Note: Descriptions are shown in the official language in which they were submitted.
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PROA efl AND LONG-LASTING INAZIZENZA VA CC/NE
RELATED APPLICATIONS
[0001] This application claims priority to U.S. Ser. No.
62/830,442 filed on 6 April 2019,
which is incorporated herein in its entirety.
FIELD OF THE DISCLOSURE
[0002] This application pertains generally to a monovalent
influenza pharmaceutical
formulation for intranasal administration that induces a combined mucosal,
humoral and cell-
mediated protective immune response in human subjects and provides
seroprotection against
Influenza A and Influenza B subtypes for an extended period of time.
BACKGROUND OF THE DISCLOSURE
[0003] Influenza is one of the most common viral
respiratory infections, leading to significant
morbidity and mortality. The US Centers for Disease Control and Prevention
recommends that
everyone in the US over 6 months of age receives an annual influenza
vaccination. Vaccine
effectiveness can vary greatly from year to year, and in many years overall
protection is poor.
[0004] Influenza viruses are enveloped ribonucleic acid
viruses belonging to the family of
Orthomyxoviridae and are divided into three distinct types on the basis of
antigenic differences of
internal structural proteins (Lamb RA, Krug RM. Orthomyxoviridae: The Viruses
and Their
Replication. In: Fields Virology, Editors-in-Chief: Knipe DM and Howley PM.
4th Edition.
Philadelphia, PA: Lippincott Williams and Wilkins, Publishers; 2001;1487-
1531). Two influenza
types, Type A and B, are responsible for yearly epidemic outbreaks of
respiratory illness in humans
and are further classified based on the structure of two major external
glycoproteins, hemagglutinin
(HA) and neurarninidase (NA). Type B viruses, which are largely restricted to
the human host,
have a single HA and NA subtype. In contrast, numerous HA and NA Type A
influenza subtypes
have been identified to date. Type A strains infect a wide variety of avian
and mammalian species.
[0005] Type A and B influenza variant strains emerge as a
result of frequent antigenic change,
principally from mutations in the HA and NA glycoproteins. These variant
strains may arise
through one of two mechanisms: selective point mutations in the viral genome
[Palese P. Garcia-
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Sastre A. Influenza vaccines: present and future. The Journal of Clinical
Investigation. 2002;110:9-
13; Nakajima S. Nobusawa E, Nalcajima K. Variation in response among
individuals to antigenic
sites on the HA protein of human influenza virus may be responsible for the
emergence of drift
strains in the human population. Virology. 2000;274:220-2311 or from
reassortment between two
co-circulating strains [Holmes EC, Ghedin E, Miller N. Taylor J, Bao Y, St.
George K. Grenfell
BT, Salzberg SL, Fraser CM, Lipman DJ, Taubenberger JK. Whole-genome analysis
of human
influenza A virus reveals multiple persistent lineages and reassortment among
recent 113N2
viruses. PLoS Biology. 2005;3:1579-1589; Barr IG, Komadina N, Hurt AC,
Iannello P. Tomasov
C, Shaw R, Durrant C, Sjogren H, Hampson AW. An influenza A(H3) reassortant
was epidemic
in Australia and New Zealand in 2003. Journal of Medical Virology. 2005;76:391-
3971.
[0006] Since 1977, influenza A virus subtypes HINI and
H3N2, and influenza B viruses have
been in global circulation in humans. The current U.S. licensed inactivated
trivalent and
quadrivalent (containing two strain lineages of influenza B virus) vaccines
are formulated to
prevent influenza illness caused by these influenza viruses. Because of the
frequent emergence of
new influenza variant strains, the antigenic composition of influenza vaccines
need to be evaluated
yearly, and the influenza vaccines are reformulated almost every year. The
immune response
elicited by previous vaccination may not be protective against new variants.
[0007] Changes in influenza virus formulation and
manufacturing are essential to protect the
public from the significant morbidity and mortality associated with seasonal
influenza infections.
The National Institute of Allergy and Infectious Diseases (NIAID) recently
developed a strategic
plan to guide basic research and development of more effective influenza
vaccines. [Erbelding EJ,
et al. A universal influenza vaccine: The strategic plan for the National
Institute of Allergy and
Infectious Diseases. J Infect Dis. 2018; 218;347-54] This plan highlighted
multiple weaknesses of
currently available vaccines, including strain mismatch exacerbated by egg
passage, inadequate
durability of immune response, poor cellular immune responses, and inadequate
tissue-resident
immunity [Erbelding et al., 2018]. Vaccines produced in eggs are not only more
likely to be
antigenically dissimilar the corresponding strains in circulation than vaccine
produced in tissue
culture [Seqirus presents favorable outcomes data for adjuvanted trivalent
influenza vaccine
(FLUADO) at 6th annual IDWeek. Seqiris Web site] but are also associated with
longer
manufacturing timelines and supply chain risks and induce allergic response in
many individuals.
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[0008] Most currently licensed vaccines are based on
circulating influenza strains adapted to
grow in chicken eggs. In general, these vaccines are well tolerated but
provide limited protection
to influenza viruses that are not well matched to the vaccine strains. An
intranasal live attenuated
influenza virus (LAW) vaccine has been licensed since 2003, but its use is
limited to older children
and adults up to age 49 years. Intranasal vaccines may be preferred over
parenteral vaccines due
to the ease of administration and decreased discomfort associated with
administration. A
significant fraction of the US population manifests fear of needles (McLenon
J. et al. The fear of
needles: A systematic review and meta-analysis (2019; Jan) J. Adv. Nurs.;
75(1):30-42). Recent
post-marketing studies have shown declining effectiveness, and it was not
recommended for use
in the 2016-2017 and 2017-2018 influenza seasons (CDC website). While data
from 2010-2011
through 2016-2017 indicated that LAW lacked effectiveness among 2 through 17-
year-olds
against H1N1pdm09 influenza viruses (2009 H1N1) in the U.S., LAW was effective
against
influenza B viruses, and was similarly effective against 113N2 viruses as
inactivated influenza
vaccines. For the 2018-2019 season, the manufacturer of LAIV4 included a new
H1N1 vaccine
component. Some data suggest this will result in improved effectiveness of
LAIV4 against H1N1.
However, no published effectiveness estimates for this vaccine component
against H1N1 viruses
are yet available.
[0009] Moreover, a recently published study of the 2013-
2014 LAW vaccine showed weak
systemic antibody responses [King JP, McLean HQ, Meece JK, et al. Vaccine
failure and serologic
response to live attenuated and inactivated influenza vaccines in children
during the 2013-2014
season. Vaccine. 2018; 36:1214-9]. For all types of influenza vaccines,
effectiveness can vary
greatly from year to year, and in many years overall protection is poor.
According to the CDC, the
average overall adjusted vaccine effectiveness for influenza seasons has been
approximately 40%
from 2005 to 2015 and was < 20% in the 2014-2015 influenza season.
[0010] Cellular immunity to influenza may reduce disease
severity and contagiousness in those
infected, mucosal immune responses provide protection against influenza at the
initial site of
infection and both may be able to protect against infection in the absence of
seroprotective levels
of serum HAI antibody [Gould VMW, Francis JN, Anderson KJ, et al. Nasal IgA
provides
protection against human influenza challenge in volunteers with low serum
influenza antibody
titre. Front Microbiol. 2017;8:900; McMichael AJ, Gotch FM, Noble GR, et al.
Cytotoxic T-cell
immunity to influenza. N Engl J Med. 1983;309:13-7; Seibert CW, Rahmat S,
Krause JC, et al.
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Recombinant IgA is sufficient to prevent influenza virus transmission in
guinea pigs. J Vito!.
2013;87:7793-804; Wilkinson TM, Li CK, Chui CS, et at Preexisting influenza
specific CD4+ T
cells correlate with disease protection against influenza challenge in humans.
Nat Med.
2012;18:274-80J. However, no single influenza vaccine induces those combined
responses of the
immune system arms to provide long term seroprotection against influenza A and
influenza B
subtypes. Therefore, a need remains for an influenza vaccine that induces long
term
seroprotection, along with a combined immune response of the mucosal, humoral
and cell-mediate
types.
SUMMARY OF THE DISCLOSURE
[0010] Herein some embodiments provided include compositions and methods for
inducing long
term systemic immune protection against influenza A and influenza B virus
subtypes in human
subjects.
[0011] In embodiments provided herein is a monovalent influenza pharmaceutical
formulation
suitable for a single dose intranasal administration to a human subject. In
certain embodiments,
the formulation comprises an effective amount of at least 10" viral particle
(vp) of replication
deficient adenovirus vector that contains and expresses influenza virus
hemagglutinin antigen
codon optimized for the human subject, wherein the effective amount induces a
combined
mucosal, humoral (i.e., ex-mucosal antibodies, such as in blood or serum), and
T cell immune
response that is preferably protective from influenza infection; and, a
pharmaceutically acceptable
diluent or carrier. In embodiments, the formulation is configured to provide
seroprotection to the
human subject of an HAI antibody titer >40 for at least 12 months against the
influenza virus.
[0012] In certain embodiment provided herein is influenza pharmaceutical
formulation suitable
for a single dose intranasal administration to a human subject, wherein the
formulation comprises
an effective amount of at least 109 viral particles (vp) of replication
deficient adenovirus vector
that contains and expresses influenza virus hemagglutinin antigen codon
optimized for the human
subject, wherein the effective amount induces a combined mucosal and humoral
immune response
that is preferably protective, preferably being configured to provide
seroprotection to the human
subject of an HAI antibody titer >40 for at least 12 months against the
influenza virus; and, a
pharmaceutically acceptable diluent or carrier. In certain embodiments, the
HAT antibody titer is
at least 50.
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[0013] In certain embodiments, the influenza virus hemagglutinin antigen is
from an Influenza A
virus. In embodiments, the Influenza A virus is subtype H1N1.
[0014] In certain other embodiments provided herein are method of inducing a
combined mucosal,
humoral and T cell immune response that is preferably protective in a human
subject against
influenza virus. In preferred embodiments, the methods comprise administering
intranasally to a
human subject a single dose of the present influenza pharmaceutical
formulation, wherein the
administration induces serum antibodies, mucosal antibodies and T cells
against influenza virus
whereby the human subject is seroprotected for at least 12 months.
In preferred embodiments, the seroprotection lasts at least 13 months, at
least 14 months, or longer.
In embodiments, administration induces an HAI antibody titer of at least 50
for at least 12 months
post administration.
[0015] In some embodiments, the present disclosure provides methods that
provide a combined
mucosal, humoral and T cell immune response that is preferably protective
against Influenza A
virus. In embodiments, the Influenza virus A is subtype H1N1 and/or H3N2. In
embodiments,
the present methods provide a combined mucosal, humoral and T cell immune
response that is
preferably protective against Influenza B virus. In certain embodiments, the
present methods
provide a combined mucosal, humoral and T cell immune response that is
preferably protective
against Influenza A virus subtypes and Influenza B virus infection.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] The accompanying drawings, which are incorporated into and constitute a
part of this
specification, illustrate one or more embodiments of the present disclosure
and, together with the
detailed description and examples sections, serve to explain the principles
and implementations of
the disclosure.
[0018] Figure 1 shows the schematic diagram of the adenoviral vector
containing the influenza
virus HA gene, wherein the numbers refer to base pair number in wild-type Ad5
sequence,
GenBank ID AY339865.1.
[0019] Figure 2 shows the serum antibody (hemagglutination-inhibiting
antibody, HAI)
(humoral) response at day 29 post administration induced following single
administration of the
present monovalent influenza vaccine composition with 100% seroprotection at
two dose levels,
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wherein the antibodies are sufficient to prevent an infection from influenza
virus. Fluzone is an
inactivated quadrivalent high dose seasonal influenza vaccine.
[0020] Figure 3 shows in table format the serum antibodies (HAI, MN) measured
at day 29 post
administration of the present monovalent influenza vaccine composition,
wherein serum
neutralizing antibodies were measured in a microneutralization ("MN") assay
and the serum
hemagglutination inhibiting antibodies measured in a HAI assay are presented
as a geometric mean
titer ("GMT").
[0021] Figure 4 shows T cell immunity (cell mediated immune) response induced
following
single administration of the present monovalent influenza vaccine composition.
