U.S. patent application number 17/579879 was filed with the patent office on 2022-05-05 for method for the safe induction of immunity against rsv.
The applicant listed for this patent is Janssen Vaccines & Prevention B.V.. Invention is credited to Benoit Christophe Stephan CALLENDRET, Olivier GODEAUX, Jerald C. SADOFF, Myra Noorely WIDJOJOATMODJO, Kristi Lynn WILLIAMS.
Application Number | 20220133878 17/579879 |
Document ID | / |
Family ID | |
Filed Date | 2022-05-05 |
United States Patent
Application |
20220133878 |
Kind Code |
A1 |
WIDJOJOATMODJO; Myra Noorely ;
et al. |
May 5, 2022 |
METHOD FOR THE SAFE INDUCTION OF IMMUNITY AGAINST RSV
Abstract
Methods of inducing a safe immune response against respiratory
syncytial virus (RSV) in a human subject in need thereof, including
administering to the subject a composition including recombinant
adenovirus including a nucleic acid encoding an RSV Fusion (F)
protein including the amino acid sequence of SEQ ID NO: 1, and a
pharmaceutically acceptable carrier, in a total dose of from about
1.times.101.degree. to about 2.times.10'' viral particles (vp), are
described.
Inventors: |
WIDJOJOATMODJO; Myra Noorely;
(Zeist, NL) ; GODEAUX; Olivier; (Louvain-La-Neuve,
BE) ; WILLIAMS; Kristi Lynn; (Chalfont, PA) ;
CALLENDRET; Benoit Christophe Stephan; (Den Haag, NL)
; SADOFF; Jerald C.; (Amsterdam, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Janssen Vaccines & Prevention B.V. |
Leiden |
|
NL |
|
|
Appl. No.: |
17/579879 |
Filed: |
January 20, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16642082 |
Feb 26, 2020 |
11229695 |
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PCT/EP2018/074710 |
Sep 13, 2018 |
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17579879 |
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62558994 |
Sep 15, 2017 |
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International
Class: |
A61K 39/155 20060101
A61K039/155; A61P 31/14 20060101 A61P031/14; C12N 7/00 20060101
C12N007/00 |
Claims
1. A method of inducing a safe immune response against respiratory
syncytial virus (RSV) in a human subject in need thereof,
comprising administering to the human subject a composition
comprising a replication-deficient recombinant adenovirus
comprising a nucleic acid encoding an RSV Fusion (F) protein
comprising the amino acid sequence of SEQ ID NO: 1, and a
pharmaceutically acceptable carrier, wherein the recombinant
adenovirus is administered in a total dose of from about
1.times.10.sup.10 to about 2.times.10.sup.11 viral particles
(vp).
2. The method according to claim 1, wherein the total dose is from
about 5.times.10.sup.10 to about 1.times.10.sup.11 viral particles
(vp).
3. The method according to claim 1, wherein the immune response
comprises the induction of antibodies specifically binding to RSV F
protein.
4. The method according to claim 1, wherein the immune response
comprises the induction of RSV neutralizing antibodies.
5. The method according to claim 1, wherein the immune response
comprises the induction of antibodies specific for the RSV F
protein in the pre-fusion conformation and antibodies specific for
the RSV F protein in the post-fusion conformation, and wherein the
geometric mean titer (GMT) increase of antibodies specific for RSV
F protein in the pre-fusion conformation is higher than the
geometric mean titer (GMT) increase of antibodies specific for RSV
F protein in the post-fusion conformation, in enzyme linked
immunosorbent assays (ELISAs).
6. The method according to claim 1, wherein the ratio between the
geometric mean titer (GMT) increase of post-fusion F specific
antibodies as measured in ELISA and the geometric mean titer (GMT)
increase of neutralizing antibodies as measured in a VNA assay is
reduced after administration of said composition as compared to
said ratio before administration of said composition.
7. The method according to claim 1, wherein the immune response
further comprises a cellular response as indicated by IFNgamma
producing T cells as measured in an IFN.gamma. ELISPOT in response
to stimulation with a pool of peptides covering the RSV F protein
of SEQ ID NO: 1, and/or by measurement of CD4 and CD8 T-cell
subsets expressing IFN.gamma., IL-2 and TNF.alpha. by intracellular
staining (ICS) after stimulation with a pool of peptides covering
the RSV F protein of SEQ ID NO: 1.
8. The method according to claim 1, wherein the subject is a human
of 60 years or older.
9. The method according to claim 1, wherein the nucleic acid
encoding the RSV F protein comprises the nucleic acid sequence of
SEQ ID NO: 2.
10. The method according to claim 1, wherein the recombinant
adenovirus is a human adenovirus.
11. The method according to claim 10, wherein the recombinant
adenovirus is of serotype 26 or 35.
12. The method according to claim 1, wherein the composition is
administered intranasally.
13. The method according to claim 1, further comprising
administering to the human subject the composition one year after
the initial administration of the composition.
14. A method of inducing a safe immune response against respiratory
syncytial virus (RSV) in a human subject in need thereof,
comprising administering to the human subject a composition
comprising a replication-deficient recombinant adenovirus serotype
26 (Ad26) comprising the polynucleotide sequence of SEQ ID NO: 2,
and a pharmaceutically acceptable carrier, wherein the recombinant
Ad26 is administered in a total dose of from about
1.times.10.sup.10 to about 2.times.10.sup.11 viral particles
(vp).
15. The method according to claim 14, wherein the total dose is
from about 5.times.10.sup.10 to about 1.times.10.sup.11 viral
particles (vp).
16. The method according to claim 15, wherein the composition is
administered intranasally.
17. The method according to claim 14, wherein the composition is
administered intranasally.
18. The method according to claim 14, wherein the subject is a
human of 60 years or older.
19. The method according to claim 14, further comprising
administering to the human subject the composition one year after
the initial administration of the composition.
20. The method according to claim 14, wherein the immune response
comprises the induction of RSV neutralizing antibodies.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a continuation of U.S. application Ser.
No. 16/642,082, filed on Feb. 26, 2020, which is a Section 371 of
International Application No. PCT/EP2018/074710, filed Sep. 13,
2018, which was published in the English language on Mar. 21, 2019,
under International Publication No. WO 2019/053109 A1 which claims
priority under 35 U.S.C. .sctn. 119(e) to U.S. Provisional
Application No. 62/558,994, filed Sep. 15, 2017, the disclosures of
which are incorporated herein by reference in their entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] This application contains a sequence listing, which is
submitted electronically via EFS-Web as an ASCII formatted sequence
listing with a file name "Sequence_Listing_004852_159US2", creation
date of Jan. 7, 2022, and having a size of about 7.6 kB. The
sequence listing submitted via EFS-Web is part of the specification
and is herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The invention relates to methods for safely inducing
effective immunity against
[0004] RSV. In particular, the invention relates to adenovirus
serotype 26 expression vectors expressing RSV F protein providing
the safe induction of immunity against multiple strains of RSV in
human subjects.
BACKGROUND OF THE INVENTION
[0005] Respiratory syncytial virus (RSV) is a highly contagious
childhood pathogen of the respiratory tract which is believed to be
responsible for 200,000 childhood deaths annually. In children
younger than 2 years, RSV accounts for approximately 50% of the
hospitalizations due to respiratory infections, with a peak of
hospitalization occurring at 2-4 months of age. It has been
reported that almost all children will have experienced infection
with RSV by the age of two, and repeated infection during life is
attributed to low natural immunity. In the elderly, the RSV disease
burden is similar to those caused by non-pandemic influenza A
infections.
[0006] RSV is a paramyxovirus, belonging to the subfamily of
pneumovirinae. Its genome encodes for various proteins, including
the membrane proteins known as RSV Glycoprotein (G) and RSV fusion
(F) protein which are the major antigenic targets for neutralizing
antibodies.
[0007] Unlike the RSV G protein, the F protein is conserved between
RSV strains; which makes it an attractive vaccine candidate able to
elicit broadly neutralizing antibodies. The F protein is a
transmembrane protein and it is incorporated in the virion membrane
from cellular membrane during virus budding. The RSV F protein
facilitates infection by fusing the viral and host-cell membranes.
In the process of fusion, the F protein refolds irreversibly from a
labile pre-fusion conformation to a stable post-fusion
conformation. The protein precursor, F0, requires cleavage during
intracellular maturation by a furin-like protease. There are two
furin sites, cleavage of which results in removal of a p27 peptide
and formation of two domains: an N-terminal F2 domain and a
C-terminal F1 domain (FIG. 1). The F2 and F1 domains are connected
by two cystine bridges. Antibodies against the fusion protein can
prevent virus uptake in the cell and thus have a neutralizing
effect. Besides being a target for neutralizing antibodies, RSV F
contains cytotoxic T cell epitopes (Pemberton et al, 1987, J. Gen.
Virol. 68: 2177-2182).
[0008] Despite 50 years of research, there is still no licensed
vaccine against RSV. One major obstacle to the vaccine development
is the legacy of vaccine-enhanced disease in a clinical trial in
the 1960s with a formalin-inactivated (FI) RSV vaccine. FI-RSV
vaccinated children were not protected against natural infection
and infected children experienced more severe illness than
non-vaccinated children, including two deaths. This phenomenon is
referred to as `enhanced disease`.
