U.S. patent application number 16/924032 was filed with the patent office on 2020-10-22 for computationally optimized broadly reactive antigens for influenza.
This patent application is currently assigned to University of Pittsburgh - Of the Commonwealth System of Higher Education. The applicant listed for this patent is University of Pittsburgh - Of the Commonwealth System of Higher Education. Invention is credited to Brendan M. Giles, Ted M. Ross.
Application Number | 20200330584 16/924032 |
Document ID | / |
Family ID | 1000004939888 |
Filed Date | 2020-10-22 |
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United States Patent
Application |
20200330584 |
Kind Code |
A1 |
Ross; Ted M. ; et
al. |
October 22, 2020 |
COMPUTATIONALLY OPTIMIZED BROADLY REACTIVE ANTIGENS FOR
INFLUENZA
Abstract
The development of a computationally optimized influenza HA
protein that elicits broadly reactive immune response to all H5N1
influenza virus isolates is described. The optimized HA protein was
developed through a series of HA protein alignments, and subsequent
generation of consensus sequences, for clade 2 H5N1 influenza virus
isolates. The final consensus HA amino acid sequence was reverse
translated and optimized for expression in mammalian cells.
Influenza virus-like particles containing the optimized HA protein
are an effective vaccine against H5N1 influenza virus infection in
animals.
Inventors: |
Ross; Ted M.; (Athens,
GA) ; Giles; Brendan M.; (Centennial, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
University of Pittsburgh - Of the Commonwealth System of Higher
Education |
Pittsburgh |
PA |
US |
|
|
Assignee: |
University of Pittsburgh - Of the
Commonwealth System of Higher Education
Pittsburgh
PA
|
Family ID: |
1000004939888 |
Appl. No.: |
16/924032 |
Filed: |
July 8, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16128917 |
Sep 12, 2018 |
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16924032 |
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15463960 |
Mar 20, 2017 |
10098946 |
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16128917 |
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14511930 |
Oct 10, 2014 |
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15463960 |
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13822844 |
Mar 13, 2013 |
8883171 |
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PCT/US2011/051072 |
Sep 9, 2011 |
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14511930 |
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61403407 |
Sep 14, 2010 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2039/5258 20130101;
C12N 2760/16123 20130101; C12N 2760/16323 20130101; A61K 39/00
20130101; C07K 2319/00 20130101; C12N 2760/16122 20130101; A61K
2039/545 20130101; C07K 14/11 20130101; A61K 2039/55505 20130101;
C12N 2800/22 20130101; A61K 2039/575 20130101; C12N 7/00 20130101;
A61K 39/145 20130101; C12N 2760/16223 20130101; C07K 14/005
20130101; C12N 2760/16234 20130101; C12Y 302/01018 20130101; C12N
2760/16334 20130101; C12N 2760/16134 20130101; C12N 9/2402
20130101; C12N 2760/16143 20130101; A61K 2039/54 20130101; C12N
2760/16171 20130101; A61K 39/39 20130101; C12N 15/86 20130101; A61K
39/12 20130101; A61K 2039/552 20130101 |
International
Class: |
A61K 39/145 20060101
A61K039/145; A61K 39/12 20060101 A61K039/12; C07K 14/005 20060101
C07K014/005; C07K 14/11 20060101 C07K014/11; A61K 39/39 20060101
A61K039/39; C12N 7/00 20060101 C12N007/00; C12N 9/24 20060101
C12N009/24; C12N 15/86 20060101 C12N015/86 |
Claims
1. A method of eliciting a broadly reactive immune response against
influenza virus in a subject, comprising generating an optimized
influenza virus polypeptide sequence comprising the steps of: (i)
obtaining the amino acid sequences of the polypeptide from a group
of influenza virus isolates, wherein the influenza virus isolates
are from the same subtype; (ii) organizing the amino acid sequences
of the polypeptide from the group of influenza virus isolates by
clade or sub-clade and then by geographical region within each
clade or sub-clade; (iii) aligning the amino acid sequences within
each geographical region to generate primary consensus sequences,
wherein each geographic region is represented by a primary
consensus sequence; (iv) aligning the primary consensus sequences
to generate secondary consensus sequences, wherein each clade or
sub-clade is represented by a secondary consensus sequence; and (v)
aligning the secondary consensus sequences, thereby generating the
optimized influenza virus polypeptide sequence; and administering
the optimized influenza virus polypeptide to the subject.
2. The method of claim 1, wherein generating the optimized
influenza virus polypeptide sequence further comprises: (vi)
reverse translating the optimized influenza virus polypeptide
sequence to generate a coding sequence; and (vii) optimizing the
coding sequence for expression in mammalian cells.
3. The method of claim 1, comprising administering a virus-like
particle (VLP) comprising the optimized influenza virus polypeptide
sequence to the subject.
4. The method of claim 3, wherein the influenza virus is an H5N1
virus and the VLP elicits a broadly reactive immune response
against H5N1 influenza.
5. The method of claim 4, wherein the VLP elicits a protective
immune response against at least 80% of known H5N1 isolates.
6. The method of claim 1, wherein the geographical region is a
continent.
7. The method of claim 1, wherein the geographical region is a
country.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This is a continuation of U.S. application Ser. No.
16/128,917, filed Sep. 12, 2018, which is a continuation of U.S.
application Ser. No. 15/463,960, filed Mar. 20, 2017, issued as
U.S. Pat. No. 10,098,946 on Oct. 16, 2018, which is a divisional of
U.S. application Ser. No. 14/511,930, filed Oct. 10, 2014, now
abandoned, which is a divisional of U.S. application Ser. No.
13/822,844, filed Mar. 13, 2013, issued as U.S. Pat. No. 8,883,171
on Nov. 11, 2014, which is the U.S. National Stage of International
Application No. PCT/US2011/051072, filed Sep. 9, 2011, published in
English under PCT Article 21(2), which claims the benefit of U.S.
Provisional Application No. 61/403,407, filed Sep. 14, 2010. The
above-listed applications are herein incorporated by reference in
their entirety.
FIELD
[0002] This disclosure concerns an optimized influenza
hemagglutinin protein that elicits broadly reactive immune
responses to H5N1 virus isolates and its use as a vaccine.
BACKGROUND
[0003] Influenza virus is a member of Orthomyxoviridae family.
There are three subtypes of influenza viruses, designated influenza
A, influenza B, and influenza C. The influenza virion contains a
segmented negative-sense RNA genome, which encodes the following
proteins: hemagglutinin (HA), neuraminidase (NA), matrix (M1),
proton ion-channel protein (M2), nucleoprotein (NP), polymerase
basic protein 1 (PB1), polymerase basic protein 2 (PB2), polymerase
acidic protein (PA), and nonstructural protein 2 (NS2). The HA, NA,
M1, and M2 are membrane associated, whereas NP, PB1, PB2, PA, and
NS2 are nucleocapsid associated proteins. The M1 protein is the
most abundant protein in influenza particles. The HA and NA
proteins are envelope glycoproteins, responsible for virus
attachment and penetration of the viral particles into the cell,
and the sources of the major immunodominant epitopes for virus
neutralization and protective immunity. Both HA and NA proteins are
considered the most important components for prophylactic influenza
vaccines.
[0004] Each year, seasonal influenza causes over 300,000
hospitalizations and 36,000 deaths in the U.S. alone (Simonsen et
al., Lancet Infect Dis 7:658-66, 2007). The emergence of the novel
H1N1 influenza virus in 2009 demonstrated how quickly a new
influenza pandemic can sweep across the world. The spread of highly
pathogenic H5N1 viruses in birds and coincident infections in
humans have raised the concerns that H5N1 viruses may cause a new
pandemic in humans. Vaccination is an effective method to prevent
influenza infection. There are two influenza vaccine approaches
licensed in the United States; the inactivated, split vaccine and
the live-attenuated virus vaccine. Inactivated vaccines can
efficiently induce humoral immune responses but generally only poor
cellular immune responses. Thus, a need exists for a broadly
protective influenza virus vaccine.
SUMMARY
[0005] Disclosed herein is the development of an optimized
influenza HA protein that elicits broadly reactive immune response
to H5N1 influenza virus isolates. The optimized HA protein was
developed through a series of HA protein alignments, and subsequent
generation of consensus sequences for clade 2 H5N1 influenza virus
isolates (FIG. 1). The final consensus HA amino acid sequence was
reverse translated and optimized for expression in mammalian cells.
The optimized HA coding sequence is set forth herein as SEQ ID NO:
1, and the optimized HA protein sequence is set forth herein as SEQ
ID NO: 2.
[0006] Provided herein is an isolated nucleic acid molecule
comprising a nucleotide sequence encoding an optimized influenza HA
polypeptide, wherein the nucleotide sequence encoding the HA
polypeptide is at least 94%, at least 95%, at least 96%, at least
97%, at least 98% or at least 99% identical to SEQ ID NO: 1.
Optimized influenza HA polypeptides encoded by the nucleic acid
molecule, vectors comprising the nucleic acid molecule, and host
cells containing the disclosed vectors are also provided
herein.
[0007] Further provided is an optimized influenza HA polypeptide,
wherein the amino acid sequence of the polypeptide is at least 99%
identical to SEQ ID NO: 2. Also provided are fusion proteins
comprising the optimized HA polypeptide, virus-like particles
(VLPs) containing the optimized HA polypeptides, and compositions
comprising the optimized HA polypeptide.
[0008] Collections of plasmids are also provided herein. In some
embodiments, the collections of plasmids include a plasmid encoding
an influenza NA, a plasmid encoding an influenza MA, and a plasmid
encoding the optimized HA protein disclosed herein.
[0009] Further provided is a method of eliciting an immune response
to influenza virus in a subject by administering the optimized
influenza HA protein, fusion proteins containing the optimized
influenza HA, or VLPs containing the optimized influenza HA, as
disclosed herein. Also provided is a method of immunizing a subject
against influenza virus by administering to the subject VLPs
containing the optimized influenza HA protein disclosed herein.
[0010] The foregoing and other objects and features of the
disclosure will become more apparent from the following detailed
description, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE FIGURES
[0011] FIGS. 1A-1B: COBRA HA Design. (FIG. 1A) Schematic
illustrating the design of the COBRA HA molecule. The phylogenetic
tree was inferred from hemagglutinin amino acid sequences using the
maximum likelihood method and clade/sub-clade groupings were
identified. Primary consensus sequences were generated for each
outbreak group. Secondary consensus sequences were then generated
for each sub-clade using the primary sequences as input. The
secondary consensus sequences were then aligned and the resulting
consensus, designated COBRA, was generated. (FIG. 1B) Phylogenetic
analysis of the COBRA HA. The unrooted phylogenetic tree was
inferred from hemagglutinin amino acid sequences from human H5N1
infections isolated from 2004 to 2009 and the clade/sub-clade
groupings are indicated. The star represents the COBRA HA sequence
relative to human H5N1 infections.
[0012] FIGS. 2A-2C: COBRA HA Functional Characterization. COBRA HA
was translated in vitro and the cell culture lysates were analyzed
by SDS-PAGE (FIG. 2A). Lane designations: 1) H5N1 recombinant HA;
2) COBRA HA; 3) Expression vector; 4) H5N1 reassortant virus. The
COBRA HA (lane 2) migrates at its expected molecular weight
confirming expression of the synthetic protein. COBRA HA VLPs were
prepared in various amounts, serially diluted, and incubated with
1% erythrocytes to evaluate receptor binding (FIG. 2B). HA titer
was determined as the last well in which the RBCs remained
suspended in a lattice structure. COBRA HA and control lentiviral
pseudoparticles packaging a CMV-Luc gene were generated in HEK 293T
cells and used to infect MDCK cells with or without trypsin (FIG.
2C). Particle fusion was determined by luciferase production by
infected cells.
[0013] FIGS. 3A-3F: COBRA HA Mouse Dosing Immunogenicity. BALB/c
mice (n=5/group) were vaccinated at 0 and 3 weeks with blood
collected at 14 to 21 days after each vaccination. Vaccines were
formulated at high (1.5 .mu.g HA), and low (0.03 .mu.g HA) doses,
with and without Imject.RTM. alum, and delivered intramuscularly.
Total IgG at week 5 was determined via ELISA for each vaccine group
(FIGS. 3A and 3B). Values represent the geometric mean titer
(.+-.95% confidence interval) of log.sub.10 transformed endpoint
titers. IgG isotypes were evaluated via ELISA for each vaccine
group (FIGS. 3C and 3D). Values represent the mean OD.sub.450 of a
1:200 dilution of serum. Hemagglutination inhibition (HAI) serum
antibody titer for each vaccine group was determined at week 5
using representative reassortant viruses (FIGS. 3E and 3F). Values
represent the geometric mean titer (.+-.95% confidence interval) of
log 2 transformed titers. The dotted line represents the 1:40
titer. Significant differences were determined by two-way ANOVA
with Bonferroni's post-test to evaluate differences between the
vaccine formulations for each test antigen. A p value of less than
0.05 was considered significant.
[0014] FIGS. 4A-4D: COBRA HA Mouse Dosing Efficacy. BALB/c mice
(n=5/group) were vaccinated with COBRA HA VLPs with or without
adjuvant. Mice were infected with 5.times.10.sup.3 PFU of the
highly pathogenic clade 2.2 H5N1 virus A/Whooper
Swan/Mongolia/244/2005. Mice were followed to monitor weight loss
(FIGS. 4A and 4B) and sickness (FIGS. 4C and 4D). Sickness score
was determined by evaluating activity (0=normal, 1=reduced,
2=severely reduced), hunched back (0=absent, 1=present) and ruffled
fur (0=absent, 1=present). All mock vaccinated mice reached the
experimental endpoint and required euthanasia by 6 days post
infection.
[0015] FIGS. 5A-5B: Mouse Comparison Immunogenicity. BALB/c mice
(n=20/group) were vaccinated at 0 and 3 weeks with blood collected
at 14 to 21 days after each vaccination. Vaccines were formulated
at a high dose (3 .mu.g HA) with Imject.RTM. alum and delivered
intramuscularly. Total IgG at week 5 was determined via ELISA for
each vaccine group (FIG. 5A). Values represent the geometric mean
titer (.+-.95% confidence interval) of log.sub.10 transformed
endpoint titers. Hemagglutination inhibition (HAI) serum antibody
titer for each vaccine group was determined at week 5 using
representative reassortant viruses (FIG. 5B). Values represent the
geometric mean titer (.+-.95% confidence interval) of log 2
transformed titers. The dotted line represents the 1:40 titer.
Significant differences were determined by two-way ANOVA with
Bonferroni's post-test to evaluate differences between the vaccine
formulations for each test antigen. A p value of less than 0.05 was
considered significant.
[0016] FIGS. 6A-6B: Mouse Comparison Efficacy. BALB/c mice
(n=20/group) were vaccinated with VLPs and adjuvant. Mice were
infected with 5.times.10.sup.3 PFU of the highly pathogenic clade
2.2 H5N1 virus A/Whooper Swan/Mongolia/244/2005. Mice were followed
to monitor weight loss (FIG. 6A) and sickness (FIG. 6B). Sickness
score was determined by evaluating activity (0=normal, 1=reduced,
2=severely reduced), hunched back (0=absent, 1=present) and ruffled
fur (0=absent, 1=present). All mock (adjuvant-only) vaccinated mice
reached the experimental endpoint and required euthanasia by 6 days
post infection.
[0017] FIGS. 7A-7B: Ferret Immunogenicity. Ferrets (n=9/group) were
vaccinated with VLPs (15 .mu.g HA) with Imject.RTM. alum at weeks 0
and 3 and serum collected at week 5. Total IgG at week 5 was
determined via ELISA for each vaccine group (FIG. 7A). Values
represent the geometric mean titer (.+-.95% confidence interval) of
login transformed endpoint titers. Hemagglutination inhibition
(HAI) serum antibody titer for each vaccine group was determined at
week 5 using representative reassortant viruses (FIG. 7B). Values
represent the geometric mean titer (.+-.95% confidence interval) of
log 2 transformed titers. The dotted line represents the 1:40
titer. Significant differences were determined by two-way ANOVA
with Bonferroni's post-test to evaluate differences between the
vaccine formulations for each test antigen. A p value of less than
0.05 was considered significant.
[0018] FIGS. 8A-8E: Ferret Efficacy. Ferrets (n=9/group) were
vaccinated with VLPs formulated with adjuvant. Ferrets were
challenged with 1.times.10.sup.6 PFU of the highly pathogenic clade
2.2 H5N1 virus A/Whooper Swan/Mongolia/244/2005. Animals were
monitored daily for weight loss (FIG. 8A), survival (FIG. 8B),
temperature (FIG. 8C) and clinical symptoms (FIG. 8D). Relative
sickness scores were determined by measuring lethargy (0-3), runny
nose (0-1), sneezing (0-1), loss of appetite (0-1) and diarrhea
(0-1). Animals reaching experimental endpoint were euthanized
according to institutional guidelines. Nasal washes were collected
serially post infection and virus titers determined via plaque
assay (FIG. 8E). Statistical significance was determined using a
two-way ANOVA with Bonferroni's post test. A p value of less than
0.05 was considered significant.
[0019] FIG. 9: Phylogenetic diversity of H5N1 influenza. The
unrooted phylogenetic tree was inferred from HA amino acid
sequences derived from 8 to 10 representative isolates in all
clades and sub-clades and the COBRA HA using the maximum likelihood
method. Clade/sub-clade clusters were identified and are indicated
in the shaded ovals. The star identifies where the COBRA antigen is
located relative to the various representative isolates. Sequences
were aligned with MUSCLE 3.7 software and the alignment was refined
by Gblocks 0.91b software. Phylogeny was determined using the
maximum likelihood method with PhyML software. Trees were rendered
using TreeDyn 198.3 software (Dereeper et al., Nucleic Acids Res
36:W465-W469, 2008). The NCBI accession numbers for the HA
sequences used in phylogeny inference were obtained through the
Influenza Virus Resource (Bao et al., J Virol 82:596-601,
2008).
[0020] FIGS. 10A-10F: Serology. Total IgG at week 3 (FIG. 10A) and
week 6 (FIG. 10B) was determined via ELISA for each vaccine group.
Each collected antiserum was assayed for antibody binding to
representative HA molecules from clade 2.1 (A/Indonesia/5/2005),
clade 2.2 (A/Whooper Swan/Mongolia/244/2005), and clade 2.3
(A/Anhui/1/2005). Values represent the geometric mean titer
(.+-.95% confidence interval) of log.sub.10 transformed endpoint
titers. Statistical significance of the antibody titer data was
determined using a two-way analysis of variance (ANOVA) followed by
Bonferroni's post-test to analyze differences between each vaccine
group for each of the different antigens that were tested
(multiparametric). Significance was defined as p<0.05.
Statistical analyses were performed with GraphPad Prism software.
HAI titer for each vaccine group was determined at week 3 (FIG.
10C) and week 6 (FIG. 10D) using representative H5N1 influenza
viruses: clade 2.1 (A/Indonesia/5/2005), clade 2.2 (A/Whooper
swan/Mongolia/244/2005), and clade 2.3 (A/Anhui/1/2005). Values
represent the geometric mean titer (.+-.95% confidence interval) of
log 2 transformed titers. The dotted line represents the 1:40
titer. Significant differences were determined by two-way ANOVA
with Bonferroni's post-test to evaluate differences between the
vaccine formulations for each test antigen. A p value of less than
0.05 was considered significant. The number of monkeys that
responded with a titer greater than 1:40 is listed above each bar.
Neutralizing antibody at week 3 (FIG. 10E) and week 6 (FIG. 10F)
was determined via microneutralization assay for each vaccine
group. Values represent the geometric mean titer (.+-.95%
confidence interval).
[0021] FIG. 11: HAI serum antibody titers from vaccinated monkeys
against a panel of clade 0, 1, 2, 4, and 7 isolates. HAI titer for
each vaccine group was determined at week 9 using H5N1 influenza
viruses. Values represent the geometric mean titer (.+-.95%
confidence interval) of log 2 transformed titers. Significant
differences were determined by two-way ANOVA with Bonferroni's
post-test to evaluate differences between the vaccine formulations
for each test antigen. A p value of less than 0.05 was considered
significant as described in FIG. 10.
[0022] FIGS. 12A-12D: Vaccine induced serum antibody responses.
BALB/c mice (n=30/group) or Fitch ferrets (n=6/group) were
vaccinated at 0 and 3 weeks with blood collected 14 to 21 days
after each vaccination. Total IgG after the second vaccination was
determined via ELISA for each vaccine group (FIGS. 12A and 12C).
Receptor blocking antibody titers after the second vaccination were
determined via hemagglutination inhibition (HAI) for each vaccine
group (FIGS. 12B and 12D). Values represent the geometric mean of
the reciprocal dilution (+/-95% confidence interval) of the last
positive well. Significant differences between COBRA and polyvalent
vaccines were determined by a two-tailed Student's T test and a p
value of less than 0.05 was considered significant (*).
[0023] FIGS. 13A-13D: Highly pathogenic Clade 2.2 challenge.
Vaccinated BALB/c mice (n=5/group) were infected with
5.times.10.sup.3 PFU of the highly pathogenic clade 2.2 H5N1 virus
A/Whooper Swan/Mongolia/244/2005 (WS/05). Mice were monitored daily
for weight loss (FIG. 13A) and sickness (FIG. 13B). Vaccinated
Fitch ferrets (n=6/group) were infected with 1.times.10.sup.6 PFU
of the highly pathogenic clade WS/05 virus. Ferrets were monitored
daily for weight loss (FIG. 13C) and sickness (FIG. 13D). Values
represent mean (+/-SEM) for each group.
[0024] FIGS. 14A-14B: Clade 2.2 viral loads. Vaccinated BALB/c mice
(n=15/group) were infected with 5.times.10.sup.3 PFU of the highly
pathogenic clade 2.2 H5N1 virus A/Whooper Swan/Mongolia/244/2005
(WS/05). Cohorts of mice (n=5/group) were sacrificed at 1, 3 and 5
days post infection, lungs harvested, and viral load determined by
plaque assay (FIG. 14A). Vaccinated Fitch ferrets (n=6/group) were
infected with 1.times.10.sup.6 PFU of the highly pathogenic WS/05
virus. Nasal washes were collected and viral load determined by
plaque assay (FIG. 14B). Values represent mean (+/-SEM) viral titer
for each group. Significant differences between COBRA and
polyvalent vaccines were determined by a two-tailed Student's T
test and a p value of less than 0.05 was considered significant
(*).
[0025] FIGS. 15A-15B: Histopathology of infected lungs. Vaccinated
BALB/c mice (n=15/group) were infected with 5.times.10.sup.3 PFU of
the highly pathogenic clade 2.2 H5N1 virus A/Whooper
Swan/Mongolia/244/2005 (WS/05). Cohorts of mice (n=5/group) were
sacrificed at 3 days post infection and in situ hybridization (ISH)
for influenza matrix protein (MP) was performed on sections from
paraffin embedded lung tissue (FIG. 15A). Severity of influenza ISH
foci was accessed in the bronchi (FIG. 15B). Scoring: 0=no
definitive signal; 1=occasional focus; 2=focus in most fields;
3=more than one focus per field.
[0026] FIGS. 16A-16C: Clade 1 challenge. Vaccinated BALB/c mice
(n=4/group) were infected with 5.times.10.sup.3 PFU of reassortant
virus containing the HA and NA genes from the clade 1 H5N1 virus
A/Vietnam/1203/2004 (VN/04). Mice were monitored daily for weight
loss (FIG. 16A) and sickness (FIG. 16B). Values represent mean
(+/-SD) for each group. An additional cohort of vaccinated mice
(n=3/group) were infected and lungs were harvested 3 days post
infection for analysis of viral burden (FIG. 16C). Values represent
mean (+/-SEM) viral titer for each group.
[0027] FIGS. 17A-17B: Post-challenge cellular immune responses.
Vaccinated BALB/c mice (n=3/group) were infected with
5.times.10.sup.3 PFU of reassortant virus containing the HA and NA
genes from the clade 1 H5N1 virus A/Vietnam/1203/2004 (VN/04). Mice
were sacrificed 6 days post infection, lungs were harvested and the
numbers of antibody secreting cells (FIG. 17A) and IFN-.gamma.
producing cells (FIG. 17B) were determined by ELISpot assay. Values
represent the mean (+/-SEM) spots for each group.
[0028] FIGS. 18A-18B: Passive transfer clade 1 challenge. BALB/c
mice (n=10/group) were vaccinated at 0 and 3 weeks with blood
collected 14 to 21 days after each vaccination. Serum collected
after the second vaccination was pooled for each vaccine group and
administered to naive recipient mice (n=5/group). One day after
passive transfer, recipient mice were infected with
5.times.10.sup.3 PFU of reassortant virus containing the HA and NA
genes from the clade 1 H5N1 virus A/Vietnam/1203/2004 (VN/04). Mice
were monitored daily for weight loss (FIG. 18A) and sickness (FIG.
18B). Values represent mean (+/-SD) for each group. Significant
differences were determined by two-way ANOVA with Bonferroni's
post-test to evaluate differences between vaccines at each day. A p
value of less than 0.05 was considered significant (*).
SEQUENCE LISTING
[0029] The nucleic and amino acid sequences listed in the
accompanying sequence listing are shown using standard letter
abbreviations for nucleotide bases, and three letter code for amino
acids, as defined in 37 C.F.R. 1.822. Only one strand of each
nucleic acid sequence is shown, but the complementary strand is
understood as included by any reference to the displayed strand.
The Sequence Listing is submitted as an ASCII text file, created on
Jul. 7, 2020, 51.0 KB, which is incorporated by reference herein.
In the accompanying sequence listing:
[0030] SEQ ID NOs: 1 and 2 are the nucleotide and amino acid
sequences, respectively, of a codon-optimized influenza HA
(designated COBRA).
[0031] SEQ ID NOs: 3 and 4 are the nucleotide and amino acid
sequences, respectively, of a codon-optimized influenza NA.
[0032] SEQ ID NOs: 5 and 6 are the nucleotide and amino acid
sequences, respectively, of a codon-optimized influenza M1.
[0033] SEQ ID NO: 7 is the nucleotide sequence of a plasmid
encoding a codon-optimized influenza HA.
[0034] SEQ ID NO: 8 is the nucleotide sequence of a plasmid
encoding a codon-optimized influenza NA.
[0035] SEQ ID NO: 9 is the nucleotide sequence of a plasmid
encoding a codon-optimized influenza M1.
[0036] SEQ ID NO: 10 is the amino acid sequence of a T cell epitope
in H5 HA (HAs33).
[0037] SEQ ID NO: 11 is the amino acid sequence of an ovalbumin T
cell epitope (Ova257).