[0022] Figure 5 shows mucosal IgA antibody (mucosal) response induced
following single
administration of the present monovalent influenza vaccine composition.
[0023] Figure 6 shows extended seroprotection via measurement of serum
antibody (HA!) at
amounts sufficient to prevent an infection from influenza virus. The figure
shows the geometric
mean (95% Confidence Interval) hemagglutination inhibition titer against
influenza
A/California/07/2009(H1N1) to end point day 181 by dose of the present
monovalent influenza
vaccine composition.
[0024] Figure 7 shows geometric mean (GMR) (95% confidence interval)
microneutralization
titer against Influenza A/California/07/2009(H1N1) to end point day 181 by
dose of the present
monovalent influenza vaccine composition.
[0025] Figure 8 shows the cellular immune response to the present monovalent
influenza vaccine
composition by dose. Abbreviations: CI = confidence interval; GM = geometric
mean titer; LS =
least squares; SFU = spot-forming units; vp = viral particles. a. The analysis
of covariance uses
log-transformed level as dependent variable, dose group as a factor, and
baseline log-transformed
analysis as a covariate. Differences of LS mean estimates and 95% CIs were
back-transformed
to the original scale, resulting in a ratio of the geometric means; b. Post-
hoc analysis; c. The
number and percentage of subjects with 3-fold rise since Baseline and 25
SFU/106 cells greater
than Baseline; and, d. From Fisher's exact test.
[0026] Figure 9 shows humoral immune response induced by the present
monovalent influenza
vaccine composition (e.g., NasoVAX) at a dose of 11x1011 vp and Fluzone0
Groups on days 29,
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91 and 181 post administration, with the HAT presented as a geometric mean
ratio ("GMR") along
with the seroconversion rate ("SCR") and the percentage of subjects with a HAT
titer > 1:40 ("a"),
and the seroprotection rate ("SPR"), wherein the percentage of subjects with
either a baseline HAI
titer, 1:10 and postvaccination titer > 1:40 ("b) (which is 4 times the assay
lower limit of
quantification) are included in the last row.
[0027] Figure 10A shows the HA! titer ("GMT") over 13 months induced by the
present
monovalent influenza vaccine composition (e.g., NasoVAX), wherein 8/15
subjects in the high
dose group (11x1011 vp) returned at about 13 months for evaluation with an
average of 13.5 months
from administration to measurement of HA! titer.
[0028] Figure 10B shows seroprotection and seroconversion rates induced by the
present
monovalent influenza vaccine composition (e.g., NasoVAX), demonstrating the
seroprotection
and seroconversion rates are identical between study days 15 and 400 (days
post administration of
the present monovalent influenza vaccine composition). The immune response was
intact at 13
months with the rate of seroprotection and the rate of seroconversion
unchanged.
[0029] Figure 11 shows a dose-dependent vector shedding that is absent at 2
weeks post-
administration (of the present monovalent influenza vaccine composition (e.g.,
NasoVAX)) with
no replication competent virus found (as determined via polymerase chain
reaction ("PCR") assay)
and anti-vector antibody presented as GMR at Day 29 vs baseline wherein only a
2.3-fold induction
after 1 month at highest dose was demonstrated. The present monovalent
influenza vaccine
composition demonstrates a transient shedding (Advector) with limited anti-
vector (Ad-vector)
immune response.
[0030] Figure 12 shows the effect on NasoVAX immunogenicity (high dose;
11x1011 vp) of pre-
existing anti-vector (Ad5) immunity as measured for humoral ("HAI" or
microneutralization
"MN" at day 29), mucosal ("IgA" at day 29) and cellular ("ELISpot" at day 8)
wherein no
difference in an immune response between Ad5 seronegative or Ad5 seropositive
subjects was
observed. Median titer of Ad5+ subjects (seropositive) was 22-fold above the
lower limit of
quantification (LLOQ), wherein seroconversion is typically about 4-fold over
background or the
LLOQ assay.
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DETAILED DESCRIPTION OF THE INVENTION
[0031] Introduction
[0032] The present invention provides compositions and methods for inducing
long term systemic
immune protection against influenza A virus subtypes and influenza B viruses
in human subjects.
In certain embodiments provided herein is a influenza pharmaceutical
formulation (preferably
monovalent) suitable for a single dose intranasal administration to a human
subject. In
embodiments, the formulation comprises an effective amount of at least 1011
viral particle (vp) of
replication deficient adenovirus vector that contains and expresses influenza
virus hemagglutinin
antigen codon optimized for the human subject, wherein the effective amount
induces a combined
mucosal, humoral and T cell immune response, which is preferably protective
against influenza
infection; and, a pharmaceutically acceptable diluent or carrier.
[0033] In other embodiments, is provided an influenza pharmaceutical
formulation suitable for a
single dose intranasal administration to a human subject, wherein the
formulation comprises an
effective amount of at least 109 viral particles (vp) of replication deficient
adenovirus vector that
contains and expresses influenza virus hemagglutinin antigen codon optimized
for the human
subject, wherein the effective amount induces a combined mucosal and humoral
immune response,
which is preferably protective against influenza infection, and is some
preferred embodiments
configured to provide seroprotection to the human subject of an HAI antibody
titer >40 for at least
12 months against the influenza virus; and, a pharmaceutically acceptable
diluent or carrier
[0034] In certain embodiments is provided a method for inducing a combined
mucosal, Immoral
and T cell immune response, preferably protective, in a human subject against
influenza virus. In
certain embodiments, the methods comprise administering intranasally to a
human subject a single
dose of a present influenza pharmaceutical formulation, wherein the
administration induces serum
antibodies, mucosal antibodies and T cells against influenza virus whereby the
human subject is
seroprotected for at least 12 months.
[0035] Applicants have developed an adenoviral vector (e.g., Ad5-vectored),
intranasal influenza
vaccine produced in tissue culture (NasoVAX). Adenovirus is a naturally
occurring respiratory
virus that has been used frequently as a vector to introduce genetic material
into cells. By
incorporating the influenza HA gene into replication-deficient (RD) adenovirus
(Ad-HA) and
applying the Ad-HA into the nose (intranasal route of administration), the
adenoviral vector can
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transduce the HA gene into cells of the nasal mucosa, leading to transient
expression of the
encoded HA protein. NasoVAX is delivered intranasally, where we herein
demonstrate the vaccine
composition induced both local and long-lasting systemic immune responses. See
Example 3.
Subsequent production of the HA antigen in normal human epithelial cells
allows for an immune
response against the HA antigen as it occurs in natural circulating influenza
virus. Moreover,
Applicants have shown the use of the adenoviral vector, when administered
intranasally, even in
subjects seropositive for Ad5, bypasses the adenovims immunity of the subjects
and thereby its
effects are not adversely affected by a pre-existing immune response against
the vector. See
Figure 13. In embodiments, the present intranasal influenza vaccine
(comprising an adenoviral
vector) can be administered repeatedly (e.g., as a seasonal influenza vaccine
administered about
once every 11-14 months) without inducing a significant immune response
against the viral vector.
[0036] The clinical study disclosed herein was designed to evaluate the safety
and immunogenicity
of a monovalent A/California/04/2009(H1N1)-like strain version of NasoVAX.
Exploratory
endpoints were included to evaluate the breadth of antibody response and the
ability of NasoVAX
to induce cellular and mucosal immune responses. See Example 2 and 3. The
trial results disclosed
herein demonstrate the present monovalent influenza pharmaceutical formulation
satisfies the
unmet needs identified by NIA1D [Erbelding et at., 2018].
[0037] As understood by one of skill in the art, the measurement of HAT
antibodies is used as a
surrogate of protection wherein a HAI antibody titer of >40 measured in post
vaccination serum
demonstrates induced seroprotection by the administered influenza vaccine
[Trombetta CM. et at.;
Overview of Serological Techniques for Influenza Vaccine Evaluation: Past,
Present and Future
Vaccines (Basel) (2014) Dec. 2(4): 707-734]. A "surrogate of protection" means
an immune
marker that can substitute for the clinical end point and thus, can be used to
reliably predict vaccine
efficacy. In the case of influenza vaccines, antibodies measured in a
hemagglutination inhibition
(HAT) assay is a surrogate of protection, wherein the HAI assay is based on
the ability of
antibodies, if present in the serum, to prevent agglutination between
erythrocytes and viral
hemagglutinin. It is generally understood that subjects with a HAI antibody
titer of >40 are
protected from represented Influenza A virus subtypes and Influenza B virus.
In other words, an
HAI antibody titer of 40 (or greater) is generally considered as a
"protective" threshold level,
beyond which there is a 50% or greater reduction in the possibility of
contracting an influenza
infection. An HAI titer equal to or greater than 40 is used as an
immunological correlate of
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protection and is regarded as the best currently available parameter for
predicting protection from
natural infection, according to FDA guidelines for pandemic influenza vaccines
(Noah et al.
Qualification of the hemagglutination inhibition assay in support of pandemic
influenza vaccine
licensure. Clin. Vaccine Immunol. 2009;16:558-566).
[0038] In a separate study, Applicants have shown the present intranasal
influenza vaccine
(NasoVAX) is stable for about 3 months at an ambient temperature, such as room
temperature
(e.g.,15 to 30 C, preferably 20-25 C). See Example 7. In embodiments, the
present intranasal
influenza vaccine can be stored, or shipped, without the need for
refrigeration or specific storage
conditions. In certain embodiments, the present intranasal influenza vaccine
comprises influenza
antigens present in an Influenza A pandemic virus strain and may be shipped
directly to the user
i.e., vaccinee, for intranasal administration.
[0039] Definitions
[0040] As used herein, the terms "a" or "an" are used, as is common in patent
documents, to
include one or more than one, independent of any other instances or usages of
"at least one" or
"one or more."
[0041] As used herein, the term "or" is used to refer to a nonexclusive or,
such that "A or B"
includes "A but not B," "B but not A," and "A and B," unless otherwise
indicated.
[0042] As used herein, the term "about" is used to refer to an amount that is
approximately, nearly,
almost, or in the vicinity of being equal to or is equal to a stated amount,
e.g., the state amount
plus/minus about 5%, about 4%, about 3%, about 2% or about 1%.
[0043] The compositions, formulations and methods of the present invention may
comprise,
consist essentially of, or consist of the components and ingredients of the
present invention as well
as other ingredients described herein. As used herein, "consisting essentially
of' means that the
compositions, formulations and methods may include additional steps,
components or ingredients,
but only if the additional steps, components or ingredients do not materially
alter the basic and
novel characteristics of the claimed compositions, formulations and methods.
[0044] It should also be noted that, as used in this specification and the
appended claims, the term
"configured" describes a system, apparatus, or other structure that is
constructed or configured to
perform a particular task or adopt a particular configuration. The term
"configured" can be used
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interchangeably with other similar phrases such as arranged and configured,
constructed and
arranged, adapted and configured, adapted, constructed, manufactured and
arranged, and the like.
[0045] As used herein, an "adjuvant" refers to a substance that enhances the
body's immune
response to an antigen. In embodiments, the present monovalent influenza
pharmaceutical
formulation is a non-adjuvanted vaccine composition.
[0046] By "administration" is meant introducing a vaccine composition of the
present disclosure
into a subject; it may also refer to the act of providing a composition of the
present disclosure to a
subject (e.g., by prescribing). The term "therapeutically effective amount" as
used herein refers
to that amount of the compound being administered which will induce a
combined, mucosal,
humoral and cell mediated immune response. The term also refers to an amount
of the present
compositions that will relieve or prevent to some extent one or more of the
symptoms of the
condition to be treated. In reference to conditions/diseases that can be
directly treated with a
composition of the disclosure, a therapeutically effective amount refers to
that amount which has
the effect of preventing the condition/disease from occurring in a mammal that
may be predisposed
to the disease but does not yet experience or exhibit symptoms of the
condition/disease
(prophylactic treatment), alleviation of symptoms of the condition/disease,
diminishment of extent
of the condition/disease, stabilization (e.g., not worsening) of the
condition/disease, preventing the
spread of condition/disease, delaying or slowing of the condition/disease
progression, amelioration
or palliation of the condition/disease state, and combinations thereof. The
term "effective amount"
refers to that amount of the compound being administered which will produce a
reaction that is
distinct from a reaction that would occur in the absence of the compound.