[0009] Since the trial with the FI-RSV vaccine, various approaches
to generate an RSV vaccine have been pursued. Attempts include
classical live attenuated cold passaged or temperature sensitive
mutant strains of RSV, (chimeric) protein subunit vaccines, peptide
vaccines and RSV proteins expressed from recombinant viral vectors,
including adenoviral vectors. Although some of these vaccines
showed promising pre-clinical data, no vaccine has been licensed
for human use due to safety concerns or lack of efficacy.
[0010] The most potent RSV neutralizing antibodies bind to a
particular site (site zero) on the RSV F protein which is only
exposed when the RSV protein is in its pre-fusion conformation
which makes this particular conformation very attractive as a
vaccine antigen. However, the F protein in its pre-fusion
conformation is very unstable and readily undergoes a major
conformational shift to the post-fusion conformation. Due to its
instability the pre-fusion conformation thus has a propensity to
prematurely refold into the stable postfusion conformation. This
phenomenon is an intrinsic feature of the protein both in solution
and on the surface of the virions.
[0011] Vaccine candidates based on the RSV F protein have failed
due to problems with e.g. stability, purity, reproducibility, and
potency. Indeed, despite many efforts to produce a vaccine against
RSV that contain pre-fusion forms of RSV F protein, no stable
pre-fusion RSV F polypeptides have been described that have been
tested in humans.
[0012] Therefore, a need still exists for safe and effective
vaccines and methods of vaccinating against RSV. The present
invention aims at providing such methods for vaccinating against
RSV in a safe manner.
SUMMARY OF THE INVENTION
[0013] According to the present invention it has for the first time
been demonstrated that recombinant adenovectors expressing RSV F
protein stabilized in the pre-fusion comformation induce a safe and
effective (immunogenic) immune response against RSV when
administered to human subjects.
[0014] The invention thus provides a method of inducing a safe
immune response against respiratory syncytial virus (RSV) in a
human subject in need thereof, comprising administering to the
subject a composition comprising recombinant adenovirus comprising
nucleic acid encoding an RSV Fusion (F) protein comprising the
amino acid sequence of SEQ ID NO: 1, and a pharmaceutically
acceptable carrier, in a total dose of from about 1.times.10.sup.10
to about 2.times.10.sup.11 viral particles (vp) of adenovirus.
[0015] In a further aspect, the invention provides a vaccine
composition comprising recombinant adenovirus comprising nucleic
acid encoding an RSV Fusion (F) protein comprising the amino acid
sequence of SEQ ID NO: 1, and a pharmaceutically acceptable
carrier, in a total dose of about 1.times.10.sup.10 to about
2.times.10.sup.11 viral particles (vp), for use in a method of
inducing a safe immune response against respiratory syncytial virus
(RSV) in a subject in need thereof.
[0016] In certain embodiments of the invention, the total dose is
from about 5.times.10.sup.10 to about 1.times.10.sup.11 viral
particles (vp) of adenovirus. In certain embodiments, the dose is
about 5.times.10.sup.10 or about 1.times.10.sup.11 vp of
adenovirus.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1: Schematic representation of the RSV F protein. A
protein precursor includes F2 and F1 domains and p27 peptide that
is removed from the mature proteins by cleavage with furin-like
proteases. The cleavage sites are indicated by arrows. The numbers
on top of the boxes indicate amino acid positions in the full
length protein excluding signal peptide. In the F1, structural
elements are shown: fusion peptide (FP), refolding region 1 (RR1)
including heptad repeat A (HRA) and refolding region 2 (RR2)
including heptad repeat B (HRB).
[0018] FIG. 2: Relative surface expression of F protein variants.
Full length variants of F protein were expressed in HEK293T cells.
The cell were stained with anti-RSV F antibody (CR9503) and
analyzed by Flow Cytometry (FACS). The Mean Fluorescent Intensity
(MFI) values were calculated and normalized to MFI of control F
wild type (Fwt)-transfected cell sample. The MFI of Fwt was set to
1. Bars represent mean values, error bars represent range of
values.
[0019] FIG. 3: Fraction of pre-fusion F protein on cell surface.
Full length variants of F protein were expressed in HEK293T cells.
The cell were stained with anti-RSV F antibody CR9503 and
anti-pre-fusion RSV F antibody CR9501 and analyzed by Flow
Cytometry. The Mean Fluorescent Intensity (MFI) values were
calculated and MFI measured by CR9501 was normalized to MFI
measured by CR9503. The normalized MFI values indicate fraction of
the pre-F on the cell surface. Bars represent mean values, error
bars represent range of values.
[0020] FIG. 4: Relative surface expression of F protein variants.
Soluble versions with the transmembrane region and cytoplasmic
region deleted (Fsl) and full length variants of F protein were
expressed in HEK293T cells. The expression level of the soluble
protein was measured in the culture supernatant by octet and the
full length variants were tested by cell staining using anti-RSV F
antibody (CR9503) and analyzed by Flow Cytometry. The Mean
Fluorescent Intensity (MFI) values were calculated and normalized
to MFI of control F wild type (Fwt)-transfected cell sample. The
MFI of Fwt was set to 1. Bars represent mean values, error bars
represent range of values.
[0021] FIG. 5: Temperature stability of the F protein variants.
Full length variants of F protein were expressed in HEK293T cells.
After heat-shock, the cell were stained with anti-RSV F antibody
(CR9501--solid lines and CR9503--dashed lines) and analyzed by Flow
Cytometry. The percentage of cells positive for the staining was
determined. Symbols represent mean values, error bars represent
range of values. (A) The percentage of cells positive for the
staining was determined. (B) The Mean Fluorescent Intensity (MFI)
values were calculated and normalized to MFI of 37 C cell sample.
The MFI of 37.degree. C. sample was set to 1. Dotted lines
correspond to background staining at 60.degree. C.
[0022] FIG. 6: Stability of the F proteins. PreF is more stable
than FA2 protein in prefusion conformation on the cell surface in
the heat-stress assay. A549 cells were infected with Ad26 and Ad35
comprising the insert FA2 (wt RSV F, grey bars) or prefusion F
stabilized insert (preF2.2, black bars) at the indicated MOI. The
cells were temperature treated at the indicated temperature for 15
minutes before staining. Top: percentage of cells presenting
prefusion F on their surface (detected by CR9501 antibody); bottom:
percentage of cells presenting any form of the F protein, prefusion
and post fusion (detected by CR9503 antibody). The values were
normalized to 37.degree. C. samples. All bars represent a single
measurement.
[0023] FIG. 7: Ad26.RSV.preF2.1 and F2.2 elicit a cellular immune
response after single administration in mice. Horizontal bars
depict the geometric mean of the response within a group. The
background level is calculated as the 95% percentile of the spot
forming units (SFU) observed in non-stimulated splenocytes, and is
indicated with a dotted line.
[0024] FIG. 8: Ad26.RSV.preF2.1 and F2.2 induce increased virus
neutralizing antibodies when compared to Ad26.RSV.FA2, after single
immunization in mice. Balb/c mice (n=4 per group) were immunized
with the indicated dose of 108 to 1010 viral particles (vp)
Ad26.RSV.FA2 or Ad26.RSV.preF2.1 or Ad26.RSV.preF2.2, or with
formulation buffer, and humoral immune responses were assayed in
the serum isolated 8 weeks after immunization. (A) Virus
neutralizing antibodies were determined against RSV A Long using a
micro-neutralization assay with an ELISA based read out. Titers are
given as the log2 value of the IC50. (B) Pre-fusion or post-fusion
F antibody titers were determined by ELISA, and the ratio between
pre- and post-fusion F antibodies for all samples that showed pre-
and post-fusion F titers above lower limit of quantification (LLoQ)
was calculated. (C) Subclass ELISA was performed using post-fusion
RSV F A2 as coating reagent, and the IgG2a/IgG1 ratio (log10) is
plotted. Ratio's observed for Th1 (serum derived from animals
immunized with RSV F expression Adenoviral vectors) and Th2 (serum
derived from FI-RSV immunized animals) references samples are
indicated with dashed lines. The LLoQ is indicated with a dotted
lines (panels A), and horizontal bars represent the mean responses
per group.
[0025] FIG. 9: Ad26.RSV.preF2.2 elicits antibody responses that
neutralize a wide range of RSV isolates. Sera from Balb/c mice that
were immunized with 1010 viral particles (vp) Ad26.RSV.FA2 (n=3) or
Ad26.RSV.preF (n=4) or formulation buffer (n=2) were used in virus
neutralization assays (micro-neutralization assay with an ELISA
based read out) with the RSV A (upper panels) and B strains (lower
panels) indicated. Titers are given as the log2 value of the IC50,
and horizontal bars represent the mean response per group. LLoQ is
indicated with a dashed line.