DETAILED DESCRIPTION
I. Abbreviations
[0038] ASC: antibody secreting cell
[0039] DPI: days post infection
[0040] HA: hemagglutinin or hemagglutination assay
[0041] HAI: hemagglutination inhibition
[0042] hRBC: horse red blood cell
[0043] IFU: infectious unit
[0044] LD.sub.50: lethal dose 50
[0045] M1: matrix protein 1
[0046] MN: microneutralization
[0047] MOI: multiplicity of infection
[0048] NA: neuraminidase
[0049] PFU: plaque form unit
[0050] RDE: receptor destroying enzyme
[0051] TCID: tissue culture infectious dose
[0052] tRBC: turkey red blood cell
[0053] VLP: virus-like particle
II. Terms and Methods
[0054] Unless otherwise noted, technical terms are used according
to conventional usage. Definitions of common terms in molecular
biology may be found in Benjamin Lewin, Genes V, published by
Oxford University Press, 1994 (ISBN 0-19-854287-9); Kendrew et al.
(eds.), The Encyclopedia of Molecular Biology, published by
Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A.
Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive
Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8).
[0055] In order to facilitate review of the various embodiments of
the disclosure, the following explanations of specific terms are
provided:
[0056] Adjuvant: A substance or vehicle that non-specifically
enhances the immune response to an antigen. Adjuvants can include a
suspension of minerals (alum, aluminum hydroxide, or phosphate) on
which antigen is adsorbed; or water-in-oil emulsion in which
antigen solution is emulsified in mineral oil (for example,
Freund's incomplete adjuvant), sometimes with the inclusion of
killed mycobacteria (Freund's complete adjuvant) to further enhance
antigenicity.
[0057] Immunostimulatory oligonucleotides (such as those including
a CpG motif) can also be used as adjuvants (for example, see U.S.
Pat. Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116;
6,339,068; 6,406,705; and 6,429,199). Adjuvants also include
biological molecules, such as costimulatory molecules. Exemplary
biological adjuvants include IL-2, RANTES, GM-CSF, TNF-.alpha.,
IFN-.gamma., G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L and 41 BBL.
[0058] Administer: As used herein, administering a composition to a
subject means to give, apply or bring the composition into contact
with the subject. Administration can be accomplished by any of a
number of routes, such as, for example, topical, oral,
subcutaneous, intramuscular, intraperitoneal, intravenous,
intrathecal and intramuscular.
[0059] Antibody: An immunoglobulin molecule produced by B lymphoid
cells with a specific amino acid sequence. Antibodies are evoked in
humans or other animals by a specific antigen (immunogen).
Antibodies are characterized by reacting specifically with the
antigen in some demonstrable way, antibody and antigen each being
defined in terms of the other. "Eliciting an antibody response"
refers to the ability of an antigen or other molecule to induce the
production of antibodies.
[0060] Antigen: A compound, composition, or substance that can
stimulate the production of antibodies or a T-cell response in an
animal, including compositions that are injected or absorbed into
an animal. An antigen reacts with the products of specific humoral
or cellular immunity, including those induced by heterologous
immunogens. In some embodiments of the disclosed compositions and
methods, the antigen is an influenza HA protein.
[0061] Attenuated: In the context of a live virus, the virus is
attenuated if its ability to infect a cell or subject and/or its
ability to produce disease is reduced (for example, eliminated)
compared to a wild-type virus. Typically, an attenuated virus
retains at least some capacity to elicit an immune response
following administration to an immunocompetent subject. In some
cases, an attenuated virus is capable of eliciting a protective
immune response without causing any signs or symptoms of infection.
In some embodiments, the ability of an attenuated virus to cause
disease in a subject is reduced at least about 10%, at least about
25%, at least about 50%, at least about 75% or at least about 90%
relative to wild-type virus.
[0062] Clade: Refers to the different categorizations of the known
influenza viruses, such as influenza A H5N1 viruses. Viruses in an
H5N1 clade are genetically related, but do not share the exact
viral genome. There are at least ten different clades of H5N1
subtypes designated in the art: clade 0 clade 1, clade 2, clade 3,
clade 4, clade 5, clade 6, clade 7, clade 8 and clade 9
(Abdel-Ghafar et al., N Engl J Med 358:261-273, 2008). Clade 2 is
further divided into sub-clades (including clade 2.1, clade 2.2,
clade 2.3, clade 2.4 and clade 2.5).
[0063] Codon-optimized: A "codon-optimized" nucleic acid refers to
a nucleic acid sequence that has been altered such that the codons
are optimal for expression in a particular system (such as a
particular species of group of species). For example, a nucleic
acid sequence can be optimized for expression in mammalian cells.
Codon optimization does not alter the amino acid sequence of the
encoded protein.
[0064] Fusion protein: A protein generated by expression of a
nucleic acid sequence engineered from nucleic acid sequences
encoding at least a portion of two different (heterologous)
proteins. To create a fusion protein, the nucleic acid sequences
must be in the same reading frame and contain to internal stop
codons. For example, a fusion protein includes an influenza HA
fused to a heterologous protein.
[0065] Geographical location or geographical region: Refers to
preselected divisions of geographical areas of the earth, for
example, by continent or other preselected territory or subdivision
(e.g., the Middle East, which spans more than one continent).
Examples of different geographical regions include countries (e.g.,
Turkey, Egypt, Iraq, Azerbaijan, China, United States), continents
(e.g., Asia, Europe, North America, South America, Oceania,
Africa), and recognized geopolitical subdivisions (such as the
Middle East).
[0066] Hemagglutinin (HA): An influenza virus surface glycoprotein.
HA mediates binding of the virus particle to a host cells and
subsequent entry of the virus into the host cell. The nucleotide
and amino acid sequences of numerous influenza HA proteins are
known in the art and are publically available, such as those
deposited with GenBank (see Table 1 for a list of GenBank Accession
Nos. of H5N1 HA sequences). HA (along with NA) is one of the two
major influenza virus antigenic determinants.
[0067] Immune response: A response of a cell of the immune system,
such as a B-cell, T-cell, macrophage or polymorphonucleocyte, to a
stimulus such as an antigen or vaccine. An immune response can
include any cell of the body involved in a host defense response,
including for example, an epithelial cell that secretes an
interferon or a cytokine. An immune response includes, but is not
limited to, an innate immune response or inflammation. As used
herein, a protective immune response refers to an immune response
that protects a subject from infection (prevents infection or
prevents the development of disease associated with infection).
Methods of measuring immune responses are well known in the art and
include, for example, measuring proliferation and/or activity of
lymphocytes (such as B or T cells), secretion of cytokines or
chemokines, inflammation, antibody production and the like.
[0068] Immunogen: A compound, composition, or substance which is
capable, under appropriate conditions, of stimulating an immune
response, such as the production of antibodies or a T-cell response
in an animal, including compositions that are injected or absorbed
into an animal. As used herein, as "immunogenic composition" is a
composition comprising an immunogen (such as an HA
polypeptide).
[0069] Immunize: To render a subject protected from an infectious
disease, such as by vaccination.
[0070] Influenza virus: A segmented negative-strand RNA virus that
belongs to the Orthomyxoviridae family. There are three types of
Influenza viruses, A, B and C. Influenza A viruses infect a wide
variety of birds and mammals, including humans, horses, marine
mammals, pigs, ferrets, and chickens. In animals, most influenza A
viruses cause mild localized infections of the respiratory and
intestinal tract. However, highly pathogenic influenza A strains,
such as H5N1, cause systemic infections in poultry in which
mortality may reach 100%. H5N1 is also referred to as "avian
influenza."
[0071] Isolated: An "isolated" biological component (such as a
nucleic acid, protein or virus) has been substantially separated or
purified away from other biological components (such as cell
debris, or other proteins or nucleic acids). Biological components
that have been "isolated" include those components purified by
standard purification methods. The term also embraces recombinant
nucleic acids, proteins or viruses, as well as chemically
synthesized nucleic acids or peptides.
[0072] Linker: One or more amino acids that serve as a spacer
between two polypeptides of a fusion protein.
[0073] Matrix (M1) protein: An influenza virus structural protein
found within the viral envelope. M1 is thought to function in
assembly and budding.
[0074] Neuraminidase (NA): An influenza virus membrane
glycoprotein. NA is involved in the destruction of the cellular
receptor for the viral HA by cleaving terminal sialic acid residues
from carbohydrate moieties on the surfaces of infected cells. NA
also cleaves sialic acid residues from viral proteins, preventing
aggregation of viruses. NA (along with HA) is one of the two major
influenza virus antigenic determinants.
[0075] Operably linked: A first nucleic acid sequence is operably
linked with a second nucleic acid sequence when the first nucleic
acid sequence is placed in a functional relationship with the
second nucleic acid sequence. For instance, a promoter is operably
linked to a coding sequence if the promoter affects the
transcription or expression of the coding sequence. Generally,
operably linked DNA sequences are contiguous and, where necessary
to join two protein-coding regions, in the same reading frame.
[0076] Optimized influenza HA protein: As used herein, "optimized
influenza HA protein" refers to the HA protein consensus sequence
generated by sequence alignments of clade 2 H5N1 influenza viruses
(as described in Example 1 below). The nucleotide sequence encoding
the optimized HA protein was further optimized for expression in
mammalian cells via codon-optimization and RNA optimization (such
as to increase RNA stability). The optimized influenza HA protein
disclosed herein (and set forth herein as SEQ ID NO: 2) is also
referred to as "COBRA."
[0077] ORF (open reading frame): A series of nucleotide triplets
(codons) coding for amino acids without any termination codons.
These sequences are usually translatable into a peptide.
[0078] Outbreak: As used herein, an influenza virus "outbreak"
refers to a collection of virus isolates from within a single
country in a given year.
[0079] Pharmaceutically acceptable vehicles: The pharmaceutically
acceptable carriers (vehicles) useful in this disclosure are
conventional. Remington's Pharmaceutical Sciences, by E. W. Martin,
Mack Publishing Co., Easton, Pa., 15th Edition (1975), describes
compositions and formulations suitable for pharmaceutical delivery
of one or more therapeutic compositions, such as one or more
influenza vaccines, and additional pharmaceutical agents.
[0080] In general, the nature of the carrier will depend on the
particular mode of administration being employed. For instance,
parenteral formulations usually comprise injectable fluids that
include pharmaceutically and physiologically acceptable fluids such
as water, physiological saline, balanced salt solutions, aqueous
dextrose, glycerol or the like as a vehicle. For solid compositions
(for example, powder, pill, tablet, or capsule forms), conventional
non-toxic solid carriers can include, for example, pharmaceutical
grades of mannitol, lactose, starch, or magnesium stearate. In
addition to biologically-neutral carriers, pharmaceutical
compositions to be administered can contain minor amounts of
non-toxic auxiliary substances, such as wetting or emulsifying
agents, preservatives, and pH buffering agents and the like, for
example sodium acetate or sorbitan monolaurate.
[0081] Plasmid: A circular nucleic acid molecule capable of
autonomous replication in a host cell.
[0082] Polypeptide: A polymer in which the monomers are amino acid
residues which are joined together through amide bonds. When the
amino acids are alpha-amino acids, either the L-optical isomer or
the D-optical isomer can be used. The terms "polypeptide" or
"protein" as used herein are intended to encompass any amino acid
sequence and include modified sequences such as glycoproteins. The
term "polypeptide" is specifically intended to cover naturally
occurring proteins, as well as those which are recombinantly or
synthetically produced. The term "residue" or "amino acid residue"
includes reference to an amino acid that is incorporated into a
protein, polypeptide, or peptide.
[0083] Conservative amino acid substitutions are those
substitutions that, when made, least interfere with the properties
of the original protein, that is, the structure and especially the
function of the protein is conserved and not significantly changed
by such substitutions. Examples of conservative substitutions are
shown below.
TABLE-US-00001 Original Residue Conservative Substitutions Ala Ser
Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln
Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met;
Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu
[0084] Conservative substitutions generally maintain (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target site, or
(c) the bulk of the side chain.
[0085] The substitutions which in general are expected to produce
the greatest changes in protein properties will be
non-conservative, for instance changes in which (a) a hydrophilic
residue, for example, seryl or threonyl, is substituted for (or by)
a hydrophobic residue, for example, leucyl, isoleucyl,
phenylalanyl, valyl or alanyl; (b) a cysteine or proline is
substituted for (or by) any other residue; (c) a residue having an
electropositive side chain, for example, lysyl, arginyl, or
histadyl, is substituted for (or by) an electronegative residue,
for example, glutamyl or aspartyl; or (d) a residue having a bulky
side chain, for example, phenylalanine, is substituted for (or by)
one not having a side chain, for example, glycine.
[0086] Preventing, treating or ameliorating a disease: "Preventing"
a disease refers to inhibiting the full development of a disease.
"Treating" refers to a therapeutic intervention that ameliorates a
sign or symptom of a disease or pathological condition after it has
begun to develop. "Ameliorating" refers to the reduction in the
number or severity of signs or symptoms of a disease.
[0087] Promoter: An array of nucleic acid control sequences which
direct transcription of a nucleic acid. A promoter includes
necessary nucleic acid sequences near the start site of
transcription. A promoter also optionally includes distal enhancer
or repressor elements. A "constitutive promoter" is a promoter that
is continuously active and is not subject to regulation by external
signals or molecules. In contrast, the activity of an "inducible
promoter" is regulated by an external signal or molecule (for
example, a transcription factor). In some embodiments herein, the
promoter is a CMV promoter.
[0088] Purified: The term "purified" does not require absolute
purity; rather, it is intended as a relative term. Thus, for
example, a purified peptide, protein, virus, or other active
compound is one that is isolated in whole or in part from naturally
associated proteins and other contaminants. In certain embodiments,
the term "substantially purified" refers to a peptide, protein,
virus or other active compound that has been isolated from a cell,
cell culture medium, or other crude preparation and subjected to
fractionation to remove various components of the initial
preparation, such as proteins, cellular debris, and other
components.
[0089] Recombinant: A recombinant nucleic acid, protein or virus is
one that has a sequence that is not naturally occurring or has a
sequence that is made by an artificial combination of two otherwise
separated segments of sequence. This artificial combination is
often accomplished by chemical synthesis or by the artificial
manipulation of isolated segments of nucleic acids, for example, by
genetic engineering techniques.
[0090] Sequence identity: The similarity between amino acid or
nucleic acid sequences is expressed in terms of the similarity
between the sequences, otherwise referred to as sequence identity.
Sequence identity is frequently measured in terms of percentage
identity (or similarity or homology); the higher the percentage,
the more similar the two sequences are. Homologs or variants of a
given gene or protein will possess a relatively high degree of
sequence identity when aligned using standard methods.
[0091] Methods of alignment of sequences for comparison are well
known in the art. Various programs and alignment algorithms are
described in: Smith and Waterman, Adv. Appl. Math. 2:482, 1981;
Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and
Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444, 1988; Higgins and
Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153,
1989; Corpet et al., Nucleic Acids Research 16:10881-10890, 1988;
and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85:2444,
1988. Altschul et al., Nature Genet. 6:119-129, 1994.
[0092] The NCBI Basic Local Alignment Search Tool (BLAST.TM.)
(Altschul et al., J. Mol. Biol. 215:403-410, 1990) is available
from several sources, including the National Center for
Biotechnology Information (NCBI, Bethesda, Md.) and on the
Internet, for use in connection with the sequence analysis programs
blastp, blastn, blastx, tblastn and tblastx.
[0093] Subject: Living multi-cellular vertebrate organisms, a
category that includes both human and non-human mammals, such as
non-human primates. In one example, a subject is one who is
infected with H5N1 or is susceptible to such infection.
[0094] Therapeutically effective amount: A quantity of a specified
agent sufficient to achieve a desired effect in a subject being
treated with that agent. For example, this may be the amount of an
influenza virus vaccine useful for eliciting an immune response in
a subject and/or for preventing infection by influenza virus.
Ideally, in the context of the present disclosure, a
therapeutically effective amount of an influenza vaccine is an
amount sufficient to increase resistance to, prevent, ameliorate,
and/or treat infection caused by influenza virus in a subject
without causing a substantial cytotoxic effect in the subject. The
effective amount of an influenza vaccine useful for increasing
resistance to, preventing, ameliorating, and/or treating infection
in a subject will be dependent on, for example, the subject being
treated, the manner of administration of the therapeutic
composition and other factors.
[0095] Transformed: A transformed cell is a cell into which has
been introduced a nucleic acid molecule by molecular biology
techniques. As used herein, the term transformation encompasses all
techniques by which a nucleic acid molecule might be introduced
into such a cell, including transfection with viral vectors,
transformation with plasmid vectors, and introduction of naked DNA
by electroporation, lipofection, and particle gun acceleration.
[0096] Vaccine: A preparation of immunogenic material capable of
stimulating an immune response, administered for the prevention,
amelioration, or treatment of disease, such as an infectious
disease. The immunogenic material may include, for example,
attenuated or killed microorganisms (such as attenuated viruses),
or antigenic proteins, peptides or DNA derived from them. Vaccines
may elicit both prophylactic (preventative) and therapeutic
responses. Methods of administration vary according to the vaccine,
but may include inoculation, ingestion, inhalation or other forms
of administration. Inoculations can be delivered by any of a number
of routes, including parenteral, such as intravenous, subcutaneous
or intramuscular. Vaccines may be administered with an adjuvant to
boost the immune response.
[0097] Vector: A vector is a nucleic acid molecule allowing
insertion of foreign nucleic acid without disrupting the ability of
the vector to replicate and/or integrate in a host cell. A vector
can include nucleic acid sequences that permit it to replicate in a
host cell, such as an origin of replication. An insertional vector
is capable of inserting itself into a host nucleic acid. A vector
can also include one or more selectable marker genes and other
genetic elements. An expression vector is a vector that contains
the necessary regulatory sequences to allow transcription and
translation of inserted gene or genes. In some embodiments of the
present disclosure, the vector encodes an influenza HA, NA or M1
protein. In some embodiments, the vector is the pTR600 expression
vector (U.S. Patent Application Publication No. 2002/0106798; Ross
et al., Nat Immunol. 1(2):102-103, 2000; Green et al., Vaccine
20:242-248, 2001).
[0098] Virus-like particle (VLP): Virus particles made up of one of
more viral structural proteins, but lacking the viral genome.
Because VLPs lack a viral genome, they are non-infectious. In
addition, VLPs can often be produced by heterologous expression and
can be easily purified. Most VLPs comprise at least a viral core
protein that drives budding and release of particles from a host
cell. One example of such a core protein is influenza M1. In some
embodiments herein, an influenza VLP comprises the HA, NA and M1
proteins. As described herein, influenza VLPs can be produced by
transfection of host cells with plasmids encoding the HA, NA and M1
proteins. After incubation of the transfected cells for an
appropriate time to allow for protein expression (such as for
approximately 72 hours), VLPs can be isolated from cell culture
supernatants. Example 1 provides an exemplary protocol for
purifying influenza VLPs from cell supernatants. In this example,
VLPs are isolated by low speed centrifugation (to remove cell
debris), vacuum filtration and ultracentrifugation through 20%
glycerol.
[0099] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
The singular terms "a," "an," and "the" include plural referents
unless context clearly indicates otherwise. Similarly, the word
"or" is intended to include "and" unless the context clearly
indicates otherwise. Hence "comprising A or B" means including A,
or B, or A and B. It is further to be understood that all base
sizes or amino acid sizes, and all molecular weight or molecular
mass values, given for nucleic acids or polypeptides are
approximate, and are provided for description. Although methods and
materials similar or equivalent to those described herein can be
used in the practice or testing of the present disclosure, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. All GenBank Accession
numbers are incorporated by reference herein as they appeared in
the database on Sep. 9, 2010. In case of conflict, the present
specification, including explanations of terms, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
III. Overview of Several Embodiments
[0100] Disclosed herein is the development of a computationally
optimized influenza HA protein that elicits broadly reactive immune
responses to H5N1 influenza virus isolates, such as the isolates
listed in Table 1. The optimized HA protein was developed through a
series of HA protein alignments, and subsequent generation of
consensus sequences, for clade 2 H5N1 influenza virus isolates
(described in detail in Example 1 below; see also FIG. 1). The
final consensus HA amino acid sequence was reverse translated and
optimized for expression in mammalian cells. Optimization of the
nucleic acid sequence included optimization of the codons for
expression in mammalian cells and RNA optimization (such as RNA
stability). The optimized HA coding sequence is set forth herein as
SEQ ID NO: 1, and the optimized HA protein sequence is set forth
herein as SEQ ID NO: 2.
[0101] Thus, provided herein is an isolated nucleic acid molecule
comprising a nucleotide sequence encoding an influenza HA
polypeptide. In some embodiments, the nucleotide sequence encoding
the HA polypeptide is at least 94%, at least 95%, at least 96%, at
least 97%, at least 98% or at least 99% identical to SEQ ID NO:
1.
[0102] In some examples, the nucleotide sequence encoding the
influenza HA polypeptide that is at least 94%, at least 95%, at
least 96%, at least 97%, at least 98%, or at least 99% identical to
SEQ ID NO: 1 lacks the start codon (nucleotides 1-3 of SEQ ID NO:
1), encoding a N-terminal methionine. In particular examples, the
nucleotide sequence encoding the influenza HA polypeptide is at
least 94% identical to nucleotides 4-1707 of SEQ ID NO: 1. In other
examples, the nucleotide sequence encoding the HA polypeptide
comprises or consists of nucleotides 4-1707 of SEQ ID NO: 1.
[0103] In some examples, the nucleotide sequence encoding the HA
polypeptide comprises SEQ ID NO: 1. In particular examples, the
nucleotide sequence encoding the HA polypeptide consists of SEQ ID
NO: 1. Also provided herein are influenza HA polypeptides encoded
by the disclosed nucleic acid molecules.
[0104] Further provided are vectors containing a nucleotide
sequence encoding an optimized HA polypeptide. In some embodiments
of the vectors provided herein, the nucleotide sequence encoding
the HA polypeptide is at least 94%, at least 95%, at least 96%, at
least 97%, at least 98% or at least 99% identical to SEQ ID NO: 1.
In some examples, the vector further includes a promoter operably
linked to the nucleotide sequence encoding the HA polypeptide. In
particular examples, the promoter is a cytomegalovirus (CMV)
promoter. In some embodiments, the nucleotide sequence of the
vector is at least 85%, at least 90%, at least 95%, at least 98% or
at least 99% identical to the nucleotide sequence of SEQ ID NO: 7.
In some examples, the nucleotide sequence of the vector comprises
the nucleotide sequence of SEQ ID NO: 7. In particular examples,
the nucleotide sequence of the vector consists of the nucleotide
sequence of SEQ ID NO: 7.
[0105] Also provided herein are influenza HA polypeptides produced
by transfecting a host cell with a vector provided herein under
conditions sufficient to allow for expression of the HA
polypeptide. Isolated cells containing the disclosed vectors are
also provided.
[0106] Also provided herein are optimized influenza HA
polypeptides. In some embodiments, the amino acid sequence of the
polypeptide is at least 99% identical to SEQ ID NO: 2. In some
examples, the amino acid sequence of the influenza HA polypeptide
that is at least 99% identical to SEQ ID NO: 2 lacks the N-terminal
methionine residue. In particular examples, the amino acid sequence
of the influenza HA polypeptide is at least 99% identical to amino
acids 2-568 of SEQ ID NO: 2. In other examples, the amino acid
sequence of the HA polypeptides comprises or consists of amino
acids 2-568 of SEQ ID NO: 2.
[0107] In some examples, the amino acid sequence of the polypeptide
comprises the amino acid sequence of SEQ ID NO: 2. In particular
examples, the amino acid sequence of the polypeptide consists of
the amino acid sequence of SEQ ID NO: 2. Fusion proteins comprising
the influenza HA polypeptides disclosed herein are also provided.
The influenza HA polypeptide can be fused to any heterologous amino
acid sequence to form the fusion protein.
[0108] Further provided herein are influenza virus-like particles
(VLPs) containing an optimized influenza HA protein disclosed
herein. In some embodiments, the HA protein of the VLP is at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99% or 100% identical to SEQ ID NO: 2. The influenza VLPs can
further include any additional influenza proteins necessary to form
the virus particle. In some embodiments, the influenza VLPs further
include influenza neuraminidase (NA) protein, influenza matrix (M1)
protein, or both.
[0109] In some embodiments of the influenza VLPs, the amino acid
sequence of the influenza NA protein is at least 85%, at least 90%,
at least 95%, at least 98% or at least 99% identical to SEQ ID NO:
4. In some examples, the amino acid sequence of the influenza NA
protein comprises SEQ ID NO: 4. In particular examples, the amino
acid sequence of the influenza NA protein consists of SEQ ID NO: 4.
In some embodiments, the amino acid sequence of the influenza M1
protein is at least 85%, at least 90%, at least 95%, at least 98%
or at least 99% identical to SEQ ID NO: 6. In some examples, the
amino acid sequence of the influenza M1 protein comprises SEQ ID
NO: 6. In particular examples, the amino acid sequence of the
influenza M1 protein consists of SEQ ID NO: 6.
[0110] Also provided is an influenza VLP containing an influenza HA
polypeptide as described herein, produced by transfecting a host
cell with a vector encoding the HA polypeptide, a vector encoding
an influenza NA protein and a vector encoding an influenza M1
protein, under conditions sufficient to allow for expression of the
HA, M1 and NA proteins.
[0111] The vectors used to express the HA, NA and M1 proteins can
be any suitable expression vectors known in the art. The vectors
can be, for example, mammalian expression vectors, or viral
vectors. In some embodiments, the vector is the pTR600 expression
vector (U.S. Patent Application Publication No. 2002/0106798,
herein incorporated by reference; Ross et al., Nat Immunol.
1(2):102-103, 2000; Green et al., Vaccine 20:242-248, 2001).
[0112] In some embodiments, the nucleotide sequence of the vector
encoding the HA polypeptide is at least 85%, at least 90%, at least
95%, at least 96%, at least 97%, at least 98% or at least 99%
identical to SEQ ID NO: 7. In some examples, the nucleotide
sequence of the vector encoding the HA polypeptide comprises SEQ ID
NO: 7. In particular examples, the nucleotide sequence of the
vector encoding the HA polypeptide consists of SEQ ID NO: 7.
[0113] In some embodiments, the nucleotide sequence of the vector
encoding the NA protein is at least 85%, at least 90%, at least
95%, at least 96%, at least 97%, at least 98% or at least 99%
identical to SEQ ID NO: 8. In some examples, the nucleotide
sequence of the vector encoding the NA protein comprises SEQ ID NO:
8. In particular examples, the nucleotide sequence of the vector
encoding the NA protein consists of SEQ ID NO: 8.
[0114] In some embodiments, the nucleotide sequence of the vector
encoding the M1 protein is at least 85%, at least 90%, at least
95%, at least 96%, at least 97%, at least 98% or at least 99%
identical to SEQ ID NO: 9. In some examples, the nucleotide
sequence of the vector encoding the M1 protein comprises SEQ ID NO:
9. In particular examples, the nucleotide sequence of the vector
encoding the M1 protein consists of SEQ ID NO: 9.
