shown). The intact structure of TT was further shown by
electrophoresis and Western blot (FIG. 1). FIG. 1 shows three
oligomeric components of tetanus toxoid protein, ranging from a
monomer of 150 kDa to a dimer of 270 kDa. Freezing/thawing and
maintaining TT at room temperature for several days had little
effect on vaccine stability, as determined by ELISA.
Example 3: Microneedle Fabrication
[0088] The microneedles penetrated the skin. To decrease dose
variability due to variable insertion depth into the skin, the TT
loading solution was concentrated at the microneedle tip. To assess
insertion efficacy, sulforhodamine B was added to the solution to
facilitate imaging of the microneedles (FIG. 2A and FIG. 2B).
Example 4
[0089] Stability of TT was tested with ELISA before and after
concentration. Microneedle patches encapsulating TT were dissolved
in ELISA buffer for quantitation of the antigen (Lf units per
patch). Tetanus toxoid patches containing unconcentrated antigen
had 0.6.+-.0.075 Lf per patch. Patches with 10.times. concentrated
TT had 5.5.+-.0.7 Lf each, which was close to the required dose for
human vaccines (5 Lf) delivered intramuscularly.
[0090] The formulation of TT microneedle patches described above
led to successful penetration of murine skin, and dissolving after
20 min (FIG. 3A, FIG. 3B, and FIG. 3C). The data demonstrated that
rigid TT patches can penetrate skin.
Example 5: Humoral Immune Responses in Mothers
[0091] A single dose of intramuscular delivery of non-adjuvanted TT
in pregnant mice induced very low TT-specific antibodies, being the
least effective vaccination approach (FIG. 4A).
[0092] In contrast, intradermal vaccination surprisingly increased
the humoral responses in pregnancy as early as 14 days, reaching
2.5-fold higher titers than for intramuscular vaccination up to
last bleed, and as early as day 7 for microneedle patch vaccination
with titers rising from 6-fold to 23-fold (p<0.001) on day 28
post-vaccination when compared to intramuscular vaccination.
[0093] Further, although pregnant mice produced overall higher
antibody titers against TT than non-pregnant mice, skin
immunization-induced antibody production showed the same
fold-differences over intramuscular vaccination between pregnant
and non-pregnant animals in second (day 14) and third bleed (day
28).
[0094] In contrast, for the ID vs. IM induced titers in pregnant
and non-pregnant animals, the differences surprisingly improved
two-fold in pregnancy. This effect was even more pronounced in mice
immunized with microneedle patches with 6-fold higher MN vs. ID
ratios between pregnant and non-pregnant populations (FIG. 4B,
Table 1). Without wishing to be bound by any particular theory, the
skin may be under regulatory control of pregnancy hormones.
Example 6: Passive Immune Response in Offspring of Vaccinated
Mothers
[0095] Serum samples were collected from offspring of mothers
vaccinated during pregnancy at various time points following
weaning at 3 weeks-old. The samples were analyzed for the levels of
serum anti-TT IgG antibodies. While the titers gradually decreased
in both groups over time, there was a surprising 10-fold difference
in antibody levels between the MN and IM groups by week 3
(p<0.001) and a 5-fold difference between MN and ID groups
(p<0.001) indicating a greater passive immune response as a
result of microneedle vaccination (FIG. 5A). Similar differences
were observed in IgG1 isotypes reflecting a Th2 immune profile
(FIG. 5B).
[0096] In contrast to MN vaccination that produced IgG2a
antibodies, none of the other vaccine delivery approaches elicited
any significant IgG2a responses. As a result, the IgG1/IgG2a ratio
which indicates a Th1 or a Th2 bias varied from very low in MN
immunization (IgG1/IgG2a=23), suggesting activation of both arms of
immunity, to very high in ID immunization, suggesting a
preponderance of Th2 responses, which is more effective against
toxins (IgG1/IgG2a=200).
Example 7: Protective Immunity of Vaccinated Mice Against Lethal
Dose of Tetanus Toxin
[0097] Twenty-eight days after immunization, adult mice vaccinated
were challenged with 50.times.LD.sub.50 tetanus toxin and monitored
for stages T1-T3 of paralysis for 4 days. Mice showing stage T2-T3
were euthanized according to IACUC protocol. A 100% survival rate
was observed in both MN and ID vaccinated mice whereas the IM
immunized group survived by 40% (FIG. 6A). Similar effect of MN and
ID immunization was observed in mice that received TT vaccination
during pregnancy. An 84% survival rate was observed in mice
immunized with microneedles, 70% survival rate in intradermally
immunized mice while a 20% survival rate was observed in mice that
were immunized intramuscularly (FIG. 6B). Anti-tetanus NT titer is
shown in FIG. 6C.