[0047] In embodiments, an effective amount of the present monovalent influenza
pharmaceutical
formulation comprises at least 109 infectious units (ifu) of a replication
deficient adenoviral vector
containing and expressing influenza virus hemagglutinin antigen codon
optimized for the human
subject.
[0048] As used herein, the term "ambient temperature" is the air temperature
for storing the
present monovalent influenza pharmaceutical formulation. In embodiments, the
ambient
temperature is a room temperature, such as selected from any temperature
within the range from
about 15 to 30 C, preferably from about 20 to 25 C.
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[0049] As used herein, the term "human adenovirus" is intended to encompass
all human
adenoviruses of the Adenoviridae family, which include members of the
Mastadenovirus genera.
To date, over fifty-one human serotypes of adenoviruses have been identified
(see, e.g., Fields et
al., Virology 2, Ch. 67 (3d ed., Lippincott-Raven Publishers)). The adenovirus
may be of serogroup
A. B. C, D, E. or F. The human adenovirus may be a serotype 1 (Ad 1), serotype
2 (Ad2), serotype
3 (Ad3), serotype 4 (Ad4), serotype 5 (Ad5), serotype 6 (Ad6), serotype 7
(Ad7), serotype 8 (Ad8),
serotype 9 (Ad9), serotype 10 (Ad10), serotype 11 (Ad! 1), serotype 12 (Ad12),
serotype 13
(Ad13), serotype 14 (Ad14), serotype 15 (Ad15), serotype 16 (Ad16), serotype
17 (Ad17),
serotype 18 (Ad18), serotype 19 (Ad19), serotype 19a (Adl9a), serotype 19p
(Adl9p), serotype
20 (Ad20), serotype 21 (Ad21), serotype 22 (Ad22), serotype 23 (Ad23),
serotype 24 (Ad24),
serotype 25 (Ad25), serotype 26 (Ad26), serotype 27 (Ad27), serotype 28
(Ad28), serotype 29
(Ad29), serotype 30 (Ad30), serotype 31 (Ad31), serotype 32 (Ad32), serotype
33 (Ad33),
serotype 34 (Ad34), serotype 35 (Ad35), serotype 36 (Ad36), serotype 37
(Ad37), serotype 38
(Ad38), serotype 39 (Ad39), serotype 40 (Ad40), serotype 41 (Ad41), serotype
42 (Ad42),
serotype 43 (Ad43), serotype 44 (Ad44), serotype 45 (Ad45), serotype 46
(Ad46), serotype 47
(Ad47), serotype 48 (Ad48), serotype 49 (Ad49), serotype 50 (Ad50), serotype
51 (Ad51), or
combinations thereof, but are not limited to these examples. In certain
embodiments, the
adenovirus is serotype 5 (Ad5).
[0050] As used herein, a "pharmaceutically acceptable carrier" refers to a
carrier or diluent that
does not cause significant irritation to the human subject and does not
abrogate the biological
activity and properties of the administered vaccine compositions.
[0051] As used here, the term "seroconversion" is a rate defined as the
percentage of individuals
vaccinated (administered a present vaccine formulation) who have at least a 4-
fold increase in
serum haemagglutinin inhibition (HI) titers after vaccination. As used herein
"conversion factor"
is defined as the fold increase in serum HI geometric mean titers (GMTs) after
vaccination.
[0052] As used herein, the term "seroprotection" refers to an HAI antibody
titer of 40 or greater
measured in serum from a human subject post-vaccination. The term "protection
rate" as used
herein is defined as the percentage of individuals vaccinated with a serum HAI
titer equal to or
greater than 1:40 after vaccination and is normally accepted as indicating
protection.
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[0053] As used herein, the term "seasonal influenza virus" refers to an
influenza A and/or B virus
that circulates and are responsible for seasonal flu epidemics each year.
Influenza A viruses are
divided into subtypes based on two proteins on the surface of the virus:
hemagglutinin (HA) and
neuraminidase (NA). There are 18 different hemagglutinin subtypes and 11
different
neuraminidase subtypes (H1 through H18 and Ni through N11, respectively).
While there are
potentially 198 different influenza A subtype combinations, only 131 subtypes
have been detected
in nature. Current subtypes of influenza A viruses that routinely circulate in
humans include
A(H1N1) and A(113N2). Influenza B viruses are further classified into two
lineages: B/Yamagata
and B/Victoria. Both influenza A and B viruses can be further classified into
specific clades and
sub-clades (which are sometimes called groups and sub-groups). Currently
circulating influenza
A(H1N1) viruses are related to the pandemic 2009 H1N1 virus that emerged in
the spring of 2009
and caused a flu pandemic. This virus, referred to as the "A(H1N1)pdm09
virus," and more
generally called "2009 Hi Ni," continues to circulate and contribute to
seasonal flu epidemics each
year. Of all the influenza viruses that routinely circulate and cause illness
in people, influenza
A(113N2) viruses tend to change more rapidly, both genetically and
antigenically. Influenza
A(H3N2) viruses have formed many separate, genetically different clades in
recent years that
continue to co-circulate.
[01:154] As used herein, the term "pandemic influenza virus" refers to an
influenza A virus that
circulates globally and is responsible for a global flu pandemic. Pandemics
happen when new
(novel) influenza A viruses emerge which are able to infect people easily and
spread from person
to person in an efficient and sustained way, spreading globally.
[0055] The terms "treat", "treating", and "treatment" are an approach for
obtaining beneficial or
desired clinical results. Specifically, beneficial or desired clinical results
include, but are not
limited to, alleviation of symptoms, diminishment of extent of disease,
stabilization (e.g., not
worsening) of disease, delaying or slowing of disease progression,
substantially preventing spread
of disease, amelioration or palliation of the disease state, and remission
(partial or total) whether
detectable or undetectable. In addition, "treat", "treating", and "treatment"
can also mean
prolonging survival as compared to expected survival if not receiving
treatment and/or can be
therapeutic in terms of a partial or complete cure for a disease and/or
adverse effect attributable to
the disease. As used herein, the terms "prophylactically treat" or
"prophylactically treating" refers
completely, substantially, or partially preventing a disease/condition or one
or more symptoms
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thereof in a host. Similarly, "delaying the onset of a condition" can also be
included in
"prophylactically treating" and refers to the act of increasing the time
before the actual onset of a
condition in a patient that is predisposed to the condition.
[0056] As used herein, a "vaccine" refers to a composition comprise the
adenoviral vector
containing and expressing an influenza antigen, along with other components of
a vaccine
formulation, including for example adjuvants, slow release compounds,
solvents, etc. In
embodiments of the invention vaccines improve immune responses to any antigen
regardless of
the antigen source or its function.
[0057] As referred to herein, a "vector" carries a genetic code, or a portion
thereof, for an antigen,
however it is not the antigen itself. In an exemplary aspect, a vector can
include a viral vector or
bacterial vector. As referred to herein an "antigen" means a substance that
induces a specific
immune response in a subject, including humans and/or animals. The antigen may
comprise a
whole organism, killed, attenuated or live; a subunit or portion of an
organism; a recombinant
vector containing an insert with immunogenic properties; a piece or fragment
of DNA capable of
inducing an immune response upon presentation to a host animal; a polypeptide,
an epitope, a
hapten, or any combination thereof. In various aspects, the antigen is a
virus, bacterium, a subunit
of an organism, an auto-antigen, or a cancer antigen.
[0058] Vaccine Formulation
[0059] Provided herein are influenza pharmaceutical formulations, also
referred to herein as
vaccine formulations, suitable and/or configured for a single dose intranasal
administration to a
human subject. In embodiments, the instant formulations comprise an effective
amount of at least
109 viral particle (vp) of replication deficient adenovirus vector that
contains and expresses
influenza virus hemagglutinin antigen codon optimized for the human subject
and a
pharmaceutically acceptable diluent or carrier. In exemplary embodiments the
formulation is a
monovalent influenza pharmaceutical formulation. In certain embodiments, the
adenoviral vector
is present in a formulation buffer comprising 10 m/vI TRIS, 75 inNI NaCl, 0.2%
Polysorbate 80,
5% sucrose, 1 mM MgCl2, 0.1 mM EDTA, 0.5% ethanol, 10 mM
[0060] In alternative embodiments, the present replication deficient
adenovirus vector that
contains and expresses influenza virus hemagglutinin antigen codon optimized
for the human
subject, may be combined with other influenza antigens (e.g. viral vector
expressed antigens) to
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form a multivalent influenza pharmaceutical formulation. The other components
may be included
to induce a humoral response with antibodies to a different epitope than that
presented in the instant
adenoviral vectored containing influenza virus hemagglutinin antigen. In other
embodiments, the
other component(s) may be included to induce a different arm of the immune
system, such as cell-
mediated or mucosal immune response to an influenza antigen.
[0061] In exemplary embodiments provided herein is a monovalent influenza
pharmaceutical
formulation suitable for a single dose intranasal administration to a human
subject, comprising: an
effective amount of at least 1011 viral particles (vp) of replication
deficient adenovirus vector that
contains and expresses influenza virus hemagglutinin antigen codon optimized
for the human
subject, wherein the effective amount induces a combined mucosal, humoral and
T cell protective
immune response configured to provide seroprotection to the human subject for
at least 12 months
against the influenza virus; and, a pharmaceutically acceptable diluent or
carrier.
[00621 In other exemplary embodiments provided herein is an influenza
pharmaceutical
formulation suitable for a single dose intranasal administration to a human
subject, comprising an
effective amount of at least 109 viral particles (vp) of replication deficient
adenovirus vector that
contains and expresses influenza virus hemagglutinin antigen codon optimized
for the human
subject, wherein the effective amount induces a combined mucosal and humoral
protective
immune response configured to provide seroprotection to the human subject of
an HAT antibody
titer >40 for at least 12 months against the influenza virus; and, a
pharmaceutically acceptable
diluent or carrier_
[00631 In certain embodiments, the non-replicating adenoviral viral vector is
a human adenovirus.
In alternative embodiments, the adenovirus is a bovine adenovirus, a canine
adenovirus, a non-
human primate adenovirus, a chicken adenovirus, or a porcine or swine
adenovirus. In exemplary
embodiments, the non-replicating viral vector is a human adenovirus.
[0064] In embodiments, non-replicating adenoviral vectors are particularly
useful for gene transfer
into eukaryotic cells and vaccine development, and in animal models.
[0065] In embodiments, any adenoviral vector (Ad-vector) known to one of skill
in art, and
prepared for administration to a mammal, which may comprise and express an
influenza antigen
may be used in the compositions and with the methods of this application. Such
Ad-vectors
include any of those in US Patent Nos. 6,706,693; 6,716,823; 6,348,450; or US
Patent Publ. Nos.
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2003/0045492; 2004/0009936; 2005/0271689; 2007/0178115; 2012/0276138 (herein
incorporated by reference in entirety).
[0066] In certain embodiments the recombinant adenovirus vector may be non-
replicating or
replication-deficient requiring complementing El activity for replication. In
embodiments the
recombinant adenovirus vector may include El-defective, E3-defective, and/or
E4-defective
adenovirus vectors, or the "gutless" adenovirus vector in which viral genes
are deleted. The El
mutation raises the safety margin of the vector because El-defective
adenovirus mutants are
replication incompetent in non-permissive cells. The E3 mutation enhances the
immunogenicity
of the antigen by disrupting the mechanism whereby adenovirus down-regulates
MHC class I
molecules. The E4 mutation reduces the immunogenicity of the adenovirus vector
by suppressing
the late gene expression, thus may allow repeated re-vaccination utilizing the
same vector. In
exemplary embodiments, the recombinant adenovirus vector is an El and E3
defective vector.
[0067] The "gutless" adenovirus vector replication requires a helper virus and
a special human
293 cell line expressing both Ela and Cre, a condition that does not exist in
natural environment;
the vector is deprived of viral genes, thus the vector as a vaccine carrier is
non-immunogenic and
may be inoculated for multiple times for re-vaccination. The "gutless"
adenovirus vector also
contains 36 kb space for accommodating transgenes, thus allowing co-delivery
of a large number
of antigen genes into cells. Specific sequence motifs such as the ROD motif
may be inserted into
the H-I loop of an adenovirus vector to enhance its infectivity. An adenovirus
recombinant may
be constructed by cloning specific transgenes or fragments of transgenes into
any of the adenovirus
vectors such as those described below. The adenovirus recombinant vector is
used to transduce
epidermal cells of a vertebrate in a non-invasive mode for use as an
immunizing agent. The
adenovirus vector may also be used for invasive administration methods, such
as intravenous,
intramuscular, or subcutaneous injection.