[0026] FIG. 10: Single immunization with Ad26.RSV.preF2.2 or
Ad35.RSV.preF2.2 at low doses protects cotton rats against
challenge with the homologous RSV A2. Cotton rats (Sigmodon
hispidus) (n=7 to 9 per group) were immunized with the indicated
doses (in vp/animal) of Ad26.RSV.preF2.2, Ad26.RSV.FA2 (left
panels), Ad35.RSV.preF2.2 or Ad35.RSV.FA2 (right panels) by single
intramuscular administration. Control immunizations were performed
with formulation buffer, FI-RSV, or intranasal application of a low
dose of RSV A2. At seven weeks post-immunization animals were
challenged intranasally with 105 pfu RSV A2. The lung (A-B) and
nose viral titers (C-D) were determined by plaque assay 5 days
after challenge. (E-F) Sera taken just before challenge were used
to perform a virus neutralizing assay with the RSV A Long strain
(micro-neutralization assay with an ELISA based read out). The
dotted line represents the lower level of quantification (LLoQ).
Horizontal bars represent the mean titer per group.
[0027] FIG. 11: Ad26.RSV.preF2.2 or Ad35.RSV.preF2.2 immunization
of cotton rats does not result in increased alveolitis scores after
RSV A2 challenge. Cotton rats (Sigmodon hispidus) (n=7 to 9 per
group) were immunized with the indicated doses (in vp/animal) of
Ad26.RSV.preF2.2, Ad26.RSV.FA2 (upper panel), Ad35.RSV.preF2.2, or
Ad35.RSV.FA2 (lower panel) by single intramuscular administration.
Control immunizations were performed with formulation buffer,
FI-RSV, or intranasal application of a low dose of RSV A2. At seven
weeks post-immunization animals were challenged intranasally with
105 pfu RSV A2. Alveolitis was scored by histopathological
examination of one lung lobe 5 days after challenge on a non-linear
scale from 0 to 4. The horizontal dotted line marks the maximal
score of the control animals that were pre-exposed to RSV-A2 before
challenge to mimic a natural exposure to RSV that does not lead to
ERD.
DETAILED DESCRIPTION OF THE INVENTION
[0028] Although respiratory syncytial virus (RSV) infects people
throughout life, most people fail to mount a long lasting
protective immune response. In addition, in the elderly the waning
immune response contributes to increased susceptibility to severe
disease after RSV infection causing significant morbidity and
mortality. There are indications in the literature that both
neutralizing antibody and T-cell mediated protection play a role in
preventing RSV infection. It is therefore believed that a
successful RSV vaccine, in particular for the elderly, should
increase both potent neutralizing antibody levels and induce a
robust T-cell response.
[0029] RSV infection induces virus specific antibodies which are
mainly directed against the fusion (F) protein. The most potent RSV
neutralizing antibodies found in human sera bind to a particular
site (site .PHI. or site zero) on the RSV F protein, which is only
exposed when the RSV protein is in its pre-fusion conformation
which makes this particular conformation very attractive as a
vaccine antigen. However, the F protein in its pre-fusion
conformation is very unstable and readily undergoes a major
conformational shift to the post-fusion conformation.
[0030] In the research that has led to the present invention, a
stabilized RSV F protein was prepared with a unique set of amino
acid mutations compared to the wild type RSV F antigen from the RSV
A2 strain (Genbank ACO83301.1). By demonstrating specific binding
in vitro to pre-fusion specific antibodies it was shown that indeed
a stabilized RSV F antigen in the pre-fusion conformation was
obtained.
[0031] According to the present invention, it has now been shown
that the stabilized RSV F protein maintains its pre-fusion
conformation in vivo, is immunogenic and can be safely administered
to human subjects in need thereof.
[0032] Thus, according to the present invention methods are
provided for inducing a safe immune response against RSV in a human
subject in need thereof, comprising administering to the subject a
composition comprising recombinant adenovirus comprising nucleic
acid encoding an RSV Fusion (F) protein comprising the amino acid
sequence of SEQ ID NO: 1, and a pharmaceutically acceptable
carrier, in a total dose of from about 1.times.10.sup.10 to about
2.times.10.sup.11 viral particles (vp) of adenovirus.
[0033] In a further aspect, the invention provides a vaccine
composition comprising recombinant adenovirus comprising nucleic
acid encoding an RSV Fusion (F) protein comprising the amino acid
sequence of SEQ ID NO: 1, and a pharmaceutically acceptable
carrier, in a total dose of about 1.times.10.sup.10 to about
2.times.10.sup.11 viral particles (vp), for use in a method of
inducing a safe immune response against respiratory syncytial virus
(RSV) in a subject in need thereof.
[0034] In certain embodiments of the invention, the composition
comprises recombinant adenovirus comprising nucleic acid encoding
an RSV Fusion (F) protein comprising the amino acid sequence of SEQ
ID NO: 1, and a pharmaceutically acceptable carrier, in a total
dose of from about 5.times.10.sup.10 to about
1.times.10.sup.11viral particles (vp) of adenovirus.
[0035] In certain embodiments, the dose is about 5.times.10.sup.10
or about 1.times.10.sup.11 vp of adenovirus.
[0036] According to the present invention it has been shown that
adenovirus encoding RSV F protein in the pre-fusion conformation
(in particular the RSV F protein of SEQ ID NO: 1) is highly
immunogenic. Humoral immune responses are increased as compared to
similar doses of adenovirus encoding the wild-type RSV F protein,
which is not stabilized in the pre-fusion conformation and likely
quickly transitions into the post-fusion conformation upon
expression.
[0037] In certain embodiments, the immune response comprises the
induction of antibodies directed against RSV F protein. Thus,
according to certain embodiments, the immune response comprises the
induction of antibodies that specifically bind to the RSV F
protein, as measured in an ELISA.
[0038] In certain embodiments, the immune response comprises the
induction of RSV neutralizing antibodies. In certain embodiments,
the neutralizing antibodies are capable of neutralizing RSV A and B
strains. In certain embodiments, the immune response comprises the
induction of antibodies that are capable of neutralizing RSV A and
B strains in a VNA assay.
[0039] As used herein the " induction of antibodies" means that the
level of antibodies as measured after administration of composition
comprising recombinant adenovirus expressing the RSV F protein of
SEQ ID NO: 1 is higher than the level of antibodies prior to
administration of said composition.
[0040] In certain embodiments, the immune response comprises the
induction of antibodies specific for the RSV F protein in the
pre-fusion conformation and antibodies specific for the RSV F
protein in the post-fusion conformation, wherein the geometric mean
titer (GMT) increase of antibodies specific for RSV F protein in
the pre-fusion conformation is higher than the GMT increase of
antibodies specific for RSV F protein in the post-fusion
conformation, in enzyme linked immunosorbent assays (ELISAs). The
methods of the present invention thus result in a larger increase
of antibodies that are specifically binding to RSV F protein in the
pre-fusion conformation as compared to antibodies that are
specifically binding to RSV F protein in the post-fusion
conformation. Without wishing to be bound to a particular theory,
it is believed that this results in a more effective immune
response since pre-fusion specific antibodies are thought to be
more effective in neutralizing RSV virus, and thereby more
effective in prevention RSV infection (Gilman M S, Castellanos C A,
Chen M, Ngwuta J O, Goodwin E, Moin S M, Mas V, Melero J A, Wright
PF, Graham B S, McLellan J S, Walker L M. Sci Immunol. 2016 Dec.
16; 1(6). pii: eaaj1879. doi: 10.1126/sciimmunol.aaj1879. Epub 2016
Dec. 9. Rapid profiling of RSV antibody repertoires from the memory
B cells of naturally infected adult donors)
In certain embodiments, the ratio between the GMT increase of
post-fusion F specific antibodies as measured in ELISA and the mean
geometric titer increase of neutralizing antibodies as measured in
a VNA assay is reduced after administration of said composition as
compared to said ratio before administration of said composition.
The methods of the present invention thus results in a more
favourable composition of the RSV specific humoral response that
favours neutralizing antibodies over binding antibodies.
[0041] In certain embodiments, the immune response further
comprises a cellular response as indicated by IFNgamma producing T
cells as measured in an IFNy ELISPOT in response to stimulation
with a pool of peptides covering the F protein of SEQ ID NO: 1
and/or by measurement of CD4 and CD8 T-cell subsets expressing
IFN.gamma., IL-2 and TNF.alpha. by intracellular staining (ICS)
after stimulation with a pool of peptides covering the RSV F
protein of SEQ ID NO: 1. It has been suggested that RSV specific T
cells can support the prevention of infection and limit disease;
this could be especially beneficial for the older adults since it
has been described that the cellular response may decrease with age
(Openshaw P J M, Chiu C, Culley F J, Johansson C. Annu Rev Immunol.
2017 Apr. 26; 35:501-532. doi:
10.1146/annurev-immunol-051116-052206. Epub 2017 Feb. 6. Protective
and Harmful Immunity to RSV Infection.)
[0042] By inducing a safe and immunogenic immune response, the
methods of the present invention can be used to prevent serious
lower respiratory tract disease leading to hospitalization and to
decrease the frequency of complications such as pneumonia and
bronchiolitis due to RSV infection and replication in a
subject.