[0115] Collections of plasmids are also provided herein. In some
embodiments, the collection of plasmids includes a plasmid encoding
an influenza NA, a plasmid encoding an influenza MA, and a plasmid
encoding the optimized HA protein disclosed herein. In some
embodiments, the nucleotide sequence encoding the codon-optimized
influenza HA of the HA-encoding plasmid is at least 94%, at least
95%, at least 96%, at least 97%, at least 98% or at least 99%
identical to SEQ ID NO: 1. Also provided are kits comprising the
collection of plasmids.
[0116] In some embodiments of the collections of plasmids, the
influenza NA is codon-optimized and/or the influenza M1 is
codon-optimized. In some examples, the nucleotide sequence encoding
the codon-optimized influenza NA is at least 85%, at least 90%, at
least 95%, at least 96%, at least 97%, at least 98% or at least 99%
identical to SEQ ID NO: 3. In particular examples, the nucleotide
sequence encoding the codon-optimized influenza NA comprises, or
consists of, SEQ ID NO: 3. In some examples, the nucleotide
sequence encoding the codon-optimized influenza M1 is at least 85%,
at least 90%, at least 95%, at least 96%, at least 97%, at least
98% or at least 99% identical to SEQ ID NO: 5. In particular
examples, the nucleotide sequence encoding the codon-optimized
influenza M1 comprises, or consists of, SEQ ID NO: 5.
[0117] In one non-limiting example, the plasmid encoding influenza
NA comprises SEQ ID NO: 8; the plasmid encoding influenza M1
comprises SEQ ID NO: 9; and the plasmid encoding influenza HA
comprises SEQ ID NO: 10.
[0118] In some embodiments, transfection of the collection of
plasmids into host cells under conditions sufficient to allow for
expression of the HA, NA and M1 proteins produces influenza VLPs
comprising the HA, NA and M1 proteins.
[0119] Also provided herein are compositions comprising an
optimized influenza HA protein as disclosed herein, or a fusion
protein or VLP comprising the optimized influenza HA protein. In
some embodiments, the compositions further comprise a
pharmaceutically acceptable carrier and/or an adjuvant. For
example, the adjuvant can be alum, Freund's complete adjuvant, a
biological adjuvant or immunostimulatory oligonucleotides (such as
CpG oligonucleotides).
[0120] Further provided is a method of eliciting an immune response
to influenza virus in a subject by administering an influenza HA
protein disclosed herein, fusion proteins containing the influenza
HA, or VLPs containing the influenza HA, as disclosed herein. In
some embodiments, the influenza virus is an H5N1 influenza virus.
In some embodiments, the HA protein, HA fusion protein or VLP can
be administered using any suitable route of administration, such
as, for example, intramuscular. In some embodiments, the HA
protein, fusion protein or VLP is administered as a composition
further comprising a pharmaceutically acceptable carrier and/or an
adjuvant. For example, the adjuvant can be alum, Freund's complete
adjuvant, a biological adjuvant or immunostimulatory
oligonucleotides (such as CpG oligonucleotides).
[0121] Also provided is a method of immunizing a subject against
influenza virus by administering to the subject VLPs containing the
optimized influenza HA protein disclosed herein, or administering a
composition thereof. In some embodiments of the method, the
composition further comprises a pharmaceutically acceptable carrier
and/or an adjuvant. For example, the adjuvant can be alum, Freund's
complete adjuvant, a biological adjuvant or immunostimulatory
oligonucleotides (such as CpG oligonucleotides). In some
embodiments, the VLPs (or compositions thereof) are administered
intramuscularly.
[0122] In some embodiments of the methods of eliciting an immune
response or immunizing a subject, the subject is administered at
least 1 .mu.g of the VLPs containing the optimized HA protein, such
as at least 5 .mu.g, at least 10 .mu.g, at least 15 .mu.g, at least
20 .mu.g, at least 25 .mu.g, at least 30 .mu.g, at least 40 .mu.g
or at least 50 .mu.g of the VLPs containing the optimized HA
protein, for example about 1 to about 50 .mu.g or about 1 to about
25 .mu.g of the VLPs containing the optimized HA protein. In
particular examples, the subject is administered about 5 to about
20 .mu.g of the VLPs, or about 10 to about 15 .mu.g of the VLPs. In
one specific non-limiting example, the subject is administered
about 15 .mu.g of the VLPs. However, one of skill in the art is
capable of determining a therapeutically effective amount (for
example an amount that provides protection against H5N1 influenza
virus infection) of VLPs to administer to a subject.
[0123] It is disclosed herein that administration of VLPs
comprising the COBRA HA disclosed herein (SEQ ID NO: 2) elicits
protective levels of HAI antibodies to a number of representative
clade 2 isolates and provides complete protection against lethal
challenge with a clade 2.2 H5N1 virus. In some embodiments,
administration of VLPs containing an optimized influenza HA results
in production of high HAI titers (.gtoreq.1:40) to H5N1 clade 1,
clade 2.1, clade 2.2 and clade 2.3 isolates. In some examples, the
VLPs containing an optimized influenza HA elicit high HAI titers
against clade 1 and/or clade 7 viruses. The VLPs containing an
optimized influenza HA disclosed herein elicit a broader immune
response (e.g., elicit neutralizing antibodies to a broader range
is H5N1 virus isolates, such as those from clade 1, sub-clades of
clade 2, and clade 7) than a polyvalent influenza virus
vaccine.
[0124] Also provided herein is a method of optimizing an influenza
protein sequence to elicit broadly reactive immune responses in a
subject. In the context of the present disclosure, "broadly
reactive" means the protein sequence elicits an immune response in
a subject that is sufficient to inhibit, neutralize or prevent
infection of a broad range of influenza viruses (such as most or
all influenza viruses within a specific subtype). In some
embodiments, the influenza protein is influenza HA or influenza NA.
In one example, the optimized influenza protein is capable of
eliciting a protective immune response against most or all known
H5N1 influenza virus isolates (such as those listed in Table 1),
such as about 80%, about 85%, about 90% or about 95% of known H5N1
influenza virus isolates.
[0125] In some embodiments, the method of optimizing an influenza
protein sequence includes obtaining the amino acid sequences of a
group of influenza virus isolates. For example, the group can
consist of influenza virus isolates from a specific subtype (such
as, for example, H5N1 or H1N1), and/or from one or more
clades/sub-clades of a specific influenza subtype (for example, one
or more of clades/sub-clades 0, 1, 2.1, 2.2, 2.3, 2.4, 3, 4, 5, 6,
7, 8 and 9 of H5N1). Amino acid sequences of the group of influenza
viruses are first organized by clade or sub-clade and then by
geographic location within each clade or sub-clade. The amino acid
sequences for each geographic location are aligned to generate a
primary consensus sequence for each geographical region. Grouping
virus isolates by geographical region controls for single outbreak
dominance and incomplete reporting and sequencing. The primary
consensus sequence can be generated, for example, by multiple
alignment analysis using AlignX (Vector NTI), or by any other
method known in the art. The primary geographically-based consensus
sequences for each clade or sub-clade are then aligned, and a
secondary consensus sequence is generated for each clade or
sub-clade. The secondary consensus sequences for each clade or
sub-clade are then aligned to generate the optimized, broadly
reactive, consensus sequence (see FIG. 1). In some embodiments, the
optimized influenza virus polypeptide sequence is further optimized
for expression in mammalian cells. In some examples, optimization
includes reverse translation of the optimized influenza virus
polypeptide sequence to generate a coding sequence, followed by
codon-optimization and/or optimization of the RNA (such as for
stability).
[0126] In one non-limiting example, the method of optimization
includes: (i) obtaining the amino acid sequences of the polypeptide
from a group of influenza virus isolates, wherein the influenza
virus isolates are from the same subtype; (ii) organizing the amino
acid sequences of the polypeptide from the group of influenza virus
isolates by clade or sub-clade and then by geographical region
within each clade or sub-clade; (iii) aligning the amino acid
sequences within each geographical region to generate primary
consensus sequences, wherein each geographic region is represented
by a primary consensus sequence; (iv) aligning the primary
consensus sequences to generate secondary consensus sequences,
wherein each clade or sub-clade is represented by a secondary
consensus sequence; and (v) aligning the secondary consensus
sequences to generate the optimized influenza virus polypeptide
sequence. In some cases, the method further includes (i) reverse
translating the optimized influenza virus polypeptide sequence to
generate a coding sequence; and (ii) optimizing the coding sequence
for expression in mammalian cells.
[0127] In an alternative embodiment, the primary consensus sequence
is obtained by aligning influenza protein sequences (such as HA or
NA sequences) from viral isolates from a single outbreak (a
collection of influenza virus isolates within a single country
within a given year). Thus, in one non-limiting example, the method
of optimization includes: (i) obtaining the amino acid sequences of
the polypeptide from a group of influenza virus isolates, wherein
the influenza virus isolates are from the same subtype; (ii)
organizing the amino acid sequences of the polypeptide from the
group of influenza virus isolates by clade or sub-clade and then by
outbreak; (iii) aligning the amino acid sequences within each
outbreak to generate primary consensus sequences, wherein each
outbreak is represented by a primary consensus sequence; (iv)
aligning the primary consensus sequences to generate secondary
consensus sequences, wherein each clade or sub-clade is represented
by a secondary consensus sequence; and (v) aligning the secondary
consensus sequences to generate the optimized influenza virus
polypeptide sequence. In some cases, the method further includes
(i) reverse translating the optimized influenza virus polypeptide
sequence to generate a coding sequence; and (ii) optimizing the
coding sequence for expression in mammalian cells.
VI. Influenza
[0128] Influenza viruses are segmented negative-strand RNA viruses
that belong to the Orthomyxoviridae family. There are three types
of Influenza viruses, A, B and C. Influenza A viruses infect a wide
variety of birds and mammals, including humans, horses, marine
mammals, pigs, ferrets, and chickens. In animals, most influenza A
viruses cause mild localized infections of the respiratory and
intestinal tract. However, highly pathogenic influenza A strains,
such as H5N1, cause systemic infections in poultry in which
mortality may reach 100%. Animals infected with influenza A often
act as a reservoir for the influenza viruses and certain subtypes
have been shown to cross the species barrier to humans.
[0129] Influenza A viruses can be classified into subtypes based on
allelic variations in antigenic regions of two genes that encode
surface glycoproteins, namely, hemagglutinin (HA) and neuraminidase
(NA) which are required for viral attachment and cellular release.
Currently, sixteen subtypes of HA (H1-H16) and nine NA (N1-N9)
antigenic variants are known for influenza A virus. Previously,
only three subtypes were known to circulate in humans (H1N1, H1N2,
and H3N2). However, in recent years, the pathogenic H5N1 subtype of
avian influenza A has been reported to cross the species barrier
and infect humans as documented in Hong Kong in 1997 and 2003,
leading to the death of several patients.
[0130] In humans, the avian influenza virus infects cells of the
respiratory tract as well as the intestinal tract, liver, spleen,
kidneys and other organs. Symptoms of avian influenza infection
include fever, respiratory difficulties including shortness of
breath and cough, lymphopenia, diarrhea and difficulties regulating
blood sugar levels. In contrast to seasonal influenza, the group
most at risk is healthy adults which make up the bulk of the
population. Due to the high pathogenicity of certain avian
influenza A subtypes, particularly H5N1, and their demonstrated
ability to cross over to infect humans, there is a significant
economic and public health risk associated with these viral
strains, including a real epidemic and pandemic threat. Currently,
no effective vaccines for H5N1 infection are available.
[0131] The influenza A virus genome encodes nine structural
proteins and one nonstructural (NS1) protein with regulatory
functions. The influenza virus segmented genome contains eight
negative-sense RNA (nsRNA) gene segments (PB2, PB1, PA, NP, M, NS,
HA and NA) that encode at least ten polypeptides, including
RNA-directed RNA polymerase proteins (PB2, PB1 and PA),
nucleoprotein (NP), neuraminidase (NA), hemagglutinin (subunits HA1
and HA2), the matrix proteins (M1 and M2) and the non-structural
proteins (NS1 and NS2) (Krug et al., In "The Influenza Viruses," R.
M. Krug, ed., Plenum Press, N.Y., 1989, pp. 89 152).
[0132] Influenza virus' ability to cause widespread disease is due
to its ability to evade the immune system by undergoing antigenic
change, which is believed to occur when a host is infected
simultaneously with both an animal influenza virus and a human
influenza virus. During mutation and reassortment in the host, the
virus may incorporate an HA and/or NA surface protein gene from
another virus into its genome, thereby producing a new influenza
subtype and evading the immune system.
[0133] HA is a viral surface glycoprotein generally comprising
approximately 560 amino acids and representing 25% of the total
virus protein. It is responsible for adhesion of the viral particle
to, and its penetration into, a host cell in the early stages of
infection. Cleavage of the virus HA0 precursor into the HA1 and HA2
sub-fragments is a necessary step in order for the virus to infect
a cell. Thus, cleavage is required in order to convert new virus
particles in a host cell into virions capable of infecting new
cells. Cleavage is known to occur during transport of the integral
HA0 membrane protein from the endoplasmic reticulum of the infected
cell to the plasma membrane. In the course of transport,
hemagglutinin undergoes a series of co- and post-translational
modifications including proteolytic cleavage of the precursor HA
into the amino-terminal fragment HAI and the carboxy terminal HA2.
One of the primary difficulties in growing influenza strains in
primary tissue culture or established cell lines arises from the
requirement for proteolytic cleavage activation of the influenza
hemagglutinin in the host cell.
[0134] Although it is known that an uncleaved HA can mediate
attachment of the virus to its neuraminic acid-containing receptors
on a cell surface, it is not capable of the next step in the
infectious cycle, which is fusion. It has been reported that
exposure of the hydrophobic amino terminus of HA2 by cleavage is
required so that it can be inserted into the target cell, thereby
forming a bridge between virus and target cell membrane. This
process is followed by fusion of the two membranes and entry of the
virus into the target cell.
[0135] Proteolytic activation of HA involves cleavage at an
arginine residue by a trypsin-like endoprotease, which is often an
intracellular enzyme that is calcium dependent and has a neutral pH
optimum. Since the activating proteases are cellular enzymes, the
infected cell type determines whether the HA is cleaved. The HA of
the mammalian influenza viruses and the nonpathogenic avian
influenza viruses are susceptible to proteolytic cleavage only in a
restricted number of cell types. On the other hand, HA of
pathogenic avian viruses among the H5 and H7 subtypes are cleaved
by proteases present in a broad range of different host cells.
Thus, there are differences in host range resulting from
differences in hemagglutinin cleavability which are correlated with
the pathogenic properties of the virus.
[0136] Neuraminidase (NA) is a second membrane glycoprotein of the
influenza viruses. The presence of viral NA has been shown to be
important for generating a multi-faceted protective immune response
against an infecting virus. For most influenza A viruses, NA is 413
amino acid in length, and is encoded by a gene of 1413 nucleotides.
Nine different NA subtypes have been identified in influenza
viruses (N1, N2, N3, N4, N5, N6, N7, N8 and N9), all of which have
been found among wild birds. NA is involved in the destruction of
the cellular receptor for the viral HA by cleaving terminal
neuraminic acid (also called sialic acid) residues from
carbohydrate moieties on the surfaces of infected cells. NA also
cleaves sialic acid residues from viral proteins, preventing
aggregation of viruses. Using this mechanism, it is hypothesized
that NA facilitates release of viral progeny by preventing newly
formed viral particles from accumulating along the cell membrane,
as well as by promoting transportation of the virus through the
mucus present on the mucosal surface. NA is an important antigenic
determinant that is subject to antigenic variation.
[0137] In addition to the surface proteins HA and NA, influenza
virus comprises six additional internal genes, which give rise to
eight different proteins, including polymerase genes PB1, PB2 and
PA, matrix proteins M1 and M2, nucleoprotein (NP), and
non-structural proteins NS1 and NS2 (Horimoto et al., Clin
Microbiol Rev. 14(1):129-149, 2001).
[0138] In order to be packaged into progeny virions, viral RNA is
transported from the nucleus as a ribonucleoprotein (RNP) complex
composed of the three influenza virus polymerase proteins, the
nucleoprotein (NP), and the viral RNA, in association with the
influenza virus matrix 1 (M1) protein and nuclear export protein
(Marsh et al., J Virol, 82:2295-2304, 2008). The M1 protein that
lies within the envelope is thought to function in assembly and
budding. A limited number of M2 proteins are integrated into the
virions (Zebedee, J. Virol. 62:2762-2772, 1988). They form
tetramers having H+ ion channel activity, and when activated by the
low pH in endosomes, acidify the inside of the virion, facilitating
its uncoating (Pinto et al., Cell 69:517-528, 1992). Amantadine is
an anti-influenza drug that prevents viral infection by interfering
with M2 ion channel activity, thus inhibiting virus uncoating.
[0139] NS1, a nonstructural protein, has multiple functions,
including regulation of splicing and nuclear export of cellular
mRNAs as well as stimulation of translation. The major function of
NS1 seems to be to counteract the interferon activity of the host,
since an NS1 knockout virus was viable although it grew less
efficiently than the parent virus in interferon-nondefective cells
(Garcia-Sastre, Virology 252:324-330, 1998).
[0140] NS2 has been detected in virus particles (Richardson et al.,
Arch. Virol. 116:69-80, 1991; Yasuda et al., Virology 196:249-255,
1993). The average number of NS2 proteins in a virus particle was
estimated to be 130-200 molecules. An in vitro binding assay shows
direct protein-protein contact between M1 and NS2. NS2-M1 complexes
have also been detected by immunoprecipitation in virus-infected
cell lysates. The NS2 protein is thought to play a role in the
export of RNP from the nucleus through interaction with M1 protein
(Ward et al., Arch. Virol. 140:2067-2073, 1995).
V. Influenza Proteins, VLPs and Administration Thereof
[0141] Optimized influenza HA polypeptides and influenza VLPs
comprising an optimized HA (such as the HA having the sequence set
forth as SEQ ID NO: 2) are provided herein. The optimized HA
polypeptides can be administered to elicit an immune response
against influenza. In particular examples, the optimized HA
polypeptides are administered as part of a VLP.
[0142] The influenza VLPs are made up of the HA, NA and M1
proteins. The production of influenza VLPs has been described in
the art and is within the abilities of one of ordinary skill in the
art. As described herein, influenza VLPs can be produced by
transfection of host cells with plasmids encoding the HA, NA and M1
proteins. After incubation of the transfected cells for an
appropriate time to allow for protein expression (such as for
approximately 72 hours), VLPs can be isolated from cell culture
supernatants. Example 1 below provides an exemplary protocol for
purifying influenza VLPs from cell supernatants. In this example,
VLPs are isolated by low speed centrifugation (to remove cell
debris), vacuum filtration and ultracentrifugation through 20%
glycerol.
[0143] The influenza VLPs disclosed herein can be used as influenza
vaccines to elicit a protective immune response against H5N1
influenza viruses.
[0144] Influenza HA polypeptides and VLPs comprising HA
polypeptides, or compositions thereof, can be administered to a
subject by any of the routes normally used for introducing
recombinant virus into a subject. Methods of administration
include, but are not limited to, intradermal, intramuscular,
intraperitoneal, parenteral, intravenous, subcutaneous, vaginal,
rectal, intranasal, inhalation or oral. Parenteral administration,
such as subcutaneous, intravenous or intramuscular administration,
is generally achieved by injection. Injectables can be prepared in
conventional forms, either as liquid solutions or suspensions,
solid forms suitable for solution or suspension in liquid prior to
injection, or as emulsions. Injection solutions and suspensions can
be prepared from sterile powders, granules, and tablets of the kind
previously described. Administration can be systemic or local.
[0145] Influenza VLPs, or compositions thereof, are administered in
any suitable manner, such as with pharmaceutically acceptable
carriers. Pharmaceutically acceptable carriers are determined in
part by the particular composition being administered, as well as
by the particular method used to administer the composition.
Accordingly, there is a wide variety of suitable formulations of
pharmaceutical compositions of the present disclosure.
[0146] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives may also be present such as, for example,
antimicrobials, anti-oxidants, chelating agents, and inert gases
and the like.
[0147] Some of the compositions may potentially be administered as
a pharmaceutically acceptable acid- or base-addition salt, formed
by reaction with inorganic acids such as hydrochloric acid,
hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid,
sulfuric acid, and phosphoric acid, and organic acids such as
formic acid, acetic acid, propionic acid, glycolic acid, lactic
acid, pyruvic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, and fumaric acid, or by reaction with an inorganic
base such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide, and organic bases such as mono-, di-, trialkyl and aryl
amines and substituted ethanolamines.
[0148] Administration can be accomplished by single or multiple
doses. The dose administered to a subject in the context of the
present disclosure should be sufficient to induce a beneficial
therapeutic response in a subject over time, or to inhibit or
prevent H5N1 influenza virus infection. The dose required will vary
from subject to subject depending on the species, age, weight and
general condition of the subject, the severity of the infection
being treated, the particular composition being used and its mode
of administration. An appropriate dose can be determined by one of
ordinary skill in the art using only routine experimentation.
[0149] Provided herein are pharmaceutical compositions which
include a therapeutically effective amount of the influenza VLPs
alone or in combination with a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers include, but are not limited
to, saline, buffered saline, dextrose, water, glycerol, ethanol,
and combinations thereof. The carrier and composition can be
sterile, and the formulation suits the mode of administration. The
composition can also contain minor amounts of wetting or
emulsifying agents, or pH buffering agents. The composition can be
a liquid solution, suspension, emulsion, tablet, pill, capsule,
sustained release formulation, or powder. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulations can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, and
magnesium carbonate. Any of the common pharmaceutical carriers,
such as sterile saline solution or sesame oil, can be used. The
medium can also contain conventional pharmaceutical adjunct
materials such as, for example, pharmaceutically acceptable salts
to adjust the osmotic pressure, buffers, preservatives and the
like. Other media that can be used with the compositions and
methods provided herein are normal saline and sesame oil.
[0150] The influenza VLPs described herein can be administered
alone or in combination with other therapeutic agents to enhance
antigenicity. For example, the influenza VLPs can be administered
with an adjuvant, such as Freund incomplete adjuvant or Freund's
complete adjuvant.
[0151] Optionally, one or more cytokines, such as IL-2, IL-6,
IL-12, RANTES, GM-CSF, TNF-.alpha., or IFN-.gamma., one or more
growth factors, such as GM-CSF or G-CSF; one or more molecules such
as OX-40L or 41 BBL, or combinations of these molecules, can be
used as biological adjuvants (see, for example, Salgaller et al.,
1998, J. Surg. Oncol. 68(2):122-38; Lotze et al., 2000, Cancer J.
Sci. Am. 6(Suppl 1):S61-6; Cao et al., 1998, Stem Cells 16(Suppl
1):251-60; Kuiper et al., 2000, Adv. Exp. Med. Biol. 465:381-90).
These molecules can be administered systemically (or locally) to
the host.
[0152] A number of means for inducing cellular responses, both in
vitro and in vivo, are known. Lipids have been identified as agents
capable of assisting in priming CTL in vivo against various
antigens. For example, as described in U.S. Pat. No. 5,662,907,
palmitic acid residues can be attached to the alpha and epsilon
amino groups of a lysine residue and then linked (for example, via
one or more linking residues, such as glycine, glycine-glycine,
serine, serine-serine, or the like) to an immunogenic peptide. The
lipidated peptide can then be injected directly in a micellar form,
incorporated in a liposome, or emulsified in an adjuvant. As
another example, E. coli lipoproteins, such as
tripalmitoyl-S-glycerylcysteinlyseryl-serine can be used to prime
tumor specific CTL when covalently attached to an appropriate
peptide (see, Deres et al., Nature 342:561, 1989). Further, as the
induction of neutralizing antibodies can also be primed with the
same molecule conjugated to a peptide which displays an appropriate
epitope, two compositions can be combined to elicit both humoral
and cell-mediated responses where that is deemed desirable.
[0153] Although administration of VLPs containing the optimized HA
protein, one of skill in the art would understand that it is also
possible to administer the optimized influenza HA protein itself
(in the absence of a viral particle) or as a fusion protein to
elicit an immune response in a subject.
[0154] The following examples are provided to illustrate certain
particular features and/or embodiments. These examples should not
be construed to limit the disclosure to the particular features or
embodiments described.
EXAMPLES
Example 1: A Computationally Optimized Broadly Reactive Antigen
(COBRA) Based H5N1 VLP Vaccine Elicits Broadly Reactive Antibodies
in Mice and Ferrets
[0155] This example describes the finding that mice and ferrets
vaccinated with COBRA hemagglutinin (HA) H5N1 VLPs exhibited
protective levels of HAI antibodies to representative isolates from
each sub-clade of clade 2 and were completely protected from lethal
challenge with a clade 2.2 H5N1 virus.
Materials and Methods
COBRA Hemagglutinin (HA) Construction and Synthesis
[0156] Influenza A HA amino acid sequences isolated from human H5N1
infections were downloaded from the NCBI Influenza Virus Resource
database (Bao et al., J Virol 82:596-601, 2008; see Table 1 for a
complete list of accession numbers and isolate descriptions).
Nucleotide sequences were translated into protein sequences using
the standard genetic code. All available full length sequences from
H5N1 clade 2 human infections from 2004 to 2006 were acquired and
used for subsequent consensus generations. For each round of
consensus generation, multiple alignment analysis was applied and
the consensus sequence was generated using AlignX (Vector NTI). The
final amino acid sequence, termed computationally optimized broadly
reactive antigen (COBRA), was reverse translated and optimized for
expression in mammalian cells, including codon usage and RNA
optimization (GeneArt; Regensburg, Germany). This construct was
then synthesized and inserted into the pTR600 expression vector
(U.S. Patent Application Publication No. 2002/0106798; Ross et al.,
Nat Immunol. 1(2):102-103, 2000; Green et al., Vaccine 20:242-248,
2001).
