U.S. patent application number 12/765641 was filed with the patent office on 2010-10-21 for method of conferring a protective immune response to norovirus.
This patent application is currently assigned to Ligocyte Pharmaceuticals, Inc.. Invention is credited to Thomas R. Foubert, Charles RICHARDSON, Thomas S. Vedvick.
Application Number | 20100266636 12/765641 |
Document ID | / |
Family ID | 42981144 |
Filed Date | 2010-10-21 |
United States Patent
Application |
20100266636 |
Kind Code |
A1 |
RICHARDSON; Charles ; et
al. |
October 21, 2010 |
METHOD OF CONFERRING A PROTECTIVE IMMUNE RESPONSE TO NOROVIRUS
Abstract
The present invention relates to vaccine compositions comprising
Norovirus antigens and adjuvants, in particular, mixtures of
monovalent VLPs and mixtures of multivalent VLPs, and to methods of
conferring protective immunity to Norovirus infections in a human
subject.
Inventors: |
RICHARDSON; Charles;
(Bozeman, MT) ; Vedvick; Thomas S.; (Bozeman,
MT) ; Foubert; Thomas R.; (Bozeman, MT) |
Correspondence
Address: |
COOLEY LLP;ATTN: Patent Group
Suite 1100, 777 - 6th Street, NW
WASHINGTON
DC
20001
US
|
Assignee: |
Ligocyte Pharmaceuticals,
Inc.
Bozeman
MT
|
Family ID: |
42981144 |
Appl. No.: |
12/765641 |
Filed: |
April 22, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12678813 |
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PCT/US08/76763 |
Sep 18, 2008 |
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12765641 |
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60973389 |
Sep 18, 2007 |
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60986826 |
Nov 9, 2007 |
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Current U.S.
Class: |
424/216.1 |
Current CPC
Class: |
A61P 31/14 20180101;
A61K 2039/543 20130101; A61K 39/125 20130101; A61K 2039/57
20130101; C12N 2770/16034 20130101; A61K 2039/5258 20130101; A61K
39/12 20130101; A61K 2039/55572 20130101 |
Class at
Publication: |
424/216.1 |
International
Class: |
A61K 39/125 20060101
A61K039/125; A61P 31/14 20060101 A61P031/14 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was produced with government support from the
US Army Medical Research and Material Command, under contract
number W81XWH-05-C-0135. The government may have certain rights to
the invention.
Claims
1. A method of eliciting protective immunity to a Norovirus
infection in a human comprising administering to the human a
vaccine comprising Norovirus virus-like particles (VLPs) and at
least one adjuvant.
2. The method of claim 1, wherein said Norovirus VLPs are selected
from the group consisting of Norovirus genogroup I and genogroup II
viral strains.
3. The method of claim 1, wherein said Norovirus VLPs are
monovalent VLPs.
4. The method of claim 1, wherein said Norovirus VLPs are
multivalent VLPs.
5. The method of claim 1, wherein said vaccine comprises a second
type of Norovirus VLPs.
6. The method of claim 5, wherein said first and second Norovirus
VLPs are monovalent VLPs from different genogroups.
7. The method of claim 6, wherein said first Norovirus VLPs are
Norwalk virus VLPs and said second Norovirus VLPs are VLPs
generated from expression of a consensus sequence of genogroup II
Norovirus.
8. The method of claim 1, wherein said vaccine further comprises a
delivery agent.
9. The method of claim 8, wherein the delivery agent is a
bioadhesive.
10. The method of claim 9, wherein said bioadhesive is a
mucoadhesive.
11. The method of claim 10, wherein said mucoadhesive is selected
from the group consisting of dermatan sulfate, chondroitin, pectin,
mucin, alginate, cross-linked derivatives of poly(acrylic acid),
polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides,
hydroxypropyl methylcellulose, lectins, fimbrial proteins, and
carboxymethylcellulose.
12. The method of claim 11, wherein said mucoadhesive is a
polysaccharide.
13. The method of claim 12, wherein said polysaccharide is
chitosan, chitosan salt, or chitosan base.
14. The method of claim 1, wherein the adjuvant is selected from
the group consisting of toll-like receptor (TLR) agonists,
monophosphoryl lipid A (MPL), synthetic lipid A, lipid A mimetics
or analogs, aluminum salts, cytokines, saponins, muramyl dipeptide
(MDP) derivatives, CpG oligos, lipopolysaccharide (LPS) of
gram-negative bacteria, polyphosphazenes, emulsions, virosomes,
cochleates, poly(lactide-co-glycolides) (PLG) microparticles,
poloxamer particles, microparticles, liposomes, oil-in-water
emulsion, MF59, and squalene.
15. The method of claim 14, wherein the adjuvant is a toll-like
receptor (TLR) agonist.
16. The method of claim 14, wherein the adjuvant is MPL.
17. The method of claim 1, wherein the vaccine comprises two
adjuvants.
18. The method of claim 17, wherein the two adjuvants are MPL and
alum.
19. The method of claim 1, wherein the adjuvant is not a toxin
adjuvant.
20. The method of claim 1, wherein the vaccine is in a powder
formulation.
21. The method of claim 1, wherein the vaccine is in a liquid
formulation.
22. The method of claim 1, wherein said vaccine is administered to
the human by a route selected from the group consisting of mucosal,
intranasal, parenteral, intramuscular, intravenous, subcutaneous,
intradermal, subdermal, and transdermal routes of
administration.
23. The method of claim 22, wherein said vaccine is administered
intranasally.
24. The method of claim 23, wherein the administration of the
vaccine elicits a protective immunity comprising an increase in the
serum titer of Norovirus-specific functional antibodies as compared
to the serum titer in a human not receiving the vaccine.
25. The method of claim 24, wherein the serum titer of
Norovirus-specific functional antibodies is a geometric mean titer
greater than 40 titer/mL as measured by a hemagglutination
inhibition assay.
26. The method of claim 23, wherein the administration of the
vaccine elicits a protective immunity comprising an increase in the
level of IgA Norovirus-specific antibody secreting cells in the
blood as compared to the level in a human not receiving the
vaccine.
27. The method of claim 26, wherein the IgA Norovirus-specific
antibody secreting cells are CD19+, CD27+, CD62L+, and
.alpha.4.beta.7+.
28. The method of claim 23, wherein said vaccine is administered to
the nasal mucosa by rapid deposition within the nasal passage from
one or more devices comprising the vaccine held close to the nasal
passageway.
29. The method of claim 28, wherein said vaccine is administered to
one or both nostrils.
30. The method of claim 22, wherein said vaccine is administered
intramuscularly.
31. The method of claim 30, wherein the administration of the
vaccine elicits a protective immunity comprising an increase in the
serum titer of Norovirus-specific functional antibodies as compared
to the serum titer in a human not receiving the vaccine.
32. The method of claim 31, wherein the serum titer of
Norovirus-specific functional antibodies is a geometric mean titer
greater than 40 titer/mL as measured by a hemagglutination
inhibition assay.
33. The method of claim 1, wherein the Norovirus VLPs are present
in a concentration of from about 0.01% (w/w) to about 80%
(w/w).
34. The method of claim 1, wherein the Norovirus VLPs are present
in an amount of from about 1 .mu.g to about 100 mg per dose.
35. The method of claim 34, wherein the Norovirus VLPs are present
from about 1 .mu.g to about 200 .mu.g per dose.
36. The method of claim 35, wherein the Norovirus VLPs are present
at about 50 .mu.g per dose.
37. The method of claim 35, wherein the Norovirus VLPs are present
at about 100 .mu.g per dose.
38. The method of claim 35, wherein the Norovirus VLPs are present
at about 150 .mu.g per dose.
39. The method of claim 1, wherein said vaccine confers protection
from one or more symptoms of Norovirus infection.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
application Ser. No. 12/678,813, filed Mar. 18, 2010, which is a
national stage application of International Application No.
PCT/US2008/076763, filed Sep. 18, 2008, which claims the benefit of
priority of U.S. Provisional Application No. 60/973,389, filed Sep.
18, 2007, and U.S. Provisional Application No. 60/986,826, filed
Nov. 9, 2007, all of which are herein incorporated by reference in
their entireties.
FIELD OF THE INVENTION
[0003] The invention is in the field of vaccines, particularly
vaccines for Noroviruses. In addition, the invention relates to
methods of preparing vaccine compositions and methods of inducing a
protective immune response.
BACKGROUND OF THE INVENTION
[0004] Noroviruses are non-cultivatable human Caliciviruses that
have emerged as the single most important cause of epidemic
outbreaks of nonbacterial gastroenteritis (Glass et al., 2000;
Hardy et al., 1999). The clinical significance of Noroviruses was
under-appreciated prior to the development of sensitive molecular
diagnostic assays. The cloning of the prototype genogroup I Norwalk
virus (NV) genome and the production of virus-like particles (VLPs)
from a recombinant Baculovirus expression system led to the
development of assays that revealed widespread Norovirus infections
(Jiang et al. 1990; 1992).
[0005] Noroviruses are single-stranded, positive sense RNA viruses
that contain a non-segmented RNA genome. The viral genome encodes
three open reading frames, of which the latter two specify the
production of the major capsid protein and a minor structural
protein, respectively (Glass et al. 2000). When expressed at high
levels in eukaryotic expression systems, the capsid protein of NV,
and certain other Noroviruses, self-assembles into VLPs that
structurally mimic native Norovirus virions. When viewed by
transmission electron microscopy, the VLPs are morphologically
indistinguishable from infectious virions isolated from human stool
samples.
[0006] Immune responses to Noroviruses are complex, and the
correlates of protection are just now being elucidated. Human
volunteer studies performed with native virus demonstrated that
mucosally-derived memory immune responses provided short-term
protection from infection and suggested that vaccine-mediated
protection is feasible (Lindesmith et al. 2003; Parrino et al.
1997; Wyatt et al., 1974).
[0007] Although Norovirus cannot be cultivated in vitro, due to the
availability of VLPs and their ability to be produced in large
quantities, considerable progress has been made in defining the
antigenic and structural topography of the Norovirus capsid. VLPs
preserve the authentic confirmation of the viral capsid protein
while lacking the infectious genetic material. Consequently, VLPs
mimic the functional interactions of the virus with cellular
receptors, thereby eliciting an appropriate host immune response
while lacking the ability to reproduce or cause infection. In
conjunction with the NIH, Baylor College of Medicine studied the
humoral, mucosal and cellular immune responses to NV VLPs in human
volunteers in an academic, investigator-sponsored Phase I clinical
trial. Orally administered VLPs were safe and immunogenic in
healthy adults (Ball et al. 1999; Tacket et al. 2003). At other
academic centers, preclinical experiments in animal models have
demonstrated enhancement of immune responses to VLPs when
administered intranasally with bacterial exotoxin adjuvants
(Guerrero et al. 2001; Nicollier-Jamot et al. 2004; Periwal et al.
2003; Souza et al. (2007) Vaccine, doi:
10.1016/j.vaccine.2007.09.040). However, no studies have reported
being able to achieve protective immunity against Norovirus using
any Norovirus vaccine.
SUMMARY OF THE INVENTION
[0008] The present invention provides methods of inducing
protective immunity to a Norovirus infection in a subject, in
particular a human subject, comprising administering a vaccine
comprising at least one Norovirus antigen. In one embodiment, the
antigen is a Norovirus virus-like particle (VLP). Vaccines used in
the methods of the invention may further comprise one or more
adjuvants. The Norovirus VLPs can be selected from genogroup I or
genogroup II virus or a mixture thereof. In one embodiment, the
vaccine comprises Norovirus VLPs in a concentration from about
0.01% to about 80% by weight. In another embodiment, the vaccine
comprises dosages of Norovirus VLPs from about 1 .mu.g to about 100
mg per dose. In certain embodiments, the vaccine comprises a dosage
of Norovirus VLPs of about 25 .mu.g, about 30 .mu.g, about 50
.mu.g, about 60 .mu.g, about 70 .mu.g, about 80 .mu.g, about 90
.mu.g, about 100 .mu.g, about 125 .mu.g, or about 150 .mu.g.
[0009] In some embodiments, the vaccine further comprises a
delivery agent, which functions to enhance antigen uptake, provide
a depot effect, increase antigen retention time at the site of
delivery, or enhance the immune response through relaxation of
cellular tight junctions at the delivery site. The delivery agent
can be a bioadhesive, preferably a mucoadhesive selected from the
group consisting of dermatan sulfate, chondroitin, pectin, mucin,
alginate, cross-linked derivatives of poly(acrylic acid), polyvinyl
alcohol, polyvinyl pyrollidone, polysaccharides, hydroxypropyl
methylcellulose, lectins, fimbrial proteins, and
carboxymethylcellulose. Preferably, the mucoadhesive is a
polysaccharide. More preferably, the mucoadhesive is chitosan, or a
mixture containing chitosan, such as a chitosan salt or chitosan
base.
[0010] In other embodiments, the vaccine comprises an adjuvant. The
adjuvant may be selected from the group consisting of toll-like
receptor (TLR) agonists, monophosphoryl lipid A (MPL.RTM.),
synthetic lipid A, lipid A mimetics or analogs, aluminum salts,
cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG
oligos, lipopolysaccharide (LPS) of gram-negative bacteria,
polyphosphazenes, emulsions, virosomes, cochleates,
poly(lactide-co-glycolides) (PLG) microparticles, poloxamer
particles, microparticles, endotoxins, for instance bacterial
endotoxins and liposomes. Preferably, the adjuvant is a toll-like
receptor (TLR) agonist. More preferably, the adjuvant is MPL.RTM..
In certain embodiments, the vaccine comprises two adjuvants, such
as MPL.RTM. and alum.
[0011] The methods of the present invention include administering
Norovirus vaccines formulated as a liquid or a dry powder. Dry
power formulations may contain an average particle size from about
10 to about 500 micrometers in diameter. Suitable routes for
administering the vaccine include mucosal, intramuscular,
intravenous, subcutaneous, intradermal, subdermal, or transdermal.