[0068] With respect to dosages, routes of administration, formulations,
adjuvants, and uses for
recombinant viruses and expression products therefrom, compositions of the
invention may be
used for parenteral or mucosal administration, preferably by intradermal,
subcutaneous,
intranasal or intramuscular routes. When mucosal administration is used, it is
possible to use
oral, ocular or nasal routes. In exemplary embodiments, the present vaccine
formulations are
administered intranasally.
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[0069] The formulations which comprise the adenovirus vector of interest, can
be prepared in
accordance with standard techniques well known to those skilled in the
pharmaceutical or
veterinary art. See Example 1. Such formulations can be administered in
dosages and by
techniques well known to those skilled in the clinical arts taking into
consideration such factors
as the age, sex, weight, and the route of administration. The formulations can
be administered
alone or can be co-administered or sequentially administered with
compositions, e.g., with "other"
immunological composition, or attenuated, inactivated, recombinant vaccine or
therapeutic
compositions thereby providing multivalent or "cocktail" or combination
compositions of the
invention and methods employing them. In embodiments, the formulations may
comprise
sucrose as a cryoprotectant and polysorbate-80 as a non-ionic surfactant. In
certain embodiments,
the formulations further comprise free-radical oxidation inhibitors ethanol
and histidine, the metal-
ion chelator ethylenediaminetetraacetic acid (EDTA), or other agents with
comparable activity
(e.g., block or prevent metal-ion catalyzed free-radical oxidation).
[0070] The formulations may be present in a liquid preparation for mucosal
administration, e.g.,
oral, nasal, ocular, etc., formulations such as suspensions and, preparations
for parenteral,
subcutaneous, intraderrnal, intramuscular, intravenous (e.g., injectable
administration) such as
sterile suspensions or emulsions. In such formulations the adenoviral vector
may be in
admixture with a suitable carrier, diluent, or excipient such as sterile
water, physiological saline,
or the like. The formulations can also be lyophilized or frozen. The
formulations can contain
auxiliary substances such as wetting or emulsifying agents, pI-1 buffering
agents, adjuvants,
preservatives, and the like, depending upon the route of administration and
the preparation
desired. The formulations can contain at least one adjuvant compound. In
exemplary
embodiments, the present vaccine formulations are non-adjuvanted.
[0071] Standard texts, such as "REMINGTON'S PHARMACEUTICAL SCIENCE", 17th
edition, 1985, incorporated herein by reference, may be consulted to prepare
suitable
preparations, without undue experimentation.
[0072] In embodiments, an effective amount (e.g., an amount that induces a
combined mucosal,
humoral and cell-mediated immune response) of the adenoviral vector is at
least 109 infectious
units (ifu) of a replication deficient adenoviral vector containing and
expressing influenza virus
hemagglutinin antigen codon optimized for the human subject. As understood by
one of skill in
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the art, codon optimization improves expression of heterologous genes in a
host organism. The
present influenza antigen was codon optimized for a mammalian host, which
includes a human
subject.
[0073] In certain embodiments, the present monovalent influenza formulation
comprises an
effective amount of about 109 viral particles (vp) of a replication deficient
adenoviral vector. In
exemplary embodiments, the present monovalent influenza formulation comprises
an effective
amount of about 1019 viral particles (vp) of a replication deficient
adenoviral vector. In certain
other exemplary embodiments, the present monovalent influenza formulation
comprises an
effective amount of about 1011 viral particles (vp) of a replication deficient
adenoviral vector.
[0074] In embodiments, the effective amount of adenoviral vector in the
present vaccine
formulation induces a combined influenza-specific mucosal (as demonstrated via
IgA
measurement), humoral (as demonstrated via sera HAI and microneutralization
antibodies) and
cell mediated (as demonstrated via influenza HA antigen specific T cell
activation) immune
response in a human subject against influenza virus. In embodiments, the serum
antibodies are
seroprotective for at least 6 months, at least 12 months, at least 13 month or
at least 14 months.
[0075] In embodiments, the combined immune response provides protection
against Influenza A
virus. In certain embodiments, the combined immune response induced by the
present monovalent
influenza pharmaceutical formulation provides protection against Influenza B
virus. In
embodiments, the Influenza A virus and/or Influenza B virus are "seasonal"
influenza virus which
cause seasonal epidemics, mostly during the winter months. In other
embodiments, the Influenza
A virus is a "pandemic" influenza virus, which can cause widespread illness
due to new and
different antigenic epitopes present in the virus.
[0076] Influenza A viruses are divided into subtypes based on two proteins on
the surface of the
virus: the hemagglutinin (H) and the neuraminidase (N). There are at least 18
different
hemagglutinin subtypes and at least 11 different neuraminidase subtypes. (H1
through H18 and
Ni through N11 respectively.). Influenza A viruses can be further broken down
into different
strains. Current subtypes of influenza A viruses found in people are influenza
A (H1N1) and
influenza A (113N2) viruses. Influenza B viruses are not divided into subtypes
but can be further
broken down into lineages and strains_ Currently circulating influenza B
viruses belong to one of
two lineages: B/Yamagata and BNictoria. Naming convention for influenza
viruses includes
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multiple components and follows the approach: the antigenic type (e.g., A, B,
C); the host of origin
(e.g., swine, equine, chicken, etc. For human-origin viruses, no host of
origin designation is
given.); geographical origin (e.g., Denver, Taiwan, etc.); strain number
(e.g., 15, 7, etc.); year of
isolation (e.g., 57, 2009, etc.); and, for influenza A viruses, the
hemagglutinin and neurarninidase
antigen description in parentheses (e.g., (H1N1), (H5N1).
[0077] In exemplary embodiments, the present adenoviral vector comprises a
genetic insert
encoding the HA surface protein antigen from an A/California/04/2009(H1N1)
virus. In certain
embodiments, the present adenoviral vector contains and expresses a
hemagglutinin antigen from
an H1N1 influenza A virus subtype. In other certain embodiments, the present
adenoviral vector
contains and expresses a hemagglutinin antigen from an H3N2 influenza A virus
subtype. In other
embodiments, the present adenoviral vector contains and expresses a
hemagglutinin antigen from
an influenza B virus. In certain embodiments, the present adenoviral vector
contains and expresses
a hemagglutinin antigen from a pandemic Influenza A virus. In certain other
embodiments, the
present adenoviral vector contains and expresses a hemagglutinin antigen (HA)
from a seasonal
influenza virus.
[0078] Methods of Use
[0079] Provided herein is a method of inducing a combined mucosal, humoral and
T cell protective
immune response in a human subject against influenza virus whereby the human
subject is
seroprotected for at least 6 months, at least 12 months, at least 13 months or
longer. In
embodiments, the methods comprise administering intranasally a single dose of
an effective
amount of at least 109 viral particles (vp) of a replication deficient
adenoviral vector containing
and expressing influenza virus hemagglutinin antigen codon optimized for the
human subject,
wherein the administration induces sera antibodies, mucosal antibodies and T
cells against
influenza virus.
[0080] In embodiments, the present monovalent influenza pharmaceutical
formulation is used to
provide protection against seasonal influenza virus. In certain other
embodiments, the present
monovalent influenza pharmaceutical formulation is used to provide protection
against pandemic
influenza virus.
[0081] In embodiments, the present methods provide protection against
infection by Influenza A
virus subtypes. In certain embodiments, the present methods provide protection
against infection
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by Influenza A virus subtypes H1N1 and/or H3N2. In other embodiments, the
present methods
provide protection against infection by Influenza B virus.
[0082] In embodiments, the seroprotection lasts at least about 13 months. In
certain embodiments,
the seroprotection lasts at least about 14 months, or longer.
[0083] In embodiments, the step of administering a single dose of a present
(monovalent)
influenza pharmaceutical formulation induces HAI antibodies at a titer of 50
or greater for at least
6 months, at least 12 months, at least 13 months, at least 14 months or
longer. In embodiments
the titer of HAI antibodies in a human subject 12 months post vaccination
(administration of the
present influenza pharmaceutical formulation) is at least 50, at least 60, at
least 70, at least 80, at
least 90, or at least 100.
[0084] Thus, as discussed herein and in the Examples below, NasoVAX has been
surprisingly
found to be seroprotective and to induce the production of neutralizing
antibody response rates
similar to commercial influenza vaccine (e.g. Fluzone), an antibody response
that is durable for
at least one year, strong mucosal (IgA) and cellular (e.g., T cells as
measured by ELISspot)
responses, minimal induction of anti-vector (e.g., Ad5) antibodies, to have be
little or unaffected
by pre-existing vector (e.g., Ad5) antibody, to be well-tolerated at all dose
levels tested, and to be
stable (i.e., comprise an effective amount of viral particles) after about
three months at ambient
temperature (e.g., about 20-25t).
[0085] This disclosure, then, in some embodiments, provides influenza
pharmaceutical
formulations (e.g., monovalent) suitable for a single dose intranasal
administration to a human
subject, the formulations comprising: an effective amount such as at least
about any of 106 vp, 107
VP, 108 vp, 109 vp, 1010 vp, or 1011 vp, preferably at least 109 vp or 10" vp
of replication deficient
adenovirus vector that contains and expresses influenza virus hemagglutinin
antigen (HA) codon
optimized for the human subject, wherein the effective amount induces a
combined mucosal,
humoral and T cell immune response, which is preferably protective; and, a
pharmaceutically
acceptable diluent or carrier. In some embodiments, the mucosal immune
response, which is
preferably protective either alone or in combination with the other immune
responses, is
determined by anti-hemagglutinin (HA) IgA ELISA, the humoral immune response,
which is
preferably protective either alone or in combination with the other immune
responses, is
determined by hemagglutination inhibition assay (HA!) titer and/or the
presence of neutralizing
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antibody as determined using a microneutralization assay, optionally as
measured using one or
more of the geometric mean titer (GMT), geometric mean ratio (GMR),
seroconversion rate
(SCR), seropositivity rate (SPR); and/or, the T cell immune response, which is
preferably
protective either alone or in combination with the other immune responses, is
determined by using
y-interferon ELISpot. In some embodiments, the formulation is configured to
provide
seroprotection to the human subject as determined by the subject having an HAI
antibody titer >40
for at least 12 months against the influenza virus. In some embodiments, this
disclosure provides
pharmaceutical formulations suitable for a single dose intranasal
administration to a human
subject, comprising: an effective amount of at least 109 viral particles (vp)
of replication deficient
adenovirus vector that contains and expresses influenza virus hemagglutinin
antigen (HA) codon
optimized for the human subject, wherein the effective amount induces a
combined mucosa] and
humoral protective immune response configured to provide seroprotection to the
human subject as
determined by the subject having an HAI antibody titer >40 (or, in some
embodiments, >50) for
at least 12 months against the influenza virus; and, a pharmaceutically
acceptable diluent or carrier.