TABLE-US-00002 TABLE 1 COBRA input sequences Strain Clade Accession
Host Country Year A/Indonesia/534H/2006 2.1.2 EU146737 Human
Indonesia 2006 A/Indonesia/535H/2006 2.1.2 EU146753 Human Indonesia
2006 A/Indonesia/536H/2006 2.1.2 EU146754 Human Indonesia 2006
A/Indonesia/538H/2006 2.1.2 EU146745 Human Indonesia 2006
A/Indonesia/546bH/2006 2.1.2 EU146793 Human Indonesia 2006
A/Indonesia/546H/2006 2.1.2 EU146755 Human Indonesia 2006
A/Indonesia/560H/2006 2.1.2 EU146785 Human Indonesia 2006
A/Indonesia/CDC594/2006 2.1.2 CY014272 Human Indonesia 2006
A/Indonesia/CDC595/2006 2.1.2 CY014280 Human Indonesia 2006
A/Indonesia/CDC596/2006 2.1.2 CY014288 Human Indonesia 2006
A/Indonesia/CDC597/2006 2.1.2 CY014296 Human Indonesia 2006
A/Indonesia/CDC599/2006 2.1.2 CY014303 Human Indonesia 2006
A/Indonesia/CDC599N/2006 2.1.2 CY014477 Human Indonesia 2006
A/Indonesia/CDC625/2006 2.1.2 CY014433 Human Indonesia 2006
A/Indonesia/CDC625L/2006 2.1.2 CY014465 Human Indonesia 2006
A/Indonesia/160H/2005 2.1.3 EU146648 Human Indonesia 2005
A/Indonesia/175H/2005 2.1.3 EU146640 Human Indonesia 2005
A/Indonesia/177H/2005 2.1.3 EU146680 Human Indonesia 2005
A/Indonesia/195H/2005 2.1.3 EU146656 Human Indonesia 2005
A/Indonesia/239H/2005 2.1.3 EU146664 Human Indonesia 2005
A/Indonesia/245H/2005 2.1.3 EU146672 Human Indonesia 2005
A/Indonesia/283H/2006 2.1.3 EU146681 Human Indonesia 2006
A/Indonesia/286H/2006 2.1.3 EU146688 Human Indonesia 2006
A/Indonesia/292H/2006 2.1.3 EU146713 Human Indonesia 2006
A/Indonesia/298H/2006 2.1.3 EU146697 Human Indonesia 2006
A/Indonesia/304H/2006 2.1.3 EU146705 Human Indonesia 2006
A/Indonesia/321H/2006 2.1.3 EU146721 Human Indonesia 2006
A/Indonesia/341H/2006 2.1.3 EU146729 Human Indonesia 2006
A/Indonesia/5/2005 2.1.3 EF541394 Human Indonesia 2005
A/Indonesia/542H/2006 2.1.3 EU146777 Human Indonesia 2006
A/Indonesia/567H/2006 2.1.3 EU146801 Human Indonesia 2006
A/Indonesia/569H/2006 2.1.3 EU146809 Human Indonesia 2006
A/Indonesia/583H/2006 2.1.3 EU146817 Human Indonesia 2006
A/Indonesia/604H/2006 2.1.3 EU146825 Human Indonesia 2006
A/Indonesia/7/2005 2.1.3 EU146632 Human Indonesia 2005
A/Indonesia/CDC184/2005 2.1.3 CY014197 Human Indonesia 2005
A/Indonesia/CDC194P/2005 2.1.3 CY014168 Human Indonesia 2005
A/Indonesia/CDC287E/2005 2.1.3 CY014198 Human Indonesia 2005
A/Indonesia/CDC287T/2005 2.1.3 CY014199 Human Indonesia 2005
A/Indonesia/CDC292N/2005 2.1.3 CY014200 Human Indonesia 2005
A/Indonesia/CDC292T/2005 2.1.3 CY014201 Human Indonesia 2005
A/Indonesia/CDC326/2006 2.1.3 CY014204 Human Indonesia 2006
A/Indonesia/CDC326N/2006 2.1.3 CY014202 Human Indonesia 2006
A/Indonesia/CDC326N2/2006 2.1.3 CY014203 Human Indonesia 2006
A/Indonesia/CDC326T/2006 2.1.3 CY014205 Human Indonesia 2006
A/Indonesia/CDC329/2006 2.1.3 CY014206 Human Indonesia 2006
A/Indonesia/CDC357/2006 2.1.3 CY014207 Human Indonesia 2006
A/Indonesia/CDC370/2006 2.1.3 CY014209 Human Indonesia 2006
A/Indonesia/CDC370E/2006 2.1.3 CY014210 Human Indonesia 2006
A/Indonesia/CDC370P/2006 2.1.3 CY014211 Human Indonesia 2006
A/Indonesia/CDC370T/2006 2.1.3 CY014212 Human Indonesia 2006
A/Indonesia/CDC390/2006 2.1.3 CY014213 Human Indonesia 2006
A/Indonesia/CDC523/2006 2.1.3 CY014311 Human Indonesia 2006
A/Indonesia/CDC523E/2006 2.1.3 CY014368 Human Indonesia 2006
A/Indonesia/CDC523T/2006 2.1.3 CY014376 Human Indonesia 2006
A/Indonesia/CDC582/2006 2.1.3 CY014384 Human Indonesia 2006
A/Indonesia/CDC610/2006 2.1.3 CY014393 Human Indonesia 2006
A/Indonesia/CDC623/2006 2.1.3 CY014401 Human Indonesia 2006
A/Indonesia/CDC623E/2006 2.1.3 CY014409 Human Indonesia 2006
A/Indonesia/CDC624/2006 2.1.3 CY014417 Human Indonesia 2006
A/Indonesia/CDC624E/2006 2.1.3 CY014425 Human Indonesia 2006
A/Indonesia/CDC634/2006 2.1.3 CY014441 Human Indonesia 2006
A/Indonesia/CDC634P/2006 2.1.3 CY014449 Human Indonesia 2006
A/Indonesia/CDC634T/2006 2.1.3 CY014457 Human Indonesia 2006
A/Indonesia/CDC644/2006 2.1.3 CY014518 Human Indonesia 2006
A/Indonesia/CDC644T/2006 2.1.3 CY014510 Human Indonesia 2006
A/Indonesia/CDC669/2006 2.1.3 CY014481 Human Indonesia 2006
A/Indonesia/CDC669P/2006 2.1.3 CY014489 Human Indonesia 2006
A/Indonesia/CDC699/2006 2.1.3 CY014497 Human Indonesia 2006
A/Indonesia/CDC7/2005 2.1.3 CY014177 Human Indonesia 2005
A/Indonesia/CDC739/2006 2.1.3 CY014529 Human Indonesia 2006
A/Indonesia/CDC759/2006 2.1.3 CY014543 Human Indonesia 2006
A/Indonesia/CDC835/2006 2.1.3 CY017662 Human Indonesia 2006
A/Indonesia/CDC836/2006 2.1.3 CY017670 Human Indonesia 2006
A/Indonesia/CDC836T/2006 2.1.3 CY017678 Human Indonesia 2006
A/Indonesia/CDC887/2006 2.1.3 CY017688 Human Indonesia 2006
A/Indonesia/CDC938/2006 2.1.3 CY017638 Human Indonesia 2006
A/Indonesia/CDC938E/2006 2.1.3 CY017646 Human Indonesia 2006
A/Indonesia/CDC940/2006 2.1.3 CY017654 Human Indonesia 2006
A/Indonesia/TLL001/2006 2.1.3 EU015403 Human Indonesia 2006
A/Indonesia/TLL002/2006 2.1.3 EU015404 Human Indonesia 2006
A/Indonesia/TLL003/2006 2.1.3 EU015405 Human Indonesia 2006
A/Indonesia/TLL004/2006 2.1.3 EU015406 Human Indonesia 2006
A/Indonesia/TLL005/2006 2.1.3 EU015407 Human Indonesia 2006
A/Indonesia/TLL006/2006 2.1.3 EU015408 Human Indonesia 2006
A/Indonesia/TLL007/2006 2.1.3 EU015409 Human Indonesia 2006
A/Indonesia/TLL008/2006 2.1.3 EU015410 Human Indonesia 2006
A/Indonesia/TLL009/2006 2.1.3 EU015411 Human Indonesia 2006
A/Indonesia/TLL010/2006 2.1.3 EU015412 Human Indonesia 2006
A/Indonesia/TLL011/2006 2.1.3 EU015413 Human Indonesia 2006
A/Indonesia/TLL012/2006 2.1.3 EU015414 Human Indonesia 2006
A/Indonesia/TLL013/2006 2.1.3 EU015415 Human Indonesia 2006
A/Indonesia/TLL014/2006 2.1.3 EU015416 Human Indonesia 2006
A/Djibouti/5691NAMRU3/2006 2.2 DQ666146 Human Djibouti 2006
A/Egypt/7021-NAMRU3/2006 2.2 CY062439 Human Egypt 2006
A/human/Iraq/207-NAMRU3/2006 2.2 DQ435202 Human Iraq 2006
A/Iraq/1/2006 2.2 EU146870 Human Iraq 2006 A/Iraq/659/2006 2.2
EU146876 Human Iraq 2006 A/Iraq/754/2006 2.2 EU146877 Human Iraq
2006 A/Iraq/755/2006 2.2 EU146869 Human Iraq 2006 A/Iraq/756/2006
2.2 EU146878 Human Iraq 2006 A/Turkey/12/2006 2.2 EF619982 Human
Turkey 2006 A/Turkey/15/2006 2.2 EF619989 Human Turkey 2006
A/Turkey/651242/2006 2.2 EF619990 Human Turkey 2006
A/Turkey/65596/2006 2.2 EF619998 Human Turkey 2006
A/Xinjiang/1/2006 2.2 FJ492886 Human China 2006
A/Egypt/14724-NAMRU3/2006 2.2.1 EF200512 Human Egypt 2006
A/Egypt/14725-NAMRU3/2006 2.2.1 EF200513 Human Egypt 2006
A/Egypt/2782-NAMRU3/2006 2.2.1 DQ464377 Human Egypt 2006
A/Egypt/2991-NAMRU3/2006 2.2.1 EU095023 Human Egypt 2006
A/Egypt/2992-NAMRU3/2006 2.2.1 EU095024 Human Egypt 2006
A/Egypt/902782/2006 2.2.1 EU146867 Human Egypt 2006
A/Egypt/902786/2006 2.2.1 EU146868 Human Egypt 2006 A/Anhui/1/2005
2.3.4 DQ371928 Human China 2005 A/Anhui/2/2005 2.3.4 DQ371929 Human
China 2005 A/China/2006 2.3.4 EF624256 Human China 2006
A/China/GD01/2006 2.3.4 DQ835313 Human China 2006 A/Fujian/1/2005
2.3.4 FJ492882 Human China 2005 A/Guangdong/1/2006 2.3.4 FJ492884
Human China 2006 A/Guangxi/1/2005 2.3.4 DQ371930 Human China 2005
A/human/China/GD02/2006 2.3.4 EU263981 Human China 2006
A/Hunan/1/2006 2.3.4 FJ492879 Human China 2006 A/Jiangxi/1/2005
2.3.4 FJ492885 Human China 2005 A/Shanghai/1/2006 2.3.4 AB462295
Human China 2006 A/Shenzhen/406H/2006 2.3.4 EF137706 Human China
2006 A/Sichuan/1/2006 2.3.4 FJ492881 Human China 2006
A/Vietnam/UT30850/2005 2.3.4 HM114537 Human Viet Nam 2005
A/Zhejiang/1/2006 2.3.4 FJ492880 Human China 2006
A/Zhejiang/16/2006 2.3.4 DQ643809 Human China 2006
COBRA HA Antigenic Modeling
[0157] Influenza hemagglutinin (HA) protein sequences representing
COBRA, A/Indonesia/5/2005 (Clade 2.1), A/Whooper
Swan/Mongolia/244/2005 (Clade 2.2) and A/Anhui/1/2005 (Clade 2.3)
were submitted to the 3D-JIGSAW Protein Comparative Modeling
website for rendering (Bates et al., Proteins 45(55):39-46, 2001;
Bates and Sternberg, Proteins 37(53):47-54, 1999; Contreras-Moreira
and Bates, Bioinformatics 18(8):1141-1142, 2002). Structures were
overlaid and analyzed using Swiss-Pdb viewer software (Guex and
Peitsch, Electrophoresis 18(15):2714-23, 1998). Antigenic regions
and designations are based on the original description of the
antigenic structure of the H3N2 virus A/Hong Kong/1/1968 (Wiley et
al., Nature 289(5796):373-378, 1981). No significant alterations
were observed in region B of the COBRA HA relative to the primary
influenza isolates; however, some divergent structures in HA
regions A and C were identified in primary isolates.
In Vitro Expression
[0158] COBRA HA protein expression was confirmed by transfecting
mammalian cells. Human embryonic kidney (HEK) 293T cells
(1.times.10.sup.6) were transiently transfected with 3 .mu.g of DNA
expressing COBRA. Cells were incubated for 72 hours at 37.degree.
C., supernatants were removed, the cells were lysed with 1%
Triton-X 100 and cell lysates were collected. Cell lysates were
electrophoresed on a 10% SDS-PAGE gel and transferred to a PVDF
membrane. The blot was probed with mouse polyclonal antisera pooled
from mice infected with 6:2 reassortant H5N1 viruses with the
surface glycoproteins derived from either A/Vietnam/1203/2004 or
A/Whooper Swan/244/2005, and the HA-antibody complexes were
detected using a goat anti-mouse IgG conjugated to horse radish
peroxidase (HRP) (Southern Biotech; Birmingham, Ala., USA). HRP was
detected by chemiluminescent substrate (Pierce Biotechnology;
Rockford Ill., USA) and exposed to X-ray film (ThermoFisher;
Pittsburgh, Pa., USA).
COBRA HA Functional Characterization
[0159] To determine receptor binding characteristics, virus-like
particles (VLPs) containing COBRA HA proteins were purified from
the supernatants of mammalian cell lines. HEK 293T cells were
transiently transfected with plasmids expressing HIV Gag, COBRA HA
and neuraminidase (NA, A/Thailand/1(KAN-1)/2004) and incubated for
72 hours at 37.degree. C. Supernatants were collected and VLPs were
purified via ultracentrifugation (100,000.times.g through 20%
glycerol, weight per volume) for 4 hours at 4.degree. C. The
pellets were subsequently resuspended in phosphate buffered saline
PBS, pH 7.2 and stored at -80.degree. C. until use. Protein
concentration was determined by Micro BCA.TM. Protein Assay Reagent
Kit (Pierce Biotechnology, Rockford, Ill., USA). COBRA HA VLPs were
prepared in various amounts as measured by total protein and each
individual preparation was two-fold serially diluted in v-bottom
microtiter plates. An equal volume of either 1% turkey or 1% horse
erythrocytes (RBC) (Lampire; Pipersville, Pa., USA) in PBS was
added to the diluted VLPs and incubated for 30-60 minutes at room
temperature. The HA titer was determined by the reciprocal dilution
of the last well which contained agglutinated RBC.
[0160] To determine endosomal fusion characteristics,
COBRA-pseudotyped lentiviral vectors expressing a luciferase
reporter gene were produced as described (Yang et al., J Virol
78(8):4029-4036). Briefly, 293T cells were co-transfected by using
the following plasmids: 7 .mu.g of pCMVdeltaR8.2, 7 .mu.g of
pHRCMV-Luc, 3 .mu.g pCMV/R N1(Kan-1) and 3 .mu.g pTR600 COBRA.
Cells were transiently transfected and incubated for 72 hours at
37.degree. C. Supernatants were harvested, centrifuged to clear
cell debris, filtered through a 0.22 .mu.m syringe filter,
aliquotted, and used immediately or frozen at -80.degree. C. For
fusion assays, 100 .mu.l of pseudoviruses were added to confluent
MDCK cells in 48-well plates (30,000 cells per well). Plates were
incubated at room temperature for 30 minutes, washed, and fresh
medium added. Forty-eight hours after infection, cells were lysed
in mammalian cell lysis buffer (Promega; Madison, Wis., USA). A
standard quantity of cell lysate was used in a luciferase assay
with luciferase assay reagent (Promega; Madison, Wis., USA)
according to the manufacturer's protocol.
Vaccine Preparation and Dose Determination HEK 293T cells were
transiently transfected with plasmids expressing M1 (A/Puerto
Rico/8/1934, optimized for expression in mammalian cells; SEQ ID
NO: 9), NA (A/Thailand/1(KAN-1)/2004, optimized for expression in
mammalian cells; SEQ ID NO: 8) and COBRA HA (SEQ ID NO: 7) and
incubated for 72 hours at 37.degree. C. Supernatants were collected
and cell debris removed by low speed centrifugation followed by
vacuum filtration through a 0.22 .mu.m sterile filter. VLPs were
purified via ultracentrifugation (100,000.times.g through 20%
glycerol, weight per volume) for 4 hours at 4.degree. C. The
pellets were subsequently resuspended in PBS pH 7.2 and stored in
single use aliquots at -80.degree. C. until use. Total protein
concentration was determined by Micro BCA.TM. Protein Assay Reagent
Kit (Pierce Biotechnology, Rockford, Ill., USA).
[0161] HA specific content was determined by western blot and
densitometry. Purified recombinant COBRA HA and purified VLPs were
prepared in standard total protein amounts and were electrophoresed
on a 10% SDS-PAGE gel and transferred to a PVDF membrane. The blot
was probed with mouse polyclonal antisera pooled from mice infected
with 6:2 reassortant H5N1 viruses with the surface glycoproteins
derived from either A/Vietnam/1203/2004 or A/Whooper Swan/244/2005
and the HA-antibody complexes were detected using a goat anti-mouse
IgG conjugated to horse radish peroxidase (HRP) (Southern Biotech;
Birmingham, Ala., USA). HRP was detected by chemiluminescent
substrate (Pierce Biotechnology; Rockford Ill., USA) and exposed to
X-ray film (ThermoFisher; Pittsburgh, Pa., USA). Density of bands
was determined using ImageJ software (NIH) (Abramoff et al.,
Biophotonics International 11(7):36-42, 2004). Density of
recombinant HA bands were used to calculate a standard curve and
the density of the purified VLPs was interpolated using the results
from the recombinant HA. Experiments were performed in triplicate
and multiple exposure times were analyzed for all iterations.
Codon-Optimized Influenza HA, NA and M1 Genes
[0162] The nucleotide sequences of the codon-optimized HA (SEQ ID
NO: 1), codon-optimized NA (SEQ ID NO: 3) and codon-optimized M1
(SEQ ID NO: 5) genes are set forth in the Sequence Listing. The
corresponding amino acid sequences of the encoded HA, NA and M1
proteins are set forth in the Sequence Listing as SEQ ID NO: 2, SEQ
ID NO: 4 and SEQ ID NO: 6, respectively.
Mouse Studies
[0163] BALB/c mice (Mus musculis, females, 6-8 weeks old) were
purchased from Harlan Sprague Dawley (Indianapolis, Ind., USA) and
housed in microisolator units and allowed free access to food and
water and were cared for under USDA guidelines for laboratory
animals. Mice (5 mice per group) were vaccinated with one of three
doses of purified COBRA HA VLPs (1.5 .mu.g, 0.3 .mu.g or 0.06
.mu.g), based upon HA content from a densitometry assay, via
intramuscular injection at week 0 and then boosted with the same
dose at week 3. For comparison studies, mice (20 mice per group)
were vaccinated with purified VLPs (3 .mu.g) via intramuscular
injection at week 0 and then boosted with the same dose at week 3.
Vaccines at each dose were formulated with Imject.RTM. alum
adjuvant (Imject.RTM. Alum, Pierce Biotechnology; Rockford, Ill.,
USA) according to the manufacturer's protocol or vehicle alone.
Fourteen to twenty-one days after each vaccination, blood was
collected from anesthetized mice via the retro-orbital plexus and
transferred to a microfuge tube. Tubes were centrifuged and sera
was removed and frozen at -20.+-.5.degree. C.
[0164] Three weeks after final vaccination, mice were challenged
intranasally with 5.times.10.sup.3 plaque forming units (PFU) of
the highly pathogenic H5N1 virus A/Whooper Swan/Mongolia/244/2005
(clade 2.2) in a volume of 50 .mu.l. The challenge dose represents
approximately 50LD.sub.50 in mice. After infection, mice were
monitored daily for weight loss, disease signs and death for 14
days after infection. Individual body weights, sickness scores and
death were recorded for each group on each day after inoculation.
Sickness score was determined by evaluating activity (0=normal,
1=reduced, 2=severely reduced), hunched back (0=absent, 1=present)
and ruffled fur (0=absent, 1=present) (Toapanta and Ross,
Respiratory Res 10(1):112, 2009). Experimental endpoint was defined
as >20% weight loss or display of neurological disease such as
hind limb paralysis. All H5N1 influenza virus studies were
performed under high-containment biosafety level 3 enhanced
conditions (BSL3+).
Ferret Studies
[0165] Fitch ferrets (Mustela putorius faro, female, 6-12-months of
age), influenza naive and de-scented, were purchased from Marshall
Farms (Sayre, Pa., USA). Ferrets were pair housed in stainless
steel cages (Shor-line, Kansas City, Kans., USA) containing
Sani-chips Laboratory Animal Bedding (P.J. Murphy Forest Products,
Montville, N.J., USA). Ferrets were provided with Teklad Global
Ferret Diet (Harlan Teklad, Madison, Wis., USA) and fresh water ad
libitum. The COBRA HA VLPs were diluted in PBS, pH 7.2 to achieve
final concentration. Ferrets (n=3) were vaccinated with 15 .mu.g of
purified COBRA VLPs, based upon HA content as determined by
densitometry assay, via intramuscular injection in the quadriceps
muscle in a volume of 0.25 ml at week 0 and then boosted with the
same dose at week 3. Vaccines were stored at -80.degree. C. prior
to use and formulated with Imject.RTM. alum adjuvant (Imject.RTM.
Alum; Pierce Biotechnology, Rockford, Ill., USA) immediately prior
to use. Animals were monitored for adverse events including weight
loss, temperature, loss of activity, nasal discharge, sneezing and
diarrhea weekly during the vaccination regimen. Prior to
vaccination, animals were confirmed by HAI assay to be seronegative
for circulating influenza A (H1N1 and H3N2) and influenza B
viruses. Fourteen to twenty-one days after each vaccination, blood
was collected from anesthetized ferrets via the anterior vena cava
and transferred to a microfuge tube. Tubes were centrifuged and
sera was removed and frozen at -20.+-.5.degree. C.
[0166] Three weeks after final vaccination, ferrets were challenged
intranasally with 1.times.10.sup.6 plaque forming units (PFU) of
the highly pathogenic H5N1 virus A/Whooper Swan/Mongolia/244/2005
(clade 2.2) in a volume of 0.5 ml in each nostril for a total
infection volume of 1 ml. After infection, ferrets were monitored
daily for weight loss, disease signs and death for 14 days after
infection. Individual body weights, sickness scores, and death were
recorded for each group on each day after inoculation. Sickness
score was determined by evaluating activity (0=normal, 1=alert and
active with stimulation, 2=alert but not active after stimulation,
3=not alert or active after stimulation), nasal discharge
(0=absent, 1=present), sneezing (0=absent, 1=present), decreased
food intake (0=absent, 1=present), diarrhea (0=absent, 1=present),
dyspnea (0=absent, 1=present) and neurological symptoms (0=absent,
1=present). Nasal washes were performed by instilling 3 ml of PBS
into the nares of anesthetized ferrets each day for 14 days after
inoculation. Washes were collected and stored at -80.degree. C.
until use. Experimental endpoint was defined as >20% weight
loss, development of neurological symptoms, or an activity score of
3 (not active or alert after stimulation). All H5N1 influenza virus
studies were performed under high-containment biosafety level 3
enhanced conditions (BSL3+).
Elisa
[0167] The ELISA assay was used to assess total antibody titer and
IgG isotype titer to the HA. High binding, 96-well polystyrene
plates (Costar; Lowell, Mass., USA) were coated overnight with 50
ng/well of recombinant HA. Coating antigens were derived from the
following representative viral isolates: A/Vietnam/1203/2004 (clade
1), A/Indonesia/5/2005 (clade 2.1), A/Whooper Swan/244/2005 (clade
2.2) and A/Anhui/1/2005 (clade 2.3). Plates were blocked with 5%
milk diluted in PBS with 0.05% Tween 20. Serum samples were diluted
in blocking buffer and added to plates. Serum was two-fold serially
diluted and allowed to incubate for 1 hour at room temperature.
Plates were washed and species specific antibody against IgG, IgG1,
IgG2a, IgG2b or IgG3 and linked to horseradish peroxidase (HRP)
(Southern Biotech; Birmingham, Ala., USA) were diluted in blocking
buffer and added to plates. Plates were incubated for 1 hour at
room temperature. Plates were washed and HRP was developed with TMB
substrate (Sigma-Aldrich; St. Louis, Mo., USA). Plates were
incubated in the dark for 15 minutes and then the reaction was
stopped with 2N H2SO.sub.4. Optical densities at a wavelength of
450 nm (OD.sub.450) were read by a spectrophotometer (BioTek;
Winooski, Vt., USA) and end point dilution titers were determined.
End point titers were determined as the reciprocal dilution of the
last well which had an OD.sub.450 above the mean OD.sub.450 plus
two standard deviations of naive animal sera.
Hemagglutination Inhibition (HAI)
[0168] The HAI assay was used to assess functional antibodies to HA
able to inhibit agglutination of horse erythrocytes. The protocol
was adapted from the CDC laboratory-based influenza surveillance
manual (Gillim-Ross and Subbarao, Clin Microbiol Rev 19(4):614-636,
2006). To inactivate non-specific inhibitors, sera were treated
with receptor destroying enzyme (RDE; Denka Seiken, Co., Japan)
prior to being tested (Bright et al., Lancet 366(9492):1175-1181,
2005; Bright et al., Virology 308(2):270-278, 2003; Bright et al.,
JAMA 295(8):891-894, 2006; Mitchell et al., Vaccine
21(9-10):902-914, 2004; Ross et al., Nat Immunol 1(2):127-131,
2000). Briefly, three parts RDE was added to one part sera and
incubated overnight at 37.degree. C. RDE was inactivated by
incubation at 56.degree. C. for .about.30 minutes. RDE-treated sera
was two-fold serially diluted in v-bottom microtiter plates. An
equal volume of reassortant virus, adjusted to approximately 8
HAU/50 .mu.l, was added to each well. The reassortant viruses
contained the internal genes from the mouse adapted strain A/Puerto
Rico/8/1934 and the surface proteins HA and NA from the following
representative viral isolates: A/Vietnam/1203/2004 (clade 1),
A/Indonesia/5/2005 (clade 2.1), A/Whooper Swan/244/2005 (clade 2.2)
and A/Anhui/1/2005 (clade 2.3). The plates were covered and
incubated at room temperature for 20 minutes followed by the
addition of 1% horse erythrocytes (hRBC) (Lampire Biologicals,
Pipersville, Pa., USA) in PBS. Red blood cells were stored at
4.degree. C. and used within 72 hours of preparation. The plates
were mixed by agitation, covered, and the RBCs were allowed to
settle for 1 hour at room temperature (Askonas B, McMichael A,
Webster R. The immune response to influenza viruses and the problem
of protection against infection. In: Beare AS, editor. Basic and
applied influenza research: CRC Press 1982: 159-188). The HAI titer
was determined by the reciprocal dilution of the last row which
contained non-agglutinated RBCs. Positive and negative serum
controls were included for each plate. All mice were negative
(HAI.ltoreq.1:10) for pre-existing antibodies to currently
circulating human influenza viruses prior to vaccination.