In particular, the route of administration may be intramuscular or
mucosal, with preferred routes of mucosal administration including
intranasal, oral, or vaginal routes of administration. In another
embodiment, the vaccine is formulated as a nasal spray, nasal
drops, or dry powder, wherein the vaccine is administered by rapid
deposition within the nasal passage from a device containing the
vaccine held close to the nasal passageway. In another embodiment,
the vaccine is administrated to one or both nostrils. In still
another embodiment, the vaccine is administered
intramuscularly.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 shows that Norwalk Virus (NV)-specific IgG is
elicited in rabbits immunized with dry powder VLPs. Rabbits were
dosed 3 times, via the intranasal route of administration, on days
1, 22 and 43 (arrows) with 50 .mu.g NV-VLP+50 .mu.g MPL. Serum from
each rabbit was tested for NV-VLP-specific IgG by ELISA on the days
indicated. Only the VLP vaccinated rabbits had NV-VLP-specific IgG,
whereas the untreated and placebo treatment groups had no
detectable antigen-specific antibodies (data not shown). Arithmetic
means of the responses are shown and expressed in U/mL (1 U.about.1
.mu.g). Bars indicate the standard error of the mean.
[0013] FIG. 2 depicts the results of ELISA assays measuring serum
IgA (panel A) and IgG (panel B) levels from human volunteers
immunized with control (adjuvant/excipient) or a vaccine
formulation containing one of three doses of Norwalk Virus VLPs (5,
15, or 50.mu.g). The geometric mean fold-increase in anti-VLP titer
is shown for each of the dosage levels at 35 days after the second
immunization (day 56). Volunteers received immunizations on days 0
and 21.
[0014] FIG. 3 shows the levels of IgA (panel A) and IgG (panel B)
antibody secreting cells (ASCs) in human volunteers receiving
vaccine formulations with the 50 .mu.g dose of Norwalk Virus VLPs
or control (adjuvant/excipient). The geometric mean (GMN) of ASCs
per 10.sup.6 peripheral blood mononuclear cells (PBMCs) is plotted
versus study day (day 7 or day 28), specifically seven days post
immunization. Volunteers received immunizations on days 0 and
21.
[0015] FIG. 4 shows Norwalk VLP-Specific IgG and IgA Geometric Mean
Antibody Titers by Group by Study. In Study 1 (Example 2),
twenty-eight adult subjects were randomized sequentially by group
to receive two doses of: (1) 5 .mu.g Norwalk VLP vaccine (solid
squares, n=5) or adjuvant control (solid diamonds, n=2); (2) 15
.mu.g Norwalk VLP vaccine (solid triangles, n=5) or adjuvant
control (n=2); or (3) 50 .mu.g Norwalk VLP vaccine (open circles,
n=10) or adjuvant control (n=4). (A) Serum IgG geometric mean
titers from Study 1 (Example 2); (B) Serum IgA geometric mean
titers from Study 1 (Example 2). In Study 2 (Example 3), sixty-one
healthy adult subjects were enrolled at four sites and randomized
2:2:1:1, respectively, to receive either two doses of: (1) 50 .mu.g
Norwalk VLP vaccine (open circles, n=20); (2) 100 .mu.g Norwalk VLP
vaccine (open triangles, n=20); (3) adjuvant control (solid
diamonds, n=10); or (4) true placebo (open diamonds, n=11)
consisting of a puff of air (no dry powder). (C) Serum IgG
geometric mean titers from Study 2; (D) Serum IgA geometric mean
titers from Study 2 (Example 3). All doses were delivered
intranasally, and the two-dose regimen was separated by 21
days.
[0016] FIG. 5 shows the geometric mean titers for Norwalk
VLP-specific hemagglutination inhibition antibody by Group in Study
2 (Example 3). Sixty-one healthy adult subjects were enrolled at
four sites and randomized 2:2:1:1, respectively, to receive either
two doses of: (1) 50 .mu.g Norwalk VLP vaccine (open circles,
n=20); (2) 100 .mu.g Norwalk VLP vaccine (open triangles, n=20);
(3) adjuvant control (solid diamonds, n=10); or (4) true placebo
(open diamonds, n=11) consisting of a puff of air (no dry powder).
All doses were delivered intranasally, and the two-dose regimen was
separated by 21 days. The HAI titer represents a measurement of
functional antibody levels.
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention relates to methods of eliciting a
protective immunity to Norovirus infections in a subject. In
particular, the present invention provides methods of administering
a vaccine comprising Norovirus VLPs and at least one adjuvant to a
human, wherein the vaccine confers protection from at least one
symptom of Norovirus infection. Additionally or alternatively, the
vaccine may further comprise at least one delivery agent.
Norovirus Antigens
[0018] The invention provides a composition comprising one or more
Norovirus antigens. By "Norovirus," "Norovirus (NOR)," "norovirus,"
and grammatical equivalents herein, are meant members of the genus
Norovirus of the family Caliciviridae. In some embodiments, a
Norovirus can include a group of related, positive-sense
single-stranded RNA, nonenveloped viruses that can be infectious to
human or non-human mammalian species. In some embodiments, a
Norovirus can cause acute gastroenteritis in humans. Noroviruses
also can be referred to as small round structured viruses (SRSVs)
having a defined surface structure or ragged edge when viewed by
electron microscopy. Included within the Noroviruses are at least
four genogroups (GI-IV) defined by nucleic acid and amino acid
sequences, which comprise 15 genetic clusters. The major genogroups
are GI and GII. GIII and GIV are proposed but generally accepted.
Representative of GIII is the bovine, Jena strain. GIV contains one
virus, Alphatron, at this time. For a further description of
Noroviruses see Vinje et al. J. Clin. Micro. 41:1423-1433 (2003).
By "Norovirus" also herein is meant recombinant Norovirus
virus-like particles (rNOR VLPs). In some embodiments, recombinant
expression of at least the Norovirus capsid protein encoded by ORF2
in cells, e.g., from a baculovirus vector in Sf9 cells, can result
in spontaneous self-assembly of the capsid protein into VLPs. In
some embodiments, recombinant expression of at least the Norovirus
proteins encoded by ORF1 and ORF2 in cells, e.g., from a
baculovirus vector in Sf9 cells, can result in spontaneous
self-assembly of the capsid protein into VLPs. VLPs are
structurally similar to Noroviruses but lack the viral RNA genome
and therefore are not infectious. Accordingly, "Norovirus" includes
virions that can be infectious or non-infectious particles, which
include defective particles.
[0019] Non-limiting examples of Noroviruses include Norwalk virus
(NV, GenBank M87661, NP.sub.056821), Southampton virus (SHV,
GenBank L07418), Desert Shield virus (DSV, U04469), Hesse virus
(HSV), Chiba virus (CHV, GenBank AB042808), Hawaii virus (HV,
GenBank U0761 1), Snow Mountain virus (SMV, GenBank U70059),
Toronto virus (TV, Leite et al., Arch. Virol. 141:865-875), Bristol
virus (BV), Jena virus (JV, AJ01099), Maryland virus (MV,
AY032605), Seto virus (SV, GenBank AB031013), Camberwell (CV,
AF145896), Lordsdale virus (LV, GenBank X86557), Grimsby virus
(GrV, AJ004864), Mexico virus (MXV, GenBank U22498), Boxer
(AF538679), C59 (AF435807), VA115 (AY038598), BUDS (AY660568),
Houston virus (HoV, AY502023), MOH (AF397156), Parris Island (PiV;
AY652979), VA387 (AY038600), VA207 (AY038599), and Operation Iraqi
Freedom (OIF, AY675554). The nucleic acid and corresponding amino
acid sequences of each are all incorporated by reference in their
entirety. In some embodiments, a cryptogram can be used for
identification purposes and is organized: host species from which
the virus was isolated/genus abbreviation/species
abbreviation/strain name/year of occurrence/country of origin.
(Green et al., Human Caliciviruses, in Fields Virology Vol. 1
841-874 (Knipe and Howley, editors-in-chief, 4th ed., Lippincott
Williams & Wilkins 2001)). Norwalk virus, Snow Mountain virus,
and Houston virus are preferred in some embodiments.
[0020] The Norovirus antigen may be in the form of peptides,
proteins, or virus-like particles (VLPs). In a preferred
embodiment, the Norovirus antigen comprises VLPs. As used herein,
"virus-like particle(s) or VLPs" refer to a virus-like particle(s),
fragment(s), aggregates, or portion(s) thereof produced from the
capsid protein coding sequence of Norovirus and comprising
antigenic characteristic(s) similar to those of infectious
Norovirus particles. Norovirus antigens may also be in the form of
capsid monomers, capsid multimers, protein or peptide fragments of
VLPs, or aggregates or mixtures thereof. The Norovirus antigenic
proteins or peptides may also be in a denatured form, produced
using methods known in the art.
[0021] The VLPs of the present invention can be formed from either
the full length Norovirus capsid protein such as VP1 and/or VP2
proteins or certain VP1 or VP2 derivatives using standard methods
in the art. Alternatively, the capsid protein used to form the VLP
is a truncated capsid protein. In some embodiments, for example, at
least one of the VLPs comprises a truncated VP1 protein. In other
embodiments, all the VLPs comprise truncated VP1 proteins. The
truncation may be an N- or C-terminal truncation. Truncated capsid
proteins are suitably functional capsid protein derivatives.
Functional capsid protein derivatives are capable of raising an
immune response (if necessary, when suitably adjuvanted) in the
same way as the immune response is raised by a VLP consisting of
the full length capsid protein.
[0022] VLPs may contain major VP1 proteins and/or minor VP2
proteins. In some embodiments, each VLP contains VP1 and/or VP2
protein from only one Norovirus genogroup giving rise to a
monovalent VLP. As used herein, the term "monovalent" means the
antigenic proteins are derived from a single Norovirus genogroup.
For example, the VLPs contain VP1 and/or VP2 from a virus strain of
genogroup I (e.g., VP1 and VP2 from Norwalk virus). Preferably the
VLP is comprised of predominantly VP1 proteins. In one embodiment
of the invention, the antigen is a mixture of monovalent VLPs
wherein the composition includes VLPs comprised of VP1 and VP2 from
a single Norovirus genogroup mixed with VLPs comprised of VP1 and
VP2 from a different Norovirus genogroup (e.g. Norwalk virus and
Houston virus) taken from multiple viral strains. Purely by way of
example the composition can contain monovalent VLPs from one or
more strains of Norovirus genogroup I together with monovalent VLPs
from one or more strains of Norovirus genogroup II. Strains may be
selected based on their predominance of circulation at a given
time. Preferably, the Norovirus VLP mixture is composed of the
strains of Norwalk and Houston Noroviruses. More preferably, the
Norovirus VLP mixture is composed of the strains of Norwalk and a
consensus sequence derived from genogroup II Noroviruses. Consensus
sequences derived from circulating Norovirus sequences and VLPs
made with such sequences are described in WO 2010/017542, which is
herein incorporated by reference in its entirety.
[0023] However, in an alternative embodiment of the invention, the
VLPs may be multivalent VLPs that comprise, for example, VP1 and/or
VP2 proteins from one Norovirus genogroup intermixed with VP1
and/or VP2 proteins from a second Norovirus genogroup, wherein the
different VP1 and VP2 proteins are not chimeric VP1 and VP2
proteins, but associate together within the same capsid structure
to form immunogenic VLPs. As used herein, the term "multivalent"
means that the antigenic proteins are derived from two or more
Norovirus genogroups or strains. Multivalent VLPs may contain VLP
antigens taken from two or more viral strains. Purely by way of
example the composition can contain multivalent VLPs comprised of
capsid monomers or multimers from one or more strains of Norovirus
genogroup I (e.g. Norwalk virus) together with capsid monomers or
multimers from one or more strains of Norovirus genogroup II (e.g.
Houston virus). Preferably, the multivalent VLPs contain capsid
proteins from the strains of Norwalk and Houston Noroviruses, or
other predominantly circulating strains at a given time.
[0024] The combination of monovalent or multivalent VLPs within the
composition preferably would not reduce the immunogenicity of each
VLP type. In particular it is preferred that there is no
interference between Norovirus VLPs in the combination of the
invention, such that the combined VLP composition of the invention
is able to elicit immunity against infection by each Norovirus
genotype represented in the vaccine. Suitably the immune response
against a given VLP type in the combination is at least 50% of the
immune response of that same VLP type when measured individually,
preferably 100% or substantially 100%. The immune response may
suitably be measured, for example, by antibody responses, as
illustrated in the examples herein.
[0025] Multivalent VLPs may be produced by separate expression of
the individual capsid proteins followed by combination to form
VLPs. Alternatively multiple capsid proteins may be expressed
within the same cell, from one or more DNA constructs. For example,
multiple DNA constructs may be transformed or transfected into host
cells, each vector encoding a different capsid protein.
Alternatively a single vector having multiple capsid genes,
controlled by a shared promoter or multiple individual promoters,
may be used. IRES elements may also be incorporated into the
vector, where appropriate. Using such expression strategies, the
co-expressed capsid proteins may be co-purified for subsequent VLP
formation, or may spontaneously form multivalent VLPs which can
then be purified.
[0026] A preferred process for multivalent VLP production comprises
preparation of VLP capsid proteins or derivatives, such as VP1
proteins, from different Norovirus genotypes, mixing the proteins,
and assembly of the proteins to produce multivalent VLPs. The VP1
proteins may be in the form of a crude extract, be partially
purified or purified prior to mixing. Assembled monovalent VLPs of
different genogroups may be disassembled, mixed together and
reassembled into multivalent VLPs. Preferably the proteins or VLPs
are at least partially purified before being combined. Optionally,
further purification of the multivalent VLPs may be carried out
after assembly.
[0027] Suitably the VLPs of the invention are made by disassembly
and reassembly of VLPs, to provide homogenous and pure VLPs. In one
embodiment multivalent VLPs may be made by disassembly of two or
more VLPs, followed by combination of the disassembled VLP
components at any suitable point prior to reassembly. This approach
is suitable when VLPs spontaneously form from expressed VP1
protein, as occurs for example, in some yeast strains. Where the
expression of the VP1 protein does not lead to spontaneous VLP
formation, preparations of VP1 proteins or capsomers may be
combined before assembly into VLPs.
[0028] Where mutivalent VLPs are used, preferably the components of
the VLPs are mixed in the proportions in which they are desired in
the final mixed VLP. For example, a mixture of the same amount of a
partially purified VP1 protein from Norwalk and Houston viruses (or
other Norovirus strains) provides a multivalent VLP with
approximately equal amounts of each protein.
[0029] Compositions comprising multivalent VLPs may be stabilized
by solutions known in the art, such as those of WO 98/44944, WO
00/45841, incorporated herein by reference.
[0030] Compositions of the invention may comprise other proteins or
protein fragments in addition to VP1 and VP2 proteins or
derivatives. Other proteins or peptides may also be co-administered
with the composition of the invention. Optionally the composition
may also be formulated or co-administered with non-Norovirus
antigens. Suitably these antigens can provide protection against
other diseases.
[0031] The VP1 protein or functional protein derivative is suitably
able to form a VLP, and VLP formation can be assessed by standard
techniques such as, for example, electron microscopy and dynamic
laser light scattering.