In some embodiments, the effective amount is at least about 1010 viral
particles (vp) or at least
about 1011 viral particles (vp) and, in further embodiments, induces a T cell
response. In preferred
embodiments, the formulation does not comprise an adjuvant. In some
embodiments, the
formulation does not comprise an adjuvant. In preferred embodiments, the HA
antigen is from an
Influenza A virus (in some preferred embodiments subtype H1N1 or H3N2) or an
Influenza B
virus. In some embodiments, the formulation comprises Tris HC1 (pH 7.4),
histidine, sucrose,
sodium chloride, magnesium chloride, polysorbate 80, ethylenediaminetetraacetk
acid, and
ethanol. In some embodiments, the formulation comprises a single dose,
preferably an intranasal
dose, of about 1x109 vp, about 1 xl0t vp, or about lx1011 vp. In some
embodiments, the
formulation is frozen. In preferred embodiments, the formulation is stable at
ambient temperature
for at least about three months. In embodiments, the ambient temperature is a
room temperature
from about 15 to 30 C, preferably from about 20 to 25 C. In some embodiments,
the formulation
is stored at about -20 C or about 4-8 C until distribution, which could
require storage at ambient
temperature. In some embodiments, the formulation is configured as a seasonal
influenza vaccine
comprising antigens from a seasonal influenza virus. In some embodiments, the
formulation is
configured as a pandemic influenza vaccine comprising antigens from an
Influenza A pandemic
virus strain. In preferred embodiments, the replication deficient adenovirus
vector is human
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adenovirus serotype 5 (Ad5). In some embodiments, the formulation is contained
within a
container that, in some embodiments, can be selected from the group consisting
of a glass vial,
nasal sprayer, droplet dispenser, aerosolizer, and atomizer. In some
embodiments, the vial can be
a multi-dose vial (i.e., a vial comprising multiple doses (e.g., each dose
comprising an effective
dose of viral particles) of the formulation) that could be used in, e.g.,
pandemic situations in
combination with a pipette and/or eye dropper and administered to subjects
dropwise). In some
embodiments, the container has contained the formulation for at least about
three months at
ambient temperature (e.g. room temperature). In some embodiments, the
formulation is
configured to contain at least about 33% (i.e., allowing for about a 0.5 log
decrease (about a three-
fold decrease)), preferably in some embodiments about 50%, infectious viral
particles after about
three months at ambient temperature (e.g. room temperature) within the
container. In some
embodiments, the formulation comprises at least about 33%, preferably about
50%, of the
infectious viral particles present as compared to a matched formulation that
has been in the same
type of container at ambient temperature for less than one month (e.g., in
some embodiments
allowing for about a 0.5 log decrease (about a three-fold decrease)). In some
embodiments, the
container is a single-use container and/or configured for intranasal
administration of the
formulation. In some embodiments, this disclosure provides for the use of such
formulations in the
preparation of a medicament for administration to a human subject to prevent
and/or treat infection
by influenza virus in the subject. In some embodiments, this disclosure
provides use of such
containers in the preparation of a medicament for administration to a human
subject to prevent
and/or treat infection by influenza in the subject. In some embodiments, this
disclosure provides
kits for the preparation of preparation of a medicament for administration to
a human subject to
prevent and/or treat infection by influenza in the subject, said kit
comprising at least one of such
formulations and/or containers.
[0086] In some embodiments, this disclosure also provides methods of inducing
a combined
mucosa!, humoral and T cell protective immune response in a human subject
against influenza
virus comprising: administering intranasally to a human subject at least a
single dose of the
influenza pharmaceutical formulation disclosed herein, wherein the
administration induces a
combined mucosa!, humoral and T cell protective immune response against
influenza virus and
the human subject is seroprotected from infection by influenza virus for at
least 12 months after
said administration. In some embodiments, the mucosal protective immune
response is determined
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by anti-hemagluttinin (HA) IgA ELISA, the humoral protective immune response
is determined
by hemagglutination inhibition assay (HAT) titer and/or presence of
neutralizing antibody as
determined using a microneutralization assay, optionally as measured using one
or more of the
geometric mean titer (GMT), geometric mean ratio (GMR), seroconversion rate
(SCR),
seropositivity rate (SPR); and/or, the T cell protective immune response is
determined by using y-
interferon ELISpot. In some embodiments, the method(s) can include
administration of multiple
doses of the formulation (e.g., during an epidemic or pandemic situation). In
preferred
embodiments, the seroprotection lasts for at least about 13 months, or at
least about 14 months. In
some embodiments, the influenza virus is Influenza A (in some preferred
embodiments subtype
H1N1 or H3N2) and/or Influenza B virus. In some embodiments, the combined
immune response
provides protection against Influenza A virus subtypes and Influenza B virus.
In some
embodiments, the influenza virus is a seasonal influenza virus. In some
embodiments, the
administration induces an HAT antibody titer of at least 50 for at least 12
months post
administration. In some embodiments, the subject exhibits anti-adenovirus
vector immunity
(preferably wherein the replication deficient adenovirus vector is human
adenovirus serotype 5
(Ad5)) prior to the administering intranasally, said immunity being determined
by hemagglutinin
inhibition assay, microneutralization assay, IgA ELISA, and/or ELIspot assay.
In preferred
embodiments, administration of the formulation does not significantly enhance
the anti-adenovirus
vector immunity of the subject (e.g., not more than about three-fold, four-
fold, five-fold or six-
fold above anti-adenovirus vector immunity of the subject present before
administration of the
formulation), said immunity in preferred embodiments being determined by
hemagglutinin
inhibition assay, microneutralization assay, IgA ELISA, and/or ELIspot assay.
In some
embodiments, the subject is seropositive for human adenovirus prior to the
administration. In
some embodiments, the method(s) can further comprise administering a single
dose of a second
influenza pharmaceutical formulation about one year after administration of at
least one dose of
the previously administered influenza pharmaceutical formulation. In some such
embodiments,
the second influenza pharmaceutical formulation comprises antigens of a
seasonal influenza that
are the same or different as that comprised by the previously administered
influenza
pharmaceutical formulation. In some embodiments, the human subject is an
adult.
[0087] Other embodiments are also contemplated herein as would be understood
by those of
ordinary skill in the art.
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[0088] EXAMPLES
[0089] The following examples are put forth so as to provide those of ordinary
skill in the art with
a complete disclosure and description of how to use the embodiments provided
herein and are not
intended to limit the scope of the disclosure nor are they intended to
represent that the Examples
below are all of the experiments or the only experiments performed. Efforts
have been made to
ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.)
but some
experimental errors and deviations should be accounted for. Unless indicated
otherwise, parts are
parts by volume, and temperature is in degrees Centigrade. It should be
understood that variations
in the methods as described can be made without changing the fundamental
aspects that the
Examples are meant to illustrate.
[0090] Example 1: Preparation of Monovalent Influenza Pharmaceutical
Formulation
(NaseVAX)
[0091] The replication deficient adenoviral vector containing and expressing
influenza virus
hemagglutinin antigen codon optimized for the human subject was prepared
following procedure
detailed in [Lui J. et al.; A protocol for rapid generation of recombinant
adenoviruses using the
AdEasy system; Nat. Protoc. (2007) 2(5):1236-47].
[0092] The present adenoviral vector is an E1/E3-deleted, replication
deficient (RD)-Ad5 vector
that expresses the protein of interest (e.g., Influenza HA) within respiratory
epithelial cells. In the
case of NasoVAX, the vector contains a genetic insert encoding the HA surface
protein antigen
from influenza type A or B. The recombinant Ad5 vector lacks the El region of
the viral genome
(nucleotides 343 to 3511), which renders the virus RD and incapable of
producing infectious virus
particles upon entry into a host cell. An additional deletion of nucleotides
28132 to 30813 in the
E3 region of the vector removes genes that are involved in evading the host
immune response and
are dispensable for virus replication. An expression cassette consisting of a
cytomegalovirus
transcriptional enhancer/promoter to drive the expression of the HA gene, a
bioengineered HA
gene, and a Simian Virus 40 polyadenylation signal has been inserted in place
of the El gene
sequences. Figure 1 provides a schematic diagram of the RD-Ad5 vector and
identifies those
sequences from the parent adenovirus genome that are retained in the vector.
[0093] In the present disclosure, the vector contained a genetic insert
encoding the HA surface
protein antigen from an A/California/04/2009(H1N1)-like strain of influenza
(AdcoCA09.HA).
24
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[0094] NasoVAX was manufactured by propagation of the RD-Ad5 vector in
replication-
permissive PER.C6 cells, followed by purification of the virus from the
infected cell harvest, and
the final product included the following excipients: Tris HC1 (pH 7.4),
histidine, sucrose, sodium
chloride, magnesium chloride, polysorbate 80, ethylenediaminetetraacetic acid,
and ethanol.
[0095] NasoVAX was supplied in single-use glass vials each containing a
nominal volume of 03
mL of a sterile, frozen suspension of vaccine formulated to deliver the
nominal doses of lx 109 vp
(batch number: 17142001), 1x101 vp (batch number: 17143001), or lx1011 vp
(batch number:
17144001).
[0096] Example 2: Study Protocol: Single-Ascending-Dose Study of Immunogenkity
of
NasoVAX
[0097] Provided herein is a clinical study protocol wherein 60 healthy adults
were randomized to
an A/California 2009-based monovalent NasoVAX (present monovalent vaccine
composition)
formulation at doses of 109, 1010, or 1011 viral particles or saline placebo,
all given as a 0.5 mL
dose split approximately as 0.25 ml nasal spray in each nostril.
Table 1
Study Design
Number of Subjects
Cohort Dose (vp)
NasoVAX Placebo
1 1x109
15 5
2 1x1010
15 5
3 1x1011
15 5
Study Total Target 45 15
Total 60
[0098] The objectives of this study included: 1) To evaluate the humoral
immune response to
NasoVAX when administered by intranasal spray at a single dose of 1x109, lx101
, or lx1011 vp;
2) To evaluate the cellular immune response to NasoVAX when administered by
intranasal spray
at a single dose of 1x109, 1x10' , or 1x10'1 vp; 3) To evaluate the mucosal
immune response
NasoVAX when administered by intranasal spray at a single dose of 1x109, lx101
, or lx1011 vp;
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and, 4) To evaluate the humoral immune response against non-represented
influenza strains after
NasoVAX administration.
[0099] Subjects were followed for safety, including solicited local and
systemic side effects.
Immune measures included hemagglutination inhibition (HAI) and neutralizing
antibody (MN) at
days one (1), 15, 29, 90 arid 180, and y-interferon ELISpot at day 1 and 8. A
parallel cohort of 20
similar subjects were dosed with Fluzonee injectable influenza vaccine
containing an A/California
2009 component and had assessments at the same timepoints.
[001.00] This study was a Phase 2a, randomized, double-
blind, placebo-controlled trial to
evaluate the safety and immunogenicity of NasoVAX (monovalent Adco.CA.HA),
i.e., NasoVAX,
in healthy adults 18 to 49 years of age. Subjects were screened within 28 days
of randomization
(Day 1). Sixty subjects who met all inclusion and no exclusion criteria and
provided written
informed consent were enrolled into three (3) sequential cohorts of 20
subjects each defined by the
vaccine dose (1x109, lx i0' , and lx1011 vp). Within each cohort and its
sentinel cohort, subjects
were randomized in a 3:1 ratio to receive one (1) intranasal dose of NasoVAX
or placebo on day
one (Day 1).
[0033] A sentinel cohort of five (5) subjects from each cohort was dosed.
Dosing of the remainder
of each cohort proceeded after the last sentinel subject completed Day eight
(8) if no events
meeting stopping criteria had occurred. A serum sample was collected from each
subject for HAI
and microneutralization assays against influenza A/California/07/2009(H1N1) [a
strain
homologous to the one used for NasoVAX (monovalent AdcoCA.09.HA)] pre-dose on
Day 1 and
on Days 4, 8, 15, 29, 91, and 181; Ad5 antibody and HAI and
microneutralization assays against
non-represented influenza strains were also performed on the Day 1 and Day 29
samples. A whole
blood sample was collected from each subject and processed to isolate PBMCs
for evaluation of
T-cell responses by ELISpot pre-dose on Day 1 and on Day 8.
[0034] A nasopharyngeal swab sample was collected from each subject at
Screening (a time point
prior to administration) and on Days 4, 8, 15, 29, and 91 to measure
concentration of the Ad5
vector for assessment of vaccine vector shedding by quantitative polymerase
chain reaction assay.
Once a negative result was obtained for a given subject, later samples were
not tested. ELISA for
measurement of IgA was also performed on the swab samples from Screening and
Day 29 for
evaluation of mucosal immune response.
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[00101] Immunogenicity: Immunology analyses were
conducted using the Per-protocol
(PP) Population as the primary analysis population. Analyses based on the
Intent-to-treat (ITT)
Population were undertaken and presented only if >5% of subjects in any 1 dose
group were
excluded from the PP Population.
[00102] Analysis of covariance (ANCOVA) was used in the
analysis of the antibody titer
at each postbaseline visit, with log-transformed antibody titer as dependent
variable, dose group
as a factor, and Baseline log-transformed level as a covariate. Comparisons of
postbaseline log-
transformed antibody titer was conducted for each NasoVAX dose group against
the placebo group
and against the Fluzone group, if applicable. From the ANCOVA analyses, least
east squares
(LS) means and 95% CI of the LS means of dose group, difference of LS means,
and 95% CI of
the difference in LS means were obtained. Back-transforming the difference of
LS mean estimates
and their 95% CIs to the original scale results in a ratio of the geometric
means; these ratios were
reported in the summary.