Plaque Assay
[0169] Madin-Darby Canine Kidney (MDCK) cells were plated
(5.times.10.sup.5) in each well of a 6-well plate. Samples were
diluted (final dilution factors of 10.degree. to 10.sup.-6) and
overlayed onto the cells in 100 .mu.l of DMEM supplemented with
penicillin-streptomycin and incubated for 1 hour. Samples were
removed, cells were washed twice and media was replaced with 2 ml
of L15 medium plus 0.8% agarose (Cambrex; East Rutherford, N.J.,
USA) and incubated for 72 hours at 37.degree. C. with 5% CO2.
Agarose was removed and discarded. Cells were fixed with 10%
buffered formalin, and then stained with 1% crystal violet for 15
minutes. Following thorough washing in dH.sub.2O to remove excess
crystal violet, plates were allowed to dry, plaques counted, and
the plaque forming units (PFU)/m1 were calculated.
Statistical Analysis
[0170] Statistical significance of the antibody data was determined
using a two-way analysis of variance (ANOVA) with Bonferroni's
post-test to analyze differences between each vaccine group for the
different test antigens (multiparametric). Differences in weight
loss, sickness score, and viral titers were analyzed by two-way
ANOVA, followed by Bonferroni's post test for each vaccine group at
multiple time points. Significance was defined as p<0.05.
Statistical analyses were done using GraphPad Prism software.
Results
Computationally Optimized Broadly Reactive Antigen Design
[0171] To address the challenge of antigenic diversity present in
H5N1 influenza, a computationally optimized broadly reactive
antigen (COBRA) was designed. For the first step of antigen
generation, 129 unique hemagglutinin (HA) sequences were downloaded
from the NCBI Influenza Virus Resource (IVR) sequence database (Bao
et al., J Virol 82:596-601, 2008) representing clade 2 H5N1 viruses
isolated from human infections between 2004 and 2006. The sequences
were first grouped into phylogenetic sub-clades and then further
divided into individual outbreak groups within each sub-clade based
upon geographic location and time of isolation. HA amino acid
sequences for each individual outbreak group were aligned and the
most common amino acid at each position was determined resulting in
primary consensus sequences representing each outbreak group within
each sub-clade (FIG. 1A). Primary consensus sequences within each
sub-clade were then aligned and the most common amino acid was
chosen resulting in secondary consensus sequences representing each
sub-clade (FIG. 1A). The secondary consensus sequences were aligned
and the most common amino acid at each position was selected
resulting in the final consensus sequence referred to as clade 2
COBRA HA (FIG. 1A). Phylogenetic analysis of the COBRA HA with all
human isolates of H5N1 HA proteins indicated that COBRA retained a
clade 2-like sequence without being grouped specifically within any
clade 2 sub-clade cluster (FIG. 1B). Furthermore, a BLAST search
using the COBRA HA sequence revealed that it is a unique sequence
that has not been isolated from the environment.
Characterization of COBRA
[0172] Since COBRA is a fully synthetic protein, the retention of
natural hemagglutinin function was confirmed. Initially, COBRA
expression was verified by transient transfection of mammalian
cells. Analysis of the total cell lysate demonstrated that the
COBRA HA migrates at its predicted molecular weight of
approximately 73 kDa (FIG. 2A). Because the poly-basic cleavage
site was retained in the COBRA HA sequence, both HA0 and the HA1
subunits were detected by immunoblot at similar molecular weights
as recombinant HA and the HA on the H5N1 virion (FIG. 2A).
Virus-like particles (VLPs) with COBRA HA on the surface bound
sialic acid in a dose-dependent manner and this binding was
specific to COBRA, since empty lentiviral core alone did not bind
to the receptor (FIG. 2B).
[0173] To determine if the COBRA HA was functional, the protein was
pseudotyped onto lentiviral Gag.sub.p24 to generate pseudoparticles
(Nefkens et al., J Clin Virol 39(1):27-33, 2007; Haynes et al.,
Vaccine 27(4):530-541, 2009). COBRA HA containing pseudoparticles
mediated cell fusion as efficiently as H5N1 control pseudoparticles
without the requirement for trypsin. In contrast, H1N1
pseudoparticles did require trypsin and pseudoparticles without
surface HA produced luciferase at similar levels as the cell only
controls (FIG. 2C). Taken together, these results demonstrate that
although the COBRA HA is a synthetic protein not found in nature,
it retains all of the functions of a natural hemagglutinin
protein.
Mouse Dosing Immunizations
[0174] Mice (BALB/c; n=5) were vaccinated (week 0 and 3) via
intramuscular injection with purified COBRA VLPs at either a high
dose (1.5 .mu.g HA) or low dose (0.3 .mu.g HA) with and without
Imject.RTM. alum adjuvant. At week 5, all COBRA VLP-vaccinated
animals had anti-HA antibodies that recognized heterologous
recombinant HA derived from both clade 1 and also sub-clades of
clade 2 (FIGS. 3A and 3B). Imject.RTM. alum significantly increased
anti-HA antibody titers in both low and high dose groups as
compared to the non-adjuvanted groups (p<0.01). The IgG isotype
subclasses elicited by the VLP vaccines against a clade 2.1 coating
antigen consisted mainly of IgG1 and IgG2a, indicating a mixed T
helper response (FIGS. 3C and 3D). Similar results were found for
additional coating antigens representing clade 1, clade 2.2 and
clade 2.3. In addition to recognizing HA, antibodies were also
evaluated for the ability to block virus from binding its receptor
via inhibition of viral-induced agglutination of horse erythrocytes
(HAI). All mice administered Imject.RTM. alum adjuvanted vaccines,
regardless of dose, had HAI titers.gtoreq.1:40 to viruses
expressing HA from clades 2.1 and 2.2 and 90% of the mice had
titers.gtoreq.1:40 to a clade 2.3 representative virus (FIGS. 3E
and 3F). Non-adjuvanted vaccines elicited generally lower HAI
antibody titers with 100% of high dose animals achieving
titers.gtoreq.1:40 only against clade 2.1 viruses. Imject.RTM. alum
adjuvanted vaccines elicited significantly higher HAI antibody
titers to clade 2.2 and clade 2.3 viruses regardless of dose as
compared to non-adjuvanted vaccines (p<0.05 for high dose and
p<0.001 for low dose, respectively). None of the vaccines
elicited high HAI titer antibodies to a clade 1 virus.
Mouse Dosing Challenge
[0175] Mice that received the COBRA VLP vaccines or mock vaccinated
control mice were challenged intranasally with a lethal dose of
clade 2.2 H5N1 highly pathogenic avian influenza
(A/Mongolia/whooper swan/244/2005) to evaluate the protective
efficacy of the different COBRA vaccine formulations. All COBRA
vaccinated mice, regardless of dose or the presence of adjuvant,
were protected from weight loss and death following lethal
challenge, while all mock vaccinated animals rapidly lost weight
and required euthanasia by day 6 post infection (FIGS. 4A and 4B).
Additionally, COBRA VLP vaccinated mice had no signs of disease,
while mock vaccinated animals developed such symptoms as ruffled
fur, hunched back, and lethargy (FIGS. 4C and 4D).
Mouse Comparison Immunizations
[0176] To determine if the COBRA HA vaccine elicits a broader
antibody response compared to a vaccine derived from a primary
isolate, an additional set of mice were vaccinated with either
COBRA VLPs or clade 2.2 (A/Mongolia/whooper swan/244/2005) VLPs.
Mice (BALB/c; n=20) were vaccinated (week 0 and 3) via
intramuscular injection with either COBRA VLPs or clade 2.2 VLPs at
a high dose (3 .mu.g HA) with Imject.RTM. alum adjuvant. At week 5,
all COBRA VLP-vaccinated mice and all clade 2.2 VLP-vaccinated mice
had anti-HA antibodies that recognized heterologous recombinant HA
derived from both clade 1 and various sub-clades of clade 2 (FIG.
5A). Although no significant differences were found in total IgG
titers between vaccine groups, COBRA VLP-vaccinated animals had
higher HAI antibody titers against all viruses tested as compared
to clade 2.2 VLP-vaccinated animals (p<0.01; FIG. 5B).
Furthermore, COBRA VLP-vaccinated animals had an increased
frequency of HAI titers of .gtoreq.1:40 compared to clade 2.2
VLP-vaccinated animals (Table 2).
TABLE-US-00003 TABLE 2 Mouse seroconversion frequency Vaccine
Antigen Clade 1.sup.a Clade 2.1.sup.b Clade 2.2.sup.c Clade
2.3.sup.d COBRA 45% (9/20) 100% (20/20) 100% (20/20) 100% (20/20)
Clade 2.2.sup.c 0% (0/20) 0% (0/20) 0% (0/20) 0% (0/20) Percentage
of VLP-vaccinated animals achieving an HAI titer of .gtoreq.1:40 to
each test antigen. .sup.aA/Vietnam/1203/2004
.sup.bA/Indonesia/5/2005 .sup.cA/Whooper Swan/Mongolia/244/2005
.sup.dA/Anhui/1/2005
Mouse Comparison Challenge
[0177] Mice that received the COBRA VLP vaccine, clade 2.2 VLP
vaccine or mock vaccinated control mice were challenged
intranasally with a lethal dose of clade 2.2 H5N1 highly pathogenic
avian influenza (A/Mongolia/whooper swan/244/2005) to evaluate the
protective efficacy of the VLP vaccines. All VLP-vaccinated mice
were protected from weight loss and death following lethal
challenge while all mock vaccinated animals rapidly lost weight and
required euthanasia by day 6 post infection (FIG. 6A).
Additionally, VLP vaccinated mice did not show signs of disease,
while mock vaccinated animals developed ruffled fur, hunched back,
and lethargy (FIG. 6B). Even though the clade 2.2 VLP was matched
to the challenge virus, no significant differences were found
between COBRA VLP and clade 2.2 VLP vaccinated mice in any of the
parameters analyzed indicating that the COBRA VLP vaccine protected
animals as efficiently as the homologous vaccine.
Ferret Comparison Immunizations
[0178] Ferrets are the most relevant model for influenza disease
and as such the COBRA vaccine was tested in this more rigorous
animal model. Ferrets (Fitch; n=9) were vaccinated (week 0 and 3)
via intramuscular injection with COBRA VLPs or clade 2.2 VLPs at a
high dose (15 .mu.g HA) with Imject.RTM. alum adjuvant. Serum was
collected from ferrets at week 5 and antibody responses to the
COBRA vaccines were evaluated. All vaccinated ferrets had anti-HA
antibodies that recognized heterologous recombinant HA derived from
both clade 1 and also sub-clades of clade 2 (FIG. 7A). No
significant difference in anti-HA antibody was found between the
COBRA VLP vaccine and the clade 2.2 VLP vaccine for any of the
antigens tested (p>0.05). In addition to recognizing HA,
antibodies were also evaluated for HAI activity. COBRA
VLP-vaccinated animals had higher HAI antibody titers against clade
2.1 and clade 2.3 viruses as compared to clade 2.2 VLP-vaccinated
animals (p<0.01 FIG. 7B). Similar to the mice, COBRA
VLP-vaccinated ferrets displayed an increased rate of achieving HAI
titers.gtoreq.1:40 when compared to clade 2.2 VLP-vaccinated
ferrets (Table 3).
TABLE-US-00004 TABLE 3 Ferret seroconversion frequency Vaccine
Antigen Clade 1.sup.a Clade 2.1.sup.b Clade 2.2.sup.c Clade
2.3.sup.d COBRA 0% (0/9) 78% (7/9) 56% (5/9) 56% (5/9) Clade
2.2.sup.c 0% (0/9) 0% (0/9) 22% (2/9) 0% (0/9) Percentage of
VLP-vaccinated animals achieving an HAI titer of .gtoreq.1:40 to
each test antigen. .sup.aA/Vietnam/1203/2004
.sup.bA/Indonesia/5/2005 .sup.cA/Whooper Swan/Mongolia/244/2005
.sup.dA/Anhui/1/2005
Ferret Comparison Challenge
[0179] Ferrets that received the COBRA VLP vaccines, clade 2.2 VLP
vaccines or mock vaccinated control animals were challenged
intranasally with clade 2.2 H5N1 highly pathogenic avian influenza
(A/Mongolia/whooper swan/244/2005) to evaluate the protective
efficacy of the COBRA vaccine in the ferret model of influenza
infection. All VLP vaccinated ferrets were protected from weight
loss and death following viral challenge, while all mock vaccinated
animals rapidly lost weight and 78% (7/9) of mock vaccinated
animals required euthanasia by day 7 post-infection (FIGS. 8A and
8B). Additionally, both COBRA VLP-vaccinated and clade
2.2-vaccinated ferrets were protected from acute fever and failed
to develop significant signs of disease while mock vaccinated
animals had an elevated body temperature and developed such
symptoms as lethargy, diarrhea and decreased food and water intake
(FIGS. 8C and 8D). In addition to monitoring outward signs of
disease progression, nasal washes were collected for determination
of viral replication in the upper respiratory tract. Ferrets
vaccinated with either COBRA VLPs or clade 2.2 VLPs did not have
detectable virus at any point after infection, while mock
vaccinated animals had high levels of viral replication for the
first five days of the infection (FIG. 8E). No significant
differences were found between COBRA VLP and clade 2.2 VLP
vaccinated ferrets in any of the challenge parameters analyzed
confirming the findings in mice that the COBRA VLP vaccine
protected animals as efficiently as the homologous vaccine.
[0180] The percent identity of COBRA HA and the test antigens used
in the mouse and ferret studies described above are shown in Table
4.
TABLE-US-00005 TABLE 4 Percent Identity of Test Antigens Vaccine
Antigen Clade 1.sup.a Clade 2.1.sup.b Clade 2.2.sup.c Clade
2.3.sup.d COBRA 97% 97% 95% 97% Clade 2.2.sup.c 94% 97% 100% 94% HA
amino acid sequences were aligned and percent identity across the
entire protein was determined for the vaccine immunogens compared
to the representative test antigens. .sup.aA/Vietnam/1203/2004
.sup.bA/Indonesia/5/2005 .sup.cA/Whooper Swan/Mongolia/244/2005
.sup.dA/Anhui/1/2005
Example 2: A Computationally-Optimized HA VLP Vaccines Elicits
Broadly-Reactive Antibodies that Protect Monkeys from H5N1
Infection
[0181] This example describes the finding that a COBRA clade 2 HA
H5N1 VLP elicits broad humoral immunity against multiple H5N1
isolates from different clades.
Materials and Methods
Expression and Purification of Virus-Like Particles
[0182] The COBRA HA sequence is described above in Example 1. 293T
cells were transiently transfected with plasmids expressing HA, M1,
and NA in low serum media, incubated for 72 h at 37.degree. C., and
purified by ultracentrifugation through a 20% glycerol cushion as
previously described (Giles and Ross, Vaccine 29:3043-3054, 2011).
All VLP vaccines were engineered using the same NA from
A/Thailand/1(KAN-1)/2004. HA content was quantified as previously
described (Giles and Ross, Vaccine 29:3043-3054, 2011). Two
different VLP preparations were purified, each containing one of
the HA influenza gene products: WS/05 or the COBRA HA.
Primate Immunizations and H5N1 Challenges
[0183] Cynomolgus macaques (Macaca fascicularis, male, 3-5 years
old) were vaccinated with 15 .mu.g (based upon HA content) of
purified COBRA HA VLPs (n=7) or WS/05 VLPs (n=7), via intramuscular
injection at weeks 0, 3 and 6. Vaccines at each dose were
formulated with alum adjuvant (Imject.RTM. Alum, Pierce
Biotechnology; Rockford, Ill., USA) immediately prior to use.
Twenty-one days after each vaccination, blood was collected from
anesthetized macaques. All procedures were in accordance with the
NRC Guide for the Care and Use of Laboratory Animals.
[0184] Three weeks after final vaccination, macaques were placed
into BSL3+ isolator units (Bioqual, Inc., Rockville, Md.) and then
challenged by a multi-route of infection (ocular, nasal, tracheal)
as previously described (Kobasa et al., Nature 445:319-323, 2007;
Kuiken et al., Vet Pathol 40:304-310, 2003; Rimmelzwaan et al.,
Avian Dis 47:931-933, 2003) using 1.times.10.sup.6 plaque forming
units (PFU) of the highly pathogenic H5N1 virus, A/Whooper
Swan/Mongolia/244/2005 (clade 2.2), at each location. Monkeys were
monitored daily for weight loss, signs of disease, and mortality
until 7 days after infection. Individual body weights, sickness
scores (based upon lethargy, temperature change, nasal discharge,
lack of appetite, dehydration, lack of responsiveness), and death
were recorded for each group.
[0185] Nasal and tracheal washes were performed at day 0, 1, 3, 5,
and 7 post-infection. In addition, subsets of monkeys were
sacrificed following administration of anesthesia and necropsies
were performed according to standard procedures for assessment of
gross pathologic and histopathologic changes, as well as the extent
of virus replication.
Serological Assays
[0186] A quantitative ELISA was performed to assess anti-HA
specific IgG in immune serum as previously described (Bright et
al., PLoS One 3:e1501, 2008; Giles and Ross, Vaccine 29:3043-3054,
2011). The hemagglutination inhibition (HAI) assay was used on sera
treated with receptor destroying enzyme (RDE; Denka Seiken, Co.,
Japan) prior to being tested (Bright et al., Vaccine 25:3871-3878,
2007; Mitchell et al., Vaccine 21:902-914, 2003; Bright et al.,
PLoS One 3:e1501, 2008) to assess functional antibodies to the HA
able to inhibit agglutination of horse red blood (Askonas B,
McMichael A, Webster R. The immune response to influenza viruses
and the problem of protection against infection. In: Beare AS,
editor. Basic and applied influenza research: CRC Press 1982:
159-188). The protocol was adapted from the CDC laboratory-based
influenza surveillance manual and performed as previously described
(Gillim-Ross and Subbarao, Clin Microbiol Rev 19:614-636, 2006;
Bright et al., PLoS One 3:e1501, 2008). The HAI titer was
determined by the reciprocal dilution of the last row which
contained non-agglutinated RBC. Positive and negative serum
controls were included for each plate. All monkeys were negative
(HAI.ltoreq.1:10) for pre-existing antibodies to currently
circulating human influenza viruses prior to vaccination. Serum
neutralizing antibody titers were determined by microneutralization
(MN) assays performed on Madin Darby Canine Kidney (MDCK) cells
following the procedure until CPE was observed (Rowe et al., J Clin
Microbiol 37:937-943, 1999). Cells were then fixed in 10% formalin
and stained with 1% crystal violet to quantify CPE. The
neutralizing antibody titers are expressed as the reciprocal of the
highest dilution of serum that gave 50% neutralization of 100
TCID.sub.50 of virus in MDCK cells. Geometric mean neutralizing
antibody titers were calculated for each group.
Histopathologic Evaluation and Immunohistochemical Analysis
[0187] Formalin-inflated lungs and trachea were fixed in 10%
neutral buffered formalin. Cross-sections of upper and lower left
and right lung lobes and trachea were made, concentrating on
gross-lesions. Tissue was paraffin-embedded and 6 .mu.m sections
were stained with hematoxylin and eosin for histologic evaluation.
Sequential sections were processed for immunohistochemistry or in
situ hybridization (ISH). Immunohistochemistry was performed as
described previously (Bissel et al., Am J Pathol 160:927-941, 2002)
using an immunoperoxidase method with a polyclonal antibody (Maine
Biotechnology Services, Portland, Me.) directed against influenza A
virus. A biotinylated donkey anti-goat IgG (Rockland
Immunochemicals, Gilbertsville, Pa.) was used as the secondary
antibody. ISH was performed as described previously (Fallert et
al., J Virol Methods 99:23-32, 2002) using a 35S-labeled riboprobe
synthesized using templates derived from 760 bp of influenza
A/California/04/2009 matrix protein.
Results
Vaccine Induced Antibody Responses
[0188] Cynomolgus macaques were vaccinated with COBRA VLPs or WS/05
VLPs formulated with Imject.RTM. alum at 0, 3 and 6 weeks. At week
3 post-vaccination, all COBRA VLP-vaccinated animals had anti-HA
antibodies that recognized recombinant HA derived from three
sub-clades of clade 2, which were boosted at week 6 (FIGS. 10A and
10B). There was no statistical difference (p>0.05) in the
anti-HA titers elicited against any of the HA proteins, except
monkeys vaccinated with COBRA VLPs had a statistically higher titer
against the Indo/05 HA (clade 2.1) compared with monkeys vaccinated
with the WS/05 VLP (derived from clade 2.2) on week 6.
A Single COBRA VLP Vaccination Induced High Titer HAI and MN
Antibodies to Clade 2 H5N1 Viruses
[0189] Monkeys vaccinated with COBRA VLPs (but not with WS/05 VLPs)
had HAI activity against viruses representing all three clade 2
sub-clades after a single vaccination (FIG. 10C). Four to six
monkeys responded to the COBRA VLP vaccine with an HAI
titer.gtoreq.1:40 for the all of the various test antigens. In
contrast, 4 of 7 monkeys vaccinated with the WS/05 VLP responded to
the homologous clade 2.2 virus, but none of these vaccinated
monkeys responded to the clade 2.1 or 2.3 virus. Following a second
vaccination, almost all the monkeys vaccinated with either vaccine
responded to all three viruses (FIG. 11D). These results were
confirmed by microneutralization assay (FIGS. 11E and 11F).
However, monkeys vaccinated with COBRA VLPs showed boosted HAI
titers to all three clade 2 viruses (FIG. 11).
COBRA VLPs Induced HAI Antibodies that Recognize Broader Numbers of
H5N1 Viruses
[0190] In order to determine if the COBRA HA elicited antibodies
that recognized a broader number of H5N1 isolates, serum was
collected and tested for the ability to inhibit influenza virus
induced hemagglutination of red blood cells in vitro. Antisera
collected from both vaccinated and unvaccinated monkeys were then
tested against a broad panel of H5N1 viruses representing not only
sub-clades of clade 2, but also non-clade 2 H5N1 virus strains (0,
1, 4, and 7) by HAI. Monkeys vaccinated with the COBRA VLP had high
average HAI titers against all clade 2 isolates, regardless of
sub-clade (FIG. 11). In general, all 7 monkeys responded to the
COBRA VLP vaccine and seroconverted with an HAI titer.gtoreq.1:40
against all the clade 2 viruses. In contrast, monkeys vaccinated
with the WS/05 VLP vaccine had lower HAI titers against clade 2
viruses (FIG. 10) and fewer number of monkeys responded to the
vaccine. Of the 10 clade 2 viruses tested in the HAI assay, WS/05
VLP vaccinated monkeys responded more poorly (fewer than 4 of 7
monkeys) to 4 of the isolates and none of these monkeys had
antibodies that responded to the Dk/HU/02 (clade 2.1.1) or
Eg/3300/08 (clade 2.2.1) isolates. The COBRA VLPs elicited
significantly higher HAI titers against almost all of the clade 2
viruses than the WS/05 VLPs (FIG. 11).
[0191] In addition to clade 2 isolates, a minimum of five COBRA VLP
vaccinated monkeys had HAI antibodies against both clade 1 and 7
virus isolates (FIG. 11). In comparison, almost none of the WS/05
VLP vaccinated monkeys had HAI antibodies against clade 1 and clade
7 viruses. None of the monkeys, regardless of the vaccine, had
antibodies that responded to the clade 0 or 4 isolates. All mock
vaccinated monkeys did not recognize any of the H5N1 isolates.
Challenge of Vaccinated and Unvaccinated Primates with H5N1 Clade
2.2 Virus
[0192] Three weeks after final vaccination, both VLP vaccinated and
mock-vaccinated monkeys were transferred to ABSL3+ isolator units
and then challenged with highly pathogenic H5N1 virus, A/Whooper
Swan/Mongolia/244/2005 (clade 2.2) (1.times.10.sup.6pfu), by a
multi-route (ocular, nasal, tracheal, oral) of infection (Kobasa et
al., Nature 445:319-323, 2007; Kuiken et al., Vet Pathol
40:304-310, 2003; Rimmelzwaan et al., Avian Dis 47:931-933, 2003).
There was no significant weight loss or mortality in any of the
monkeys over the 7 day period of observation. Unvaccinated monkeys
had an elevated temperature of .about.2.degree. C. that was
sustained for 5 days post-infection and higher gross pathology
scores by day 3 post-infection (Table 5).
TABLE-US-00006 TABLE 5 Lung pathology, temperature and viral titer
of vaccinated macaques Lung Pathology Elevated Score temperature
Peak Viral Titer (pfu/ml) Vaccine (day 3) (days) (day) Mock 5.3
1.9.degree. C. Nasal wash: 2.2-2.5 (5 DPI) (1-5 DPI) Trachea wash:
2.0-4.4 (3 DPI) WS/05 VLP 3.3 1.1.degree. C.-1.3.degree. C. Nasal
wash: <2 (1-5 DPI) Trachea wash: <2 COBRA 2.1 1.3.degree. C.
Nasal wash: <2 VLP (2 DPI) Trachea wash: <2
[0193] The lungs of unvaccinated monkeys had mild to moderate acute
pneumonia with alveolar pulmonary exudate by day 3 post-infection
by H&E staining. ISH showed focal collections of H5N1 infected
cells present at day 3 post-infection in alveolar spaces and to a
lesser extent in bronchial epithelium. These results were similar
to unvaccinated monkeys infected with the clade 1 H5N1 virus,
A/Vietnam/1203/2004. In contrast, monkeys vaccinated with either
the COBRA VLP or the WS/05 VLP vaccine had a reduced gross
pathology scores of 2.1-3.3 at day 3 post-infection with a milder
increase in body temperature (1.1-1.3.degree. C.) that spiked
between days 2-3 post-infection and then returned to pre-infection
temperatures. Vaccinated animals had fewer H5N1 infected cells that
were detected primarily on day 1 post-infection (Table 6).
TABLE-US-00007 TABLE 6 H5N1 lung infection scores Alveolar
Submucosal Vaccine infection score infection score 1 3 5 1 3 5 1 3
5 Mock 1.00 0.05 0 1.10 0.48 0.25 0 0 0 WS/05 VLP 0.05 0 0 0.55
0.10 0 0 0 0 COBRA 0 0 0 0.60 0.03 0.05 0 0 0 VLP
[0194] ISH for influenza was performed on tissue sections of from
upper and lower left and right lung. A semi-quantitative scoring
system was developed to evaluate the presence of influenza infected
cells. Scores were then averaged: 0.2=rare or occasional cells but
<5% of fields; 1=>1/2 to 1/4 low power fields; 2=>1/4 low
power fields; 3=essentially all low power fields.