Antigen Preparation
[0032] The antigenic molecules of the present invention can be
prepared by isolation and purification from the organisms in which
they occur naturally, or they may be prepared by recombinant
techniques. Preferably the Norovirus VLP antigens are prepared from
insect cells such as Sf9 or H5 cells, although any suitable cells
such as E. coli or yeast cells, for example, S. cerevisiae, S.
pombe, Pichia pastori or other Pichia expression systems, mammalian
cell expression such as CHO or HEK systems may also be used. When
prepared by a recombinant method or by synthesis, one or more
insertions, deletions, inversions or substitutions of the amino
acids constituting the peptide may be made. Each of the
aforementioned antigens is preferably used in the substantially
pure state.
[0033] The procedures of production of norovirus VLPs in insect
cell culture have been previously disclosed in U.S. Pat. No.
6,942,865, which is incorporated herein by reference in its
entirety. Briefly, a cDNA from the 3' end of the genome containing
the viral capsid gene (ORF2) and a minor structural gene (ORF3)
were cloned. The recombinant baculoviruses carrying the viral
capsid genes were constructed from the cloned cDNAs. Norovirus VLPs
were produced in Sf9 or H5 insect cell cultures.
Adjuvants
[0034] The invention further provides a composition comprising
adjuvants for use with the Norovirus antigen. Most adjuvants
contain a substance designed to protect the antigen from rapid
catabolism, such as aluminum hydroxide or mineral oil, and a
stimulator of immune responses, such as Bordatella pertussis or
Mycobacterium tuberculosis derived proteins. Suitable adjuvants are
commercially available as, for example, Freund's Incomplete
Adjuvant and Complete Adjuvant (Pifco Laboratories, Detroit,
Mich.); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.);
aluminum salts such as aluminum hydroxide gel (alum) or aluminum
phosphate; salts of calcium, iron or zinc; an insoluble suspension
of acylated tyrosine acylated sugars; cationically or anionically
derivatized polysaccharides; polyphosphazenes; biodegradable
microspheres; and Quil A.
[0035] Suitable adjuvants also include, but are not limited to,
toll-like receptor (TLR) agonists, monophosphoryl lipid A (MPL),
synthetic lipid A, lipid A mimetics or analogs, aluminum salts,
cytokines, saponins, muramyl dipeptide (MDP) derivatives, CpG
oligos, lipopolysaccharide (LPS) of gram-negative bacteria,
polyphosphazenes, emulsions, virosomes, cochleates,
poly(lactide-co-glycolides) (PLG) microparticles, poloxamer
particles, microparticles, liposomes, oil-in-water emulsions, MF59,
and squalene. In some embodiments, the adjuvants are not
bacterially-derived exotoxins. Preferred adjuvants include
adjuvants which stimulate a Th1 type response such as 3DMPL or
QS21.
[0036] Monophosphoryl Lipid A (MPL), a non-toxic derivative of
lipid A from Salmonella, is a potent TLR-4 agonist that has been
developed as a vaccine adjuvant (Evans et al. 2003). In
pre-clinical murine studies intranasal MPL has been shown to
enhance secretory, as well as systemic, humoral responses
(Baldridge et al. 2000; Yang et al. 2002). It has also been proven
to be safe and effective as a vaccine adjuvant in clinical studies
of greater than 120,000 patients (Baldrick et al., 2002; 2004). MPL
stimulates the induction of innate immunity through the TLR-4
receptor and is thus capable of eliciting nonspecific immune
responses against a wide range of infectious pathogens, including
both gram negative and gram positive bacteria, viruses, and
parasites (Baldrick et al. 2004; Persing et al. 2002). Inclusion of
MPL in intranasal formulations should provide rapid induction of
innate responses, eliciting nonspecific immune responses from viral
challenge while enhancing the specific responses generated by the
antigenic components of the vaccine.
[0037] Accordingly, in one embodiment, the present invention
provides a composition comprising monophosphoryl lipid A (MPL.RTM.)
or 3 De-O-acylated monophosphoryl lipid A (3D-MPL.RTM.) as an
enhancer of adaptive and innate immunity. Chemically 3D-MPL.RTM. is
a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6
acylated chains. A preferred form of 3 De-O-acylated monophosphoryl
lipid A is disclosed in European Patent 0 689 454 B1 (SmithKline
Beecham Biologicals SA), which is incorporated herein by reference.
In another embodiment, the present invention provides a composition
comprising synthetic lipid A, lipid A mimetics or analogs, such as
BioMira's PET Lipid A, or synthetic derivatives designed to
function like TLR-4 agonists.
[0038] In certain embodiments, the vaccine comprises two adjuvants.
A combination of adjuvants may be selected from those described
above. In one particular embodiment, the two adjuvants are MPL.RTM.
and alum. In another particular embodiment, the two adjuvants are
MPL.RTM. and oil.
[0039] The term "effective adjuvant amount" or "effective amount of
adjuvant" will be well understood by those skilled in the art, and
includes an amount of one or more adjuvants which is capable of
stimulating the immune response to an administered antigen, i.e.,
an amount that increases the immune response of an administered
antigen composition, as measured in terms of the IgA levels in the
nasal washings, serum IgG or IgM levels, or B and T-Cell
proliferation). Suitably effective increases in immunoglobulin
levels include by more than 5%, preferably by more than 25%, and in
particular by more than 50%, as compared to the same antigen
composition without any adjuvant.
Delivery Agent
[0040] The invention also provides a composition comprising a
delivery agent which functions to enhance antigen uptake, provide a
depot effect, or increase antigen retention time at the site of
delivery (e.g., delay expulsion of the antigen). Such a delivery
agent may be a bioadhesive agent. In particular, the bioadhesive
may be a mucoadhesive agent such as chitosan, a chitosan salt, or
chitosan base (e.g., chitosan glutamate).
[0041] Chitosan, a positively charged linear polysaccharide derived
from chitin in the shells of crustaceans, is a bioadhesive for
epithelial cells and their overlaying mucus layer. Formulation of
antigens with chitosan increases their contact time with the nasal
membrane, thus increasing uptake by virtue of a depot effect (Ilium
et al. 2001; 2003; Davis et al. 1999; Bacon et al. 2000; van der
Lubben et al. 2001; 2001; Lim et al. 2001). Chitosan has been
tested as a nasal delivery system for several vaccines, including
influenza, pertussis and diphtheria, in both animal models and
humans (Ilium et al. 2001; 2003; Bacon et al. 2000; Jabbal-Gill et
al. 1998; Mills et al. 2003; McNeela et al. 2004). In these trials,
chitosan was shown to enhance systemic immune responses to levels
equivalent to parenteral vaccination. In addition, significant
antigen-specific IgA levels were also measured in mucosal
secretions. Thus, chitosan can greatly enhance a nasal vaccine's
effectiveness. Moreover, due to its physical characteristics,
chitosan is particularly well suited to intranasal vaccines
formulated as powders (van der Lubben et al. 2001; Mikszta et al.
2005; Huang et al. 2004).
[0042] Accordingly, in one embodiment, the present invention
provides an antigenic or vaccine composition adapted for intranasal
administration, wherein the composition includes antigen and an
effective amount of adjuvant. In preferred embodiments, the
invention provides an antigenic or vaccine composition comprising
Norovirus antigen such as Norovirus VLP, in combination with at
least one delivery agent, such as chitosan, and at least one
adjuvant, such as MPL.RTM., CPG oligos, alum, oil, imiquimod,
gardiquimod, or synthetic lipid A or lipid A mimetics or
analogs.
[0043] The molecular weight of the chitosan may be between 10 kDa
and 800 kDa, preferably between 100 kDa and 700 kDa and more
preferably between 200 kDa and 600 kDa. The concentration of
chitosan in the composition will typically be up to about 80%
(w/w), for example, 5%, 10%, 30%, 50%, 70% or 80%. The chitosan is
one which is preferably at least 75% deacetylated, for example
80-90%, more preferably 82-88% deacetylated, particular examples
being 83%, 84%, 85%, 86% and 87% deacetylation.
Vaccine and Antigenic Formulations
[0044] The compositions of the invention can be formulated for
administration as vaccines or antigenic formulations. As used
herein, the term "vaccine" refers to a formulation which contains
Norovirus VLPs or other Norovirus antigens of the present invention
as described above, which is in a form that is capable of being
administered to a vertebrate and which induces a protective immune
response sufficient to induce immunity to prevent and/or ameliorate
an infection and/or to reduce at least one symptom of an infection
and/or to enhance the efficacy of another dose of VLPs or antigen.
As used herein, the term "antigenic formulation" or "antigenic
composition" refers to a preparation which, when administered to a
vertebrate, e.g. a mammal, will induce an immune response. As used
herein, the term "immune response" refers to both the humoral
immune response and the cell-mediated immune response. The humoral
immune response involves the stimulation of the production of
antibodies by B lymphocytes that, for example, neutralize
infectious agents, block infectious agents from entering cells,
block replication of said infectious agents, and/or protect host
cells from infection and destruction. The cell-mediated immune
response refers to an immune response that is mediated by
T-lymphocytes and/or other cells, such as macrophages, against an
infectious agent, exhibited by a vertebrate (e.g., a human), that
prevents or ameliorates infection or reduces at least one symptom
thereof. In particular, "protective immunity" or "protective immune
response" refers to immunity or eliciting an immune response
against an infectious agent, which is exhibited by a vertebrate
(e.g., a human), that prevents or ameliorates an infection or
reduces at least one symptom thereof. Specifically, induction of a
protective immune response from administration of the vaccine is
evident by elimination or reduction of the presence of one or more
symptoms of gastroenteritis or a reduction in the duration or
severity of such symptoms. Clinical symptoms of gastroenteritis
from Norovirus include nausea, diarrhea, loose stool, vomiting,
fever, and general malaise. A protective immune response that
reduces or eliminates disease symptoms will reduce or stop the
spread of a Norovirus outbreak in a population. Vaccine preparation
is generally described in Vaccine Design ("The subunit and adjuvant
approach" (eds Powell M. F. & Newman M. J.) (1995) Plenum Press
New York). The compositions of the present invention can be
formulated, for example, for delivery to one or more of the oral,
gastro-intestinal, and respiratory (e.g. nasal) mucosa. The
compositions of the present invention can be formulated, for
example, for delivery by injection.
[0045] Where the composition is intended for delivery to the
respiratory (e.g. nasal) mucosa, typically it is formulated as an
aqueous solution for administration as an aerosol or nasal drops,
or alternatively, as a dry powder, e.g. for rapid deposition within
the nasal passage. Compositions for administration as nasal drops
may contain one or more excipients of the type usually included in
such compositions, for example preservatives, viscosity adjusting
agents, tonicity adjusting agents, buffering agents, and the like.
Viscosity agents can be microcrystalline cellulose, chitosan,
starches, polysaccharides, and the like. Compositions for
administration as dry powder may also contain one or more
excipients usually included in such compositions, for example,
mucoadhesive agents, bulking agents, and agents to deliver
appropriate powder flow and size characteristics. Bulking and
powder flow and size agents may include mannitol, sucrose,
trehalose, and xylitol.
[0046] Where the composition is intended for intramuscular (i.m.)
injection, it is typically formulated as a liquid suspension
comprised of Norovirus VLPs and an adjuvant. In one embodiment, the
adjuvant may be MPL.RTM.. In another embodiment, an i.m.-formulated
vaccine may have more than one adjuvant. In a preferred embodiment,
an i.m.-formulated vaccine is formulated with Aluminum Hydroxide
(e.g. alum) and Monophosphoryl Lipid A (MPL.RTM.). Administration
of an i.m.-formulated vaccine can be by needle and syringe, as is
well known in the art.
[0047] In one embodiment, the Norovirus vaccine or antigenic
formulation of the present invention contains one or more Norovirus
genogroup antigen(s) as the immunogen, an adjuvant such as
MPL.RTM., a biopolymer such as chitosan to promote adhesion to
mucosal surfaces, and bulking agents such as mannitol and sucrose.
For example, the Norovirus vaccine may be formulated as 10 mg of a
dry powder containing one or more Norovirus genogroup antigen(s)
(e.g., Norwalk virus, Houston virus, Snow Mountain virus), MPL.RTM.
adjuvant, chitosan mucoadhesive, and mannitol and sucrose as
bulking agents and to provide proper flow characteristics. The
formulation may comprise about 7.0 mg (25 to 90% w/w range)
chitosan, about 1.5 mg mannitol (0 to 50% w/w range), about 1.5 mg
sucrose (0 to 50% w/w range), about 25 .mu.g MPL.RTM. (0.1 to 5%
w/w range), and about 100 .mu.g Norovirus antigen (0.05 to 5% w/w
range).
[0048] Norovirus antigen may be present in a concentration of from
about 0.01% (w/w) to about 80% (w/w). In one embodiment, Norovirus
antigens can be formulated at dosages of about 5 .mu.g, about 15
.mu.g, about 25 .mu.g , about 50 .mu.g, about 100 .mu.g, about 150
.mu.g, about 200 .mu.g, about 500 .mu.g, and about 1 mg per 10 mg
dry powder formulation (0.05, 0.15, 0.25, 0.5, 1.0, 1.5, 2.0, 5.0,
and 10.0% w/w) for administration into both nostrils (10 mg per
nostril) or about 10 .mu.g, about 30 .mu.g, about 50 .mu.g, about
100 .mu.g, about 200 .mu.g, about 300 .mu.g, about 400 .mu.g, about
1 mg, and about 2 mgs (0.1, 0.3, 0.5, 1.0, 2.0, 3.0, 4.0, 10.0 and
20.0% w/w) per 20 mg dry powder formulation for administration into
one nostril. The formulation may be given in one or both nostrils
during each administration. There may be a booster administration 1
to 12 weeks after the first administration to improve the immune
response. The content of each Norovirus antigen in the vaccine and
antigenic formulations may be in the range of 1 .mu.g to 100 mg,
preferably in the range 1-1000 .mu.g, more preferably 5-500 .mu.g,
most typically in the range 10-200 .mu.g. Total Norovirus antigen
administered at each dose can be either about 10 .mu.g, about 25
.mu.g, about 30 .mu.g, about 50 .mu.g, about 60 .mu.g, about 70
.mu.g, about 80 .mu.g, about 90 .mu.g, about 100 .mu.g, about 125
.mu.g, about 150 .mu.g, about 175 .mu.g, about 200 .mu.g, about 250
.mu.g, about 300 .mu.g, about 400 .mu.g, about 500 .mu.g, or about
1000 .mu.g. The total vaccine dose can be administered into one
nostril or can be split in half for administration to both
nostrils. Dry powder characteristics are such that less than 10% of
the particles are less than 10 .mu.m in diameter. Mean particle
sizes range from 10 to 500 .mu.m in diameter.