[0043] As used herein, the terms nucleic acid, nucleic acid
molecule, nucleic acid or nucleotide sequence, and polynucleotide
are used interchangeably and all refer to the linear biopolymers
(chains) made from nucleotides, including DNA and RNA. It will be
understood by a skilled person that numerous different nucleic acid
molecules can encode the same polypeptide as a result of the
degeneracy of the genetic code. It is also understood that skilled
persons may, using routine techniques, make nucleotide
substitutions that do not affect the polypeptide sequence encoded
by the polynucleotides described there to reflect the codon usage
of any particular host organism in which the polypeptides are to be
expressed. Therefore, unless otherwise specified, a "nucleic acid
molecule encoding an amino acid sequence" includes all nucleotide
sequences that are degenerate versions of each other and that
encode the same amino acid sequence. Nucleotide sequences that
encode proteins and RNA may include introns. Sequences herein are
provided from 5' to 3' direction, as custom in the art.
[0044] In certain embodiments, the nucleic acid molecule encoding
the RSV pre-fusion F protein are codon optimized for expression in
mammalian cells, such as human cells. Methods of codon-optimization
are known and have been described previously (e.g. WO 96/09378). In
a preferred embodiment, the nucleic acid molecule encoding the RSV
pre-fusion F protein comprises the nucleic acid sequence of SEQ ID
NO: 2. In certain embodiments, the nucleic acid encoding the RSV F
protein consists of the the nucleic acid sequence of SEQ ID NO:
2.
[0045] According to the invention, the subject in need thereof is a
human subject. Preferably, the subject is an elderly subject, i.e.
a human of 60 years and older. According to the present invention
it has been shown that administration of recombinant adenovirus
comprising nucleic acid encoding the RSV F protein in the
pre-fusion conformation is safe and immunogenic, i.e. results in a
potent humoral and cellular response, also in older subjects which
may have a weakened immune system.
[0046] In certain embodiments, the recombinant adenovirus (also
referred to as adenoviral vector) is a human recombinant
adenovirus. The preparation of recombinant adenoviral vectors is
well known in the art. The term `recombinant` for an adenovirus, as
used herein implicates that it has been modified by the hand of
man, e.g. it has altered terminal ends actively cloned therein
and/or it comprises a heterologous gene, i.e. it is not a naturally
occurring wild type adenovirus.
[0047] In certain embodiments, the recombinant adenovirus according
to the invention is deficient in at least one essential gene
function of the E1 region, e.g. the E1a region and/or the E1b
region, of the adenoviral genome that is required for viral
replication. In certain embodiments, the adenovirus according to
the invention is deficient in at least part of the non-essential E3
region. In certain embodiments, the adenovirus is deficient in at
least one essential gene function of the E1 region and at least
part of the non-essential E3 region. The adenoviral vector can be
"multiple deficient," meaning that the adenoviral vector is
deficient in one or more essential gene functions in each of two or
more regions of the adenoviral genome. For example, the
aforementioned E1-deficient or E1-, E3-deficient adenoviral vectors
can be further deficient in at least one essential gene of the E4
region and/or at least one essential gene of the E2 region (e.g.,
the E2A region and/or E2B region).
[0048] In certain embodiments, the adenovirus is a human adenovirus
of the serotype 26 or 35. The vaccines according to the invention
based on these serotypes appear more potent than the ones described
in the prior art that were based on Ad5, since those failed to
provide complete protection against RSV challenge replication after
a single intramuscular administration (Kim et al, 2010, Vaccine 28:
3801-3808; Kohlmann et al, 2009, J Virol 83: 12601-12610; Krause et
al, 2011, Virology Journal 8:375). The serotype of the invention
further generally has a low seroprevalence and/or low pre-existing
neutralizing antibody titers in the human population. Recombinant
adenoviral vectors of these serotypes with different transgenes are
evaluated in clinical trials, and thus far shows to have an
excellent safety profile. Preparation of rAd26 vectors is
described, for example, in WO 2007/104792 and in Abbink et al.,
(2007) Virol 81(9): 4654-63. Exemplary genome sequences of Ad26 are
found in GenBank Accession EF 153474 and in SEQ ID NO:1 of WO
2007/104792. Preparation of rAd35 vectors is described, for
example, in U.S. Pat. No. 7,270,811, in WO 00/70071, and in Vogels
et al., (2003) J Virol 77(15): 8263-71. Exemplary genome sequences
of Ad35 are found in GenBank Accession AC_000019 and in FIG. 6 of
WO 00/70071.
[0049] A recombinant adenovirus according to the invention may be
replication-competent or replication-deficient. In certain
embodiments, the adenovirus is replication deficient, e.g. because
it contains a deletion in the E1 region of the genome. As known to
the skilled person, in case of deletions of essential regions from
the adenovirus genome, the functions encoded by these regions have
to be provided in trans, preferably by the producer cell, i.e. when
parts or whole of E1, E2 and/or E4 regions are deleted from the
adenovirus, these have to be present in the producer cell, for
instance integrated in the genome thereof, or in the form of
so-called helper adenovirus or helper plasmids. The adenovirus may
also have a deletion in the E3 region, which is dispensable for
replication, and hence such a deletion does not have to be
complemented.
[0050] For non-subgroup C E1-deficient adenoviruses such as Ad35
(subgroup B) or Ad26 (subgroup D), it is preferred to exchange the
E4-orf6 coding sequence of these non-subgroup C adenoviruses with
the E4-orf6 of an adenovirus of subgroup C such as Ad5. This allows
propagation of such adenoviruses in well known complementing cell
lines that express the E1 genes of Ad5, such as for example 293
cells or PER.C6 cells (see, e.g. Havenga et al, 2006, J. Gen.
Virol. 87: 2135-2143; WO 03/104467, incorporated in its entirety by
reference herein). In certain embodiments, an adenovirus that can
be used is a human adenovirus of serotype 35, with a deletion in
the E1 region into which the nucleic acid encoding RSV F protein
antigen has been cloned, and with an E4 orf6 region of Ad5. In
certain embodiments, the adenovirus in the vaccine composition of
the invention is a human adenovirus of serotype 26, with a deletion
in the E1 region into which the nucleic acid encoding RSV F protein
antigen has been cloned, and with an E4 orf6 region of Ad5.
[0051] In alternative embodiments, there is no need to place a
heterologous E4orf6 region (e.g. of Ad5) in the adenoviral vector,
but instead the E1-deficient non-subgroup C vector is propagated in
a cell line that expresses both E1 and a compatible E4orf6, e.g.
the 293-ORF6 cell line that expresses both E1 and E4orf6 from Ad5
(see e.g. Brough et al, 1996, J Virol 70: 6497-501 describing the
generation of the 293-ORF6 cells; Abrahamsen et al, 1997, J Virol
71: 8946-51 and Nan et al, 2003, Gene Therapy 10: 326-36 each
describing generation of E1 deleted non-subgroup C adenoviral
vectors using such a cell line).
[0052] Alternatively, a complementing cell that expresses E1 from
the serotype that is to be propagated can be used (see e.g. WO
00/70071, WO 02/40665).
[0053] For subgroup B adenoviruses, such as Ad35, having a deletion
in the E1 region, it is preferred to retain the 3' end of the E1B
55K open reading frame in the adenovirus, for instance the 166 bp
directly upstream of the pIX open reading frame or a fragment
comprising this such as a 243 bp fragment directly upstream of the
pIX start codon (marked at the 5' end by a Bsu36I restriction site
in the Ad35 genome), since this increases the stability of the
adenovirus because the promoter of the pIX gene is partly residing
in this area (see, e.g. Havenga et al, 2006, J. Gen. Virol. 87:
2135-2143; WO 2004/001032, incorporated by reference herein).
[0054] The nucleic acid encoding the RSV F protein of SEQ ID NO: 1
can be introduced into the adenovirus for instance by standard
molecular biology techniques. It can for instance be cloned into a
deleted E1 or E3 region of an adenoviral vector. The nucleic acid
(or transgene) is generally operably linked to expression control
sequences. This can for instance be done by placing the nucleic
acid encoding the RSV F protein under the control of a promoter.
Further regulatory sequences may be added. Many promoters can be
used for expression of a transgene(s), and are known to the skilled
person. A non-limiting example of a suitable promoter for obtaining
expression in eukaryotic cells is a CMV-promoter (U.S. Pat. No.
5,385,839), e.g. the CMV immediate early promoter, for instance
comprising nt. -735 to +95 from the CMV immediate early gene
enhancer/promoter. A polyadenylation signal, for example the bovine
growth hormone polyA signal (U.S. Pat. No. 5,122,458), may be
present behind the transgene(s).
[0055] In certain embodiments, the recombinant adenovectors of the
invention comprise as the 5' terminal nucleotides the nucleotide
sequence: CTATCTAT. These embodiments are advantageous because such
vectors display improved replication in production processes,
resulting in batches of adenovirus with improved homogeneity, as
compared to vectors having the original 5' terminal sequences
(generally CATCATCA) (see also PCT publication no. WO 2013/135615
and U.S. Pat. No. 8,932,607), incorporated in its entirety by
reference herein.
[0056] In certain embodiments the compositions comprising the
recombinant adenovirus may further comprise, or are administered
together with, one or more adjuvants. Adjuvants are known in the
art to further increase the immune response to an applied antigenic
determinant, and pharmaceutical compositions comprising adenovirus
and suitable adjuvants are for instance disclosed in WO
2007/110409, incorporated by reference herein. The terms "adjuvant"
and "immune stimulant" are used interchangeably herein, and are
defined as one or more substances that cause stimulation of the
immune system. In this context, an adjuvant is used to enhance an
immune response to the RSV prefusion F proteins of the invention.