[0195] However, monkeys vaccinated with the COBRA VLP had little to
no signs of lung inflammation by H&E staining, while animals
vaccinated with the WS/05 VLP vaccine had similar signs of
inflammation as non-vaccinated monkeys (Table 7). In addition,
unvaccinated monkeys had high titers of virus in both the nasal and
tracheal washes between days 3 and 5 post-infection. In contrast,
no virus was detected in either vaccinated groups.
TABLE-US-00008 TABLE 7 Lung involvement and inflammation scores %
lung Bronchial Alveolar involvement.sup.a inflammation.sup.b
inflammation.sup.b Vaccine 1 3 5 1 3 5 1 3 5 Mock 0.38 1.13 1.25
0.63 0.75 1.25 0.63 1.00 1.25 (0-1) (0-2) (0-2) (0-1) (0-2) (0-2)
(0-1) (0-2) (0-2) WS/05 VLP 0.75 1.50 0.88 1.00 1.42 0.63 1.00 1.25
1.00 (0-2) (0-3) (0-3) (1) (1-2) (0-2) (0-2) (0-2) (0-2) COBRA VLP
0.88 0.50 0.38 1.13 0.75 0.88 1.13 0.67 0.25 (0-2) (0-2) (0-2)
(1-2) (0-2) (0-2) (0-2) (0-2) (0-1) .sup.a% Lung involvement.
Tissue sections from upper and lower left and right lung were
evaluated for percent area demonstrating pneumonia. Scores were
then averaged. Range in parentheses. 0 = <10%, 1 = 10-24%, 2 =
25-50%, 3 = >50%. .sup.bBronchial and alveolar inflammation
scores. Tissue sections from upper and lower left and right lung
were evaluated for presence of bronchial inflammation and
denudation and alveolar immune cell infiltration. Scores were then
averages: 0 = absent, 1 = present, 2 = abundant.
Example 3: Comparison of Protective Efficacy by Vaccination with
Computationally Optimized HA and Polyvalent HA Based H5N1 VLP
Vaccines
[0196] This example describes a comparison of the COBRA HA vaccine
to a polyvalent H5N1 vaccine. The results demonstrate that a single
COBRA antigen elicits broader antibodies and is more effective than
a polyvalent mixture of primary antigens.
Materials and Methods
Vaccine Antigens and Preparation
[0197] The design and characterization of the computationally
optimized broadly reactive antigen (COBRA) is described in Example
1. Polyvalent vaccine HA antigens were derived via reverse
transcription from the following 6:2 reassortant H5N1 viruses:
A/Indonesia/5/2005 (clade 2.1; IN/05), A/Whooper
Swan/Mongolia/244/2005 (clade 2.2; WS/05) and A/Anhui/1/2005 (clade
2.3; AN/05). All HA antigens were cloned into the pTR600 expression
vector.
[0198] Virus-like particles (VLPs) were generated by transiently
transfecting HEK 293T cells with plasmids expressing M1 (A/Puerto
Rico/8/1934), NA (A/Thailand/1(KAN-1)/2004), and a single HA for
each preparation. Cells were incubated for 72 h at 37.degree. C.
after which supernatants were harvested. Cell debris was cleared by
low speed centrifugation followed by vacuum filtration through a
0.22 .mu.m sterile filter. VLPs were purified by
ultracentrifugation (100,000.times.g through 20% glycerol, weight
to volume) for 4 hours at 4.degree. C. Pellets were then
resuspended in PBS pH 7.2 and stored in single use aliquots at
-80.degree. C. until use. Total protein concentration was
determined by MicroBCA.TM. Protein Assay Reagent Kit (Pierce
Biotechnology, Rockford, Ill., USA). HA specific content of each
VLP was determined by scanning densitometry as described previously
(Giles and Ross, Vaccine 29:3043-3054, 2011). Briefly, purified HA
matched to each VLP was electrophoresed with purified VLPs,
transferred to a PVDF membrane and probed by western blot with
H5-specific antisera. The relative density of the HA band in the
purified protein lanes was used to calculate a standard curve and
the density of the HA in the VLP lanes was interpolated. In total,
four different VLP preparations were purified and HA content
quantified independently, each containing one of the three
wild-type influenza gene products (IN/05, WS/05, AN/05) or the
COBRA HA.
Mouse Studies
[0199] BALB/c mice (Mus musculis, females, 6-8 weeks) were
purchased from Harlan Sprague Dawley, (Indianapolis, Ind., USA) and
housed in microisolator units and allowed free access to food and
water and were cared for under USDA guidelines for laboratory
animals. Mice were vaccinated with purified COBRA VLPs (3 .mu.g HA)
or a polyvalent formulation of VLPs consisting of 1 .mu.g HA each
IN/05, WS/05 and AN/05 (3 .mu.g HA total) via intramuscular
injection at week 0 and then boosted at week 3. Vaccines were
formulated with Imject.RTM. alum adjuvant (Imject.RTM. Alum, Pierce
Biotechnology; Rockford, Ill., USA) according to the manufacturer's
protocol. Fourteen to twenty-one days after each vaccination, blood
was collected from anesthetized mice via the retro-orbital plexus
and transferred to a microfuge tube. Tubes were centrifuged and
sera was removed and frozen at -20.+-.5.degree. C.
[0200] Three weeks after final vaccination, mice were challenged
intranasally with 5.times.10.sup.3 plaque forming units (PFU) of
either highly pathogenic wild type H5N1 virus A/Whooper
Swan/Mongolia/244/2005 (n=20/group) or 6:2 reassortant virus with
internal genes from the mouse adapted virus A/Puerto Rico/8/1934
and the surface proteins HA and NA from A/Vietnam/1203/2004
(n=10/group) in a total volume of 50 .mu.l. Challenge doses for
both viruses were established independently and represent
approximately 50LD.sub.50. After infection, mice were monitored
daily for weight loss, disease signs and death for 14 days after
infection. Individual body weights, sickness scores and death were
recorded for each group on each day after inoculation. Sickness
score was determined by evaluating activity (0=normal, 1=reduced,
2=severely reduced), hunched back (0=absent, 1=present) and ruffled
fur (0=absent, 1=present) (Toapanta and Ross, Respiratory Res
10(1):112, 2009). Experimental endpoint was determined by >20%
weight loss or display of neurological disease such as hind limb
paralysis. All highly pathogenic wild type H5N1 influenza virus
studies were performed under high-containment biosafety level 3
enhanced conditions (BSL3+).
Ferret Studies
[0201] Fitch ferrets (Mustela putorius faro, female, 6-12-months of
age), influenza naive and descented, were purchased from Marshall
Farms (Sayre, Pa., USA). Ferrets were pair housed in stainless
steel cages (Shor-line, Kansas City, Kans., USA) containing
Sani-chips Laboratory Animal Bedding (P.J. Murphy Forest Products,
Montville, N.J., USA). Ferrets were provided with Teklad Global
Ferret Diet (Harlan Teklad, Madison, Wis., USA) and fresh water ad
libitum. The VLPs were diluted in PBS, pH 7.2 to achieve final
concentration. Ferrets (n=6) were vaccinated with purified COBRA
VLPs (15 .mu.g HA) or a polyvalent formulation of VLPs consisting
of 5 .mu.g HA each IN/05, WS/05 and AN/05 (15 .mu.g HA total) via
intramuscular injection at week 0 and then boosted at week 3.
Vaccines were formulated with Imject.RTM. alum adjuvant
(Imject.RTM. Alum, Pierce Biotechnology; Rockford, Ill., USA)
immediately prior to use according to the manufacturer's protocol.
Animals were monitored for adverse events including weight loss,
temperature, loss of activity, nasal discharge, sneezing and
diarrhea weekly during the vaccination regimen. Prior to
vaccination, animals were confirmed by HAI assay to be seronegative
for circulating influenza A (H1N1 and H3N2) and influenza B
viruses. Fourteen to twenty-one days after each vaccination, blood
was collected from anesthetized ferrets via the anterior vena cava
and transferred to a microfuge tube. Tubes were centrifuged and
sera was removed and frozen at -20.+-.5.degree. C.
[0202] Three weeks after final vaccination, ferrets were challenged
intranasally with 1.times.10.sup.6 plaque forming units (PFU) of
the highly pathogenic H5N1 virus A/Whooper Swan/Mongolia/244/2005
(clade 2.2) in a volume of 0.5 ml in each nostril for a total
infection volume of 1 ml. After infection, ferrets were monitored
daily for weight loss, disease signs and death for 14 days after
infection. Individual body weights, sickness scores, and death were
recorded for each group on each day after inoculation. Sickness
score was determined by evaluating activity (0=normal, 1=alert and
active after stimulation, 2=alert but not active after stimulation,
3=neither active nor alert after stimulation), nasal discharge
(0=absent, 1=present), sneezing (0=absent, 1=present), decreased
food intake (0=absent, 1=present), diarrhea (0=absent, 1=present),
dyspnea (0=absent, 1=present) and neurological symptoms (0=absent,
1=present) as previously described (Giles and Ross, Vaccine
29:3043-3054, 2011). Experimental endpoint was defined as >20%
weight loss, development of neurological disease or an activity
score of 3 (neither active nor alert after stimulation). Nasal
washes were performed by instilling 3 ml of PBS into the nares of
anesthetized ferrets each day for 14 days after inoculation. Washes
were collected and stored at -80.degree. C. until use. All highly
pathogenic wild type H5N1 influenza virus studies were performed
under high-containment biosafety level 3 enhanced conditions
(BSL3+).
ELISA Assay
[0203] The ELISA assay was used to assess total antibody titer to
the HA. High binding, 96-well polystyrene plates (Costar; Lowell,
Mass., USA) were coated overnight with 50 ng/well of recombinant
HA. Coating antigens were derived from the following representative
viral isolates: A/Vietnam/1203/2004 (clade 1), A/Indonesia/5/2005
(clade 2.1), A/Whooper Swan/Mongolia/244/2005 (clade 2.2) and
A/Anhui/1/2005 (clade 2.3). Plates were blocked with 5% milk
diluted in PBS with 0.05% Tween 20. Serum samples were diluted in
blocking buffer and added to plates. Serum was two-fold serially
diluted and allowed to incubate for 1 hour at room temperature.
Plates were washed and species specific antibody against IgG linked
to horseradish peroxidase (HRP) was diluted in blocking buffer and
added to plates. Plates were incubated for 1 hour at room
temperature. Plates were washed and HRP was developed with TMB
substrate (Sigma-Aldrich; St. Louis, Mo., USA). Plates were
incubated in the dark for 15 minutes and then the reaction was
stopped with 2N H2504. Optical densities at a wavelength of 450 nm
(OD.sub.450) were read by a spectrophotometer (BioTek; Winooski,
Vt., USA) and end point dilution titers were determined as the
reciprocal dilution of the last well which had an OD.sub.450 above
the mean OD.sub.450 plus two standard deviations of naive animal
sera.
Hemagglutination Inhibition (HAI) Assay
[0204] The HAI assay was used to assess functional antibodies to
the HA able to inhibit agglutination of horse erythrocytes. The
protocol was adapted from the CDC laboratory-based influenza
surveillance manual (Gillim-Ross and Subbarao, Clin Microbiol Rev
19(4):614-636, 2006). To inactivate non-specific inhibitors, sera
were treated with receptor destroying enzyme (RDE; Denka Seiken,
Co., Japan) prior to being tested. Briefly, three parts RDE was
added to one part sera and incubated overnight at 37.degree. C. RDE
was inactivated by incubation at 56.degree. C. for .about.30 min.
RDE treated sera was two-fold serially diluted in v-bottom
microtiter plates. An equal volume of reassortant virus, adjusted
to approximately 8 HAU/50.sub.111, was added to each well. The
reassortant viruses contained the internal genes from the mouse
adapted strain A/Puerto Rico/8/1934 and the surface proteins HA and
NA from the following representative viral isolates:
A/Vietnam/1203/2004 (clade 1), A/Indonesia/5/2005 (clade 2.1),
A/Whooper Swan/Mongolia/244/2005 (clade 2.2) and A/Anhui/1/2005
(clade 2.3). The plates were covered and incubated at room
temperature for 20 minutes followed by the addition of 1% horse
erythrocytes (HRBC) (Lampire Biologicals, Pipersville, Pa., USA) in
PBS. Red blood cells were stored at 4.degree. C. and used within 72
h of preparation. The plates were mixed by agitation, covered, and
the RBCs were allowed to settle for 1 h at room temperature
(Askonas B, McMichael A, Webster R. The immune response to
influenza viruses and the problem of protection against infection.
In: Beare AS, editor. Basic and applied influenza research: CRC
Press 1982: 159-188). The HAI titer was determined by the
reciprocal dilution of the last well which contained
non-agglutinated RBC. Positive and negative serum controls were
included for each plate. All mice and ferrets were negative
(HAI.ltoreq.1:10) for pre-existing antibodies to currently
circulating human influenza viruses prior to vaccination.
Plaque Assay
[0205] For mouse infections, lung virus titers were evaluated. For
ferret infections, nasal wash virus titers were used to assess
viral burden. Both lungs and nasal wash virus titers were
determined using a plaque assay (Tobita et al., Med Microbiol
Immunol 162:23-27, 1975; Tobita et al., Med Microbiol Immunol
162:9-14, 1975). Briefly, lungs from mice infected with virus were
harvested post infection, snap-frozen and stored at -80.degree. C.
until use. Samples were thawed, weighed and single cell suspensions
were prepared via passage through a 70 .mu.m mesh (BD Falcon,
Bedford, Mass., USA) in an appropriate volume of DMEM supplemented
with penicillin-streptomycin (iDEME) as to achieve 100 mg/ml final
concentration. Cell suspensions were centrifuged at 2000 rpm for 5
minutes and the supernatants were collected.
[0206] Madin-Darby Canine Kidney (MDCK) cells were plated
(5.times.10.sup.5) in each well of a 6 well plate. Samples (lung
supernatants for mice and nasal washes for ferrets) were diluted
(dilution factors of 1.times.10.sup.1 to 10.sup.6) and overlayed
onto the cells in 100 .mu.l of iDMEM and incubated for 1 hour.
Virus-containing medium was removed and replaced with 2 ml of L15
medium plus 0.8% agarose (Cambrex, East Rutherford, N.J., USA) and
incubated for 96 hours at 37.degree. C. with 5% CO2. Agarose was
removed and discarded. Cells were fixed with 10% buffered formalin,
and then stained with 1% crystal violet for 15 minutes. Following
thorough washing in dH2O to remove excess crystal violet, plates
were allowed to dry, plaques counted, and the plaque forming units
(PFU)/g for or PFU/ml for nasal washes were calculated.
Histopathological Analysis
[0207] Left lobes of lungs from infected mice were collected 4 days
post-infection and placed into 10% buffered formalin. After
fixation, lungs were paraffin embedded and 6 .mu.m sections were
prepared for histopathological analysis. For in situ hybridization
(ISH), vectors containing 760 bp of Influenza/California/04/2009
matrix protein were linearized to create antisense and sense
templates. .sup.35S-labeled riboprobes were generated using
MAXIscript in vitro transcription kit (Ambion, Austin, Tex.). ISH
was performed as described before (Bissel et al., Brain Pathol,
Accepted Article doi: 10.1111/j.1750-3639.2010.00514.x). Control
riboprobes did not hybridize to lung tissue at any time point
post-infection and non-infected tissue did not show hybridization
with viral probes. Hybridized slides were assessed and scored for
abundance of foci.
Cellular Assays
[0208] The number of anti-influenza specific cells secreting
interferon gamma (IFN-.gamma.) was determined by enzyme-linked
immunospot (ELISpot) assay (R&D systems, Minneapolis, Minn.,
USA) following the manufacturer's protocol. Mice were sacrificed at
6 days post infection (DPI) and spleens and lungs were harvested
and prepared in single cell suspensions. Briefly, pre-coated
anti-IFN.gamma. plates were blocked with RPMI plus 10% FCS and
antibiotics (cRPMI) for 30 minutes at room temperature. Media was
removed from wells and 10.sup.5 cells were added to each well.
Cells were stimulated with purified recombinant HA from
ANietnam/1203/2004 (truncated at residue 530; 1 .mu.g/well),
inactivated 6:2 reassortant virus A/Vietnam/1203/2004 (1:100
dilution of inactivated stock; 100 .mu.l/well) or the
immunodominant H2-K.sup.d CD8.sup.+ T cell epitope in H5 HA:
HA.sub.533 (IYSTVASSL; SEQ ID NO: 10; 1 .mu.g/well) (Pepscan
Presto, Leystad, Netherlands). Additional wells were stimulated
with PMA (50 ng/well) and ionomycin (500 ng/well) as positive
controls or Ova257 (SIINFEKL; SEQ ID NO: 11; 1 .mu.g/well) (Pepscan
Presto, Leystad, Netherlands) as negative controls. Additionally,
IL-2 (10 U/ml) was added to each well. Plates were incubated at
37.degree. C. for 48 hours. After incubation, plates were washed
four times with R&D wash buffer and were incubated at 4.degree.
C. overnight with biotinylated anti-mouse IFN.gamma.. Plates were
washed as before and incubated at room temperature for 2 hours with
streptavidin conjugated to alkaline phosphatase. Plates were washed
as before and spots were developed by incubating at room
temperature for 1 hour in the dark with BCIP/NBT chromogen
substrate. The plates were washed extensively with DI H.sub.2O and
allowed to dry overnight prior to spots being counted using an
ImmunoSpot ELISpot reader (Cellular Technology Ltd., Cleveland,
Ohio, USA).
[0209] The number of anti-HA and anti-NA specific antibody
secreting cells was determined by B cell ELISpot assay as
previously described (Joo et al., Vaccine 28:2186-2194, 2009;
Sasaki et al., PLoS ONE 3:e2975, 2008; Sasaki et al., J Virol
81:215-228, 2007). Mice were sacrificed at 6 DPI and spleens and
lungs were harvested and prepared in single cell suspensions.
Briefly, 0.45 .mu.m PVDF membrane plates (Millipore, Billerica,
Mass., USA) were coated with either purified recombinant HA from
A/Vietnam/1203/2004 or purified recombinant NA from
A/Thailand/1(KAN-1)/2004 (250 ng/well) and incubated at 4.degree.
C. overnight. Plates were washed three times with PBS and blocked
with cRPMI for at 37.degree. C. for 3-4 hours. Media was removed
from wells and 10.sup.5 cells were added to each well. Plates were
incubated at 37.degree. C. for 48 hours. After incubation, plates
were washed as before and incubated at room temperature for 2 hours
with horse radish peroxidase conjugated anti-mouse IgG or IgA
(Southern Biotech, Birmingham, Ala., USA). Plates were washed as
before and spots were developed at room temperature for 1 hour in
the dark with detection substrate (NovaRED.TM.; Vector Labs,
Burlingame, Calif., USA). The plates were washed extensively with
DI H.sub.2O and allowed to dry overnight prior to spots being
counted using an ImmunoSpot ELISpot reader (Cellular Technology
Ltd., Cleveland, Ohio, USA).
Passive Transfer of Sera
[0210] Serum from vaccinated mice was pooled and passively
transferred into 9 week old recipient BALB/c mice (n=5/group).
Equal amounts of serum from each mouse in a particular vaccine
group were pooled and heat inactivated for 30 minutes at 56.degree.
C. 200 .mu.l of pooled and inactivated serum was transferred to
recipient mice via IP injection. 24 hours post transfer, mice were
infected with 6:2 reassortant virus with internal genes from the
mouse adapted virus A/Puerto Rico/8/1934 and surface antigens from
A/Vietnam/1203/2004.
Statistical Analysis
[0211] Statistical significance of the antibody and cellular
immunology data was determined using a two-tailed Student's T test
to analyze differences between COBRA and polyvalent vaccine groups
for each of the different test antigens. Differences in weight loss
and sickness score were analyzed by two-way ANOVA, followed by
Bonferroni's post test for each vaccine group at multiple time
points (multiparametric). Statistical significance of viral titer
data was evaluated using a two-tailed Student's T test on
Log.sub.10 transformed values. Significance was defined as
p<0.05. Statistical analyses were done using GraphPad Prism
software.
Results
Immunogenicity in Mice and Ferrets
[0212] BALB/c mice were vaccinated twice via intramuscular
injection with either purified COBRA or polyvalent VLPs and two
weeks after the second vaccination serum was analyzed for antibody
responses. All vaccinated mice had high titer anti-HA antibodies
that bound to recombinant HA derived from both clade 1 and various
sub-clades of clade 2 (FIG. 12A). Although both COBRA and
polyvalent vaccines elicited similar total IgG, COBRA vaccinated
animals had higher HAI antibody titers for all viruses tested
(p<0.001; FIG. 12B). In addition to higher HAI titer, COBRA
vaccinated mice had an increased frequency of HAI titers
.gtoreq.1:40 for all viruses tested, including those which were
components of the polyvalent formulation (Table 8).
[0213] To confirm the results from mice in a more rigorous animal
model, ferrets were vaccinated twice via intramuscular injection
with either COBRA or polyvalent vaccines. Serum was collected two
weeks after the second vaccination and antibody responses were
evaluated. Similar to the mice, all vaccinated ferrets had anti-HA
antibodies that bound to diverse recombinant HA and the relative
total IgG titers were equivalent for both COBRA and polyvalent
vaccines (FIG. 12C). COBRA vaccinated ferrets demonstrated
increased HAI antibody titers compared to polyvalent vaccinated
animals against all viruses tested, however only the antibodies to
the clade 2.1 virus were significantly different (p<0.05; FIG.
12D). Furthermore, COBRA vaccinated animals displayed an increased
rate of achieving an HAI titer of .gtoreq.1:40 in comparison to the
polyvalent vaccinated ferrets for all test antigens (Table 8).
TABLE-US-00009 TABLE 8 Seroconversion frequency Species Vaccine
antigen Clade 1 Clade 2.1 Clade 2.2 Clade 2.3 Mouse COBRA 60%
(18/30) 100% (30/30) 100% (30/30) 100% (30/30) Polyvalent 3.3%
(1/30) 70% (21/30) 50% (15/30) 53% (16/30) Ferret COBRA 33% (2/6)
67% (4/6) 50% (3/6) 50% (3/6) Polyvalent 0% (0/6) 33% (2/6) 0%
(0/6) 0% (0/6)
Wild Type Clade 2.2 Challenge
[0214] To confirm protective efficacy against highly pathogenic
H5N1 infection, vaccinated animals were challenged with a lethal
dose of the wild-type clade 2.2 isolate A/Whooper
Swan/Mongolia/244/2005. All VLP vaccinated mice were protected from
weight loss and death while mock vaccinated animals rapidly lost
weight and reached experimental end-point by 6 days post infection
(DPI; FIG. 13A). COBRA and polyvalent vaccinated mice both had a
mean maximum weight loss of 4% at 12 and 13 DPI, respectively.
Additionally, all VLP vaccinated mice failed to develop any overt
signs of disease while mock vaccinated mice developed visible
illness (FIG. 13B).
[0215] Similar to the mice, all VLP vaccinated ferrets were
protected from death following a lethal challenge. Vaccinated
ferrets demonstrated mild weight loss in response to the infection
with COBRA vaccinated animals having mean maximum weight loss of
5.5% at 2 DPI and polyvalent vaccinated animals losing 6.8% at 3
DPI (FIG. 13C). Both groups rapidly recovered weight and failed to
develop any significant signs of disease (FIG. 13D). Furthermore,
VLP vaccinated animals did not demonstrate any temperature spikes
while mock vaccinated animals had an elevated temperature of
.about.3.degree. C. for 1-3 DPI.
[0216] To evaluate vaccine efficacy with a more sensitive output
than morbidity and mortality, the viral burden of infected animals
was also determined. Both COBRA and polyvalent vaccinated mice had
reduced lung viral titers as quickly as 1 DPI when compared to mock
vaccinated animals. Furthermore, COBRA vaccinated mice did not have
detectable virus by 3 DPI while polyvalent vaccinated mice
demonstrated prolonged viral replication with 1.8.times.10.sup.3
PFU/g at 3 DPI (p<0.05; FIG. 14A). Additionally, both VLP
vaccines prevented extra-pulmonary spread of the virus while mock
vaccinated animals had detectable virus in both kidney and liver by
3 DPI. Control of virus replication in ferrets was similar to that
observed in mice, although complete clearance of the virus was
delayed (FIG. 14B). All VLP vaccinated animals had decreased
recovery of virus in nasal washes compared to mock vaccinated
ferrets at all timepoints tested (p<0.05). COBRA vaccinated
animals did not have detectable virus by 5 DPI. In contrast, virus
replication did not reach undetectable levels until 9 DPI in
polyvalent vaccinated ferrets.
Histopathology of Infected Lungs
[0217] To evaluate the location and severity of influenza viral
antigen and viral replication, ISH for influenza A MP was scored on
3 DPI lung sections. COBRA vaccinated animals had rare bronchial
epithelium infection (FIGS. 15A and 15B). Animals receiving
polyvalent vaccines had occasional bronchial epithelium infection
that was comparable to the COBRA vaccinated animals (FIGS. 15A and
15B). This was in contrast to significant bronchial epithelium
infection and replication observed in mock animals (FIGS. 15A and
15B).
Reassortant Clade 1 Challenge
[0218] Having established the clade 2.2 protective profile of both
the COBRA and polyvalent vaccines, the efficacy of these vaccines
against a more divergent clade 1 challenge in mice was evaluated.
COBRA and polyvalent vaccinated mice were challenged with 6:2
reassortant virus containing the HA and NA proteins from the clade
1 virus A/Vietnam/1203/2004. All VLP vaccinated animals were
protected from weight loss and death while mock vaccinated animals
rapidly lost weight and reached experimental endpoint by 7 DPI
(FIG. 16A). Furthermore, vaccinated mice also did not develop any
signs of disease throughout the course of the study (FIG. 16B).
Lungs were harvested at 3 DPI for determination of viral burden
(FIG. 16C). COBRA vaccinated animals did not have detectable virus
while polyvalent animals had 1.1.times.10.sup.3 PFU/g virus
(p=0.12). Importantly, both vaccines had significantly less
recoverable virus than mock vaccinated animals at 3 DPI
(p<0.01).
Post-Challenge Cellular Immune Responses
[0219] The magnitude of influenza specific cellular immune
responses in the lungs post-infection was evaluated via ELISpot
assay for both antibody secreting cells (ASC) and IFN-.gamma.
producing cells. Vaccinated mice were infected with reassortant
A/Vietnam/1203/2994 virus as before and lungs were harvested at 6
DPI. COBRA and polyvalent vaccinated animals had statistically
equivalent numbers of both IgG and IgA ASC specific for HA from the
challenge virus (p>0.05; FIG. 17A). No ASC were detected in mock
vaccinated animals indicating that the 6 DPI time point is likely
representative of a recall response. Additionally, the majority of
the ASC response to infection was specific for HA as lower numbers
of cells were detected for the NA component of the vaccines.