[0049] In another embodiment, the antigenic and vaccine
compositions can be formulated as a liquid for subsequent
administration to a subject. A liquid formulation intended for
intranasal administration would comprise Norovirus genogroup
antigen(s), adjuvant, and a delivery agent such as chitosan. Liquid
formulations for intramuscular (i.m.) administration would comprise
Norovirus genogroup antigen(s), adjuvant, and a buffer, without a
delivery agent (e.g., chitosan). In one embodiment, a liquid
formulation for i.m. administration comprises Norovirus genogroup
antigen(s), MPL.RTM., alum, and a buffer. In another embodiment, a
liquid formulation for i.m. administration comprises Norovirus
genogroup antigen(s), MPL.RTM., oil, and a buffer.
[0050] Preferably the antigenic and vaccine compositions
hereinbefore described are lyophilized and stored anhydrous until
they are ready to be used, at which point they are reconstituted
with diluent. Alternatively, different components of the
composition may be stored separately in a kit (any or all
components being lyophilized). The components may remain in
lyophilized form for dry formulation or be reconstituted for liquid
formulations, and either mixed prior to use or administered
separately to the patient. For dry powder administration, the
vaccine or antigenic formulation may be preloaded into an
intranasal delivery device and stored until use. Preferably, such
intranasal delivery device would protect and ensure the stability
of its contents.
[0051] The lyophilization of antigenic formulations and vaccines is
well known in the art. Typically the liquid antigen is freeze dried
in the presence of agents to protect the antigen during the
lyophilization process and to yield a cake with desirable powder
characteristics. Sugars such as sucrose, mannitol, trehalose, or
lactose (present at an initial concentration of 10-200 mg/mL) are
commonly used for cryoprotection of protein antigens and to yield
lyophilized cake with desirable powder characteristics.
Lyophilizing the compositions theoretically results in a more
stable composition. While the goal of most formulation processes is
to minimize protein aggregation and degradation, the inventors have
discovered that the presence of aggregated antigen enhances the
immune response to Norovirus VLPs (see Examples 3 and 4).
Therefore, the inventors have developed methods by which the
percentage of aggregation of the antigen can be controlled during
the lyophilization process to produce an optimal ratio of
aggregated antigen to intact antigen to induce a maximal immune
response.
[0052] Thus, the invention also encompasses a method of making
Norovirus antigen formulations comprising (a) preparing a
pre-lyophilization solution comprising Norovirus antigen, sucrose,
and chitosan, wherein the ratios of sucrose to chitosan are from
about 0:1 to about 10:1; (b) freezing the solution with liquid
nitrogen; and (c) lyophilizing the frozen solution at ambient
temperature for 48-72 hours, wherein the final lyophilized product
contains a percentage of said Norovirus antigen in aggregated form.
In one embodiment, the pre-lyophilization solution further
comprises a bulking agent. In another embodiment, said bulking
agent is mannitol.
[0053] Appropriate ratios of sucrose and chitosan to yield desired
percentages of aggregation can be determined by the following
guidelines. A pre-lyophilization mixture containing a weight ratio
of sucrose to chitosan in a range from about 2.5:1 to about 10:1
will yield greater than 95% intact Norovirus antigen
post-lyophilization (i.e. less than 5% aggregated antigen). A range
of sucrose to chitosan weight ratios of about 1:1 to about 2.1:1
will yield about 50% to about 90% intact Norovirus antigen (i.e.
about 10% to about 50% aggregated antigen). Weight ratios of 0:1
sucrose to chitosan will produce less than 30% of intact Norovirus
antigen. Omission of both sucrose and chitosan will produce less
than 5% intact antigen (i.e. greater than 95% aggregated antigen).
Using these guidelines, the skilled artisan could adjust the
sucrose to chitosan weight ratios in the pre-lyophilization mixture
to obtain the desired amount of aggregation necessary to produce an
optimal immune response.
[0054] In addition, the inclusion of sucrose and chitosan to the
pre-lyophilization solution promotes the stability of the intact
Norovirus antigen over time. The ratio of aggregated antigen/intact
antigen in the formulation does not increase when stored as a dry
powder for a period of about 12 months or greater. Thus, this
lyophilization procedure ensures stable formulations with
predictable and controllable ratios of aggregated to intact
Norovirus antigen.
Methods of Stimulating an Immune Response
[0055] The amount of antigen in each antigenic or vaccine
formulation dose is selected as an amount which induces a robust
immune response without significant, adverse side effects. Such
amount will vary depending upon which specific antigen(s) is
employed, route of administration, and adjuvants used. In general,
the dose administered to a patient, in the context of the present
invention should be sufficient to effect a protective immune
response in the patient over time, or to induce the production of
antigen-specific antibodies. Thus, the composition is administered
to a patient in an amount sufficient to elicit an immune response
to the specific antigens and/or to prevent, alleviate, reduce, or
cure symptoms and/or complications from the disease or infection,
and thus reduce or stop the spread of a Norovirus outbreak in a
population. An amount adequate to accomplish this is defined as a
"therapeutically effective dose."
[0056] For a substantially pure form of the Norovirus antigen, it
is expected that each dose will comprise about 1 .mu.g to 10 mg,
preferably about 15-500 .mu.g for each Norovirus antigen in the
formulation. In a typical immunization regime employing the
antigenic preparations of the present invention, the formulations
may be administered in several doses (e.g. 1-4), each dose
containing 1-1000 .mu.g of each antigen. Total Norovirus antigen
administered at each dose can be either about 10 .mu.g, about 25
.mu.g, about 30 .mu.g, about 50 .mu.g, about 60 .mu.g, about 70
.mu.g, about 80 .mu.g, about 90 .mu.g, about 100 .mu.g, about 125
.mu.g, about 150 .mu.g, about 175 .mu.g, about 200 .mu.g, about 250
.mu.g, about 300 .mu.g, about 400 .mu.g, about 500 .mu.g, or about
1000 .mu.g. The dose will be determined by the immunological
activity the composition produced and the condition of the patient,
as well as the body weight or surface areas of the patient to be
treated. The size of the dose also will be determined by the
existence, nature, and extent of any adverse side effects that may
accompany the administration of a particular composition in a
particular patient.
[0057] The antigenic and vaccine formulations of the present
invention may be administered via a non-mucosal or mucosal route.
These administrations may include in vivo administration via
parenteral injection (e.g. intravenous, subcutaneous, and
intramuscular) or other traditional direct routes, such as
buccal/sublingual, rectal, oral, nasal, topical (such as
transdermal and ophthalmic), vaginal, pulmonary, intraarterial,
intraperitoneal, intraocular, or intranasal routes or directly into
a specific tissue. Alternatively, the vaccines of the invention may
be administered by any of a variety of routes such as oral,
topical, subcutaneous, mucosal, intravenous, intramuscular,
intranasal, sublingual, transcutaneous, subdermal, intradermal and
via suppository. In one embodiment, the vaccine is administered by
an intramuscular route of administration. Administration may be
accomplished simply by direct administration using a needle,
catheter or related device, at a single time point or at multiple
time points.
[0058] In a preferred embodiment, the antigenic and vaccine
formulations of the present invention are administered by the
intranasal route. Immunization via the mucosal surfaces offers
numerous potential advantages over other routes of immunization.
The most obvious benefits are 1) mucosal immunization does not
require needles or highly-trained personnel for administration, and
2) immune responses are raised at the site(s) of pathogen entry, as
well as systemically (Isaka et al. 1999; Kozlowski et al. 1997;
Mestecky et al. 1997; Wu et al. 1997).
[0059] In a further aspect, the invention provides a method of
eliciting an IgA mucosal immune response and an IgG systemic immune
response by administering (preferably intranasally) to a mucosal
surface of the patient an antigenic or vaccine composition
comprising one or more Norovirus antigens, at least one effective
adjuvant and/or at least one delivery agent. In one embodiment, the
immune response is a highly-biased mucosal response, i.e.
characterized by a large increase in serum IgA relative to serum
IgG. For instance, in some embodiments, the immune response is
characterized by a ratio of serum IgA mean fold rise titer to serum
IgG mean fold rise titer of from about 1.5:1 to about 3:1.
[0060] In a further aspect, the invention provides a method of
boosting a pre-existing mucosal immune response by administering
the vaccine of the invention parenterally, including but not
limited to the intramuscular route.
[0061] The present invention also contemplates the provision of
means for dispensing formulations of Norovirus antigens
hereinbefore defined, and at least one adjuvant or at least one
delivery agent as hereinbefore defined. A dispensing device for
intranasal formulations may, for example, take the form of an
aerosol delivery system, and may be arranged to dispense only a
single dose, or a multiplicity of doses. Such a device would
deliver a metered dose of the vaccine or antigenic formulation to
the nasal passage. Other examples of appropriate devices include,
but are not limited to, droppers, swabs, aerosolizers, insufflators
(e.g. Valois Monopowder Nasal Administration Device, single dose
Bespak UniDose DP dry powder intranasal delivery device),
nebulizers, and inhalers. The devices may deliver the antigenic or
vaccine formulation by passive means requiring the subject to
inhale the formulation into the nasal cavity. Alternatively, the
device may actively deliver the formulation by pumping or spraying
a dose into the nasal cavity. The antigenic formulation or vaccine
may be delivered into one or both nostrils by one or more such
devices. Administration could include two devices per subject (one
device per nostril). In a preferred embodiment, the antigenic or
vaccine formulation is administered to the nasal mucosa by rapid
deposition within the nasal passage from a device containing the
formulation held close to the nasal passageway. For intraparenteral
formulations (e.g. intramuscular formulations), a dispensing device
can be a syringe equipped with a needle or an autoinjector.
[0062] Actual dose of active ingredient (Norovirus antigen) may be
about 5-1000 .mu.g. In certain embodiments, the actual dose of
Norovirus antigen per device is about 50 .mu.g or about 100 .mu.g.
In certain embodiments, the actual dose of Norovirus antigen per
device is about 150 .mu.g or about 300 .mu.g. In other embodiments,
the actual dose of Norovirus antigen per device is about 300 .mu.g
or about 600 .mu.g.
[0063] The invention also provides a method of generating
antibodies to one or more Norovirus antigens, said method
comprising administration of a vaccine or antigenic formulation of
the invention as described above to a subject. These antibodies can
be isolated and purified by routine methods in the art. The
isolated antibodies specific for Norovirus antigens can be used in
the development of diagnostic immunological assays. These assays
could be employed to detect a Norovirus in clinical samples and
identify the particular virus causing the infection (e.g. Norwalk,
Houston, Snow Mountain, etc.). Alternatively, the isolated
antibodies can be administered to subjects susceptible to Norovirus
infection to confer passive or short-term immunity.
[0064] The invention provides methods for eliciting protective
immunity to a Norovirus infection in a subject comprising
administering a vaccine to the subject, wherein said vaccine
comprises Norovirus VLPs and at least one adjuvant. In one
embodiment, the subject is a human and the vaccine confers
protection from one or more symptoms of Norovirus infection.
Although others have reported methods of inducing an immune
response with Norovirus antigens (see U.S. Patent Application
Publication No. US 2007/0207526), no one has demonstrated the
induction of a protective immune response in humans. Unlike several
vaccines currently licensed in the U.S. where effectiveness of the
vaccine correlates with serum antibodies, studies have shown that
markers of an immune response, such as increased titers of serum
antibodies against Norwalk virus, are not associated with
protective immunity in humans (Johnson et al. (1990) J. Infectious
Diseases 161: 18-21). Moreover, another study examining Norwalk
viral challenge in humans indicated that susceptibility to Norwalk
infection was multifactorial and included factors such as secretor
status and memory mucosal immune response (Lindesmith et al. (2003)
Nature Medicine 9: 548-553). Because Norovirus is not able to be
cultured in vitro, no viral neutralization assays are currently
available. A functional assay which serves as a substitute for the
neutralization assay is the hemagglutination inhibition (HAI)
assay. HAI measures the ability of Norovirus vaccine-induced
antibodies to inhibit the agglutination of antigen-coated red blood
cells by Norovirus VLPs because Norovirus VLPs bind to red blood
cell antigens. This assay is also known as a carbohydrate blocking
assay, as it is indicative of the functional ability of antibodies
to block binding of the virus or VLPs to blood group antigen
carbohydrates on a red blood cell. In this assay, a fixed amount of
Norovirus VLPs is mixed with a fixed amount of red blood cells and
serum from immunized subjects. If the serum sample contains
functional antibodies, the antibodies will compete with the VLPs
for binding to the red blood cells, thereby inhibiting the
agglutination of the red blood cells. As used herein, "functional
antibodies" refer to antibodies that are capable of inhibiting the
interaction between Norovirus particles and red blood cell
antigens. The serum titer of Norovirus-specific functional
antibodies can be measured by the HAI assay described above. An
increase in the level of Norovirus-specific functional antibodies
can be an indicator of a protective immune response. Thus, in one
embodiment, the administration of the vaccine elicits a protective
immunity comprising an increase in the serum titer of
Norovirus-specific functional antibodies as compared to the serum
titer in a human not receiving the vaccine. The serum titer of
Norovirus-specific functional antibodies indicative of a protective
immune response is preferably a geometric mean titer greater than
40, 50, 75, 100, 125, 150, 175, or 200 titer/mL as measured by the
HAI assay. In one embodiment, the serum titer of Norovirus-specific
functional antibodies is a geometric mean titer greater than 40
titer/mL as measured by the HAI assay. In another embodiment, the
serum titer of Norovirus-specific functional antibodies is a
geometric mean titer greater than 100 titer/mL as measured by the
HAI assay.