Examples of suitable adjuvants include aluminium salts such as
aluminium hydroxide and/or aluminium phosphate; oil-emulsion
compositions (or oil-in-water compositions), including
squalene-water emulsions, such as MF59 (see e.g. WO 90/14837);
saponin formulations, such as for example QS21 and
Immunostimulating Complexes (ISCOMS) (see e.g. U.S. Pat. No.
5,057,540; WO 90/03184, WO 96/11711, WO 2004/004762, WO
2005/002620); bacterial or microbial derivatives, examples of which
are monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL),
CpG-motif containing oligonucleotides, ADP-ribosylating bacterial
toxins or mutants thereof, such as E. coli heat labile enterotoxin
LT, cholera toxin CT, and the like. It is also possible to use
vector-encoded adjuvant, e.g. by using heterologous nucleic acid
that encodes a fusion of the oligomerization domain of C4-binding
protein (C4bp) to the antigen of interest (e.g. Solabomi et al,
2008, Infect Immun 76: 3817-23). In certain embodiments the
compositions of the invention comprise aluminium as an adjuvant,
e.g. in the form of aluminium hydroxide, aluminium phosphate,
aluminium potassium phosphate, or combinations thereof, in
concentrations of 0.05-5 mg, e.g. from 0.075-1.0 mg, of aluminium
content per dose.
[0057] Administration of the compositions can be performed using
standard routes of administration. Non-limiting embodiments include
parenteral administration, such as by injection e.g. intradermal,
intramuscular, etc, or subcutaneous, transcutaneous, or mucosal
administration, e.g. intranasal, oral, and the like. Intranasal
administration has generally been seen as a preferred route for
vaccines against RSV. The most important advantage of the live
intrasal strategy is the direct stimulation of local respiratory
tract immunity and the lack of associated disease enhancement.
Intranasal administration is a suitable preferred route according
to the present invention as well. The advantage of intramuscular
administration is that it is simple and well-established. In one
embodiment of the invention the composition is administered by
intramuscular injection, e.g. into the deltoid muscle of the arm,
or vastus lateralis muscle of the thigh. The skilled person knows
the various possibilities to administer a composition, e.g. a
vaccine in order to induce an immune response to the antigen(s) in
the vaccine.
[0058] Recombinant adenovirus can be prepared and propagated in
host cells, according to well known methods, which entail cell
culture of the host cells that are infected with the adenovirus.
The cell culture can be any type of cell culture, including
adherent cell culture, e.g. cells attached to the surface of a
culture vessel or to microcarriers, as well as suspension
culture.
[0059] Most large-scale suspension cultures are operated as batch
or fed-batch processes because they are the most straightforward to
operate and scale up. Nowadays, continuous processes based on
perfusion principles are becoming more common and are also suitable
(see e.g. WO 2010/060719, and WO 2011/098592, both incorporated by
reference herein, which describe suitable methods for obtaining and
purifying large amounts of recombinant adenoviruses).
[0060] Producer cells are cultured to increase cell and virus
numbers and/or virus titers. Culturing a cell is done to enable it
to metabolize, and/or grow and/or divide and/or produce virus of
interest according to the invention. This can be accomplished by
methods as such well known to persons skilled in the art, and
includes but is not limited to providing nutrients for the cell,
for instance in the appropriate culture media. Suitable culture
media are well known to the skilled person and can generally be
obtained from commercial sources in large quantities, or
custom-made according to standard protocols. Culturing can be done
for instance in dishes, roller bottles or in bioreactors, using
batch, fed-batch, continuous systems and the like. Suitable
conditions for culturing cells are known (see e.g. Tissue Culture,
Academic Press, Kruse and Paterson, editors (1973), and R. I.
Freshney, Culture of animal cells: A manual of basic technique,
fourth edition (Wiley-Liss Inc., 2000, ISBN 0-471-34889-9).
[0061] A producer cell (sometimes also referred to in the art and
herein as `packaging cell` or `complementing cell` or `host cell`)
that can be used can be any producer cell wherein a desired
adenovirus can be propagated. For example, the propagation of
recombinant adenovirus vectors is done in producer cells that
complement deficiencies in the adenovirus. Such producer cells
preferably have in their genome at least an adenovirus E1 sequence,
and thereby are capable of complementing recombinant adenoviruses
with a deletion in the E1 region. Any E1-complementing producer
cell can be used, such as human retina cells immortalized by E1,
e.g. 911 or PER.C6 cells (see U.S. Pat. No. 5,994,128),
E1-transformed amniocytes (See EP patent 1230354), E1-transformed
A549 cells (see e.g. WO 98/39411, U.S. Pat. No. 5,891,690),
GH329:HeLa (Gao et al, 2000, Human Gene Therapy 11: 213-219), 293,
and the like. In certain embodiments, the producer cells are for
instance HEK293 cells, or PER.C6 cells, or 911 cells, or IT293SF
cells, and the like.
[0062] Typically, the adenovirus will be exposed to the appropriate
producer cell in a culture, permitting uptake of the virus.
Usually, the optimal agitation is between about 50 and 300 rpm,
typically about 100-200, e.g. about 150, typical DO is 20-60%, e.g.
40%, the optimal pH is between 6.7 and 7.7, the optimal temperature
between 30 and 39.degree. C., e.g. 34-37.degree. C., and the
optimal MOI between 5 and 1000, e.g. about 50-300. Typically,
adenovirus infects producer cells spontaneously, and bringing the
producer cells into contact with rAd particles is sufficient for
infection of the cells. Generally, an adenovirus seed stock is
added to the culture to initiate infection, and subsequently the
adenovirus propagates in the producer cells. This is all routine
for the person skilled in the art.
[0063] After infection of an adenovirus, the virus replicates
inside the cell and is thereby amplified, a process referred to
herein as propagation of adenovirus. Adenovirus infection results
finally in the lysis of the cells being infected. The lytic
characteristics of adenovirus therefore permits two different modes
of virus production.
[0064] The first mode is harvesting virus prior to cell lysis,
employing external factors to lyse the cells. The second mode is
harvesting virus supernatant after (almost) complete cell lysis by
the produced virus (see e.g. U.S. Pat. No. 6,485,958, describing
the harvesting of adenovirus without lysis of the host cells by an
external factor). It is preferred to employ external factors to
actively lyse the cells for harvesting the adenovirus.
[0065] Methods that can be used for active cell lysis are known to
the person skilled in the art, and have for instance been discussed
in WO 98/22588, p. 28-35. Useful methods in this respect are for
example, freeze-thaw, solid shear, hypertonic and/or hypotonic
lysis, liquid shear, sonication, high pressure extrusion, detergent
lysis, combinations of the above, and the like. In one embodiment
of the invention, the cells are lysed using at least one detergent.
Use of a detergent for lysis has the advantage that it is an easy
method, and that it is easily scalable.
[0066] Detergents that can be used, and the way they are employed,
are generally known to the person skilled in the art. Several
examples are for instance discussed in WO 98/22588, p. 29-33.
Detergents can include anionic, cationic, zwitterionic, and
nonionic detergents. The concentration of the detergent may be
varied, for instance within the range of about 0.1%-5% (w/w). In
one embodiment, the detergent used is Triton X-100.
[0067] Nuclease may be employed to remove contaminating, i.e.
mostly from the producer cell, nucleic acids. Exemplary nucleases
suitable for use in the present invention include Benzonase.RTM.,
Pulmozyme.RTM., or any other DNase and/or RNase commonly used
within the art. In preferred embodiments, the nuclease is
Benzonase.RTM., which rapidly hydrolyzes nucleic acids by
hydrolyzing internal phosphodiester bonds between specific
nucleotides, thereby reducing the viscosity of the cell lysate.
Benzonase.RTM. can be commercially obtained from Merck KGaA (code
W214950). The concentration in which the nuclease is employed is
preferably within the range of 1-100 units/ml. Alternatively, or in
addition to nuclease treatment, it is also possible to selectively
precipitate host cell DNA away from adenovirus preparations during
adenovirus purification, using selective precipitating agents such
as domiphen bromide (see e.g. U.S. Pat. No. 7,326,555; Goerke et
al., 2005, Biotechnology and bioengineering, Vol. 91: 12-21; WO
2011/045378; WO 2011/045381).
[0068] Methods for harvesting adenovirus from cultures of producer
cells have been extensively described in WO 2005/080556.
[0069] In certain embodiments, the harvested adenovirus is further
purified. Purification of the adenovirus can be performed in
several steps comprising clarification, ultrafiltration,
diafiltration or separation with chromatography as described in for
instance WO 05/080556, incorporated by reference herein.
Clarification may be done by a filtration step, removing cell
debris and other impurities from the cell lysate. Ultrafiltration
is used to concentrate the virus solution. Diafiltration, or buffer
exchange, using ultrafilters is a way for removal and exchange of
salts, sugars and the like. The person skilled in the art knows how
to find the optimal conditions for each purification step. Also WO
98/22588, incorporated in its entirety by reference herein,
describes methods for the production and purification of adenoviral
vectors. The methods comprise growing host cells, infecting the
host cells with adenovirus, harvesting and lysing the host cells,
concentrating the crude lysate, exchanging the buffer of the crude
lysate, treating the lysate with nuclease, and further purifying
the virus using chromatography.