[00103] Categorical data derived from the assay data
(e.g., SPR, SCR, responder rate) were
tabulated by counts and percentages per dose group, as well as the 95% Clopper-
Pearson exact CI
of the percentage. In addition, comparisons of responders in each NasoVAX dose
group against
the placebo group and against the Fluzone group, if applicable, were
conducted using Fisher's
exact test.
[00104] An analysis of median change from Baseline for
Day 8 ELISpot SFU was added
because median is the conventional parameter for descriptive analysis of raw
ELISpot data and to
account for differing Baseline group mean values.
[00105] Scatterplots of Baseline Ad5 antibody levels
along the x-axis were presented with
each of the following variables along the y-axis: HAI antibody titer at Day
29, ELIS pot response
at Day 8, and IgA titer at Day 29. The scatterplot analysis of effect of pre-
existing Ad5 serum
antibody levels on NasoVAX immunogenicity were uninformative, so post-hoc
subgroup analysis
by Baseline Ad5 serostatus (positive, defined as titer > LLOQ, or negative) of
Day 29 HAI assay
GMR, Day 29 microneutralization assay GMR, median Day 8 change from Baseline
in ELISpot
SRI, and Day 29 IgA GMR was performed.
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[00106] Example 3: Combined Mucosa!, Humoral and Cell-
Mediated Immune
Response Induced by Monovalent Influenza Pharmaceutical Formulation (NasoVax)
[00107] As described in Example 2, NasoVAX (monovalent
AdcoCA0911A) administered
by intranasal spray at a single dose of 1x109, 1x10' , or lx1011 vp elicited a
combined Immoral
and mucosal immune responses and at a dose of lx1011 a combined immune
response including a
cellular response (e.g., T cells).
[00108] In certain embodiments provide herein is a
monovalent influenza pharmaceutical
formulation suitable for a single dose intranasal administration to a human
subject, comprising: an
effective amount of at least 1011 viral particle (vp) of replication deficient
adenovirus vector that
contains and expresses influenza virus hemagglutinin antigen codon optimized
for the human
subject, wherein the effective amount induces a combined mucosal, humoral and
T cell protective
immune response; and, a pharmaceutically acceptable diluent or carrier.
[00109] In certain other embodiments provided herein is
an influenza pharmaceutical
formulation suitable for a single dose intranasal administration to a human
subject, comprising: an
effective amount of at least 109 viral particles (vp) of replication deficient
adenovirus vector that
contains and expresses influenza virus hemagglutinin antigen codon optimized
for the human
subject, wherein the effective amount induces a combined mucosal and humoral
protective
immune response configured to provide seroprotection to the human subject of
an HAI antibody
titer >40 for at least 12 months against the influenza virus; and, a
pharmaceutically acceptable
diluent or carrier.
[00110] Also provided in certain embodiments is a method
of inducing a combined mucosal,
humoral and T cell protective immune response in a human subject against
influenza virus
whereby the human subject is seroprotected for at least 6 months. In
embodiments, the method
comprises administering intranas ally to the human subject a single dose of
any present influenza
pharmaceutical formulation, wherein the administration induces serum
antibodies, mucosal
antibodies and T cells against influenza virus.
[00111] NasoVAX (monovalent AdcoCA09.HA) was
administered by intranasal spray at a
single dose of 1x109, lx101 , or 1x1011 vp and found to elicit a humoral
immune response to
influenza A/California/07/2009(H1N1). All post-vaccination immunology results
in the
NasoVAX groups were superior to the placebo group from Day 15 through Day 181.
For instance,
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a clear dose-effect was observed in the NasoVAX groups for HAI assay geometric
mean titer
(GMT), geometric mean ratio (GMR), seroconversion rate (SCR), and
seroprotection rate (SPR);
and for microneutralization GMT, GMR, and responder rates (2-fold and 4-fold).
Except for HAI
SCR in the overall group, all results in the NasoVAX lx1011 vp and overall
groups at Day 29 were
statistically significantly higher than those in the placebo group (P < 0.05).
For the
microneutralization assay, Day 29 results in the lx1011 vp NasoVAX group were
similar to those
in the Fluzone group; in the HAI assay, Day 29 results in the Fluzone group
were generally
higher than in the NasoVAX groups. However, mean GMTs in the NasoVAX groups
remained
stable over time, while those in the Fluzone group decrease substantially
through Day 181. A
post-hoc subgroup analysis of HAI and microneutralization GMTs by Baseline Ad5
serostatus
showed some suppression of immune response to the hemagglutinin genetic insert
in the Ad5
vector at the two lower doses, but this effect was apparently overcome in the
highest dose group.
See Figures 2-9.
[00112] NasoVAX elicited a HA-specific cellular immune
response. Day 8 ELISpot results
in the NasoVAX groups were higher than in the placebo group, and the
difference was statistically
significant in the 1x!0" vp group (95% CI for LS GM SFUs ratio vs placebo:
3.5, 102.2;
nonoverlapping 95% CIs for median change from Baseline; P = 0.0028 for
responder rate), and
overall in the NasoVAX group (95% CI for LS GM SFUs ratio vs placebo: 1.6,
24.5; P = 0.0351
for responder rate). Results in the lx 109 vp and lx101 vp dose groups were
slightly lower than in
the Fluzone group, while results in the lx1011 vp dose groups were
substantially higher than in
the Fluzone group.
[00113] NasoVAX elicited HA-specific mucosal
immunogenicity not seen in the subjects
who received Fluzone . GMRs at Day 29 showed mucosal response in all the
NasoVAX groups
and no response in the Fluzone or placebo groups; the difference was
statistically significant for
the lx101 vp and lx1011 vp dose groups (95% CI: 1.6, 3.3 and 1.2, 2.9,
respectively). Pre-existing
Ad5 irnmunogenicity had no apparent effect on mucosal inununogenicity.
[00114] Humoral Immune Response
[00115] Humoral immune response (i.e., antibodies in the
blood or serum) to the influenza
HA protein as measured by HAI is a recognized correlate of protection for
influenza (Clinical Data
Needed to Support the Licensure of Seasonal Inactivated Influenza Vaccines,
May 2007).
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Protection can be demonstrated via hemagglutination inhibition (HI) antibody
response to a viral
strain included in the vaccine, wherein GMT and SCR (defined as the percentage
of subjects with
either a pre-vaccination HI titer <1:10 and a post-vaccination HI titer > 1:40
or a pre-vaccination
HI titer > 1:10 and a minimum four-fold rise in post-vaccination HI antibody
titer) are measured
to be in either a non-inferiority study or a placebo controlled study. For a
non-inferiority study
in-ununogenicity trial an antibody responses to a new vaccine as compared to
an authorized
influenza vaccine): 1) the upper bound of the two-sided 95% CI on the ratio of
the GMTs (GMT
of U.S. licensed vaccine divided by GMT of new vaccine) should not exceed 1.5;
and, 2) the upper
bound of the two-sided 95% CI on the difference between the SCRs (SCR for U.S.
licensed vaccine
less SCR for new vaccine) should not exceed 10 percentage points.
[00116]
For a placebo-controlled
immunogenicity trial, HI antibody response to the new
vaccine may demonstrate protection when a percentage of subjects achieve an HI
antibody titer >
1:40.
[00117]
A blood sample was
collected in a serum separation tube from each subject at day
-28 to day -1, day 1, 2, 4, 8, 15, 29, 91 and 181 for evaluation of humoral
response. HAI and
microneutralization assays against A/California/07/2009(H1N1) were performed
on all samples
collected. After collection, samples were allowed to clot at room temperature
for 30 minutes to 1
hour before centrifugation. The resulting serum component of the blood was
transferred into
aliquots in cryovials and stored at -80 C for 24 to 48 hours before shipment
on dry ice to the testing
laboratories. Samples were stored at -80 C before testing.
[00118]
For the HAI assay, up to 9
samples were analyzed on each 96-well V-bottom
microtiter plate (the test plate) along with quality (QC) controls on each
plate consisting of positive
(reference antiserum), negative, virus, and cell quality controls. A single QC
plate was included in
each assay and contained quality controls and a viral back titer (VBT) to
confirm virus
concentration on a 96-well V-bottom microtiter plate. Samples were treated
with receptor
destroying enzyme in accordance with the laboratory Standard Operation
Procedure. Both test
plates and QC plates were covered and incubated for 120 to 140 minutes at room
temperature
(15 C to 30 C). 50 pi, of 1% horse red blood cells was then added to every
well on all plates and
incubated for 30 to 60 minutes at room temperature. Each well was then read
for agglutination or
non-agglutination and evaluated in accordance with the following criteria: 1)
The titer was
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determined by the last well in the column that showed non-agglutination and
was assigned the
value of the reciprocal of that dilution. Serum dilutions were designated as
1:10 to 1:1280. A
negative titer was assigned a titer of 5. Titers > 1280 were assigned a titer
of 1280 for use in GMT
calculation; and, 2) The VBT on the QC plate had to be 4 HA units/25 pL 1
dilution for a valid
assay. Samples were analyzed in triplicate for all assays.
[00119] For the microneutralization assay, up to nine
(9) samples were analyzed on nach
96-well flat-bottom microtiter plate (the test plate) along with QC on each
plate consisting of a
positive (reference antiserum), virus, and cell controls. At least three (3)
QC plates were included
in each assay and contained quality controls and a VBT to confirm virus
concentration on a 96-
well flat-bottom microtiter plate. Fifty microliters (50 pL) of diluted virus
(diluted to target a VBT
within the range of 1.17 to 9.38xTCID50 [the amount of virus required to kill
50% of infected hosts
or to produce a cytopathic effect in 50% of inoculated tissue culture
cells]/50 pL) was added to all
wells in all columns containing serum and the wells designated as the virus
control; no virus was
added to cell control wells. Both test plates and QC plates were incubated at
37 C - 2 C with 0%
to 5% CO2) for 2 to 2.5 hours. One hundred microliters (100 pL) of MDCK cells
(1.5x105 cells/mL
in diluent, 50% to 99% confluent) were added to all wells on every plate.
Plates were incubated
for 19 to 21 hours at 37 C 2 C with 5% 1% CO2. Media was removed from all
plates and each
well washed with 100 pL Dulbecco's phosphate-buffered saline (DPBS,lx
concentration). DPBS
was removed, and 100 tuLL of fixative was added to each well. Plates were
covered and incubated
at room temperature (15 C to 30 C) for 10 to 15 minutes. Fixative was removed,
and plates were
air-dried before washing 3 times with 200 pL/well of wash buffer. 100 mL of
diluted primary
antibody was added to each well, and plates were incubated for 1 to 1.25 hours
at room
temperature. Plates were washed four times with 200 mL/well of wash buffer,
and 100 mL of
diluted secondary antibody was added to each well. Plates were incubated for 1
to 1.25 hours at
room temperature. Plates were washed four times with 200 mL/well of wash
buffer, and 100 mL
fresh substrate was added (in low light) to each well followed by incubation
for 15 to 25 minutes
in low light at room temperature. Stop solution (100 mL) was immediately added
to each well, and
absorbance was read at 490 to 495 nm. The resulting data were evaluated in
accordance with the
following criteria: 1) the plate cut-off value for each plate was calculated
according to the
following equation: (average of virus control wells ¨ average of cell control
wells)/2; and, 2) serum
samples with an absorbance value below the plate cut-off were positive for
neutralizing antibody,
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whereas those above the plate cut-off were negative for neutralizing antibody.
VBT values with
an absorbance value above the plate cut-off were positive for virus
replication. VBT values with
an absorbance value below the plate cut-off were negative for virus
replication. The titer was
calculated as the reciprocal of the highest dilution scored as a positive. A
negative titer (all
absorbance values above the plate cut-off) was assigned a value of 5.
Dilutions were designated
1:10 to 1:1280. Titers > 1280 were assigned a titer of 1280 for use in GMT
calculations but were
reported as > 1280 in the data tables. Exceptions included: absorbance value
that crossed over
the plate cut-off value (value fell below the cut-off, rose above it (noted as
'flag'), then fell back
below) had the titer assigned for the final absorbance value that fell below
the plate cut-off value
unless there was a? 2-fold difference between the initial fall in absorbance
and when it rose again
above it; samples with more than one flag were not accepted unless a
mechanical issue was noted
on data evaluation or the final absorbance value was more than two dilutions
from the last flag;
values that crossed over the plate cut-off in the VBT were reported as the
titer prior to the cross;
and, when evaluating absorbance values on all plates, data trending was
evaluated down and across
plates to look for indications of mechanical issues (e.g., partial or complete
clogged tip that could
result in absorbance values higher or lower than expected). These values were
taken into
consideration when evaluating and assigning the final titer of the sample.