[0220] VLP vaccine primed IFN-.gamma. secreting cells were also
evaluated after infection. IFN-.gamma. responses were equivalent
between VLP vaccine groups regardless of stimulating antigen
(p>0.05; FIG. 17B). Recombinant HA and inactivated virus were
inefficient stimulators of IFN-.gamma. production compared to the
HA533 peptide. HA533 is the immunodominant CD8.sup.+ T cell epitope
in BALB/c mice and is conserved in all HA vaccine antigens used in
this study. Overlapping peptide pools spanning the entire HA
molecule were also used to stimulate cells and no differences were
observed between COBRA and polyvalent vaccines for any of the
pools. Similar to the ASC data, no IFN-.gamma. responses were
detectable above background in mock vaccinated animals at 6
DPI.
Passive Transfer of Immune Sera
[0221] The contribution of serum factors to protection from clade 1
challenge was evaluated using a passive transfer model. Nine-week
old recipient mice were administered pooled sera via IP injection
from COBRA, polyvalent and mock vaccinated mice. The next day,
recipient mice were challenged with the clade 1 reassortant
A/Vietnam/1203/2004 virus as before. Regardless of transferred
serum, all recipient mice lost weight and became visibly ill (FIGS.
18A and 18B). COBRA serum recipient mice lost less weight than
polyvalent recipient mice with maximum losses of 5.2% (6 DPI) and
11.8% (7 DPI), respectively (p<0.05 at 7 DPI). COBRA serum
recipient mice also began to resolve the clinical symptoms more
rapidly than polyvalent recipient mice (p<0.05 at 7 DPI).
Although COBRA serum prevented recipient mice from developing
illness more efficiently than polyvalent serum, both COBRA and
polyvalent serum protected all recipient mice from death.
Conversely, all mice receiving serum from mock vaccinated mice
rapidly lost weight, became visibly ill and reached experimental
endpoint by 7 DPI.
[0222] In view of the many possible embodiments to which the
principles of the disclosed invention may be applied, it should be
recognized that the illustrated embodiments are only examples of
the disclosure and should not be taken as limiting the scope of the
invention. Rather, the scope of the invention is defined by the
following claims. We therefore claim as our invention all that
comes within the scope and spirit of these claims.
Sequence CWU 1
1
1111707DNAArtificial SequenceSynthetic polynucleotideCDS(1)..(1707)
1atg gaa aag atc gtg ctg ctg ctg gct atc gtg agc ctg gtg aag agc
48Met Glu Lys Ile Val Leu Leu Leu Ala Ile Val Ser Leu Val Lys Ser1
5 10 15gac cag att tgc atc ggc tac cac gcc aac aac agc acc gag cag
gtg 96Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr Glu Gln
Val 20 25 30gac acc atc atg gaa aag aac gtc acc gtg acc cac gcc cag
gac atc 144Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His Ala Gln
Asp Ile 35 40 45ctg gaa aag acc cac aac ggc aag ctg tgc gac ctg gac
ggc gtg aag 192Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp Leu Asp
Gly Val Lys 50 55 60ccc ctg atc ctg agg gac tgc agc gtg gcc ggc tgg
ctg ctg ggc aac 240Pro Leu Ile Leu Arg Asp Cys Ser Val Ala Gly Trp
Leu Leu Gly Asn65 70 75 80ccc atg tgc gac gag ttc atc aac gtg ccc
gag tgg agc tac atc gtg 288Pro Met Cys Asp Glu Phe Ile Asn Val Pro
Glu Trp Ser Tyr Ile Val 85 90 95gag aag gcc aac ccc gcc aac gac ctg
tgc tac ccc ggc aac ttc aac 336Glu Lys Ala Asn Pro Ala Asn Asp Leu
Cys Tyr Pro Gly Asn Phe Asn 100 105 110gac tac gag gaa ctg aag cac
ctg ctg tcc agg atc aac cac ttc gag 384Asp Tyr Glu Glu Leu Lys His
Leu Leu Ser Arg Ile Asn His Phe Glu 115 120 125aag atc cag atc atc
ccc aag agc agc tgg tcc gac cac gag gcc agc 432Lys Ile Gln Ile Ile
Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser 130 135 140agc ggc gtg
agc agc gcc tgc cca tac cag ggc agc ccc agc ttc ttc 480Ser Gly Val
Ser Ser Ala Cys Pro Tyr Gln Gly Ser Pro Ser Phe Phe145 150 155
160aga aac gtg gtg tgg ctg atc aag aag aac aac acc tac ccc acc atc
528Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Thr Tyr Pro Thr Ile
165 170 175aag agg tcc tac aac aac acc aac cag gaa gat ctg ctg gtc
ctg tgg 576Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu Val
Leu Trp 180 185 190ggc atc cac cac cct aat gac gcc gcc gaa cag acc
agg ctg tac cag 624Gly Ile His His Pro Asn Asp Ala Ala Glu Gln Thr
Arg Leu Tyr Gln 195 200 205aac ccc acc acc tac atc agc gtg ggc aca
agc acc ctg aac cag agg 672Asn Pro Thr Thr Tyr Ile Ser Val Gly Thr
Ser Thr Leu Asn Gln Arg 210 215 220ctg gtg ccc aag atc gcc acc agg
tcc aag gtg aac gga cag tcc ggc 720Leu Val Pro Lys Ile Ala Thr Arg
Ser Lys Val Asn Gly Gln Ser Gly225 230 235 240agg atg gaa ttc ttc
tgg acc atc ctg aag cct aac gac gcc atc aac 768Arg Met Glu Phe Phe
Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn 245 250 255ttc gag agc
aac ggc aac ttt atc gcc ccc gag tac gcc tac aag atc 816Phe Glu Ser
Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile 260 265 270gtg
aag aag ggc gac agc gcc atc atg aag agc gag ctg gaa tac ggc 864Val
Lys Lys Gly Asp Ser Ala Ile Met Lys Ser Glu Leu Glu Tyr Gly 275 280
285aac tgc aac acc aag tgc cag acc ccc atc ggc gcc atc aac agc agc
912Asn Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn Ser Ser
290 295 300atg ccc ttc cac aac atc cac ccc ctg acc atc ggc gag tgc
ccc aag 960Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly Glu Cys
Pro Lys305 310 315 320tac gtg aag agc aac agg ctg gtg ctg gcc acc
ggc ctg agg aac agc 1008Tyr Val Lys Ser Asn Arg Leu Val Leu Ala Thr
Gly Leu Arg Asn Ser 325 330 335ccc cag aga gag agc aga aga aag aag
agg ggc ctg ttc ggc gct atc 1056Pro Gln Arg Glu Ser Arg Arg Lys Lys
Arg Gly Leu Phe Gly Ala Ile 340 345 350gcc ggc ttc atc gag ggc ggc
tgg cag ggc atg gtg gac ggg tgg tac 1104Ala Gly Phe Ile Glu Gly Gly
Trp Gln Gly Met Val Asp Gly Trp Tyr 355 360 365ggc tac cac cac tct
aac gag cag ggc agc ggc tac gcc gcc gac aaa 1152Gly Tyr His His Ser
Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys 370 375 380gag agc acc
cag aag gcc atc gac ggc gtc acc aac aag gtg aac agc 1200Glu Ser Thr
Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser385 390 395
400atc atc gac aag atg aac acc cag ttc gag gcc gtg ggc aga gag ttc
1248Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu Phe
405 410 415aac aac ctg gaa agg cgg atc gag aac ctg aac aag aaa atg
gaa gat 1296Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys Met
Glu Asp 420 425 430ggc ttc ctg gac gtg tgg acc tac aac gcc gag ctg
ctg gtg ctg atg 1344Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu Leu
Leu Val Leu Met 435 440 445gaa aac gag agg acc ctg gac ttc cac gac
agc aac gtg aag aac ctg 1392Glu Asn Glu Arg Thr Leu Asp Phe His Asp
Ser Asn Val Lys Asn Leu 450 455 460tac gac aaa gtg cgg ctg cag ctg
agg gac aac gcc aaa gag ctg ggc 1440Tyr Asp Lys Val Arg Leu Gln Leu
Arg Asp Asn Ala Lys Glu Leu Gly465 470 475 480aac ggc tgc ttc gag
ttc tac cac aag tgc gac aac gag tgc atg gaa 1488Asn Gly Cys Phe Glu
Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu 485 490 495agc gtg agg
aac ggc acc tac gac tac ccc cag tac agc gag gaa gcc 1536Ser Val Arg
Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala 500 505 510agg
ctg aag agg gaa gag atc agc gga gtg aag ctg gaa agc atc ggc 1584Arg
Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly 515 520
525acc tac cag atc ctg agc atc tac agc acc gtc gcc agc agc ctg gcc
1632Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser Leu Ala
530 535 540ctg gct atc atg gtg gcc gga ctg agc ctg tgg atg tgc agc
aac ggc 1680Leu Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met Cys Ser
Asn Gly545 550 555 560agc ctg cag tgc agg atc tgc atc tga 1707Ser
Leu Gln Cys Arg Ile Cys Ile 5652568PRTArtificial SequenceSynthetic
Construct 2Met Glu Lys Ile Val Leu Leu Leu Ala Ile Val Ser Leu Val
Lys Ser1 5 10 15Asp Gln Ile Cys Ile Gly Tyr His Ala Asn Asn Ser Thr
Glu Gln Val 20 25 30Asp Thr Ile Met Glu Lys Asn Val Thr Val Thr His
Ala Gln Asp Ile 35 40 45Leu Glu Lys Thr His Asn Gly Lys Leu Cys Asp
Leu Asp Gly Val Lys 50 55 60Pro Leu Ile Leu Arg Asp Cys Ser Val Ala
Gly Trp Leu Leu Gly Asn65 70 75 80Pro Met Cys Asp Glu Phe Ile Asn
Val Pro Glu Trp Ser Tyr Ile Val 85 90 95Glu Lys Ala Asn Pro Ala Asn
Asp Leu Cys Tyr Pro Gly Asn Phe Asn 100 105 110Asp Tyr Glu Glu Leu
Lys His Leu Leu Ser Arg Ile Asn His Phe Glu 115 120 125Lys Ile Gln
Ile Ile Pro Lys Ser Ser Trp Ser Asp His Glu Ala Ser 130 135 140Ser
Gly Val Ser Ser Ala Cys Pro Tyr Gln Gly Ser Pro Ser Phe Phe145 150
155 160Arg Asn Val Val Trp Leu Ile Lys Lys Asn Asn Thr Tyr Pro Thr
Ile 165 170 175Lys Arg Ser Tyr Asn Asn Thr Asn Gln Glu Asp Leu Leu
Val Leu Trp 180 185 190Gly Ile His His Pro Asn Asp Ala Ala Glu Gln
Thr Arg Leu Tyr Gln 195 200 205Asn Pro Thr Thr Tyr Ile Ser Val Gly
Thr Ser Thr Leu Asn Gln Arg 210 215 220Leu Val Pro Lys Ile Ala Thr
Arg Ser Lys Val Asn Gly Gln Ser Gly225 230 235 240Arg Met Glu Phe
Phe Trp Thr Ile Leu Lys Pro Asn Asp Ala Ile Asn 245 250 255Phe Glu
Ser Asn Gly Asn Phe Ile Ala Pro Glu Tyr Ala Tyr Lys Ile 260 265
270Val Lys Lys Gly Asp Ser Ala Ile Met Lys Ser Glu Leu Glu Tyr Gly
275 280 285Asn Cys Asn Thr Lys Cys Gln Thr Pro Ile Gly Ala Ile Asn
Ser Ser 290 295 300Met Pro Phe His Asn Ile His Pro Leu Thr Ile Gly
Glu Cys Pro Lys305 310 315 320Tyr Val Lys Ser Asn Arg Leu Val Leu
Ala Thr Gly Leu Arg Asn Ser 325 330 335Pro Gln Arg Glu Ser Arg Arg
Lys Lys Arg Gly Leu Phe Gly Ala Ile 340 345 350Ala Gly Phe Ile Glu
Gly Gly Trp Gln Gly Met Val Asp Gly Trp Tyr 355 360 365Gly Tyr His
His Ser Asn Glu Gln Gly Ser Gly Tyr Ala Ala Asp Lys 370 375 380Glu
Ser Thr Gln Lys Ala Ile Asp Gly Val Thr Asn Lys Val Asn Ser385 390
395 400Ile Ile Asp Lys Met Asn Thr Gln Phe Glu Ala Val Gly Arg Glu
Phe 405 410 415Asn Asn Leu Glu Arg Arg Ile Glu Asn Leu Asn Lys Lys
Met Glu Asp 420 425 430Gly Phe Leu Asp Val Trp Thr Tyr Asn Ala Glu
Leu Leu Val Leu Met 435 440 445Glu Asn Glu Arg Thr Leu Asp Phe His
Asp Ser Asn Val Lys Asn Leu 450 455 460Tyr Asp Lys Val Arg Leu Gln
Leu Arg Asp Asn Ala Lys Glu Leu Gly465 470 475 480Asn Gly Cys Phe
Glu Phe Tyr His Lys Cys Asp Asn Glu Cys Met Glu 485 490 495Ser Val
Arg Asn Gly Thr Tyr Asp Tyr Pro Gln Tyr Ser Glu Glu Ala 500 505
510Arg Leu Lys Arg Glu Glu Ile Ser Gly Val Lys Leu Glu Ser Ile Gly
515 520 525Thr Tyr Gln Ile Leu Ser Ile Tyr Ser Thr Val Ala Ser Ser
Leu Ala 530 535 540Leu Ala Ile Met Val Ala Gly Leu Ser Leu Trp Met
Cys Ser Asn Gly545 550 555 560Ser Leu Gln Cys Arg Ile Cys Ile
56531350DNAArtificial SequenceSynthetic
polynucleotideCDS(1)..(1350) 3atg aat cct aat aag aag atc atc aca
atc gga agc atc tgc atg gtg 48Met Asn Pro Asn Lys Lys Ile Ile Thr
Ile Gly Ser Ile Cys Met Val1 5 10 15aca gga atg gtg agc ctg atg ctg
cag atc gga aat ctg atc agc atc 96Thr Gly Met Val Ser Leu Met Leu
Gln Ile Gly Asn Leu Ile Ser Ile 20 25 30tgg gtg agc cac agc atc cac
aca gga aat cag cac aag gcc gag cct 144Trp Val Ser His Ser Ile His
Thr Gly Asn Gln His Lys Ala Glu Pro 35 40 45atc agc aat aca aat ttt
ctg aca gag aag gcc gtg gcc agc gtg aag 192Ile Ser Asn Thr Asn Phe
Leu Thr Glu Lys Ala Val Ala Ser Val Lys 50 55 60ctg gcc gga aat agc
agc ctg tgc cct atc aat gga tgg gcc gtg tac 240Leu Ala Gly Asn Ser
Ser Leu Cys Pro Ile Asn Gly Trp Ala Val Tyr65 70 75 80agc aag gat
aat agc atc aga atc gga agc aag gga gat gtg ttt gtg 288Ser Lys Asp
Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp Val Phe Val 85 90 95atc aga
gag cct ttt atc agc tgc agc cac ctg gag tgc aga aca ttt 336Ile Arg
Glu Pro Phe Ile Ser Cys Ser His Leu Glu Cys Arg Thr Phe 100 105
110ttt ctg aca cag gga gcc ctg ctg aat gat aag cac agc aat gga aca
384Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His Ser Asn Gly Thr
115 120 125gtg aag gat aga agc cct cac aga aca ctg atg agc tgc cct
gtg gga 432Val Lys Asp Arg Ser Pro His Arg Thr Leu Met Ser Cys Pro
Val Gly 130 135 140gag gcc cct agc cct tac aat agc aga ttt gag agc
gtg gcc tgg agc 480Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser
Val Ala Trp Ser145 150 155 160gcc agc gcc tgc cac gat gga aca agc
tgg ctg aca atc gga atc agc 528Ala Ser Ala Cys His Asp Gly Thr Ser
Trp Leu Thr Ile Gly Ile Ser 165 170 175gga cct gat aat gga gcc gtg
gcc gtg ctg aag tac aat gga atc atc 576Gly Pro Asp Asn Gly Ala Val
Ala Val Leu Lys Tyr Asn Gly Ile Ile 180 185 190aca gat aca atc aag
agc tgg aga aat aat atc ctg aga aca cag gag 624Thr Asp Thr Ile Lys
Ser Trp Arg Asn Asn Ile Leu Arg Thr Gln Glu 195 200 205agc gag tgc
gcc tgc gtg aat gga agc tgc ttt aca gtg atg aca gat 672Ser Glu Cys
Ala Cys Val Asn Gly Ser Cys Phe Thr Val Met Thr Asp 210 215 220gga
cct agc aat gga cag gcc agc cac aag atc ttt aag atg gag aag 720Gly
Pro Ser Asn Gly Gln Ala Ser His Lys Ile Phe Lys Met Glu Lys225 230
235 240gga aag gtg gtg aag agc gtg gag ctg gat gcc cct aat tac cac
tac 768Gly Lys Val Val Lys Ser Val Glu Leu Asp Ala Pro Asn Tyr His
Tyr 245 250 255gag gag tgc agc tgc tac cct gat gcc gga gag atc aca
tgc gtg tgc 816Glu Glu Cys Ser Cys Tyr Pro Asp Ala Gly Glu Ile Thr
Cys Val Cys 260 265 270aga gat aat tgg cac gga agc aat aga cct tgg
gtg agc ttt aat cag 864Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp
Val Ser Phe Asn Gln 275 280 285aat ctg gag tac cag atc gga tac atc
tgc agc gga gtg ttt gga gat 912Asn Leu Glu Tyr Gln Ile Gly Tyr Ile
Cys Ser Gly Val Phe Gly Asp 290 295 300aat cct aga cct aat gat gga
aca gga agc tgc gga cct gtg agc agc 960Asn Pro Arg Pro Asn Asp Gly
Thr Gly Ser Cys Gly Pro Val Ser Ser305 310 315 320aat gga gcc tac
gga gtg aag gga ttt agc ttt aag tac gga aat gga 1008Asn Gly Ala Tyr
Gly Val Lys Gly Phe Ser Phe Lys Tyr Gly Asn Gly 325 330 335gtg tgg
atc gga aga aca aag agc aca aat agc aga agc gga ttt gag 1056Val Trp
Ile Gly Arg Thr Lys Ser Thr Asn Ser Arg Ser Gly Phe Glu 340 345
350atg atc tgg gac cct aat gga tgg aca gag aca gat agc agc ttt agc
1104Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp Ser Ser Phe Ser
355 360 365gtg aag cag gat atc gtg gcc atc aca gat tgg agc gga tac
agc gga 1152Val Lys Gln Asp Ile Val Ala Ile Thr Asp Trp Ser Gly Tyr
Ser Gly 370 375 380agc ttt gtg cag cac cct gag ctg aca gga ctg gat
tgc atc aga cct 1200Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp
Cys Ile Arg Pro385 390 395 400tgc ttt tgg gtg gag ctg atc aga gga
aga cct aag gag agc aca atc 1248Cys Phe Trp Val Glu Leu Ile Arg Gly
Arg Pro Lys Glu Ser Thr Ile 405 410 415tgg aca agc gga agc agc atc
agc ttt tgc gga gtg aat agc gat aca 1296Trp Thr Ser Gly Ser Ser Ile
Ser Phe Cys Gly Val Asn Ser Asp Thr 420 425 430gtg gga tgg agc tgg
cct gat gga gcc gag ctg cct ttt aca atc gat 1344Val Gly Trp Ser Trp
Pro Asp Gly Ala Glu Leu Pro Phe Thr Ile Asp 435 440 445aag tga
1350Lys4449PRTArtificial SequenceSynthetic Construct 4Met Asn Pro
Asn Lys Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Val1 5 10 15Thr Gly
Met Val Ser Leu Met Leu Gln Ile Gly Asn Leu Ile Ser Ile 20 25 30Trp
Val Ser His Ser Ile His Thr Gly Asn Gln His Lys Ala Glu Pro 35 40
45Ile Ser Asn Thr Asn Phe Leu Thr Glu Lys Ala Val Ala Ser Val Lys
50 55 60Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Asn Gly Trp Ala Val
Tyr65 70 75 80Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly Asp
Val Phe Val 85 90 95Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu
Cys Arg Thr Phe 100 105 110Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp
Lys His Ser Asn Gly Thr 115 120 125Val Lys Asp Arg Ser Pro His Arg
Thr Leu Met Ser Cys Pro Val Gly 130 135 140Glu Ala Pro Ser Pro Tyr
Asn Ser Arg Phe Glu Ser Val Ala Trp Ser145 150 155 160Ala Ser Ala
Cys His Asp Gly Thr Ser Trp Leu Thr Ile Gly Ile Ser 165 170 175Gly
Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr Asn Gly Ile Ile 180 185
190Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu Arg Thr Gln Glu
195 200 205Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr Val Met
Thr Asp 210 215 220Gly Pro Ser Asn Gly Gln Ala Ser His Lys Ile
Phe
Lys Met Glu Lys225 230 235 240Gly Lys Val Val Lys Ser Val Glu Leu
Asp Ala Pro Asn Tyr His Tyr 245 250 255Glu Glu Cys Ser Cys Tyr Pro
Asp Ala Gly Glu Ile Thr Cys Val Cys 260 265 270Arg Asp Asn Trp His
Gly Ser Asn Arg Pro Trp Val Ser Phe Asn Gln 275 280 285Asn Leu Glu
Tyr Gln Ile Gly Tyr Ile Cys Ser Gly Val Phe Gly Asp 290 295 300Asn
Pro Arg Pro Asn Asp Gly Thr Gly Ser Cys Gly Pro Val Ser Ser305 310
315 320Asn Gly Ala Tyr Gly Val Lys Gly Phe Ser Phe Lys Tyr Gly Asn
Gly 325 330 335Val Trp Ile Gly Arg Thr Lys Ser Thr Asn Ser Arg Ser
Gly Phe Glu 340 345 350Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr
Asp Ser Ser Phe Ser 355 360 365Val Lys Gln Asp Ile Val Ala Ile Thr
Asp Trp Ser Gly Tyr Ser Gly 370 375 380Ser Phe Val Gln His Pro Glu
Leu Thr Gly Leu Asp Cys Ile Arg Pro385 390 395 400Cys Phe Trp Val
Glu Leu Ile Arg Gly Arg Pro Lys Glu Ser Thr Ile 405 410 415Trp Thr
Ser Gly Ser Ser Ile Ser Phe Cys Gly Val Asn Ser Asp Thr 420 425
430Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro Phe Thr Ile Asp
435 440 445Lys5759DNAArtificial SequenceSynthetic
polynucleotideCDS(1)..(759) 5atg agc ctg ctg acc gag gtg gag aca
tac gtg ctg tcc atc atc ccc 48Met Ser Leu Leu Thr Glu Val Glu Thr
Tyr Val Leu Ser Ile Ile Pro1 5 10 15agc ggc cct ctg aag gcc gag atc
gcc cag aga ctg gaa gat gtg ttc 96Ser Gly Pro Leu Lys Ala Glu Ile
Ala Gln Arg Leu Glu Asp Val Phe 20 25 30gcc ggc aag aac acc gac ctg
gaa gtg ctg atg gaa tgg ctg aaa acc 144Ala Gly Lys Asn Thr Asp Leu
Glu Val Leu Met Glu Trp Leu Lys Thr 35 40 45aga ccc atc ctg agc cct
ctg acc aag ggc atc ctg ggc ttc gtg ttc 192Arg Pro Ile Leu Ser Pro
Leu Thr Lys Gly Ile Leu Gly Phe Val Phe 50 55 60acc ctg acc gtg ccc
agc gag aga ggc ctg cag agg cgg aga ttc gtg 240Thr Leu Thr Val Pro
Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val65 70 75 80cag aac gcc
ctg aac ggc aac ggc gac ccc aac aac atg gac aag gcc 288Gln Asn Ala
Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Lys Ala 85 90 95gtg aag
ctg tac aga aag ctg aag cgg gag atc acc ttc cac ggc gcc 336Val Lys
Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His Gly Ala 100 105
110aaa gag atc agc ctg agc tac agc gct ggc gcc ctg gcc agc tgc atg
384Lys Glu Ile Ser Leu Ser Tyr Ser Ala Gly Ala Leu Ala Ser Cys Met
115 120 125ggc ctg atc tac aac aga atg ggc gcc gtg acc acc gag gtg
gcc ttc 432Gly Leu Ile Tyr Asn Arg Met Gly Ala Val Thr Thr Glu Val
Ala Phe 130 135 140ggc ctg gtc tgc gcc acc tgc gag cag atc gcc gac
agc cag cac aga 480Gly Leu Val Cys Ala Thr Cys Glu Gln Ile Ala Asp
Ser Gln His Arg145 150 155 160tcc cac aga cag atg gtc acc acc acc
aac ccc ctg atc aga cac gag 528Ser His Arg Gln Met Val Thr Thr Thr
Asn Pro Leu Ile Arg His Glu 165 170 175aac aga atg gtg ctg gcc tct
acc acc gcc aag gcc atg gaa cag atg 576Asn Arg Met Val Leu Ala Ser
Thr Thr Ala Lys Ala Met Glu Gln Met 180 185 190gcc ggc agc agc gag
cag gcc gcc gag gct atg gaa gtc gcc tct cag 624Ala Gly Ser Ser Glu
Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln 195 200 205gct agg cag
atg gtc cag gcc atg aga acc atc ggc acc cac ccc agc 672Ala Arg Gln
Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser 210 215 220agc
tct gct ggc ctg aag aac gac ctg ctg gaa aac ctg cag gcc tac 720Ser
Ser Ala Gly Leu Lys Asn Asp Leu Leu Glu Asn Leu Gln Ala Tyr225 230
235 240cag aaa aga atg ggc gtc cag atg cag aga ttc aag tga 759Gln
Lys Arg Met Gly Val Gln Met Gln Arg Phe Lys 245
2506252PRTArtificial SequenceSynthetic Construct 6Met Ser Leu Leu
Thr Glu Val Glu Thr Tyr Val Leu Ser Ile Ile Pro1 5 10 15Ser Gly Pro
Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val Phe 20 25 30Ala Gly
Lys Asn Thr Asp Leu Glu Val Leu Met Glu Trp Leu Lys Thr 35 40 45Arg
Pro Ile Leu Ser Pro Leu Thr Lys Gly Ile Leu Gly Phe Val Phe 50 55
60Thr Leu Thr Val Pro Ser Glu Arg Gly Leu Gln Arg Arg Arg Phe Val65
70 75 80Gln Asn Ala Leu Asn Gly Asn Gly Asp Pro Asn Asn Met Asp Lys
Ala 85 90 95Val Lys Leu Tyr Arg Lys Leu Lys Arg Glu Ile Thr Phe His