[0065] In certain embodiments, the administration of the vaccine
elicits a protective immunity comprising an increase in the level
of IgA Norovirus-specific antibody secreting cells in the blood as
compared to the level in a human not receiving the vaccine. In some
embodiments, the administration of the vaccine elicits a protective
immunity comprising an increase in the level of IgA
Norovirus-specific antibody secreting cells in the blood as
compared to the level in the human before receiving the vaccine. In
one embodiment, the IgA Norovirus-specific antibody secreting cells
are CD19+, CD27+, CD62L+, and .alpha.4.beta.7+. Antibody secreting
cells with this marker profile are capable of homing to both
peripheral lymphoid tissue, such as Peyer's patch in the gut, and
mucosal lymphoid tissue, such as the gut mucosa. The inventors have
surprisingly discovered that the Norovirus vaccines of the
invention induce this dual homing population (to both peripheral
and mucosal lymphoid tissues) of IgA-secreting antibody secreting
cells when administered intranasally in humans. This result is
particularly surprising because intranasal vaccines do not
typically induce protection in the gut. In one embodiment, the
number of CD19+, CD27+, CD62L+, and .alpha.4.beta.7+ IgA antibody
secreting cells is greater than about 500, about 700, about 1,000,
about 1,500, or greater than about 2,000 cells per 1.times.10.sup.6
peripheral blood monocytes. In another embodiment, the IgA
Norovirus-specific antibody secreting cells are CD19+, CD27+,
CD62L-, and .alpha.4.beta.7+. Antibody secreting cells with this
marker profile generally exhibit homing only to mucosal sites and
can be indicative of a memory B-cell response. In some embodiments,
the number of CD19+, CD27+, CD62L-, and .alpha.4.beta.7+ IgA
antibody secreting cells is greater than about 2,000, about 2,500,
about 3,000, about 4,500, about 5,000, or greater than about 6,500
cells per 1.times.10.sup.6 peripheral blood monocytes.
[0066] Similar findings have been observed with vaccines for other
viruses, such as rotavirus. For rotavirus vaccines, there is
controversy over whether serum antibodies are directly involved in
protection or merely reflect recent infection (Jiang, 2002; Franco,
2006). Defining such correlates of protection is particularly
difficult in the context of diarrheal diseases such as rotavirus or
norovirus, where preclinical studies inferring protection may be
multifaceted with contributions from mucosal immunity (such as
intestinal IgA), cytokine elaboration, and cell mediated immunity.
The difficulty in measuring such immune responses during clinical
development, and the lack of correlation to serum antibody
measurements, requires that the effectiveness of a vaccine for
these types of viruses can only be demonstrated through human
clinical challenge experiments.
[0067] As mentioned above, administration of the vaccine of the
present invention prevents and/or reduces at least one symptom of
Norovirus infection. Symptoms of Norovirus infection are well known
in the art and include nausea, vomiting, diarrhea, and stomach
cramping. Additionally, a patient with a Norovirus infection may
have a low-grade fever, headache, chills, muscle aches, and
fatigue. The invention also encompasses a method of inducing a
protective immune response in a subject experiencing a Norovirus
infection by administering to the subject a vaccine formulation of
the invention such that at least one symptom associated with the
Norovirus infection is alleviated and/or reduced. A reduction in a
symptom may be determined subjectively or objectively, e.g., self
assessment by a subject, by a clinician's assessment or by
conducting an appropriate assay or measurement (e.g. body
temperature), including, e.g., a quality of life assessment, a
slowed progression of a Norovirus infection or additional symptoms,
a reduced severity of Norovirus symptoms or suitable assays (e.g.
antibody titer, RT-PCR antigen detection, and/or B-cell or T-cell
activation assay). An effective response may also be determined by
directly measuring (e.g., RT-PCR) virus load in stool samples,
which reflects the amount of virus shed from the intestines). The
objective assessment comprises both animal and human
assessments.
[0068] Stability and efficacy in animal models of the vaccine and
antigenic formulations disclosed herein are reported in
International Application No. PCT/US07/79929, which is herein
incorporated by reference in its entirety.
Examples
[0069] The invention will now be illustrated in greater detail by
reference to the specific embodiments described in the following
examples. The examples are intended to be purely illustrative of
the invention and are not intended to limit its scope in any
way.
Example 1
GLP Toxicity Study of Norovirus Vaccine Formulations in Rabbits
[0070] The purpose of this study was to evaluate the potential
toxicity of a Norwalk virus-virus-like particle (NV-VLP) vaccine
following three intranasal doses in rabbits. The NV-VLP vaccine
contained (per 10 mg dry powder) 25 .mu.g of a Genogroup I VLP, 25
.mu.g MPL, 7 mg chitosan glutamate, 1.475 mg mannitol, and 1.475 mg
sucrose. The study was conducted over an eight week period. The
persistence, reversibility, or delayed onset of any effects were
assessed after a four-week, no-treatment recovery interval. Sixty
New Zealand White rabbits (30/sex) were randomly assigned to three
groups (10 rabbits/sex/group). Group 1 animals were not dosed (i.e.
naive). Group 2 animals were administered 10 mg/nostril (20 mg
total) of placebo (i.e. adjuvant/excipient: MPL, chitosan, sucrose,
and mannitol). Group 3 animals were administered 10 mg/nostril (20
mg total) of NV-VLP vaccine, which represented 25 .mu.g of antigen
per nostril (50 .mu.g total). Animals in groups 2 and 3 were dosed
on study day (SD) 1, 22, and 43 by intranasal administration using
the Bespak Unidose intranasal dry powder device. Animals
(5/group/sex) were subjected to a full gross necropsy on SD 46 and
74. Parameters evaluated during the study included mortality,
clinical and cageside observations, body weights, body weight
changes, food consumption, body temperature, ophthalmology
examinations, clinical pathology (clinical chemistry, hematology,
and urinalysis), gross pathology, organ weight data, and
histopathology. The study outline is summarized in Table 1. The
conclusions of the study are summarized in Table 2.
TABLE-US-00001 TABLE 1 Study Parameters for GLP Toxicity Study of
Norwalk Vaccine Formulation SPF New Zealand White Rabbits Species
with ear tag IDs No. Animals/Sex/Dose Group 10 males and 10
females/group Total Number of Animals in Study 60 Group 1
Non-treated controls Group 2 Adjuvant/Excipient Group 3 1x maximum
human dose VLPs in Adjuvant/Excipient
TABLE-US-00002 TABLE 2 Safety and Toxicology Findings for Norwalk
Vaccine Formulation Observations No treatment related effects on
mortality, clinical or cageside observations. Body weight and body
No adverse effect on body weights or body weight changes. weight
changes Food consumption No treatment related adverse effect on
food consumption. Body temperature No treatment related adverse
effect on body temperature. Opthamology No ocular lesions were
noted in any animal over the course of the study. Clinical
Polyclonal activation of B lymphocyte populations in rabbits
Pathology receiving NV-VLP Vaccine or Adjuvant/Excipient was noted
days 3-76. Absolute monocyte values were elevated in rabbits
receiving NV-VLP Vaccine or Adjuvant/Excipient on days 3-46. There
were no treatment effects on selected urinalysis parameters. Gross
Pathology No treatment related observations. Organ weights No
adverse effects on absolute or relative organ weights.
Histopathology Varying degrees of inflammatory infiltrates, either
within the lamina propria of nasal turbinates or free within the
nasal passages, and/or hemorrhage within the nasal passages of
rabbits receiving NV-VLP Vaccine or Adjuvant/Excipient. The
observed lesions are those that would be expected in an immunologic
reaction. Lesions in both groups were limited in nature and
resolved completely by SD 74.
[0071] Cage side observations revealed no significant findings.
Hematological measures (increases in globulin and total protein)
were typical of B lymphocyte polyclonal activation and may be
attributable to adjuvant effects. Histopathology findings consisted
of varying degrees of inflammatory infiltrates, either within the
lamina propria of nasal turbinates or free within the nasal
passages, and/or mild hemorrhage in the nasal passages of rabbits
in both groups. The observed lesions would be expected in an
immunologic reaction. Lesions in both groups were limited in nature
and resolved completely by study day 74.
[0072] Serological samples analyzed by ELISA for NV-VLP specific
IgG showed measurable anti-NV-VLP titers in 30% of the immunized
animals on day 10 following a single dose (see FIG. 1). Boost
treatments on days 22 and 43 increased both the number of
seroconverted animals and levels of product-specific antibodies,
and by day 73, 90% of the immunized animals seroconverted. None of
the naive or matrix treated controls had quantifiable levels of
NV-VLP specific antibodies (data not shown).
[0073] The immune response was further characterized by evaluating
memory B-cell responses in an additional set of rabbits immunized
intranasally with the same formulation on days 1, 15 and 29. Memory
B-cell responses were measured as described in International
Application No. PCT/US07/79929, which is herein incorporated by
reference in its entirety. Tissues collected 156 days after the
last boost showed the presence of NV-VLP-specific memory B-cells in
the peripheral blood, the spleen, and most notably, in the
mesenteric lymph nodes. The antigen-specific memory B-cells in the
mesenteric lymph nodes were IgA positive. Additionally,
NV-VLP-specific antibody-secreting long-lived plasma cells were
present in the bone marrow.
Example 2
Dose Escalation Safety Study of Norwalk Vaccine Formulation in
Humans (LV01-101 Study)
[0074] A double-blind, controlled, dose-escalation phase 1 study of
the safety and immunogenicity of a Norovirus genogroup 1 vaccine
was conducted. The vaccine consisted of lyophilized Norwalk
virus-like particles (VLPs) in a dry powder matrix designed for
intranasal administration. Vaccines included healthy adult
volunteers who were H type 1 antigen secretors. The rationale for
enrollment of H type 1 antigen secretors is that H type 1 antigen
secretors are susceptible to Norwalk viral infections while
non-secretors are resistant. Saliva was collected from volunteers
to determine H type 1 antigen secretor status. As a control, 2
additional volunteers at each dosage level received matrix alone.
The dry powder matrix included 25 .mu.g MPL.RTM. adjuvant, 7 mg
chitosan, 1.5 mg mannitol, and 1.5 mg sucrose. Volunteers were
dosed on days 0 and 21 and were required to keep a 7-day diary of
symptoms after each dose. Blood for serology, antibody secreting
cells (ASC), and stool and saliva samples for mucosal antibody
evaluation were collected.
[0075] The components of the Norwalk VLP vaccine are listed in
Table 3. The vaccine is packaged in an intranasal delivery device.
Single administrations of Norwalk VLP Vaccine were packaged in a
single dose Bespak (Milton Keynes, UK) UniDose DP dry powder
intranasal delivery device. Each device delivered 10 mg of the dry
powder vaccine formulation. Each dose of vaccine consisted of two
delivery devices, one in each nostril. The total vaccine dose was
20 mg of dry power. The formulation of Adjuvant/Excipient is the
same as the Norwalk VLP Vaccine except that no Norwalk VLP antigen
is included in the formulation. The formulation of the
Adjuvant/Excipient (also referred to as dry powder matrix) is
summarized in Table 4.
TABLE-US-00003 TABLE 3 Norwalk VLP Vaccine Composition Quantity per
Molecular 10 mg dry % of Final Component class powder Formulation
Norwalk VLP Recombinant 2.5, 7.5, 25, 0.025, 0.075, 0.25, or
protein or 50 .mu.g 0.50%.sup. Monophosphoryl Phospholipid 25 .mu.g
0.25%.sup. Lipid A Chitosan Polysaccha- 7.0 mg 70% ride Mannitol
Sugar 1.5 mg 15%* Sucrose Sugar 1.5 mg 15% *Quantity of mannitol
varies slightly in different formulations to account for variation
in Norwalk VLP content.
TABLE-US-00004 TABLE 4 Adjuvant/Excipient (dry powder matrix)
Quantity per Molecular 10 mg dry % of Final Component class powder
Formulation Monophosphoryl Phospholipid 25 .mu.g 0.25%.sup. Lipid A
Chitosan Polysaccharide 7.0 mg 70% Mannitol Sugar 1.5 mg 15%
Sucrose Sugar 1.5 mg 15%
[0076] Specifically, the dose escalation of the vaccine was
conducted as follows: After appropriate screening for good health,
a group of 3 volunteers was randomized to receive either 5 .mu.g
Norwalk VLP Vaccine plus dry powder matrix (n=2) or dry powder
matrix alone (n=1) by the intranasal route. These 3 volunteers were
followed for safety for 21 days and their safety data reviewed by
the Independent Safety Monitor (ISM). Upon approval of the ISM,
these individuals received their second dose of Vaccine or matrix
on day 21, and 4 additional volunteers were randomized to receive
either 5 .mu.g VLP protein plus dry powder matrix (n=3) or matrix
alone (n=1) by the intranasal route. The ISM reviewed the safety
data from this second group and upon approval of the ISM, the
second intranasal dose was given 21 days after the first dose.
Volunteers kept a 7-day diary of symptoms after each dose. After
the ISM determined that escalation to the next higher dose was
acceptable, another group of 7 volunteers was randomized to receive
either Norwalk VLP Vaccine containing 15 .mu.g VLP protein (n=5) or
dry powder matrix alone (n=2) by the intranasal route at day 0 and
day 21. Again, 7-day symptom diaries were recorded and reviewed by
the ISM before the second dose at day 21. Finally, after review of
the safety data from the first two dosage cohorts, the ISM
determined that dose escalation was acceptable and a final group of
7 volunteers were randomized to receive either Norwalk VLP Vaccine
containing 50 .mu.g VLP protein (n=5) or dry powder matrix alone
(n=2) by the intranasal route on day 0 and day 21. Seven-day
symptom diaries and other safety data were again reviewed by the
ISM before the second dose at day 21.
[0077] The volunteers kept a daily diary of symptoms (including
local symptoms such as: nasal discharge, nasal pain/discomfort,
nasal congestion, runny nose, nasal itching, nose bleed, headache
and systemic symptoms such as: daily oral temperature, myalgia,
nausea, vomiting, abdominal cramps, diarrhea, and loss of appetite)
for 7 days after receiving Norwalk VLP Vaccine or dry powder matrix
alone. Interim medical histories were obtained at each follow-up
visit (days 7.+-.1, 21.+-.2, 28.+-.2, 56.+-.2 and 180.+-.14);
volunteers were queried about interim illness, medications, and
doctor's visits. Volunteers were asked to report all serious or
severe adverse events including events that were not solicited
during follow up visits. Volunteers had CBC and serum creatinine,
glucose, AST, and ALT assessed on days 7 and 28 (7 days after each
immunization) and, if abnormal, the abnormal laboratory test was
followed until the test became normal or stabilized.
[0078] The blinded data indicated that of the volunteers that
received the low dose (n=5) or matrix (n=2), 4 of 7 reported some
or all of the following: nasal discharge, nasal pain, stuffiness,
itching, sneezing, headache, and/or sore throat in the first 24
hours after vaccination. One volunteer reported a minor nosebleed
on each of days 1 and 6. Of the volunteers that received the middle
dose (n=5) or matrix (n=2), 5 of 7 reported mild nasal discharge,
stuffiness, itching, sneezing, and/or headache in the first 24
hours. Symptoms generally resolved in the first 72 hours, but
stuffiness persisted to day 7 in one volunteer. A summary of the
findings on the unblinded data is presented in Table 5 below, which
also includes adverse events reported in the high dose. These
findings indicate that intranasal Norovirus VLP vaccine is
associated with local, usually mild, short-lived symptoms that
appeared to be independent of VLP concentration. No differences
were seen between the adjuvant/excipient (or matrix) control group
and the Norwalk VLP vaccine groups for adverse events, hematology,
blood chemistry and/or physical examination results.