[0070] Preferably, purification employs at least one chromatography
step, as for instance discussed in WO 98/22588, p. 61-70. Many
processes have been described for the further purification of
adenoviruses, wherein chromatography steps are included in the
process. The person skilled in the art will be aware of these
processes, and can vary the exact way of employing chromatographic
steps to optimize the process. It is for instance possible to
purify adenoviruses by anion exchange chromatography steps, see for
instance WO 2005/080556 and Konz et al, 2005, Hum Gene Ther 16:
1346-1353. Many other adenovirus purification methods have been
described and are within the reach of the skilled person. Further
methods for producing and purifying adenoviruses are disclosed in
for example (WO 00/32754; WO 04/020971; U.S. Pat. Nos. 5,837,520;
6,261,823; WO 2006/108707; Konz et al, 2008, Methods Mol Biol 434:
13-23; Altaras et al, 2005, Adv Biochem Eng Biotechnol 99:
193-260), all incorporated by reference herein.
[0071] For administering to humans, the invention may employ
pharmaceutical compositions comprising the recombinant adenovirus
and a pharmaceutically acceptable carrier or excipient. In the
present context, the term "pharmaceutically acceptable" means that
the carrier or excipient, at the dosages and concentrations
employed, will not cause any unwanted or harmful effects in the
subjects to which they are administered. Such pharmaceutically
acceptable carriers and excipients are well known in the art (see
Remington's Pharmaceutical Sciences, 18th edition, A. R. Gennaro,
Ed., Mack Publishing Company [1990]; Pharmaceutical Formulation
Development of Peptides and Proteins, S. Frokjaer and L. Hovgaard,
Eds., Taylor & Francis [2000]; and Handbook of Pharmaceutical
Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press
[2000]). The purified recombinant adenovirus preferably is
formulated and administered as a sterile solution although it is
also possible to utilize lyophilized preparations. Sterile
solutions are prepared by sterile filtration or by other methods
known per se in the art. The solutions are then lyophilized or
filled into pharmaceutical dosage containers. The pH of the
solution generally is in the range of pH 3.0 to 9.5, e.g pH 5.0 to
7.5. The recombinant adenovector typically is in a solution having
a suitable pharmaceutically acceptable buffer. The solution may
further also contain a salt. Optionally stabilizing agent may be
present, such as albumin. In certain embodiments, detergent is
added. In certain embodiments, the recombinant adenovirus may be
formulated into an injectable preparation. These formulations
contain effective amounts of adenovirus, are either sterile liquid
solutions, liquid suspensions or lyophilized versions and
optionally contain stabilizers or excipients. An adenovirus vaccine
can also be aerosolized for intranasal administration (see e.g. WO
2009/117134).
[0072] For instance, adenovirus may be stored in the buffer that is
also used for the Adenovirus World Standard (Hoganson et al,
Development of a stable adenoviral vector formulation,
Bioprocessing March 2002, p. 43-48): 20 mM Tris pH 8, 25 mM NaCl,
2.5% glycerol. Another useful formulation buffer suitable for
administration to humans is 20 mM Tris, 2 mM MgCl.sub.2, 25 mM
NaCl, sucrose 10% w/v, polysorbate-80 0.02% w/v. Obviously, many
other buffers can be used, and several examples of suitable
formulations for the storage and for pharmaceutical administration
of purified (adeno)virus preparations can for instance be found in
European patent no. 0853660, U.S. Pat. No. 6,225,289 and in
international patent applications WO 99/41416, WO 99/12568, WO
00/29024, WO 01/66137, WO 03/049763, WO 03/078592, WO
03/061708.
[0073] In certain embodiments of the invention, use is made of an
adenovirus formulation as described in WO2015/040002. Thus, in a
preferred embodiment, the composition comprising the adenovirus
vector comprising a nucleic acid encoding the RSV F protein of SEQ
ID NO: 1 comprises in addition to the recombinant adenovirus; a
citrate buffer, wherein the citrate concentration is ranging
between about 5 mM and 30 mM; hydroxypropyl-beta-cyclodextrin
(HBCD), wherein the concentration of
[0074] HBCD is ranging between about 1% (w/w) and 10% (w/w); a
salt, e.g. sodium chloride in a concentration between about 20 mM
and about 200 mM; and non-ionic detergent, e.g. Polysorbate-80 in a
concentration ranging from about 0.005% (w/w) to about 0.5% (w/w);
wherein said formulation has a pH ranging between 5.5 and 6.5.
[0075] In certain embodiments, the compositions have a pH ranging
between about 5.7 and 6.3, and comprise citrate at a concentration
ranging between about 5 and 30 mM; HBCD at a concentration ranging
between 1% (w/w) and 10% (w/w); NaCl at a concentration ranging
between 20 mM and 200 mM; Polysorbate-80 at a concentration ranging
between about 0.01% (w/w) and 0.05% (w/w).
[0076] In certain embodiments, the compositions comprise citrate at
a concentration of about 15 mM; HBCD at a concentration of about 5%
(w/w); NaCl at a concentration of about 75 mM, and Polysorbate-80
at a concentration of about 0.03% (w/w).
[0077] In certain embodiments, the compositions further comprise
ethanol, wherein the ethanol concentration is ranging between about
0.1% (w/w) to 1% (w/w).
[0078] In a preferred embodiment, the compositions comprise citrate
at a concentration of about 15 mM; HBCD at a concentration of about
5% (w/w); NaCl at a concentration of about 75 mM, Polysorbate-80 at
a concentration of about 0.03% (w/w) and ethanol at a concentration
of about 0.4% (w/w). The invention is further illustrated in the
following, non-limiting, examples.
EXAMPLES
Example 1. Stabilizing the RSV F Protein in its Pre Fusion
Conformation
[0079] Plasmids encoding basic RSV F sequences were synthesized and
the amino acid substitutions were introduced in the protein by
site-directed mutagenesis. The protein variants were transiently
expressed in HEK293 cells. The relative protein expression on the
cell surface was assessed by Flow Cytometry. The stability of the F
proteins in pre-fusion conformation was evaluated in a
heat-stability assay.
[0080] The protein sequence used for RSV A2 F protein variants was
retrieved from the GenBank, accession number ACO83301.1. The amino
acid substitutions were introduced in the sequence by site-directed
mutagenesis (QuikChange II XL Site-Directed Mutagenesis Kit,
Agilent technologies). The mutagenesis primers were designed using
on-line tool PrimerX. HEK293T cells (CRL-11268) were purchased from
American Tissue Culture Collection and cultured under standard cell
culture conditions (37.degree. C., 10% CO2).
[0081] Fully human IgG1 anti-RSV F protein antibodies CR9501 and
CR9503 were constructed by cloning the heavy (VH) and light (VL)
chain variable regions into a single IgG1 expression vector.
PER.C6.RTM. cells (Crucell) were transfected with the IgG1
expression constructs and the expressed antibodies were purified
from culture supernatants using POROS Mabcapture A chromatography
(Applied Biosystems) and then buffer exchanged to 50 mM NaAc, 50 mM
NaCl, pH 5.5. Antibody concentration was measured by optical
absorption at 280 nm. Antibody quality was also confirmed by
size-exclusion chromatography (SEC), SDS-PAGE and isoelectric
focusing. The antibody CR9501 comprises VH and VL regions of 58C5
(as described in WO2011/020079) which binds specifically to RSV F
protein in its pre-fusion conformation and not to the post-fusion
conformation. CR9503 comprises VH and VL regions of motavizumab,
which recognizes both the pre-fusion and post-fusion conformation
of RSV F.
Protein Expression and Temperature Treatment
[0082] The plasmids were transiently transfected into adherent
HEK293T cells using 293fectine (Cat #12347-019) transfection
reagents (Life Technologies) according to suppliers
recommendations. 48 hours post transfection the cells were
harvested by detaching with EDTA-containing FACS buffer (no
trypsin, see next section) and cell suspension was heat-treated for
10 minutes either in a water bath or in PCR block for the
temperature stability experiments. After the heat-treatment, the
cells were prepared for the Flow Cytometry analysis.
[0083] For analysis of adeno expressed F proteins, A549 cells were
infected with Ad26 virus at a MOI of 10 000 or 5000 and Ad35
viruses at a MOI of 5000, 2500 or 1.250. After 48 h, the cells were
detached and heat treated for 15 minutes at 37.degree. C.,
50.degree. C. and 56.degree. C. Upon heat treatment cells were
stained using CR9501-Alexa647 or CR9503-Alexa647 and Propidium
Iodide (PI). After staining, the cells were fixed and analyzed
using the BD FACS CANTO II cell analyzer.
Flow Cytometry Analysis
[0084] For each staining, the following controls were included: 1)
negative control sample. i.e. cells that were not subjected to any
treatment and not stained with any antibody labeled with a
fluorophore; 2) positive control samples, i.e. cells that are
stained with only one fluorophore (one of each that are used for
the experiment).