[0098] The results show that skin antigen delivery surprisingly
outperformed intramuscular immunization, despite the different
effects they exerted in antibody or cell mediated immunity.
[0099] The results show that intramuscular immunization conferred
50% lower protection in pregnant animals than non-pregnant
controls, whereas skin immunization-induced survival was only
15-30% lower in pregnant population when compared to non-pregnant
cohort.
[0100] Potency of transplacentally transferred maternal anti-TT IgG
antibodies to their fetuses was tested in 3 week old pups after
challenge with 8.times.LD.sub.50 of tetanus toxin subcutaneously
injected and observed for stages T1-T3 of paralysis for 4 days. A
13% survival rate was observed in pups born to pregnant mice
immunized intramuscularly with 5 Lf Tetanus Toxoid, whereas a 100%
survival rate was observed in pups born to mice immunized with the
same dose using microneedles and 87.5% to offspring born to mice
intradermally immunized (FIG. 7A).
[0101] The results of this protection study demonstrate
surprisingly increased potency and effectiveness due to passive
immunity elicited by skin immunization, as compared to conventional
antigen delivery. Six week old pups from microneedle immunized mice
were fully protected, as compared to the ID group and IM groups
that survived 20% and 0%, respectively (FIG. 7B). The results
demonstrated surprising longevity of passive immunity elicited by
skin immunization with microneedle patches.
Example 8
[0102] This study demonstrated improved protective immunity in
healthy adult female mice. For the second experiment, adult female
mice (8 weeks old) were immunized once with 5 Lf units of
non-adjuvanted tetanus toxoid (Research Institute of India) either
intradermally or intramuscularly and sera were collected on days 7,
14 and 28 post-vaccination.
[0103] The humoral immune responses of each treatment group were
IgG and IgM binding antibody titers were assessed with mouse
anti-tetanus toxin/toxoid ELISA kits (Alpha Diagnostic
International, San Antonio, Tex.). For total IgG (FIG. 8A), both
vaccinated groups exhibited significant increases in antibody
titers after 14 days compared to the nearly undetectable naive
controls. However, after 28 days, skin immunized (ID) mice
continued to increase while IM immunized mice decreased resulting
in an over 2-fold difference in IgG antibody titers between the
groups (p<0.05). For IgM antibody titers both groups showed
slight increases in IgM production (FIG. 8B), but remained
statistically insignificant from the naive controls after 28
days.
[0104] At 28 days post-immunization, the vaccinated groups and
naive (unvaccinated) controls were infected with 10.times.LD.sub.50
tetanus toxin (Sigma-Aldrich, St. Louis, Mo.) by subcutaneous
injection in the hind-leg and monitored for four days in order to
assess the protective immune response after lethal challenge. Mice
were characterized based on European Pharmacopeia's scale of
paralysis and symptoms were recorded each day. All naive controls
were euthanized within 24 hours after exhibiting either paresis of
the hind leg while still maintaining the ability to walk (stage T2)
or paralysis of the hind leg and lacked the ability to function
(stage T3). There was a 100% survival rate for both vaccinated
groups during the challenge period (FIG. 9A). However, mice from
both groups (60% IM, 40% ID) appeared to have slight stiffness of
the toxin-injected hind leg symptomatic of the T1 stage before
fully recovering (FIG. 9B).
[0105] Skin immunization during pregnancy is a novel approach to
boost the immune response in both mother and fetus. Tetanus toxoid
skin immunizations in adult mice demonstrated a significant
increase in IgG antibody titers compared to routinely applied
intramuscular injection without the use of alum adjuvant or a
secondary booster shot and more likely result in increased passive
transfer during pregnancy and nursing.
[0106] All publications and patents and literature specifically
mentioned herein are incorporated by reference for all
purposes.
[0107] It is understood that this invention is not limited to the
particular methodology, protocols, materials, and reagents
described, as these may vary. It is also to be understood that the
terminology used herein is for the purpose of describing particular
embodiments only, and is not intended to limit the scope of the
present invention, which will be encompassed by the appended
claims.
[0108] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. As well,
the terms "a" (or "an"), "one or more" and "at least one" can be
used interchangeably herein. It is also to be noted that the terms
"comprises," "comprising", "containing," "including", and "having"
can be used interchangeably.
[0109] Without further elaboration, it is believed that one skilled
in the art can, based on the above description, utilize the present
invention to its fullest extent. The following specific embodiments
are, therefore, to be construed as merely illustrative, and not
limitative of the remainder of the disclosure in any way
whatsoever.
[0110] All of the features disclosed in this specification may be
combined in any combination. Each feature disclosed in this
specification may be replaced by an alternative feature serving the
same, equivalent, or similar purpose.
* * * * *