TABLE-US-00005 TABLE 5 Number of Volunteers with Adverse Events to
Norwalk VLP Vaccine or Adjuvant/Excipient Adjuvant/ Reported
Adverse Excipient Low Dose Mid Dose High Dose Events (N = 6) (N =
5) (N = 5) (N = 5)* Nose and Throat Nasal Stuffiness 4 2 3 1 Nasal
Itching 3 3 2 2 Nasal Discharge 3 3 4 3 Nasal Pain -- 2 1 2
Sneezing 3 2 1 3 Nose Bleed -- 1 1 -- Sore Throat/URI -- 1 -- 1
Itchy Sore Throat -- 1 -- -- Burning in Nose/ -- 1 -- 1 Throat
Chest Cough 2 -- -- -- Chest discomfort -- -- -- 1 Systemic
Headache 2 2 1 1 Malaise 3 2 -- 1 Nausea -- 1 -- 1 Abdominal Cramp
1 -- -- 1 Laboratory ALT/AST -- 1 -- -- AST 1 -- -- -- ALT -- -- --
1 Alk Phos -- -- -- 1 Gastrointestinal Diarrhea -- 1 1 Loss of
appetite 1 -- 1 -- No Adverse Events Reported -- -- 1 2 *One
subject in cohort 3 did not receive the second dose
[0079] Blood was collected before immunization and on days 7.+-.1,
21.+-.2, 28.+-.2, 56.+-.2, and 180.+-.14 to measure serum
antibodies to Norwalk VLP Vaccine by enzyme-linked immunosorbent
assays (ELISA). Before and on day 7 after administration of each
dose of Vaccine or dry powder matrix alone peripheral blood
lymphocytes were collected to detect antibody secreting cells by
ELISPOT assay. Before and on days 21.+-.2, 56.+-.2 and 180.+-.14
after vaccination, whole blood was obtained to separate cells and
freeze for future studies of cell mediated immunity, including
cytokine production in response to Norwalk VLP antigen, and
lymphoproliferation. Finally blood from volunteers receiving the
highest dose of Norwalk VLPs (50 .mu.g, third cohort described
above) was screened for memory B-cells on days 0, 21, 56 and
180.
[0080] The following methods were used to analyze the blood samples
collected from immunized individuals or individuals receiving the
dry powder matrix alone:
A. Serum Antibody Measurements by ELISA
[0081] Twenty mL of blood were collected before and at multiple
time points after vaccination for measurement of antibodies to
Norwalk virus by ELISA, using purified recombinant Norwalk VLPs as
target antigen to screen the coded specimens. Briefly, Norwalk VLPs
in carbonate coating buffer pH 9.6 were used to coat microtiter
plates. Coated plates were washed, blocked, and incubated with
serial two-fold dilutions of test serum followed by washing and
incubation with enzyme-conjugated secondary antibody reagents
specific for human IgG, IgM, and IgA. Appropriate substrate
solutions were added, color developed, plates read, and the IgG,
IgM, and IgA endpoint titers were determined in comparison to a
reference standard curve for each antibody class. A positive
response was defined as a 4-fold rise in titer after vaccination.
The geometric mean serum titers for IgG and IgA are shown at day 0,
7, 21, 28, 56, and 180 for each vaccine dose in FIGS. 4A and B,
respectively. The mean fold rise in geometric mean titer at day 56
(35 days after the second immunization) for each of the vaccine
doses is shown in FIG. 2. The results show a dose-dependent
increase in serum titers for IgG and IgA. A significant serum titer
for both IgG and IgA was observed in volunteers receiving the
vaccine containing 50 .mu.g of Norovirus antigen.
B. Antibody Secreting Cell Assays
[0082] PBMCs were collected from heparinized blood (30 mL for
cohorts 1 and 2, 25 mL for cohort 3) for ASC assays to detect cells
secreting antibodies to Norwalk VLPs. These assays were performed
on days 0, 7.+-.1, 21.+-.2, and 28.+-.2 after administration of
Norwalk VLP Vaccine or dry powder matrix alone. The response rate
and mean number of ASC per 10.sup.6 PBMC at each time point for
each dosage were described. A positive response was defined as a
post-vaccination ASC count per 10.sup.6 PBMCs that is at least 3
standard deviations (SD) above the mean pre-vaccination count for
all subjects (in the log metric) and at least 8 ASC spots, which
corresponds to the mean of medium-stimulated negative control wells
(2 spots) plus 3 SD as determined in similar assays.
[0083] The results of the ASC assays for the 50 .mu.g dose of
Norwalk VLPs are depicted in FIG. 3. Circulating IgG and IgA
antibody secreting cells were observed seven days after initial and
boost vaccinations, suggesting that the vaccine is immunogenic.
C. Measurement of Functional Antibody Response
[0084] Serum collected as described in paragraph B, above, was
further analyzed to determine the functional properties of the
anti-Norwalk virus antibodies. Serial two-fold dilutions of test
serum were analyzed with respect to their ability to inhibit
hemagglutination of red blood cells by Norwalk VLPs (a functional
assay to indicate protective immune responses). A positive response
was defined as a 4-fold rise in titer after vaccination. The serum
titers and hemagglutination inhibition titers at day 56 (35 days
post boost) for five subjects who received the 50 .mu.g dose of the
Norwalk VLPs vaccine are shown in Table 6. The results show that
seventy five percent (75%) of the individuals who exhibited a
seroconversion response as measured by serum IgG titers also
developed a functional antibody response capable of blocking the
binding receptor on human red blood cells as measured by
hemagglutination inhibition.
TABLE-US-00006 TABLE 6 Serum IgG and Hemagglutination Inhibition
(HAI) (functional) Titers on Day 0 and Day 35 Post Boost (35PB) for
Five Human Volunteers. Subject Reference Day 0 Day 35PB Serum IgG
Titers A 2,444.6 37,185.9 B 4,462.1 23,508.4 C 7,735.7 13,357.8 D
884.5 4,577.5 E 12,719.0 91,710.8 Hemagglutination Inhibition (HAI)
Titers A 8 256 B 8 256 C 512 512 D <8 8 E 128 1024
D. Measurement of Norwalk Virus-Specific Memory B-Cells
[0085] Heparinized blood was collected from cohort 3 (30 mL days 0
and 21, 50 mL days 56 and 180) to measure memory B cells on days 0,
21, 56 and 180 after vaccination using an ELISpot assay preceded by
an in vitro antigen stimulation. A similar assay was successfully
used to measure frequency of memory B cells elicited by Norwalk VLP
formulations in rabbits (See International Application No.
PCT/US07/79929, herein incorporated by reference). Peripheral blood
mononuclear cells (5.times.10.sup.6 cells/mL, 1 mL/well in 24-well
plates) are incubated for 4 days with Norwalk VLP antigen (2-10
.mu.g/mL) to allow for clonal expansion of antigen-specific memory
B cells and differentiation into antibody secreting cells. Controls
include cells incubated in the same conditions in the absence of
antigen and/or cells incubated with an unrelated antigen. Following
stimulation, cells are washed, counted and transferred to ELISpot
plates coated with Norwalk virus VLP. To determine frequency of
virus-specific memory B cells per total Ig-secreting B lymphocytes,
expanded B cells are also added to wells coated with anti-human IgG
and anti-human IgA antibodies. Bound antibodies are revealed with
HRP-labeled anti-human IgG or anti-human IgA followed by True Blue
substrate. Conjugates to IgA and IgG subclasses (IgA1, IgA2 and
IgG1-4) may also be used to determine antigen-specific subclass
responses which may be related with distinct effector mechanisms
and locations of immune priming. Spots are counted with an ELISpot
reader. The expanded cell populations for each volunteer are
examined by flow cytometry to confirm their memory B cell
phenotype, i.e. CD19+, CD27+, IgG+, IgM+, CD38+, IgD-.
E. Cellular Immune Responses
[0086] Heparinized blood (50 mL cohorts 1 and 2, 25 mL cohort 3)
was collected as coded specimens and the peripheral blood
mononuclear cells (PBMC) isolated and cryopreserved in liquid
nitrogen for possible future evaluation of CMI responses to Norwalk
VLP antigen. Assays that may be performed include PBMC
proliferative and cytokine responses to Norwalk VLP antigen and can
be determined by measuring interferon (IFN)-.gamma. and interleukin
(IL)-4 levels according to established techniques.
Example 3
Safety and Immunogenicity Study of Two Dosages of Intranasal
Norwalk VLP Vaccine in Humans (LV01-102 Study)
[0087] A randomized, double blind, multi-center study in healthy
adults was conducted to compare the safety and immunogenicity of
two dosage levels (50 .mu.g and 100 .mu.g) of a Norwalk virus-like
particle (VLP) vaccine with adjuvant/excipients and placebo
controls (empty device). The vaccine consisted of Norwalk
virus-like particles (VLPs) in a dry powder matrix designed for
intranasal administration as described in Example 2. Vaccines
included healthy adult volunteers ages 18-49 who were H type 1
antigen secretors. Saliva was collected from volunteers to
determine H type 1 antigen secretor status. Further, only subjects
whose blood type was A or O (not type B or AB) were included in the
study as those with B blood type are reported to be less
susceptible to Norwalk infection (Glass et al. (2009) N. Engl. J.
Med., Vol. 361: 1776-1785). The human volunteers were randomly
assigned to one of four groups and each group received one of the
following treatments: two 50 .mu.g doses of the Norwalk VLP vaccine
(n=20), two 100 .mu.g doses of the Norwalk VLP vaccine (n=20), two
doses of the adjuvant/excipient (n=10), or two doses of an air puff
placebo (n=11). Volunteers were dosed on days 0 and 21 and were
required to keep a 7-day diary of symptoms after each dose. Blood
for serology, antibody secreting cells (ASC), and stool and saliva
samples for mucosal antibody evaluation were collected.
[0088] The components of the vaccine are listed in Table 3 in
Example 2. The vaccine was packaged in an intranasal delivery
device. Single administrations of the Norwalk VLP vaccine were
packaged in a single dose Bespak (Milton Keynes, UK) UniDose DP dry
powder intranasal delivery device. Each device delivered 10 mg of
the dry powder vaccine formulation. Each dose of vaccine consisted
of two delivery devices, one in each nostril. The total vaccine
dose was 20 mg of dry power. Therefore, the 50 .mu.g vaccine dose
consisted of two devices that each delivered 10 mg of dry powder
formulation, wherein each 10 mg of dry powder formulation consisted
of 25 .mu.g of Norwalk VLP, 25 .mu.g MPL.RTM. adjuvant, 7 mg
chitosan, 1.5 mg mannitol, and 1.5 mg sucrose. Similarly, the 100
.mu.g vaccine dose consisted of two devices that each delivered 10
mg of dry powder formulation, wherein each 10 mg of dry powder
formulation consisted of 50 .mu.g of Norwalk VLP, 25 .mu.g MPL.RTM.
adjuvant, 7 mg chitosan, 1.5 mg mannitol, and 1.5 mg sucrose. The
formulation of Adjuvant/Excipient was the same as the Norwalk VLP
vaccine except that no Norwalk VLP antigen was included in the
formulation. The formulation of the Adjuvant/Excipient (also
referred to as dry powder matrix) is summarized in Table 4 in
Example 2. The placebo group received two empty devices (air
puffs).
[0089] The volunteers kept a daily diary of symptoms (including
local symptoms such as: nasal discharge, nasal pain/discomfort,
nasal congestion, runny nose, nasal itching, nose bleed, headache
and systemic symptoms such as: daily oral temperature, myalgia,
nausea, vomiting, abdominal cramps, diarrhea, and loss of appetite)
for 7 days after receiving either one of the two doses of the
Norwalk VLP vaccine, dry powder matrix alone, or the placebo.
Interim medical histories were obtained at each follow-up visit
(days 7+1, 21+2, 28+2, 56+2 and 180+14); volunteers were queried
about interim illness, medications, and doctor's visits. Volunteers
were asked to report all serious or severe adverse events including
events that were not solicited during follow up visits. Volunteers
had CBC and serum creatinine, glucose, AST, and ALT assessed on
days 7 and 28 (7 days after each immunization) and, if abnormal,
the abnormal laboratory test was followed until the test became
normal or stabilized.
[0090] The safety data were very similar to those described in
Table 5 for the study in Example 2. After Dose 1 or Dose 2 of the
100 .mu.g dosage of vaccine local nasal symptoms were reported by
19 of 20 subjects and 18 of 20 subjects, respectively. Likewise in
the MPL plus chitosan control group without Norwalk antigen
(adjuvant/excipient group), 10 of 10 subjects and 8 of 10 subjects
reported local nasal symptoms after Dose 1 or Dose 2, respectively.
In the true placebo group, 8 of 11 (73%) subjects and 3 of 11 (27%)
subjects who received a puff of air (no dry powder) reported local
nasal symptoms after Dose 1 or Dose 2, respectively. Headache and
malaise were the most common systemic symptoms observed across the
study groups. After Dose 1 or Dose 2 of the 100 .mu.g dosage of
vaccine headache was reported in 35% and 47.4% of subjects,
respectively. In the adjuvant/excipient (MPL plus chitosan) control
group, 30% and 22.2% of subjects reported headache after Dose 1 or
Dose 2, respectively. In the true placebo recipients, 36.4% of
subjects and 18.2% of subjects reported headache after Dose 1 and
Dose 2, respectively.
[0091] Clinical laboratory abnormalities were infrequent and
observed with similar frequency across the study groups. Severe
(Grade 3) hematologic abnormalities were not observed. Two severe
(Grade 3) chemistry abnormalities were observed; an elevated AST in
a recipient of the 50 .mu.g dosage of vaccine and a decreased
glucose in a placebo recipient. One serious adverse event not
related to the vaccine was reported in the 180 day safety period; a
hospitalization for appendectomy 111 days after the second dose of
vaccine. No new onset medically significant medical conditions were
reported in the 180 day safety period. These results demonstrate
that the Norwalk vaccine containing higher doses of antigen is well
tolerated and generally safe in human patients.