Gly Ala 100 105 110Lys Glu Ile Ser Leu Ser Tyr Ser Ala Gly Ala Leu
Ala Ser Cys Met 115 120 125Gly Leu Ile Tyr Asn Arg Met Gly Ala Val
Thr Thr Glu Val Ala Phe 130 135 140Gly Leu Val Cys Ala Thr Cys Glu
Gln Ile Ala Asp Ser Gln His Arg145 150 155 160Ser His Arg Gln Met
Val Thr Thr Thr Asn Pro Leu Ile Arg His Glu 165 170 175Asn Arg Met
Val Leu Ala Ser Thr Thr Ala Lys Ala Met Glu Gln Met 180 185 190Ala
Gly Ser Ser Glu Gln Ala Ala Glu Ala Met Glu Val Ala Ser Gln 195 200
205Ala Arg Gln Met Val Gln Ala Met Arg Thr Ile Gly Thr His Pro Ser
210 215 220Ser Ser Ala Gly Leu Lys Asn Asp Leu Leu Glu Asn Leu Gln
Ala Tyr225 230 235 240Gln Lys Arg Met Gly Val Gln Met Gln Arg Phe
Lys 245 25075511DNAArtificial SequenceSynthetic construct
7cgacaatatt ggctattggc cattgcatac gttgtatcta tatcataata tgtacattta
60tattggctca tgtccaatat gaccgccatg ttgacattga ttattgacta gttattaata
120gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
ttacataact 180tacggtaaat ggcccgcctc gtgaccgccc aacgaccccc
gcccattgac gtcaataatg 240acgtatgttc ccatagtaac gccaataggg
actttccatt gacgtcaatg ggtggagtat 300ttacggtaaa ctgcccactt
ggcagtacat caagtgtatc atatgccaag tccgccccta 360ttgacgtcaa
tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttacggg
420actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg
gtgatgcggt 480tttggcagta caccaatggg cgtggatagc ggtttgactc
acggggattt ccaagtctcc 540accccattga cgtcaatggg agtttgtttt
ggcaccaaaa tcaacgggac tttccaaaat 600gtcgtaataa ccccgccccg
ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct 660atataagcag
agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt
720ttgacctcca tagaagacac cgggaccgat ccagcctccg cggccgggaa
cggtgcattg 780gaacgcggat tccccgtgcc aagagtgacg taagtaccgc
ctatagactc tataggcaca 840cccctttggc tcttatgcat gctatactgt
ttttggcttg gggcctatac acccccgctc 900cttatgctat aggtgatggt
atagcttagc ctataggtgt gggttattga ccattattga 960ccactcccct
attggtgacg atactttcca ttactaatcc ataacatggc tctttgccac
1020aactatctct attggctata tgccaatact ctgtccttca gagactgaca
cggactctgt 1080atttttacag gatggggtcc catttattat ttacaaattc
acatatacaa caacgccgtc 1140ccccgtgccc gcagttttta ttaaacatag
cgtgggatct ccacgcgaat ctcgggtacg 1200tgttccggac atgggctctt
ctccggtagc ggcggagctt ccacatccga gccctggtcc 1260catgcctcca
gcggctcatg gtcgctcggc agctccttgc tcctaacagt ggaggccaga
1320cttaggcaca gcacaatgcc caccaccacc agtgtgccgc acaaggccgt
ggcggtaggg 1380tatgtgtctg aaaatgagct cggagattgg gctcgcaccg
tgacgcagat ggaagactta 1440aggcagcggc agaagaagat gcaggcagct
gagttgttgt attctgataa gagtcagagg 1500taactcccgt tgcggtgctg
ttaacggtgg agggcagtgt agtctgagca gtactcgttg 1560ctgccgcgcg
cgccaccaga cataatagct gacagactaa cagactgttc ctttccatgg
1620gtcttttctg cagtcaccgt ccaagcttat ggaaaagatc gtgctgctgc
tggctatcgt 1680gagcctggtg aagagcgacc agatttgcat cggctaccac
gccaacaaca gcaccgagca 1740ggtggacacc atcatggaaa agaacgtcac
cgtgacccac gcccaggaca tcctggaaaa 1800gacccacaac ggcaagctgt
gcgacctgga cggcgtgaag cccctgatcc tgagggactg 1860cagcgtggcc
ggctggctgc tgggcaaccc catgtgcgac gagttcatca acgtgcccga
1920gtggagctac atcgtggaga aggccaaccc cgccaacgac ctgtgctacc
ccggcaactt 1980caacgactac gaggaactga agcacctgct gtccaggatc
aaccacttcg agaagatcca 2040gatcatcccc aagagcagct ggtccgacca
cgaggccagc agcggcgtga gcagcgcctg 2100cccataccag ggcagcccca
gcttcttcag aaacgtggtg tggctgatca agaagaacaa 2160cacctacccc
accatcaaga ggtcctacaa caacaccaac caggaagatc tgctggtcct
2220gtggggcatc caccacccta atgacgccgc cgaacagacc aggctgtacc
agaaccccac 2280cacctacatc agcgtgggca caagcaccct gaaccagagg
ctggtgccca agatcgccac 2340caggtccaag gtgaacggac agtccggcag
gatggaattc ttctggacca tcctgaagcc 2400taacgacgcc atcaacttcg
agagcaacgg caactttatc gcccccgagt acgcctacaa 2460gatcgtgaag
aagggcgaca gcgccatcat gaagagcgag ctggaatacg gcaactgcaa
2520caccaagtgc cagaccccca tcggcgccat caacagcagc atgcccttcc
acaacatcca 2580ccccctgacc atcggcgagt gccccaagta cgtgaagagc
aacaggctgg tgctggccac 2640cggcctgagg aacagccccc agagagagag
cagaagaaag aagaggggcc tgttcggcgc 2700tatcgccggc ttcatcgagg
gcggctggca gggcatggtg gacgggtggt acggctacca 2760ccactctaac
gagcagggca gcggctacgc cgccgacaaa gagagcaccc agaaggccat
2820cgacggcgtc accaacaagg tgaacagcat catcgacaag atgaacaccc
agttcgaggc 2880cgtgggcaga gagttcaaca acctggaaag gcggatcgag
aacctgaaca agaaaatgga 2940agatggcttc ctggacgtgt ggacctacaa
cgccgagctg ctggtgctga tggaaaacga 3000gaggaccctg gacttccacg
acagcaacgt gaagaacctg tacgacaaag tgcggctgca 3060gctgagggac
aacgccaaag agctgggcaa cggctgcttc gagttctacc acaagtgcga
3120caacgagtgc atggaaagcg tgaggaacgg cacctacgac tacccccagt
acagcgagga 3180agccaggctg aagagggaag agatcagcgg agtgaagctg
gaaagcatcg gcacctacca 3240gatcctgagc atctacagca ccgtcgccag
cagcctggcc ctggctatca tggtggccgg 3300actgagcctg tggatgtgca
gcaacggcag cctgcagtgc aggatctgca tcggatcctc 3360gcaatcccta
gggctgtgcc ttctagttgc cagccaaact gttgtttgcc cctcccccgt
3420gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa
atgaggaaat 3480tgcatcgcat tgtctgagta ggtgtcattc tattctgggg
ggtggggtgg ggcaggacag 3540caagggggag gattgggaag acaatagcag
gcatgctggg gatgcggtgg gctctatgtt 3600cagaacgctc ggttgccgcc
gggcgttttt tatctagagt cgacaaattc agaagaactc 3660gtcaagaagg
cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac
3720gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg
tagccaacgc 3780tatgtcctga tagcggtccg ccacacccag ccggccacag
tcgatgaatc cagaaaagcg 3840gccattttcc accatgatat tcggcaagca
ggcatcgcca tgggtcacga cgagatcctc 3900gccgtcgggc atgctcgcct
tgagcctggc gaacagttcg gctggcgcga gcccctgatg 3960ctcttcgtcc
agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc
4020gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg
tatgcagccg 4080ccgcattgca tcagccatga tggatacttt ctcggcagga
gcaaggtgag atgacaggag 4140atcctgcccc ggcacttcgc ccaatagcag
ccagtccctt cccgcttcag tgacaacgtc 4200gagcacagct gcgcaaggaa
cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc 4260ttgcagttca
ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg
4320cgctgacagc cggaacacgg cggcatcaga gcagccgatt gtctgttgtg
cccagtcata 4380gccgaatagc ctctccaccc aagcggccgg agaacctgcg
tgcaatccat cttgttcaat 4440catgcgaaac gatcctcatc ctgtctcttg
atcagatctt gatcccctgc gccatcagat 4500ccttggcggc gagaaagcca
tccagtttac tttgcagggc ttcccaacct taccagaggg 4560cgccccagct
ggcaattccg gttcgcttgc tgtccataaa accgcccagt ctagctatcg
4620ccatgtaagc ccactgcaag ctacctgctt tctctttgcg cttgcgtttt
cccttgtcca 4680gatagcccag tagctgacat tcatccgggg tcagcaccgt
ttctgcggac tggctttcta 4740cgtgaaaagg atctaggtga agatcctttt
tgataatctc atgaccaaaa tcccttaacg 4800tgagttttcg ttccactgag
cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 4860tccttttttt
ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
4920ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
gcttcagcag 4980agcgcagata ccaaatactg tccttctagt gtagccgtag
ttaggccacc acttcaagaa 5040ctctgtagca ccgcctacat acctcgctct
gctaatcctg ttaccagtgg ctgctgccag 5100tggcgataag tcgtgtctta
ccgggttgga ctcaagacga tagttaccgg ataaggcgca 5160gcggtcgggc
tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
5220cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
aagggagaaa 5280ggcggacagg tatccggtaa gcggcagggt cggaacagga
gagcgcacga gggagcttcc 5340agggggaaac gcctggtatc tttatagtcc
tgtcgggttt cgccacctct gacttgagcg 5400tcgatttttg tgatgctcgt
caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 5460ctttttacgg
ttcctgggct tttgctggcc ttttgctcac atgttgtcga c
551185769DNAArtificial SequenceSynthetic construct 8tcgcgcgttt
cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60cagcttgtct
gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
120ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta
ctgagagtgc 180accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
aaaataccgc atcagattgg 240ctattggcca ttgcatacgt tgtatccata
tcataatatg tacatttata ttggctcatg 300tccaacatta ccgccatgtt
gacattgatt attgactagt tattaatagt aatcaattac 360ggggtcatta
gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg
420cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
cgtatgttcc 480catagtaacg ccaataggga ctttccattg acgtcaatgg
gtggagtatt tacggtaaac 540tgcccacttg gcagtacatc aagtgtatca
tatgccaagt acgcccccta ttgacgtcaa 600tgacggtaaa tggcccgcct
ggcattatgc ccagtacatg accttatggg actttcctac 660ttggcagtac
atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta
720catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
accccattga 780cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
tttccaaaat gtcgtaacaa 840ctccgcccca ttgacgcaaa tgggcggtag
gcgtgtacgg tgggaggtct atataagcag 900agctcgttta gtgaaccgtc
agatcgcctg gagacgccat ccacgctgtt ttgacctcca 960tagaagacac
cgggaccgat ccagcctcca tcggctcgca tctctccttc acgcgcccgc
1020cgccctacct gaggccgcca tccacgccgg ttgagtcgcg ttctgccgcc
tcccgcctgt 1080ggtgcctcct gaactgcgtc cgccgtctag gtaagtttaa
agctcaggtc gagaccgggc 1140ctttgtccgg cgctcccttg gagcctacct
agactcagcc ggctctccac gctttgcctg 1200accctgcttg ctcaactcta
gttaacggtg gagggcagtg tagtctgagc agtactcgtt 1260gctgccgcgc
gcgccaccag acataatagc tgacagacta acagactgtt cctttccatg
1320ggtcttttct gcagtcaccg tcgtcgacac gatccgatat cgccgccacc
atgaatccta 1380ataagaagat catcacaatc ggaagcatct gcatggtgac
aggaatggtg agcctgatgc 1440tgcagatcgg aaatctgatc agcatctggg
tgagccacag catccacaca ggaaatcagc 1500acaaggccga gcctatcagc
aatacaaatt ttctgacaga gaaggccgtg gccagcgtga 1560agctggccgg
aaatagcagc ctgtgcccta tcaatggatg ggccgtgtac agcaaggata
1620atagcatcag aatcggaagc aagggagatg tgtttgtgat cagagagcct
tttatcagct 1680gcagccacct ggagtgcaga acattttttc tgacacaggg
agccctgctg aatgataagc 1740acagcaatgg aacagtgaag gatagaagcc
ctcacagaac actgatgagc tgccctgtgg 1800gagaggcccc tagcccttac
aatagcagat ttgagagcgt ggcctggagc gccagcgcct 1860gccacgatgg
aacaagctgg ctgacaatcg gaatcagcgg acctgataat ggagccgtgg
1920ccgtgctgaa gtacaatgga atcatcacag atacaatcaa gagctggaga
aataatatcc 1980tgagaacaca ggagagcgag tgcgcctgcg tgaatggaag
ctgctttaca gtgatgacag 2040atggacctag caatggacag gccagccaca
agatctttaa gatggagaag ggaaaggtgg 2100tgaagagcgt ggagctggat
gcccctaatt accactacga ggagtgcagc tgctaccctg 2160atgccggaga
gatcacatgc gtgtgcagag ataattggca cggaagcaat agaccttggg
2220tgagctttaa tcagaatctg gagtaccaga tcggatacat ctgcagcgga
gtgtttggag 2280ataatcctag acctaatgat ggaacaggaa gctgcggacc
tgtgagcagc aatggagcct 2340acggagtgaa gggatttagc tttaagtacg
gaaatggagt gtggatcgga agaacaaaga 2400gcacaaatag cagaagcgga
tttgagatga tctgggaccc taatggatgg acagagacag 2460atagcagctt
tagcgtgaag caggatatcg tggccatcac agattggagc ggatacagcg
2520gaagctttgt gcagcaccct gagctgacag gactggattg catcagacct
tgcttttggg 2580tggagctgat cagaggaaga cctaaggaga gcacaatctg
gacaagcgga agcagcatca 2640gcttttgcgg agtgaatagc gatacagtgg
gatggagctg gcctgatgga gccgagctgc 2700cttttacaat cgataagtga
gcggccgctc tagaccaggc cctggatcca gatctgctgt 2760gccttctagt
tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
2820aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc
attgtctgag 2880taggtgtcat tctattctgg ggggtggggt ggggcaggac
agcaaggggg aggattggga 2940agacaatagc aggcatgctg gggatgcggt
gggctctatg ggtacccagg tgctgaagaa 3000ttgacccggt tcctcctggg
ccagaaagaa gcaggcacat ccccttctct gtgacacacc 3060ctgtccacgc
ccctggttct tagttccagc cccactcata ggacactcat agctcaggag
3120ggctccgcct tcaatcccac ccgctaaagt acttggagcg gtctctccct
ccctcatcag 3180cccaccaaac caaacctagc ctccaagagt gggaagaaat
taaagcaaga taggctatta 3240agtgcagagg gagagaaaat gcctccaaca
tgtgaggaag taatgagaga aatcatagaa 3300ttttaaggcc atgatttaag
gccatcatgg ccttaatctt ccgcttcctc gctcactgac 3360tcgctgcgct
cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
3420cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
aggccagcaa 3480aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
tccataggct ccgcccccct 3540gacgagcatc acaaaaatcg acgctcaagt
cagaggtggc gaaacccgac aggactataa 3600agataccagg cgtttccccc
tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 3660cttaccggat
acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
3720cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
tgtgcacgaa 3780ccccccgttc agcccgaccg ctgcgcctta tccggtaact
atcgtcttga gtccaacccg 3840gtaagacacg acttatcgcc actggcagca
gccactggta acaggattag cagagcgagg 3900tatgtaggcg gtgctacaga
gttcttgaag tggtggccta actacggcta cactagaaga 3960acagtatttg
gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
4020tcttgatccg gcaaacaaac
caccgctggt agcggtggtt tttttgtttg caagcagcag 4080attacgcgca
gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
4140gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
aaaaaggatc 4200ttcacctaga tccttttaaa ttaaaaatga agttttaaat
caatctaaag tatatatgag 4260taaacttggt ctgacagtta ccaatgctta
atcagtgagg cacctatctc agcgatctgt 4320ctatttcgtt catccatagt
tgcctgactc gggggggggg ggcgctgagg tctgcctcgt 4380gaagaaggtg
ttgctgactc ataccaggcc tgaatcgccc catcatccag ccagaaagtg
4440agggagccac ggttgatgag agctttgttg taggtggacc agttggtgat
tttgaacttt 4500tgctttgcca cggaacggtc tgcgttgtcg ggaagatgcg
tgatctgatc cttcaactca 4560gcaaaagttc gatttattca acaaagccgc
cgtcccgtca agtcagcgta atgctctgcc 4620agtgttacaa ccaattaacc
aattctgatt agaaaaactc atcgagcatc aaatgaaact 4680gcaatttatt
catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg
4740aaggagaaaa ctcaccgagg cagttccata ggatggcaag atcctggtat
cggtctgcga 4800ttccgactcg tccaacatca atacaaccta ttaatttccc
ctcgtcaaaa ataaggttat 4860caagtgagaa atcaccatga gtgacgactg
aatccggtga gaatggcaaa agcttatgca 4920tttctttcca gacttgttca
acaggccagc cattacgctc gtcatcaaaa tcactcgcat 4980caaccaaacc
gttattcatt cgtgattgcg cctgagcgag acgaaatacg cgatcgctgt
5040taaaaggaca attacaaaca ggaatcgaat gcaaccggcg caggaacact
gccagcgcat 5100caacaatatt ttcacctgaa tcaggatatt cttctaatac
ctggaatgct gttttcccgg 5160ggatcgcagt ggtgagtaac catgcatcat
caggagtacg gataaaatgc ttgatggtcg 5220gaagaggcat aaattccgtc
agccagttta gtctgaccat ctcatctgta acatcattgg 5280caacgctacc
tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc
5340gatagattgt cgcacctgat tgcccgacat tatcgcgagc ccatttatac
ccatataaat 5400cagcatccat gttggaattt aatcgcggcc tcgagcaaga
cgtttcccgt tgaatatggc 5460tcataacacc ccttgtatta ctgtttatgt
aagcagacag ttttattgtt catgatgata 5520tatttttatc ttgtgcaatg
taacatcaga gattttgaga cacaacgtgg ctttcccccc 5580ccccccatta
ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
5640gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
gtgccacctg 5700acgtctaaga aaccattatt atcatgacat taacctataa
aaataggcgt atcacgaggc 5760cctttcgtc 576994598DNAArtificial
SequenceArtificial construct 9cgacaatatt ggctattggc cattgcatac
gttgtatcta tatcataata tgtacattta 60tattggctca tgtccaatat gaccgccatg
ttgacattga ttattgacta gttattaata 120gtaatcaatt acggggtcat
tagttcatag cccatatatg gagttccgcg ttacataact 180tacggtaaat
ggcccgcctc gtgaccgccc aacgaccccc gcccattgac gtcaataatg
240acgtatgttc ccatagtaac gccaataggg actttccatt gacgtcaatg
ggtggagtat 300ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
atatgccaag tccgccccta 360ttgacgtcaa tgacggtaaa tggcccgcct
ggcattatgc ccagtacatg accttacggg 420actttcctac ttggcagtac
atctacgtat tagtcatcgc tattaccatg gtgatgcggt 480tttggcagta
caccaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
540accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
tttccaaaat 600gtcgtaataa ccccgccccg ttgacgcaaa tgggcggtag
gcgtgtacgg tgggaggtct 660atataagcag agctcgttta gtgaaccgtc
agatcgcctg gagacgccat ccacgctgtt 720ttgacctcca tagaagacac
cgggaccgat ccagcctccg cggccgggaa cggtgcattg 780gaacgcggat
tccccgtgcc aagagtgacg taagtaccgc ctatagactc tataggcaca
840cccctttggc tcttatgcat gctatactgt ttttggcttg gggcctatac
acccccgctc 900cttatgctat aggtgatggt atagcttagc ctataggtgt
gggttattga ccattattga 960ccactcccct attggtgacg atactttcca
ttactaatcc ataacatggc tctttgccac 1020aactatctct attggctata
tgccaatact ctgtccttca gagactgaca cggactctgt 1080atttttacag
gatggggtcc catttattat ttacaaattc acatatacaa caacgccgtc
1140ccccgtgccc gcagttttta ttaaacatag cgtgggatct ccacgcgaat
ctcgggtacg 1200tgttccggac atgggctctt ctccggtagc ggcggagctt
ccacatccga gccctggtcc 1260catgcctcca gcggctcatg gtcgctcggc
agctccttgc tcctaacagt ggaggccaga 1320cttaggcaca gcacaatgcc
caccaccacc agtgtgccgc acaaggccgt ggcggtaggg 1380tatgtgtctg
aaaatgagct cggagattgg gctcgcaccg tgacgcagat ggaagactta
1440aggcagcggc agaagaagat gcaggcagct gagttgttgt attctgataa
gagtcagagg 1500taactcccgt tgcggtgctg ttaacggtgg agggcagtgt
agtctgagca gtactcgttg 1560ctgccgcgcg cgccaccaga cataatagct
gacagactaa cagactgttc ctttccatgg 1620gtcttttctg cagtcaccgt
ccaagcttag atctgccacc atgagcctgc tgaccgaggt 1680ggagacatac
gtgctgtcca tcatccccag cggccctctg aaggccgaga tcgcccagag
1740actggaagat gtgttcgccg gcaagaacac cgacctggaa gtgctgatgg
aatggctgaa 1800aaccagaccc atcctgagcc ctctgaccaa gggcatcctg
ggcttcgtgt tcaccctgac 1860cgtgcccagc gagagaggcc tgcagaggcg
gagattcgtg cagaacgccc tgaacggcaa 1920cggcgacccc aacaacatgg
acaaggccgt gaagctgtac agaaagctga agcgggagat 1980caccttccac
ggcgccaaag agatcagcct gagctacagc gctggcgccc tggccagctg
2040catgggcctg atctacaaca gaatgggcgc cgtgaccacc gaggtggcct
tcggcctggt 2100ctgcgccacc tgcgagcaga tcgccgacag ccagcacaga
tcccacagac agatggtcac 2160caccaccaac cccctgatca gacacgagaa
cagaatggtg ctggcctcta ccaccgccaa 2220ggccatggaa cagatggccg
gcagcagcga gcaggccgcc gaggctatgg aagtcgcctc 2280tcaggctagg
cagatggtcc aggccatgag aaccatcggc acccacccca gcagctctgc
2340tggcctgaag aacgacctgc tggaaaacct gcaggcctac cagaaaagaa
tgggcgtcca 2400gatgcagaga ttcaagtgat gagatatccc tcagagaggg
gatcctcgca atccctaggg 2460ctgtgccttc tagttgccag ccaaactgtt
gtttgcccct cccccgtgcc ttccttgacc 2520ctggaaggtg ccactcccac
tgtcctttcc taataaaatg aggaaattgc atcgcattgt 2580ctgagtaggt
gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
2640tgggaagaca atagcaggca tgctggggat gcggtgggct ctatgttcag
aacgctcggt 2700tgccgccggg cgttttttat ctagagtcga caaattcaga
agaactcgtc aagaaggcga 2760tagaaggcga tgcgctgcga atcgggagcg
gcgataccgt aaagcacgag gaagcggtca 2820gcccattcgc cgccaagctc
ttcagcaata tcacgggtag ccaacgctat gtcctgatag 2880cggtccgcca
cacccagccg gccacagtcg atgaatccag aaaagcggcc attttccacc
2940atgatattcg gcaagcaggc atcgccatgg gtcacgacga gatcctcgcc
gtcgggcatg 3000ctcgccttga gcctggcgaa cagttcggct ggcgcgagcc
cctgatgctc ttcgtccaga 3060tcatcctgat cgacaagacc ggcttccatc
cgagtacgtg ctcgctcgat gcgatgtttc 3120gcttggtggt cgaatgggca
ggtagccgga tcaagcgtat gcagccgccg cattgcatca 3180gccatgatgg
atactttctc ggcaggagca aggtgagatg acaggagatc ctgccccggc
3240acttcgccca atagcagcca gtcccttccc gcttcagtga caacgtcgag
cacagctgcg 3300caaggaacgc ccgtcgtggc cagccacgat agccgcgctg
cctcgtcttg cagttcattc 3360agggcaccgg acaggtcggt cttgacaaaa
agaaccgggc gcccctgcgc tgacagccgg 3420aacacggcgg catcagagca
gccgattgtc tgttgtgccc agtcatagcc gaatagcctc 3480tccacccaag
cggccggaga acctgcgtgc aatccatctt gttcaatcat gcgaaacgat
3540cctcatcctg tctcttgatc agatcttgat cccctgcgcc atcagatcct
tggcggcgag 3600aaagccatcc agtttacttt gcagggcttc ccaaccttac
cagagggcgc cccagctggc 3660aattccggtt cgcttgctgt ccataaaacc
gcccagtcta gctatcgcca tgtaagccca 3720ctgcaagcta cctgctttct
ctttgcgctt gcgttttccc ttgtccagat agcccagtag 3780ctgacattca
tccggggtca gcaccgtttc tgcggactgg ctttctacgt gaaaaggatc
3840taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
gttttcgttc 3900cactgagcgt cagaccccgt agaaaagatc aaaggatctt
cttgagatcc tttttttctg 3960cgcgtaatct gctgcttgca aacaaaaaaa
ccaccgctac cagcggtggt ttgtttgccg 4020gatcaagagc taccaactct
ttttccgaag gtaactggct tcagcagagc gcagatacca 4080aatactgtcc
ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
4140cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
cgataagtcg 4200tgtcttaccg ggttggactc aagacgatag ttaccggata
aggcgcagcg gtcgggctga 4260acggggggtt cgtgcacaca gcccagcttg
gagcgaacga cctacaccga actgagatac 4320ctacagcgtg agctatgaga
aagcgccacg cttcccgaag ggagaaaggc ggacaggtat 4380ccggtaagcg
gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
4440tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
atttttgtga 4500tgctcgtcag gggggcggag cctatggaaa aacgccagca
acgcggcctt tttacggttc 4560ctgggctttt gctggccttt tgctcacatg ttgtcgac
4598109PRTArtificial SequenceSynthetic peptide 10Ile Tyr Ser Thr
Val Ala Ser Ser Leu1 5118PRTArtificial SequenceSynthetic peptide
11Ser Ile Ile Asn Phe Glu Lys Leu1 5
* * * * *