[0085] The cells were resuspended in the Flow Cytometry (FC Buffer,
5 mM EDTA, 1% FBS in PBS) and distributed in volumes of 50 .mu.l of
the cell suspension per well in a 96-well plate with a lid (U- or
V-bottom plates). Two-step or one-step protocols were used for
staining.
[0086] In case of the two-step protocol 50 .mu.l of the first Abs
(or buffer for negative controls) was added to the wells and
incubated at RT for 30 min. Biotinylated CR9501 and CR9503 were
used at 2 .mu.g/ml (final concentration in a well 1 .mu.g/ml).
After incubation, the cells were washed 2 times with the FC buffer.
Afterwards 50 .mu.l of Streptavidin-APC (Molecular Probes cat
#SA1005, 0.1 mg/ml is used at 1:100) or buffer for negative
controls was added to the wells and incubated at RT for 30min. The
cells were washed again 2 times with the FC buffer. After the last
wash, the cells were resuspended in 100 .mu.l of FC
buffer+/-live-dead stain (PI from Invitrogen, cat #P1304MP, used at
2 .mu.g/ml) and incubated at RT for 15 minutes. The cells were
centrifuged at 200 g (1000 rpm) for 5 min., the buffer with PI was
removed and the cells were resuspended in 150 .mu.l of the FC
buffer.
[0087] In case of a one-step protocol, CR9501 and CR9503 antibodies
were labeled with fluorescent probe Alexa647 (Molecular Probes, cat
#A-20186) according to manufacturer's instructions. Cells were
stained according to the protocol above excluding the
Streptavidin-APC step.
[0088] From the live cell population, the percentage of cells
positive for CR9501/CR9503 antibody binding was determined. The
cells positive for CR9503 binding express RSV F protein on their
surface. The cells positive for CR9501 binding express pre-fusion
RSV F on their surface.
[0089] The intensity of the antibody staining (Median fluorescence
intensity--MFI) is proportional to the amount of F protein on the
cell surface. MFI was calculated from the live cell population
expressing F protein.
Results
Surface Cell Expression of the Full Length F Protein Variants
[0090] A subset of mutations that was previously identified to
increase expression or stability of the RSV F protein ectodomain in
pre-fusion conformation was introduced in the wild type full length
RSV A2 F sequence (accession number Genbank ACO83301). The
mutations were introduced alone or in multiple combinations, and
the effect on protein expression and stability was assessed.
[0091] The expression level of the protein was measured as mean
fluorescence intensity (MFI) by Flow Cytometry after staining with
the CR9503 antibody that is recognizing both pre-fusion and
post-fusion F protein. The combination of the two amino acid
substitutions that were previously described for stabilization of
the soluble RSV pre-F protein (i.e. N67I and S215P) also increased
the expression level of the full length RSV F protein by 2.3-fold,
relative to wilde type full length RSV F (FIG. 2).
[0092] A prominent increase in expression was observed for variants
with 3 amino acid substitutions combined. Interestingly,
combination of more than three mutations in one variant did not
further increase protein expression. This may be due to limited
capacity of the cellular membrane to accommodate multiple copies of
F protein.
[0093] The amount of the pre-fusion F on the surface of the cell
was assessed by staining with pre-fusion specific antibody CR9501
(FIG. 3). Transfection of the cells with all F variants resulted in
a more or less similar amount of pre-fusion F protein on the cell
surface. Presence of the transmembrane domain stabilizes the full
length protein to certain extent and therefore differences in the
pre-fusion stability are not as apparent under ambient conditions
between the full length F proteins. Therefore the heat-stability
assay was developed to better discriminate stability of full length
variants, as described below.
[0094] The A2 strain that was used as a parental sequence for the
previously described F protein variants (WO2014/174018 and
WO2014/202570) is a cell line adapted laboratory strain which has
accumulated two unique and rare mutations (i.e. of Lysine 66 and
Isoleucine 76). In the present invention, these two residues were
mutated to match the natural clinical isolates (K66E, I76V). The
K66E and I76V mutations were included in selected protein designs.
In comparison to variants with Lys66 and Ile76, variants with
glutamate at 66 (K66E) have a tendency to express slightly higher.
Addition of valine at residue 76 (a double substitution of K66E and
I76V) does not influence expression level when compared to variants
with K66E substitution alone (FIG. 4).
Stability of the Full Length F Protein Variants on the Cell
Surface
[0095] In ambient conditions on a short time scale, no significant
difference in stability of pre-fusion conformation was observed
between full length F variants with the different combinations of
stabilizing mutations. An elevated temperature is known to serve as
an efficient in vitro trigger for refolding of RSV F protein from
pre-fusion to post-fusion conformation. Therefore, a heat-shock
assay was established and used to assess stability of the
membrane-bound full length proteins. Shortly, the HEK293T cells
were transfected with the F protein constructs and used for the
assay 48 hours after transfection. The cells were detached from
cell culture dishes and the cell suspension was heat-treated at
increasing temperatures for 10 minutes. After the heat-treatment,
cells were stained with the anti-RSV F antibodies and analyzed by
Flow Cytometry. The Flow Cytometry data was analyzed in two
different ways. The percentage of the cells, positive for staining
with the anti-F antibodies was analyzed, and also mean fluorescence
intensity (MFI) of the positive cells was calculated (FIG. 5).
[0096] Both staining with CR9501 (antibody recognizing only
pre-fusion F protein) and CR9503 (antibody recognizing both pre-
and post-fusion F protein) were used in the Flow Cytometry assays.
CR9503 antibody served as a positive control. In case when F
protein loses pre-fusion conformation but still is on the surface
of the cell, the protein is still detected with the CR9503
antibody. Loss of staining with both antibodies indicates that
protein is not available on the cell surface for antibody binding,
e.g. due to aggregation.
[0097] Full length proteins with three of more amino acid
substitutions were tested in the assay and compared to the wild
type RSV F. The expression of these variants was the highest and
therefore these variants were preferred candidates. All of the
proteins contained the N67I and S215P substitutions, and one or two
extra stabilizing mutations were added.
[0098] The unmodified wild type protein had a rather stable
staining with CR9503 antibody. The MFI of the CR9503 staining was
elevated at higher temperatures however the spread of values was
also very high. This indicated that no protein aggregation occurred
after the heat-shock. Half of the pre-fusion conformation was lost
after incubation of cells at approximately 55.degree. C., after
incubation of at 60.degree. C. all pre-fusion conformation was lost
as was demonstrated by decreased CR9501 binding to the wild type F
samples after heat-shock at increasing temperatures.
[0099] All tested pre-fusion F protein variants were more stable
than the wild type RSV F with majority of the CR9501 staining
retaining also after treatment at higher temperatures (FIGS. 5 and
6). Proteins with K498R amino acid substitution were less stable
than the others. Addition of the K66E mutation further stabilized
the proteins as also variants with K498R amino acid substitution
became as stable as others and no loss of the pre-fusion
conformation was observed at 60.degree. C. Only selected
combinations of the stabilizing mutations were tested with K66E and
I76V combined. All four tested proteins were stable when percentage
of positive cells was analyzed, however when MFI was analyzed
variant with K498R showed clear decrease in CR9501 binding after
treatment with 60.degree. C., indicating that this variant is less
stable when evaluated in the temperature stress assay.
[0100] In conclusion, a combination of three stabilizing mutations
(including N67I and S215P) was considered sufficient for high
expression level and stability. The D486N mutations was selected as
a third stabilizing mutation because of its position in the protein
structure. K66E and I76V were included in the as they did not have
negative effect on the protein expression and stability but made
the sequence closer to naturally occurring ones.
[0101] The pre-fusion RSV F protein with the mutations K66E, N67I,
I76V, S215P and D486N (F2.2) (SEQ ID NO: 1) was selected for the
construction of adenoviral vectors. This protein was shown to be
stable in the pre-fusion conformation in the temperature stability
assay up to 60.degree. C., and could be expressed in high
levels.
Example 2. Preparation of Adenoviral Vectors
Cloning RSV F Gene Into E1 Region of Ad35 and Ad26
[0102] The nucleic acid sequences, coding for the pre-fusion F
proteins of the invention were gene optimized for human expression
and synthesized, by Geneart. A Kozak sequence (5' GCCACC 3') was
included directly in front of the ATG start codon, and two stop
codons (5' TGA TAA 3') were added at the end of the RSV.pre-F
coding sequence. The RSV.pre-F genes were inserted in the
pAdApt35BSU plasmid and in the pAdApt26 plasmid via HindIII and
Xbal sites.
Cell Culture
[0103] PER.C6 cells (Fallaux et al., 1998, Hum Gene Ther 9:
1909-1917) were maintained in Dulbecco's modified Eagle's medium
(DMEM) with 10% fetal bovine serum (FBS), supplemented with 10 mM
MgCl.sub.2.
Adenovirus Generation, Infections and Passaging
[0104] All adenoviruses were generated in PER.C6.RTM. cells by
single homologous recombination and produced as previously
described (for rAd35: Havenga et al., 2006, J. Gen. Virol. 87:
2135-2143; for rAd26: Abbink et al., 2007, J. Virol. 81:
4654-4663). Briefly, PER.C6 cells were transfected with Ad vector
plasmids, using Lipofectamine according to the instructions
provided by the manufacturer (Life Technologies). For rescue of
e.g. Ad35 vectors carrying the RSV.pre-F transgenes expression
cassette, the pAdApt35BSU.RSV.pre-F plasmid and
pWE/Ad35.pIX-rITR.dE3.5orf6 cosmid were used, whereas for Ad26
vectors carrying the RSV.pre-F transgene expression cassette, the
pAdApt26.RSV.pre-F plasmid and pWE.Ad26.dE3.5orf6.cosmid were used.