[0092] To analyze the immunogenicity of the Norwalk vaccine, blood
was collected before immunization and on days 7+1, 21+2, 28+2,
56+2, and 180+14 to measure serum antibodies to the Norwalk VLP
vaccine by enzyme-linked immunosorbent assays (ELISA). Before and
on day 7 after administration of each dose of vaccine, dry powder
matrix alone, or placebo, peripheral blood lymphocytes were
collected to detect antibody secreting cells by ELISPOT assay.
Before and on days 21+2, 56+2 and 180+14 after vaccination, whole
blood was obtained to separate cells and freeze for future studies
of cell mediated immunity, including cytokine production in
response to Norwalk VLP antigen, and lymphoproliferation. Blood was
screened for memory B-cells on days 0, 21, 56 and 180.
[0093] Methods used to analyze the blood samples collected from
immunized individuals, or individuals receiving the dry powder
matrix alone or placebo are described in detail in Example 2.
[0094] Serum samples were collected before immunization and on days
7, 21, 28, 56, and 180 days after intranasal administration of the
first dose of vaccine. The second dose of vaccine was administered
on day 21. Purified Norwalk VLPs were used as the target antigen to
detect specific serum IgG and IgA endpoint antibody titers
determined in comparison to a reference standard curve for each
antibody class as previously described (Tacket et al. (2003) Clin.
Immunol., Vol. 108:241-247; Gray et al. (1994) J. Clin. Microbiol.,
Vol. 32:3059-63). Geometric mean titers (GMTs), geometric mean of
fold rises (GMFRs) and seroconversion rates (.gtoreq.4-fold rises)
were determined. Norwalk VLP-specific IgG and IgA antibody
seroconversion rates and GMFRs are presented in Table 7. No
subjects developed .gtoreq.4-fold rises in serum IgM antibody. As
shown in Table 7, 12 of 19 subjects (63%) in the 100 .mu.g group
seroconverted with IgG antibodies at day 56 and 15 of 19 subjects
(79%) seroconverted with IgA antibodies. The GMTs pre- and
post-vaccination are presented in FIGS. 4C and D. Both vaccine
groups (50 and 100 .mu.g) induced strong serum IgA and IgG
responses that were significantly higher than the two control
groups.
TABLE-US-00007 TABLE 7 Anti-Norwalk VLP Specific IgG and IgA
Antibody Seroconversion Rates (% .gtoreq.4-fold rise) and Geometric
Mean Fold Rise (GMFR) by Group at Day 56 (35 Days Post-Vaccination
2) Compared to Baseline Pre-Vaccination Serum IgG .gtoreq. Serum
IgA .gtoreq. 4-fold rise Serum IgG 4-fold rise Serum IgA n/N (%)
GMFR n/N (%) GMFR 50 .mu.g Norwalk Vaccine 10/18 (56%) 4.6 (2.5,
8.6) 13/18 (72%) 7.6 (4.2, 13.8) 100 .mu.g Norwalk Vaccine 12/19
(63%) 4.8 (3.2, 7.1) 15/19 (79%) 9.1 (4.7, 17.6) MPL plus chitosan
control 0/9 (0%) 1.1 (0.9, 1.4) 0/9 (0%) 1.0 (0.8, 1.3)
(adjuvant/excipient) Placebo Control 0/11 (0%) 0.9 (0.8, 1.1) 0/11
(0%) 1.2 (0.9, 1.5)
[0095] To ascertain the functional antibody response in the various
immunization groups, sera was obtained from immunized patients at
various points following immunization and analyzed for its ability
to inhibit hemagglutination of red blood cells by Norwalk VLPs (a
functional assay which indicates protective immune responses) as
described in Example 2. Hemagglutination inhibition (HAI) titers
were calculated as the inverse of the highest dilution that
inhibited hemagglutination, with a compact negative RBC pattern
(button of RBCs). The vaccine-induced antibodies were also examined
in their capacity to inhibit hemagglutination (HAI) of O-type human
RBCs by Norwalk VLP. HAI titers (GMTs, GMFRs and .gtoreq.4-fold
rises) are presented in Table 8 and the Norwalk-specific GMTs are
presented in FIG. 5. Among subjects who received the 100 .mu.g
dosage of vaccine, the geometric mean HAI antibody titers peaked
after the second dose with a GMFR of 9.1 (CI 4.0, 20.7) and
seroconversion occurred in 73.7% of these subjects. The HAI titer
can be a good indicator of protective immunity because this
measurement reflects the level of functional antibodies that likely
block Norovirus entry. The results of these experiments show that
the Norovirus vaccine, especially at the 100 .mu.g dosage, can
induce a significant HAI titer in humans following immunization
suggesting that the vaccine likely induces a protective
immunity.
TABLE-US-00008 TABLE 8 Specific Hemagglutination Inhibition
Antibody Geometric Mean Titers, Geometric Mean Fold Rises, and
Seroconversion Rates by Group Day 56 (35 Days Post Day 180 (159
Days Post Day 21 (Pre-Vaccination 2) Vaccination 2) Vaccination 2)
% .gtoreq. % .gtoreq. % .gtoreq. Baseline 4- 4- 4- (Pre- Fold Fold
Fold Vaccination) GMFR Rise GMT GMFR Rise GMT GMFR Rise GMT GMT
(95% (95% (95% (95% (95% (95% (95% (95% N (95% CI) N (95% CI) CI)
CI) N CI) CI) CI) N CI) CI) CI) 50 .mu.g 18 13.2 18 32.0 2.4 33.3
18 52.8 4.0 38.9 17 54.4 4.0 47.1 Norwalk (8.6, (14.9, (1.2, (13.3,
(25.8, (2.0, (17.3, (25.4, (1.9, (23.0, Vaccine 20.1) 68.9) 4.8)
59.0) 108.2) 7.9) 64.3) 116.4) 8.3) 72.2) 100 .mu.g 19 25.7 19
111.9 4.4 63.2 19 234.9 9.1 73.7 19 151.6 5.9 57.9 Norwalk (14.7,
(41.7, (2.1, (38.4, (79.7, (4.0, (48.8, (56.9, (2.9, (33.5, Vaccine
44.9) 300.5) 9.0) 83.7) 692.8) 20.7) 90.9) 403.9) 12.1) 79.7) MPL 9
6.9 (4.1, 9 9.3 (5.2, 1.4 0.0 9 9.3 1.4 0.0 9 8.6 1.3 11.1 plus
11.5) 16.7) (1.0, (0.0, (4.7, (1.0, (0.0, (4.9, (0.9, (0.3,
Chitosan 1.8) 33.6) 18.7) 1.8) 33.6) 15.2) 1.8) 48.2) Placebo 11
12.4 11 19.3 1.6 9.1 11 21.9 1.8 9.1 10 19.7 1.7 10.0 Control (5.7,
(7.8, (0.9, (0.2, (8.2, (0.9, (0.2, (7.7, (1.0, (0.3, 27.3) 48.0)
2.7) 41.3) 58.7) 3.5) 41.3) 50.2) 2.9) 44.5)
[0096] ASC assays were conducted to detect circulating mononuclear
cells secreting IgG and IgA antibodies to Norwalk VLPs (Tacket et
al. (2003) Clin. Immunol., Vol. 108:241-247). Twenty-five mL of
heparinized blood were collected from each subject on days 0, 7,
21, and 28 (prior to and 7 days after administration of the first
and second dose of vaccine or controls). The response rate and mean
number of ASCs per 10.sup.6 peripheral blood mononuclear cells
(PBMCs) were assessed. A positive response was defined as a
post-vaccination ASC count that consisted of at least >8 spots
per 10.sup.6 PBMCs and was at least 3 standard deviations (SD)
above the mean pre-vaccination count for all subjects. Norwalk
VLP-specific IgG and IgA circulating ASC were detected at day 7,
waned at day 21 (immediately prior to Dose 2), and reappeared at
study day 28, seven days after Dose 2 (Table 9). In Study 1
(Example 2), seven (39%) of 18 subjects who were evaluated and
received any vaccine dosage developed rises in specific IgA ASC at
day 7, and 10 (53%) of 19 subjects had ASC responses at day 28
(Table 9). In this study (Example 3), all 10 subjects evaluated
(100%) who received 50 or 100 .mu.g of vaccine developed IgA ASCs
at day 7 and at day 28.
TABLE-US-00009 TABLE 9 Norwalk VLP-Specific IgA Antibody Secreting
Cell (ASC) Response Rate and ASC Geometric Mean Response by Group
by Study IgA ASC GM of cells per 10.sup.6 IgA ASC Response Rate
PBMC Day 7 Day 21 Day 28 Day 7 Day 21 Day 28 Study 1 (Example 2) 5
.mu.g 0/3 (0%) 0/5 (0%) 1/5 (20%) 0.5 (--) 0.36 (--) 0.9 (--)
Norwalk Vaccine 15 .mu.g 0/5 (0%) 0/5 (0%) 4/5 (80%) 0.6 (--) 0.35
(--) 12.1 Norwalk Vaccine 50 .mu.g 7/10 (70%) 1/10 (10%) 5/9 (56%)
9.2 1.50 8.0 Norwalk Vaccine MPL plus 0/8 (0%) 0/8 (0%) 0/8 (0%)
0.2 (--) 0.19 (--) 0.4 (--) Chitosan Control Study 2 (Example 3) 50
.mu.g 5/5 (100%) 0/5 (0%) 5/5 (100%) 50.2 0.3 (--) 16.5 Norwalk
Vaccine 100 .mu.g 5/5 (100%) 1/5 (20%) 5/5 (100%) 138.3 1.6 71.1
Norwalk Vaccine MPL plus 0/3 (0%) 0/3 (0%) 0/3 (0%) 0.1 (--) 0.1
(--) 0.1 (--) Chitosan Control Placebo 0/2 (0%) 0/2 (0%) 0/2 (0%)
0.1 (--) 0.1 (--) 0.1 (--) Control The geometric mean
pre-administration (Day 0) ASC responses were all <1. The symbol
(--) indicates a negative response.
Discussion
[0097] Norwalk VLP-specific IgG and IgA seroconversion rates and
GMFRs are presented in Table 10, and the kinetics of antibody
production (GMTs before and after vaccination) are presented in
FIG. 4. In Study 1 (Example 2), the seroconversion rates showed a
dose-dependent response with increased titers as the dosage of
vaccine antigen increased; a logistic regression with dose as a
continuous variable results in a chi-sq p-value <0.01 for IgG
seroconversion rates and a chi-sq p-value <0.02 for IgA
seroconversion rates. In Study 2 (Example 3), 12 (63%) of 19
subjects in the 100 .mu.g group seroconverted for IgG antibodies
and 15 (79%) of 19 subjects seroconverted for IgA antibodies at day
56 (Table 10). The 100 .mu.g group developed higher titers than the
50 .mu.g group but the differences were not statistically
significant. Both vaccine groups developed higher serum IgG and IgA
responses than the two control groups; a logistic regression with
dose as a continuous variable results in a chi-sq p-value <0.001
for IgG and IgA seroconversion rates. No subjects developed
.gtoreq.4-fold rises in serum IgM antibody (data not shown).
TABLE-US-00010 TABLE 10 Norwalk VLP-Specific IgG and IgA Antibody
Seroconversion Rates (percent of subjects with .gtoreq.4-fold rise)
and Geometric Mean Fold Rise (GMFR) by Group by Study at Day 56 (35
Days Post-Vaccination 2) Compared to Pre-Vaccination Serum IgG
.gtoreq. Serum IgA .gtoreq. 4-fold rise Serum IgG 4-fold rise Serum
IgA n/N (%) GMFR n/N (%) GMFR Study 1 (Example 2) 5 .mu.g Norwalk
Vaccine 0/5 (0%) 0.9 0/5 (0%) 1.2 15 .mu.g Norwalk Vaccine 2/5
(40%) 1.9 2/5 (40%) 2.5 50 .mu.g Norwalk Vaccine 7/9 (78%) 4.7 5/9
(56%) 4.5 MPL plus chitosan control 1/8 (13%) 1.0 0/8 1.0 Study 2
(Example 3) 50 .mu.g Norwalk Vaccine 10/18 (56%) 4.6 (2.5, 8.6)
13/18 (72%) 7.6 (4.2, 13.8) 100 .mu.g Norwalk Vaccine 12/19 (63%)
4.8 (3.2, 7.1) 15/19 (79%) 9.1 (4.7, 17.6) MPL plus chitosan
control 0/9 (0%) 1.1 (0.9, 1.4) 0/9 (0%) 1.0 (0.8, 1.3) Placebo
Control 0/11 (0%) 0.9 (0.8, 1.1) 0/11 (0%) 1.2 (0.9, 1.5)
[0098] The immunogenicity of the adjuvanted Norwalk VLP vaccine as
measured by serum IgG and IgA antibodies and circulating Norwalk
IgG and IgA specific ASCs is notable. Mucosal priming via the nasal
mucosa was supported by the ASC responses in the peripheral blood.
ASCs appear transiently in the circulation after naive B
lymphocytes at an inductive site are exposed to antigen (e.g.,
nasal associated lymphoid tissue). ASCs return to the mucosa as
immune effector cells. Norwalk IgA-specific ASC responses were
observed 7 days after the first dose of the 100 .mu.g dosage of
vaccine in all five subjects evaluated with a geometric mean of 138
cells/10.sup.6 peripheral blood mononuclear cells. These numbers
are higher than what was previously observed after administration
of oral non-adjuvanted Norwalk VLP vaccine or after ingestion of
non-adjuvanted Norwalk VLP antigen in edible transgenic plants
(Tacket et al. (2003) Clin. Immunol., Vol. 108:241-247; Tacket et
al. (2000) J. Infect. Dis., Vol. 182:302-305). These ASC counts are
also relatively higher when compared to those induced by oral
vaccines as well as to wild-type challenges with enteric organisms
(Tacket et al. (2003) Clin. Immunol., Vol. 108:241-247; Kotloff et
al. (2001) Infec. Immun., Vol. 69:3581-3590; Kotloff et al. (2000)
Infect. Immun., Vol. 68:1034-1039; McKenzie et al. (2007) Vaccine,
Vol. 25:3684-3691; and Kotloff et al. (2007) Human Vaccines, Vol.
3:268-275).