[00120] Test plates and the assay were acceptable if the
following criteria were met: 1)
Positive QC values on individual test plates were within ( ) 1 dilution of the
calculated median
value for the assay, or the individual plate was rejected; 2) The VBT median
value determined for
all accepted QC plates for an assay was in the range of 1.17 to 9.38, or the
assay was rejected; and,
3) At least 5 of the 9 serum samples on a test plate match at least 1 of their
respective replicates,
or all 9 test samples were reanalyzed. Samples were analyzed in triplicate for
all assays.
[00121] All post-vaccination results in the NasoVAX
groups were higher than in the
placebo group from Day 15 through Day 181. A clear dose effect was seen in the
NasoVAX groups
for all the endpoints measured. Except for HAI SCR in the overall group, all
humoral
imrnunogenicity results in the NasoVAX lx10" vp and overall groups at Day 29
were statistically
significantly higher than those in the placebo group. See Figure 2, 3,6 and 7.
32
CA 03132697 2021- 10-6
C
w
-
w ALT2026.PCT
r.,
0
0
-,
r.,
0
- Table 1
9
o)
0
Humoral Immune Response to NasoVAK at Day 29 by Dose Group
0
NO
0
bi
Naso VAX
-9..
tsi
1..1
1X109VP 1 x1010 vp
1x1011 vp Overall Placebo Fluzone
o
=.1
A
Statistic N:15 N:15
N:15 N : 45 N:15 N m 20
o
HAI Assay
LS GMT (95% CI)a 87.2 136.1
164.0 124.8 31.3 277.7
(52.7, 144.3)
(81,7, 226.6) (99.0, 271.6) (92.3,
167.0) (18.9, 52.0) (179.4, 429.9)
2
LS GMT ratio vs .8 4.3
5.2 4.0
placebo (95% Cl)a (1.4, 5.7) (2.1, 9.0)
(2.6, 10.7) (2.2,7.2)
LS GMT ratio vs 0.3 0.5
0.6 0.4
Fluzone (95% Cl) a
(0.2, 0.6) (0.3, 1.0)
(0.3, 1.2) (0.3, 0.8)
2.1 2.2 4.3 2.7
0.9 5.7
GMR (95% Cl)
(1.1,4.2) (1.2, 4.3)
(1.5, 12.0) (1.8,4.2) (0.7, 1.2) (2.9,
11.2)
SCRb (95% Cl) 13.3% 26.7%
33.3% 24.4% 0.0% 50.0%
(1.7%, 40.5%) (7.8%, 55.1%)
(11.8%, 61.6%) (12.9%, 39.5%) (0.0%, 21.8%) (27.2%,
72.8%)
P value vs placeboc 0.4828 0.0996
0.0421 0.0505
P value vs Fluzonee 0.0340 0.2958
0.4916 0.0506
SPRd (95% Cl) 80.0% 100.0%
100.0% 93.3% 53.3% 95.0%
(51.9%, 95.7%) (78.2%, 100.0%)
(78.2%, 100.0%) (81.8%, 98.6%) (26.6%, 78.7%) (75.1%,
99.9%) 9:1
n
P value vs placeboe 0.2451 0.0063
0.0063 0.0013
P value vs Fluzonee 0.2924 1.000
1.000 1.000
t..)
co
t4
o
Microneutralization Assay
a-D
b.)
44.9 113.1 142.5 89.8
17.8 162.8 er\
cc
GMT (95% CI)
4.
we
(21.8, 92.3)
(58.0, 220.8) (93.6, 217.1) (62.6, 128.8)
(9.1, 35.0) (95.8, 276.6)
33
C
0,
..., ALT2026.PCT
N,
c,
co
....
r.,
0
rõ
17
2.1 2.5
5.2 3.0 1.1 6.2
GMR (95% Cl)
(!.O,4.2) (1.3,4.8)
(2.2, 12.0) (2.0,4.5) (1.0,1.2)
(2.8,13.5) 0
0
Responder rate/2- 40.0% 46.7/0
73.3% 53.3% 0.0%
70.0% t...=
=
fold rise (95% CI) (16.3%, 67.7%) (21.3%, 73,4%)
(44.9%, 92.2%) (37.9%, 68.3%) (0.0%, 21.8%) (45.7%,
88.1%) ta
-C2
b.)
P value vs placeboc 0.0169 0.0063
<0.0001 0.0001
1-1
o
1-1
P value vs Fluzonec 0.0966 0.1871
1.0000 0.2785
.k.
Responder rate/4- 13.3% 26.7%
53.3% 31.1% 0.0%
50.0%
fold rise (95% Cl) (1.7%, 40.5%) (7.8%, 55.1%)
(26.6%, 78,7%) (17.6%, 44.6%) (0.0%, 21.8%) (27.2%,
72.8%)
P value vs placeboo 0.4828 0.0996
0.0022 0.0130
P value vs Fluzonec 0.0340 0.2958
1.0000 0.1716
Bold values are statistically higher than values in placebo group.
Abbreviations: Cl = confidence interval; GMT = geometric mean titer; GMR =
geometric mean ratio; HAI = hemagglutination inhibition; LS = least squares;
SCR = reconversion rate; SPR = seroprotection rate; vp = viral particles.
a. The analysis of covariance uses log-transformed level as dependent
variable, dose group as a factor, and Baseline log-transformed analysis as a
covariate.
Differences of LS mean estimates and 95% Cls were back-transformed to the
original scale, resulting In a ratio of the geometric means.
b. The percentage of subjects with a HAI titer z 1:40
c. From Fishers exact test
d. The percentage of subjects with either a Baseline HAI titer < 1:10 and a
postvaccination titer z 1:40 (which is 4 times the assay lower limit of
quantitation), or
a Baseline HAI titer z 1:10 and a 4-fold increase in postvaccination HAI titer
relative to Baseline
9:1
n
1-;
ct
t4
=
ta
4=
i
ba
cr,
ce
46
we
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[00122] Cellular Immune Response
[00123] A blood sample was collected in a heparinized
tube from each subject at day -28 to
day -1, day 1, 2,4, 8, 15, 29, 91 and 181 for evaluation of HA-specific T-cell
response by ELISpot.
[00124] PBMCs were isolated by standard Ficoll-Hypaque
gradient density technique with
centrifugation steps adjusted to obtain optimal yield and viability with site
equipment. PBMCs
were cryopreserved at a density of 107 cells/mL and stored at -80 C before
shipment on dry ice to
the testing laboratory. Samples were stored on liquid nitrogen before testing.
[00125] After thawing, PBMCs were added at a
concentration of 0.2x106 cells per well to a
96-well polyvinyl difluoride membrane ELISpot plate coated with the capture
interferon gamma
(IFN-7) antibody. PBMCs were stimulated for 18 hours in the presence of 7
pooled HA-derived
peptides, positive and negative controls. HA-derived peptide pools (19 to 20
peptides each) were
prepared from 139 short peptides (14 or 15 amino-acid long, 11 amino-acid
overlap) derived from
the HA sequence of the A/California/04/2009(H1N1) strain. Positive controls
used a CEF
(cytomegalovims, Epstein Barr virus and influenza virus) peptide pool and
phytohemagglutinin.
Negative controls were based on irrelevant human myelin oligoclendrocyte
glycoprotein peptide
pool and media alone. All conditions were tested in triplicate except media
alone, which was tested
in sextuplicate. After revelation of the IFN-y ELISpot plate, the number of
spots was counted with
an automated ELISpot counter and expressed as SF0/106PBMCs.
[00126] Figures 4 and 8 summarize the cellular immune
response (SFUs from ELISpot) at
Day 8. Baseline geometric mean SFU/106 cells varied widely among the dose
groups and,
therefore, a post-hoc analysis of change from Baseline was added. Post-
vaccination results in the
NasoVAX groups were higher than in the placebo group, and the difference was
statistically
significant in the 1 x1011 vp, overall NasoVAX groups for LS GM SFUs and
responder rate, and
in the lx1011 vp group for median change from Baseline.
[00127] Mucosal Immune Response
[00128] Nasopharyngeal swab samples collected at
Screening and Day 29 and used for
evaluation of mucosal response by anti-HA IgA ELISA. After nasopharyngeal swab
sample
collection, the swab was frozen at -80 C before being shipped on dry ice to
the testing laboratory
and stored at -20 C before use.
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[00129] Influenza A/California/04/2009(H1N1) HA protein
was adsorbed onto the surface
of microtiter wells. Nasal swab extract (i.e., sample) was incubated with HA
(i.e., allowed to bind
to HA), and a biotinylated detection antibody added to the wells to detect the
captured IgA (i.e.,
mucosal IgA). The anti-HA IgA titer was determined using a cut-off absorbance-
based method.
The total IgA per sample was also quantitated using the Human IgA ELISA
Quantitation Set, E80-
102 (Bethyl Laboratories; Montgomery, TX). The results were reported as a
ratio of HA-specific
IgA (U/mL) to total Ig A (pg/m1) to allow for direct comparison between
different nasal swab
samples. Results were transferred electronically into the study database.
[00130] Table 2 and Figure 5 summarize mucosal immune
response to vaccine strain HA
protein (IgA by ELISA) at Day 29. Unlike for humoral antibodies (i.e.,
antibodies present in
blood), mucosal IgA GMTs were similar across all groups at Baseline (range:
1.8 to 3.0). GMRs
at Day 29 showed no mucosal response in the placebo group and mucosal response
in all the
NasoVAX groups; the difference to placebo was statistically significant for
the lx101 vp and the
lx1011 vp groups dose groups.
Table 2
Mucosal Immune Response to Naso VAX at Day 29 by Dose Group
Naso VAX
1x10 vp 1x10" vp 1x1011 vp
Overall Placebo Flu zone
Statistic N = 15 N = 15 N = 15
N = 45 N = 15 N = 20
3.3 5.6 5.4 4.6 2.4
1.8
GMT (95% CI)
(2.1, 5.1) (3.8, 8.3) (3.2,
9.4) (3.6, 6.0) (2.0, 2.9) (1.2, 2.3)
1.4 2.3 1.8 1.8 1.0
1.0
GMR (95% Cl)
(0.9, 2.0) (1.6, 3.3) (1.2,
2.9) (1.1, 1.2) (0.8, 1.3) (0.8, 1.2)
Abbreviations: Cl = confidence interval; GMT = geometric mean titer; GMR =
geometric mean ratio; vp = viral
particles.
[00131] Discussion
[00132] The humoral, cellular, and mucosal
immunogenicity elicited by NasoVAX
(monovalent AdcoCA09.FIA) administered by intranasal spray at a single dose of
1x109, lx101 ,
or lx1011 vp showed the potential to address some of the shortcomings in
current influenza vaccine
options noted in the recent NIA1D review (Erbelding EJ, et al. (2018)),
specifically inadequate
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durability of immune response, poor cellular response, and lack of tissue-
resident (La, mucosal)
immunity. In this study, the humoral immune response (including that measured
by the HAI assay,
the standard correlate of protection) remained stable in the NasoVAX groups
through six (6)
months post-dose, while response in the Fluzone group decreased substantially
over that time
period.
[00133] Cellular and mucosal immunogenicity may play a
role in reduction of influenza
duration, severity, and transmissibility (Gould, et al. Nasal IgA provides
protection against human
influenza challenge in volunteers with low serum influenza antibody titre.
Front Microbiol.
2017;8:900; McMichael, et al. Cytotoxic T-cell immunity to influenza. N Engl J
Med.
1983;309:13-7; Seibert, et al. Recombinant IgA is sufficient to prevent
influenza virus
transmission in guinea pigs. J Virol. 2013;87:7793-804; Wilkinson, et al.