[0099] ASC were observed in the circulation 7 days after
immunization. To investigate the expression of homing molecules
known to direct their migration to mucosal and peripheral lymphoid
tissues, PBMCs from 5 subjects were stained and sorted
simultaneously into 4 defined subsets and assessed for their
ability to secrete Norwalk IgG and IgA as described above (Table
11). The majority of IgA ASCs were observed in two main subsets:
CD19+ CD27+ CD62L+, integrin .alpha.4/.beta.7+, i.e., expressing
both peripheral lymphoid tissue and mucosal homing molecules
(.about.700 to .about.10,700 ASC/10.sup.6 sorted cells); and CD19+
CD27+ CD62L- integrin .alpha.4/.beta.7+, i.e., expressing
exclusively mucosal homing molecules (.about.2,500 to .about.6,700
ASC/10.sup.6 sorted cells). The latter was observed in 3 of 4
vaccines (Table 11).
TABLE-US-00011 TABLE 11 Norwalk VLP Specific IgA and IgG Cell
Surface Receptor Homing Markers Group P* N/MB* M/P* M* IgA (total #
of cells/10.sup.6) Placebo 0 0 0 4 50 .mu.g Vaccine 0 2 1,000 5,197
100 .mu.g Vaccine 95 14 1,999 2,666 100 .mu.g Vaccine 33 0 717 0
100 .mu.g Vaccine 0 15 10,739 6,668 IgG (total # of cells/10.sup.6)
Placebo 0 0 0 0 50 .mu.g Vaccine 0 0 450 742 100 .mu.g Vaccine 0 0
157 0 100 .mu.g Vaccine 0 0 282 0 100 .mu.g Vaccine 0 0 667 0
Sorted subpopulation phenotype CD19+ CD19+ CD19+ CD19+ CD27+ CD27-
CD27+ CD27+ CD62L+ CD62L+ CD62L- .alpha.4.beta.7- .alpha.4.beta.7+
.alpha.4.beta.7+ P* - Peripheral homing memory B/plasma cell N/MB*
- Naive mature B cell M/P* - Mucosal and peripheral homing memory
B/plasma cell M* - Mucosal homing memory B/plasma cell
[0100] In contrast, the IgG ASC, with the exception of 1 vaccine,
were of a single phenotype: CD19+ CD27+ CD62L+ integrin
.alpha.4/.beta.7+, and the frequencies (.about.150 to .about.670
ASC/10.sup.6 sorted cells) were considerably lower than those
observed for IgA ASC. One vaccine dosed at the 50 .mu.g level
exhibited IgG ASC subsets bearing both mucosal and peripheral
lymphoid tissues homing receptors (Table 11).
[0101] No IgG or IgA ASC exhibited a phenotype associated with
either naive B cells (CD19+, CD27-) or B.sub.M (CD19+, CD27+) cells
expressing CD62L in the absence of integrin .alpha.4/.beta.7, which
would presumably home exclusively to peripheral lymphoid
tissues.
[0102] The intranasal monovalent adjuvanted Norwalk VLP vaccine was
generally well tolerated and immunogenic. A second dose of vaccine
provided increased serologic antibody responses whereas the peak
increase in ASC responses occurred after Dose 1. HAI antibody (a
functional measurement) increased only at the highest dosage tested
and the fold increase (9-fold) was similar to that of the serum
IgA. Mucosal priming via the nasal mucosa was supported by the
presence of high frequencies of IgA and IgG ASCs in peripheral
blood. ASCs appear transiently in the circulation after naive B
lymphocytes at an inductive site are exposed to a foreign antigen
(e.g., vaccine-primed B cells at the nasal associated lymphoid
tissue return to the mucosa as immune effector cells). Norwalk
IgA-specific ASC responses were observed 7 days after the first
immunization in all five subjects that received the 100 .mu.g
vaccine dose, with a geometric mean of 138 cells/10.sup.6 PBMCs.
The ASC numbers reported in this study are higher than what has
been previously observed after administration of oral
non-adjuvanted Norwalk VLP vaccine or after ingestion of
non-adjuvanted Norwalk VLP antigen in edible transgenic plants
(Tacket et al. (2003) Clin Immunol., Vol. 108:241-7; and Tacket et
al. (2000) J. Infect. Dis., Vol. 182:302-305). These ASC counts are
also generally higher than those induced by live oral vaccines or
by wild-type challenges with enteric organisms (Tacket et al.
(2003) Clin Immunol., Vol. 108:241-7; Kotloff et al. (2001) Infec.
Immun., Vol. 69:3581-3590; Kotloff et al. (2000) Infect. Immun.,
Vol. 68:1034-1039; McKenzie et al. (2007) Vaccine, Vol.
25:3684-3691; and Kotloff et al. (2007) Human Vaccines, Vol.
3:268-275). Thus, these mucosally-primed ASCs in combination with
the serum IgG and IgA antibodies may contribute to protection.
Graham et al. (J Infect Dis, Vol. 170:34-43, 1994) reported a
35-fold mean increase in serum antibody titers in a population of
41 Norwalk virus infected subjects following virus challenge. Gray
et al. (J Clin Microbiol, Vol. 32:3059-63, 1994) evaluated a subset
of these sera for IgG and IgA by ELISA and observed Norwalk IgG
peak titers of approximately 15,900 from days 15 to 90 and IgA peak
titers of approximately 12,600 from days 24 and 90. A comparison of
IgG and IgA between challenged and vaccinated subjects is
planned.
[0103] These results show for the first time the presence of
circulating Norwalk-specific IgA and IgG ASC following intranasal
vaccination with adjuvanted VLP. Hence, it was important to study
the homing characteristics of these effector cells. It is widely
accepted that CD62L is a key molecule implicated in the initial
phase of migration through high endothelial venules (HEV) in
lymphoid tissues, including lymph nodes and Peyer's Patches,
through binding to the peripheral lymph node addressins (PNad) and
the mucosal addressin cell adhesion molecule (MAdCAM-1) present in
the HEV vascular endothelium, which results in tethering and
rolling (Brandtzaeg et al. (2005) Immunol Rev, Vol. 206:32-63;
Bargatze et al. (1995) Immunity, Vol. 3:99-108; and Shyjan et al.
(1996) J. Immunol., Vol. 156:2851-7). In contrast, at mucosal
effector sites, although a number of adhesion molecules are
involved, vascular adhesion specificity is mediated by integrin
.alpha.4/.beta.7 interacting with the MAdCAM-1 addressin
(Brandtzaeg et al. (2005) Immunol Rev, Vol. 206:32-63; Bargatze et
al. (1995) Immunity, Vol. 3:99-108; and Shyjan et al. (1996) J.
Immunol., Vol. 156:2851-7). Thus, cells expressing integrin
.alpha.4/.beta.7, but not CD62L, are destined to home to the gut
mucosa, whilst cells expressing CD62L, but not integrin
.alpha.4/.beta.7, are destined to home to peripheral and mesenteric
lymph nodes. As an example, a previous study evaluating B cell
surface markers in response to acute rotavirus infections showed
that cells homing to the gut mucosa were CD27+ integrin
.alpha.4/.beta.7+ CD62L+/- (Jaimes et al. (2004) J. Virol., Vol.
78:10967-10976).
[0104] We observed that intranasal immunization with an adjuvanted
Norovirus VLP vaccine elicited circulating VLP-specific IgA and IgG
ASC with different homing potentials. While IgA specific ASC
exhibited homing receptors likely to endow them with the ability to
home to both, the gut mucosa (CD19+ CD27+ integrin
.alpha.4/.beta.7+ CD62L-) and peripheral lymphoid tissues (CD19+
CD27+ integrin .alpha.4/.beta.7+ CD62L+), IgG ASC expressed homing
receptors that support homing to peripheral lymphoid tissues (CD19+
CD27+ integrin .alpha.4/.beta.7+ CD62L+). The fact that intranasal
immunization was able to elicit ASC with such diverse homing
profile, including the gut mucosa, is noteworthy and demonstrates
that this route has the capacity to induce potent systemic and
mucosal immune responses, including effector cells active at a
distant site of infection (i.e. the gastrointestinal tract in the
case of Norovirus virus). However, intranasal immunization was not
effective in inducing significant levels of specific IgG or IgA ASC
with the potential to home exclusively to peripheral lymphoid
tissues (Brandtzaeg et al. (2005) Immunol Rev, Vol. 206:32-63).
Example 4
Norwalk Virus Challenge Study in Humans Immunized with Norwalk
Virus VLP Vaccine Formulation (LV01-103 Study)
[0105] A multi-site, randomized, double-blind, placebo-controlled
Phase 1-2 challenge study was conducted in 80 human volunteers
immunized with the Norwalk VLP vaccine described in Example 2
above. Eligible subjects included those 18-50 years of age, in good
health, who express the H type-1 oligosaccharide (as measured by
positive salivary secretor status) and who are other than Type B or
AB blood type. Subjects who are non H type-1 secretors or who have
Type B or AB blood are reported to be more resistant to infection
with Norwalk virus and were excluded from the study. At least 80%
of volunteers were expected to be eligible based on these two
criteria.
[0106] Following screening, eligible volunteers who meet all
acceptance criteria were randomized (1:1) into one of two equal
sized cohorts with approximately 40 volunteers in each cohort.
Cohort 1 is immunized with Norwalk VLP and cohort 2 receives
placebo. Volunteers were immunized with 10 mg Norwalk VLP vaccine
in each nostril (20 mg total dry powder) or placebo. Each 10 mg of
Norwalk VLP vaccine contained 50 .mu.g of Norwalk VLP, 7 mg
chitosan, 25 .mu.g MPL.RTM., 1.5 mg of sucrose and approximately
1.5 mg of mannitol. Thus, each volunteer in cohort 1 received a
total dosage of 100 .mu.g of Norwalk VLP antigen at each
immunization. Volunteers received vaccine or placebo on study days
0 and 21.
[0107] The safety of the Norwalk virus VLP vaccine compared to
placebo was assessed. Volunteers kept a diary for 7 days following
each immunization with the vaccine or placebo to document the
severity and duration of adverse events. Serious adverse events
(SAEs) and the occurrence of any significant new medical conditions
was followed for 6 months after the last dose of vaccine or placebo
and for 4 months after the challenge with infectious virus.
[0108] All volunteers were challenged with infectious Norwalk virus
between 21 to 42 days after the second dose of vaccine or placebo
(between study days 42 and 56). Each volunteer received at or >
than the 50% Human Infectious Dose (HID 50), i.e. the amount of
infectious virus that is expected to cause disease in at least 50%
of volunteers in the placebo group. The HID 50 is between about 48
and about 480 viral equivalents of the Norwalk virus. The Norwalk
virus was mixed with sterile water and given orally. The
inoculation was preceded by ingestion of 500 mg sodium bicarbonate
in water, to prevent breakdown of the virus by stomach acid and
pepsin. A second ingestion of sodium bicarbonate solution (500 mg
sodium bicarbonate in water) was taken 5 minutes after oral
inoculation of the infectious virus. The volunteers remained at the
challenge facility for at least 4 days and at least 18 hours after
symptoms/signs of acute gastroenteritis (vomiting, diarrhea, loose
stool, abdominal pain, nausea, and fever) were absent.
[0109] Several metrics were monitored to determine the efficacy of
the Norwalk VLP vaccine in preventing or reducing symptoms/signs of
acute gastroenteritis induced by the viral challenge. All
volunteers recorded their clinical symptoms of acute
gastroenteritis and these symptoms were documented by the research
staff at the study sites. Disease symptoms/signs from cohort 1
receiving the vaccine were compared to cohort 2 placebo
recipients.
[0110] Sera, saliva, and stool samples were routinely collected
from all volunteers prior to immunization with the vaccine or
placebo, and after challenge. Serum samples were analyzed by ELISA
for IgA and IgG, titers against the Norwalk VLPs. Serum samples
were also analyzed for carbohydrate blocking activity by
hemagglutination inhibition (HAI) assay. The Norwalk antigen and
Norwalk RNA were tested in stool samples by ELISA and PCR,
respectively, which indicate the presence of virus, the amount of
virus shed from the intestines, and the duration of viral shedding.
Subjects who became ill after challenge, were subject to additional
laboratory studies including serum chemistries, BUN, creatinine,
and liver function tests until symptoms/signs resolved.
[0111] Methods of collecting and analyzing the serum samples are
similar to those described in Example. Methods of collecting and
analyzing the saliva and stool samples are described below.
Collections of Stool and Saliva for Anti-Norwalk VLP sIgA
[0112] Anti-recombinant Norwalk Virus IgA is measured in stool and
saliva samples. Saliva specimens are treated with protease
inhibitors (i.e. AEBSF, leupeptin, bestatin, and aprotinin) (Sigma,
St. Louis, Mo.), stored at -70.degree. C., and assayed using a
modification of a previously described assay (Mills et al. (2003)
Infect. Immun. 71: 726-732). Stool was collected before vaccination
and after viral challenge, and specimens were stored at -70.degree.
C. until analysis. The specimens are thawed, and protease inhibitor
buffer added to prepare a 10% w/v stool suspension. Stool
supernatants are assayed for recombinant Norwalk Virus
(rNV)-specific mucosal IgA by ELISA, as described below.
[0113] Approximately 2-3 mL of whole saliva was collected before
vaccination and after viral challenge. Saliva was collected by a
commercially available device (Salivette, Sarstedt, Newton, N.C.),
in which a Salivette swab is chewed or placed under the tongue for
30-45 seconds until saturated with saliva. Saliva was collected
from the swab by centrifugation.
Measurement of Anti-Norwalk VLP in Stool and Saliva
[0114] ELISAs, utilizing plates coated with either anti-human IgA
antibody reagents or target rNV VLP antigen coatings, are performed
to determine total IgA and to titer the specific anti-VLP IgA
responses for each specimen. Total or specific IgA are revealed
with HRP-labeled anti-human IgA as described above. An internal
total IgA standard curve is included to quantify the IgA content.
Response is defined as a 4-fold rise in specific antibody.
[0115] Results from the vaccine group (cohort 1) and the placebo
group (cohort 2) are compared to assess the protective efficacy of
the vaccine against Norovirus disease overall (primary endpoint),
and/or its efficacy in ameliorating the symptoms/signs (severity
and # of days of illness) and/or the reduction of the presence, the
amount and/or the duration of virus shedding (secondary
endpoints).
[0116] The present invention is not to be limited in scope by the
specific embodiments described which are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed, various modifications of the invention,
in addition to those shown and described herein, will become
apparent to those skilled in the art from the foregoing description
and accompanying drawings using no more than routine
experimentation. Such modifications and equivalents are intended to
fall within the scope of the appended claims.
[0117] All publications, patents and patent applications mentioned
in this specification are herein incorporated by reference into the
specification to the same extent as if each individual publication,
patent or patent application was specifically and individually
indicated to be incorporated herein by reference.
[0118] Citation or discussion of a reference herein shall not be
construed as an admission that such is prior art to the present
invention.
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