Preexisting influenza
specific CD4+ T cells correlate with disease protection against influenza
challenge in humans. Nat
Med. 2012;18:274-80), characteristics that are particularly important in the
setting of pandemic
influenza. The mucosal immune response observed in these studies has the
potential to provide an
effect additive to the humoral and cellular response pathways by blocking
influenza at the site of
infection. In this study, the cellular response elicited by NasoVAX was
superior to that from
Fluzone , and mucosal immune response was elicited by NasoVAX but not by
Fluzone.
[00134] The immunogenicity results in this study clearly
indicate that the lx1011 vp dose
elicits strong humoral, cellular, and mucosal immunogenicity. The effect of
the 1x10' vp dose
was less clear, possibly because of the unusually high Baseline HAI and
microneutralization titers
in this group.
[00135] Example 4: Comparison of Induced Immune Response
by Monovalent
Influenza Pharmaceutical Formulation (NasoVAX) and Fluzone
[00136] The immunogenicity, including the ability to
induce a humoral, cellular and
mucosal immune response, was evaluated for the present monovalent influenza
pharmaceutical
formulation and compared to Fluzone . Fluzone is a high-dose quadrivalent
influenza vaccine
administered via injection for influenza A and influenza B subtypes present in
the vaccine. HAI
antibodies were measured at day 29, 91 and 181 and results reported for a HAI
assay,
seroconversion rate and seroprotection. See Figure 9.
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[00137] For the microneutralization (MN) assay, as shown
in Figure 3, Day 29 results in
the lx1011 vp NasoVAX group were similar to those in the Fluzone group; in
the HA! assay, Day
29 results in the Fluzone group were generally higher than in the NasoVAX
groups. However,
mean GMTs by both assays and HAI GMR, SCR, and SPR values in the NasoVAX
groups
remained stable over time, while those in the Fluzone group decreased
substantially through Day
181. See Figure 9.
[00138] NasoVAX (monovalent AdcoCA09.HA) administered by
intranasal spray at a
single dose of 1x109, 1x10' , or 1x10" vp elicited a humoral immune response
to influenza
A/California/07/2009(H1N1). All postvaccination results in the NasoVAX groups
were higher
than in the placebo group from Day 15 through Day 181. A clear dose effect was
seen in the
NasoVAX groups for HAI assay GMT, GMR, SCR, and SPR and for
microneutralization GMT,
GMR, and responder rates (2-fold and 4-fold). Except for HAI SCR in the
overall group, all results
in the NasoVAX 1 x1011 vp and overall groups at Day 29 were statistically
significantly higher
than those in the placebo group (P <0.05). For the microneutralization assay,
Day 29 results in the
lx1011 vp NasoVAX group were similar to those in the Fluzone group; in the
HAI assay, Day 29
results in the Fluzone group were generally higher than in the NasoVAX
groups. However, mean
GMTs in the NasoVAX groups remained stable over time, while those in the
Fluzone group
decrease substantially through Day 181.
[00139] In the cellular immunogenicity assay (ELISpot),
results in the 1x109 vp and
1 x101 vp dose groups were slightly lower than in the Fluzone group, while
results in the
1 x1011 vp dose group were substantially higher than in the Fluzone group.
See Figure 8.
[00140] NasoVAX elicited a strain-specific cellular
immune response. Day 8 ELISpot
results in the NasoVAX groups were higher than in the placebo group, and the
difference was
statistically significant in the 1x10" vp group (95% CI for LS GM SFUs ratio
vs placebo: 3.5,
102.2; nonoverlapping 95% CIs for median change from Baseline; P = 0.0028 for
responder rate)
and overall NasoVAX group (95% CI for LS GM SFUs ratio vs placebo: 1.6, 24.5;
P = 0.0351 for
responder rate). Results in the lx l0 vp and lx101 vp dose groups were
slightly lower than in the
Fluzone group while results in the 1 x1011 vp dose groups were substantially
higher than in the
Fluzone group. See Figure 8.
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[00141] In the mucosal immunogenicity assay (IgA ELISA
in nasopharyngeal swabs
samples), GMRs at Day 29 showed mucosal response in all the NasoVAX groups and
no mucosal
response in the Fluzone group; the difference was statistically significant
for the lx1010 vp and
lx 1011 vp dose group (95% CIs for GMT and GMR did not overlap with 95% CI for
the Fluzone
group). See Figure 5.
[00142] NasoVAX elicited strain-specific mucosal
immunogenicity not seen in the subjects
who received Fluzone . GMRs at Day 29 showed mucosal response in all the
NasoVAX groups
and no response in the Fluzone or placebo groups; the difference was
statistically significant for
the lx101 vp dose and 1x1011 vp dose groups (95% CI: 1.6, 3.3 and 1.2, 2.9,
respectively).
[00143] Example 5: Durability: Seroprotection for 13
months Induced by Monovalent
Influenza Pharmaceutical Formulation (NasoVAX)
[00144] The above study disclosed in Example 2 and 3 was
extended an additional six (6)
months wherein a blood sample was collected in a serum separation tube from
each available
subject between day 403 and day 433 for evaluation of humoral response. In
total eight of the 15
subjects at the 1 x 1011 vp dose group returned for evaluation after one year.
HAI and
microneutralization assays against A/California/07/2009(111N1) were performed
on all eight
samples collected. All subjects that participated in the extension study
demonstrated
seroprotection at least 13 months post administration with the present
monovalent influenza
vaccine. See Table 3 and Figure 10.
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Table 3
Seroprotection 13 to 14 months post vaccination
SUBJID
Seroprotected
Vaccine Administered Blood collection at Day
Date 365
Days HAI titer
1 11/28/2017
1/21/2019 419 226
2 12/12/2017
1/21/2019 405 56.6
3 12/13/2017
1/23/2019 406 320
4 12/13/2017
1/22/2019 405 160
12/14/2017 2/18/2019 431 320
6 12/14/2017
1/21/2019 403 320
7 12/13/2017
1/22/2019 405 226
8 12/12/2017
2/18/2019 433 80
[00145] Example 6. NasoVAX Shedding and Anti-NasoVAX
Vector Antibodies
[00146] NasoVAX was administered to human subjects as a
single intranasal dose of 109
VP, 1010 vp, or 10" vp. At four, eight and 15 days post-dose, nasopharyngeal
swab samples were
collected from each subject and the concentration of the Ad5 vector shed at
each time point
quantified by polymerase chain reaction (PCR) assay. As shown in Figure 11,
dose-dependent
shedding of administered NasoVAX vector dose was detected until day 8 post-
dose and was not
detected at day 15. No replication-competent virus was detected.
[00147] An adenovirus microneutralization (MN) assay was
carried out to determine anti-
adenovirus antibodies in subjects. Dilutions of human sera were mixed with a
consistent quantity
of a replication deficient Ad5 vector that expresses green fluorescent protein
(GFP) from a human
cytomegalovirus (CMV) promoter (termed Ad5.CMV-GFP) and incubated to allow
potential
neutralization to occur. These mixtures were then inoculated onto VERO E6
cells in a 96-well
plate and incubated for approximately three days. During this incubation,
Ad5.CMV-GFP that is
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not neutralized infects the cells and produce GFP, which was used as a readout
for the assay. The
intensity of the GFP fluorescence was compared between test samples and
controls that are either
inoculated with virus only or no virus to determine the level of
neutralization that occurred. The
reportable value for this assay is the MN50 value, corresponding to the
reciprocal of the serum
dilution that results in neutralization of 50% of the input Ad5.CMV-GFP. Fig.
11 also illustrates
the GMR of antibodies against the adenovirus vector component of NasoVAX (Ad5)
following
administration of a single intranasal dose of 109 vp, 1010 vp, or 1011 vp of
NasoVAX. As shown
therein, administration of the highest dose (10" vp) surprisingly only
resulted in about a 2.3-fold
induction of anti-Ad5 vector antibodies in subjects as compared to control.
This is an important
finding as it indicates the intranasal route of administration can be used for
repeated dosing of
NasoVAX, or potentially other Ad5-based vectors.
[00148] Fig. 12 shows the effect of pre-existing anti-
Ad5 immunity on Ad5 serostatus
following administration of a single intranasal dose (10" vp) of NasoVAX to
subjects. As shown
therein, pm-existing anti-Ad5 immunity ("Ad5 Seropositive" (median titer being
22-fold above
the lower limit of quantitation (LLOQ)) had little effect on humoral (HAI),
microneutralization
(MN), mucosal (IgA), or cellular (ELISpot) anti-Ad5 immunity following
administration of the
intranasal dose of NasoVAX. This is another important finding as it indicates
that NasoVAX can
be administered intranasally even to subject with pre-existing immunity to
Ad5.
[00149] Example 7. NasoVAX Stability at Room Temperature
[00150] This example describes the long-term stability
of NasoVAX in a liquid formulation
at room temperature. Long-term stability at room temperature is desire feature
of vaccines that
can be used in situations in which refrigeration or other means for
stabilizing a formulation may
not be available. This would be important in epidemic or pandemic situations
during which
vaccines need to be shipped to remote areas that may lack the equipment to
maintain formulations
at a cooler temperature. As shown in Tables 4 and 5 below, low dose (2 x 109
vp/mL dose) and
high dose (2 x 10" vp/rnL dose) formulations, respectively, were prepared and
maintained at 25
CM glass vials for one, three and six months. Viability of the NasoVAX vectors
was determined
using the Adenovirus Fluorescent Focus Unit (FFU) assay. Briefly, the FFU
assay is carried out
by infecting cell monolayers with the appropriate NasoVAX dilution and
incubated for 24-48
hours. The cells were then washed, inspected, fixed (e.g., ice-cold 90%
methanol for four
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minutes), and washed again. Anti-Ad5 antibody was then added at various
dilutions (antibody
omitted in control samples), followed by a detection agent (e.g., NCL-Adeno
(Novocastra,
Newcastle, UK)) under appropriate conditions (e.g., ten minutes at room
temperature with
shaking). The cells are then washed, and the total number of infectious
particles determined (e.g.,
by digital light scattering (DLS)). As shown in Tables 4 and 5, the low-dose
and high-dose
NasoVAX formulations were stable for at least three months at room
temperature.
Table 4
Stability Data for NasoVAX (2 x 109 vp/mL dose)
- Stability-
Intme-Point
Analysis
Liquid, Liquid,
Colorless; Colorless,
Liquid, Colorless;
Liquid, Colorless;
Translucent; Translucent;
Clear;
Transparent;
Appearance No visible
No visible
No visible
particulate particulate
particulate matter No visible particulate
matter
observed
matter
observed
observed observed
pH 7.5 7.5
7.7 7.5
1.2 x 109 1.1 x 109
vp by HPLC
0.9 x 109 vp/mL 1.2 x 109 vp/mL
vp/mL vp/mL
Adenovirus
Fluorescent 1.1 x 108 2.3 x 108
0.7 x 108 FFU/mL 0.1 x 108 FFU/mL
Focus Unit FFU/mL FFU/mL
(FFU) Assay
% Infectious
9% 21%
8% 0.4%
Particles
Aggregation 139.7 nm
91.9 nm 107.5 nm
66.7 nm
by DLS (23% PD)
(14% PD) (8% PD)
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Table 5
Stability Data for NasoVAX (2 x loll vp/mL dose)
= = -= =
AnalysisStability Time Point
m:::::::::
............
- -
-
Liquid, Liquid,
Colorless; Colorless; Liquid, Colorless;
Liquid, Colorless;
Translucent; Translucent; Translucent;
Translucent;
Appearance
No visible No visible No visible
No visible particulate
particulate particulate particulate matter
observed
matter matter
observed
observed observed
pH 7.6 7.5
7.5 7.6
1.3 x 1011 1.0 x 10"
vp by HPLC 0.4 x 1011 vp/mL 1.2 x 10i1 vp/nriL
vp/mL vp/mL
Adenovirus
Fluorescent 0.9 x 10m 0.9 x 1010 0.5 x 1010
0.1 x 1010 FFU/mL
Focus Unit FFU/mL FFU/mL FFU/mL
(FFU) Assay
% Infectious
7% 9%
12% 0.5%
Particles
Aggregation 118.2 nm (19%
116.9 nm (13%
by DLS
122 nm PD)
PD) 115.5 rim (14% PD)
[00151] While certain embodiments have been described in
terms of the preferred
embodiments, it is understood that variations and modifications will occur to
those skilled in the
art. Therefore, it is intended that the appended claims cover all such
equivalent variations that
come within the scope of the following claims.
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