Cells were harvested one day after full CPE, freeze-thawed,
centrifuged for 5 min at 3,000 rpm, and stored at -20.degree. C.
Next the viruses were plaque purified and amplified in PER.C6
cultured on a single well of a multiwell 24 tissue culture plate.
Further amplification was carried out in PER.C6 cultured using a
T25 tissue culture flask and a T175 tissue culture flask. Of the
T175 crude lysate, 3 to 5 ml was used to inoculate 20.times.T175
triple-layer tissue culture flasks containing 70% confluent layers
of PER.C6 cells. The virus was purified using a two-step CsCl
purification method. Finally, the virus was stored in aliquots at
-85.degree. C.
Example 3. Induction of Immunity Against RSV F Using Recombinant
Adenovirus Serotypes 26 and 35 Expressing Pre Fusion RSV F In
Vivo
[0105] The immunogenicity of Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2
was evaluated in mice, comparing cellular and humoral immune
responses to responses induced by identical doses of Ad26.RSV.FA2
(i.e. expressing the wild type RSV F protein). Balb/c mice (n=4 per
group) were immunized with the indicated dose of 10.sup.8 to
10.sup.10 viral particles (vp) Ad26.RSV.FA2 or Ad26.RSV.preF2.1 or
Ad26.RSV.preF2.2, or with formulation buffer. At 8 weeks after
prime, the number of RSV F A2 specific IFN.gamma. spot forming
units (SFU) per 10.sup.6 splenocytes was determined using ELISpot.
It was shown that Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2 induced
increased humoral immune responses in mice when compared to
Ad26.RSV.FA2, with broad neutralizing capacity and maintained
cellular responses. A single intramuscular immunization with
Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2 elicited a cellular response
(FIG. 7) which was characterized by induction of CD8+ T cells
positive for IFN.gamma., IL2 and/or TNF.alpha. (data not
shown).
[0106] The quantity and quality of the cellular responses were
comparable between Ad26.RSV.preF2.1, Ad26.RSV.preF.2.2 and
Ad26.RSV.FA2. In contrast, Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2
induced significantly higher RSV neutralizing antibody titers than
Ad26.RSV.FA2. Closer analysis of the antibody responses
demonstrated that Ad26.RSV.preF2.1 and Ad26.RSV.preF.2.2 induced
higher levels of antibodies against pre-fusion F, while post-fusion
F titers remained comparable to Ad26.RSV.FA2, resulting in
significantly increased preF/postF antibody ratios. In addition,
the IgG2a/IgG1 ratio of the antibody response remained unaltered,
demonstrating a similar Th1 skewing of the humoral response as
previously demonstrated for Ad26.RSV.FA2 (FIG. 8).
[0107] For Ad26.RSV.preF2.2 it was furthermore demonstrated that
the antibodies elicited were capable of neutralizing various RSV A
and B strains, laboratory strains as well as clinical isolates,
similar as observed for Ad26.RSV.FA2 (FIG. 9).
[0108] Subsequently, the efficacy and immunogenicity of
Ad26.RSV.preF2.2 and Ad35.RSV.preF2.2 vector constructs was
evaluated in the cotton rat model. These animals are permissive to
replication of human RSV, with peak RSV titers in the lungs at days
4 and 5. Control groups in the experiments included groups
intranasally infected with a low dose RSV A2, thereby mimicking
natural exposure, as well as groups immunized with FI-RSV, using
the original lot 100 that induced enhanced respiratory disease
(ERD) in clinical studies in the dilution that was shown to induce
ERD in cotton rats.
[0109] Single intramuscular immunization of animals with
Ad26.RSV.preF2.2 in doses ranging from 10.sup.5 to 10.sup.8
vp/animal, or Ad35.RSV.preF2.2 in doses ranging from 10.sup.6 to
10.sup.9 vp/animal resulted in complete protection of the lungs
from infection with the vaccine homologous RSV A2 strain, except
for 3 animals immunized with 10.sup.5 vp Ad26.RSV.preF2.2 (FIG. 10A
and 10B). Dose dependent protection of RSV replication in the nose
was observed for both vectors. This ranged from full protection at
10.sup.8 vp/animal, to partial protection at 10.sup.5 vp for
Ad26.RSV.preF2.2, whereas for Ad35.RSV.preF2.2, noses of animals
immunized with 10.sup.9 vp were fully protected from RSV A2, and
10.sup.6 vp resulted in partial protection (FIG. 10C and 10D) Noses
of animals immunized with Ad26.RSV.preF2.2 and Ad35.RSV.preF2.2
were better protected from RSV A2 infection than when immunized
with their respective wild type F counterparts Ad26.RSV.FA2 and
Ad35.RSV.FA2, when analyzed across dose (p=0.0003, and p=0,0001).
Protection from RSV infection was accompanied by dose-dependent
induction of virus neutralization titers against RSV A Long,
already elicited by the lowest doses of Ad26.RSV.preF2.2 or
Ad35.RSV.preF2.2 applied (FIGS. 10E and 10F). Across dose
statistical comparisons of VNA A Long titers revealed that
Ad26.RSV.preF2.2 is more immunogenic than Ad26.RSV.FA2 (p=0.0414),
whereas elicitation of VNA titers was not significantly different
between Ad35.RSV.preF2.2 and Ad35.RSV.FA2.
[0110] It was further demonstrated that Ad26.RSV.preF and
Ad35.RSV.preF do not induce histopathological signs of Enhanced
Respiratory Disease (ERD) after RSV A2 challenge, at any of the
concentrations tested. The cotton rat is the most used and best
studied model to monitor ERD. In this animal model, vaccination
with FI-RSV consistently induces ERD after RSV challenge, which is
visible by histopathological analysis of sections of the infected
lungs for parameters as alveolitits, consisting primarily of
neutrophil infiltrates, and peribronchiolitis, consisting primarily
of lymphocyte infiltrates. In cotton rats, FI-RSV-induced scores
for these parameters can be observed from day 1 after RSV
infection, and peak at 4 to 5 days after RSV challenge.
[0111] ERD was analyzed 5 days after challenge with RSV A2 by
scoring 4 parameters of pulmonary inflammatory changes
(peribronchiolitis, perivasculitis, interstitial pneumonia,
alveolitis). Immunization with FI-RSV resulted in enhanced scores
for most histopathological markers, which was especially apparent
for alveolitis (FIG. 11), the marker that was previously shown to
be the most discriminating marker for ERD. No increases in
alveolitis or any other ERD histopathological marker was observed
in animals immunized by either Ad26.RSV.preF2.2 or Ad35.RSV.preF2.2
in a prime-only regimen after RSV challenge, even at low vaccine
doses that may induce low affinity and/or low levels of antibodies
(FIG. 11). This is confirming our previous results with
Ad26.RSV.FA2 and Ad35.RSV.FA2 vectors.
[0112] According to the invention, it has thus been shown that
Ad26.RSV.preF2.2 and Ad35.RSV.preF2.2 are potent adenoviral vectors
expressing RSV F A2 which is stabilized in the pre-fusion
conformation. These vectors induce strong humoral and cellular
immune responses. The immune response elicited is protective
against RSV A2 challenge and provides a wide range of virus
neutralization in vitro against clinical and laboratory isolates of
RSV. No ERD induction was observed in cotton rats after RSV
exposure of vaccinated animals and therefore confirms the data
generated with Ad26 and Ad35 encoding for the wild type RSV F A2
antigen. Neither mice nor cotton rats showed overt signs of
reactogenicity after injection of either Ad26.RSV.preF2.2 or
Ad35.RSV.preF2.2.
Example 4. Study of RSV Vaccine Recombinant Adenovirus Serotypes 26
Expressing Pre Fusion RSV F in Humans
[0113] A Randomized, Double-blind, First-in-Human Phase 1 Study to
evaluate the safety, tolerability and immunogenicity of two
Vaccinations of Ad26.RSV.preF, one year apart, in adults aged 60
years and older in stable health was performed. This trial has been
registered at clinicaltrials.gov identifier NCT02926430.
Objectives
[0114] The primary objective of the study included assessing the
safety and tolerability of 2 single doses of either
5.times.10.sup.10 viral particles [vp] or 1.times.10.sup.11 vp of
Ad26.RSV.preF, administered intramuscularly to adults above 60
years of age. The secondary objective included assessing the
humoral and cellular immune responses as measured by virus
neutralization assay (VNA), F-protein binding antibodies (pre-F and
post-F ELISA) and intracellular cytokine staining (ICS).
Study Design
[0115] This is a single-center, randomized, placebo-controlled,
double-blind, Phase 1 study to evaluate the safety, tolerability
and immunogenicity of 2 Ad26.RSV.preF vaccinations, administered 1
year apart, in 72 male and female subjects aged 60 years and older
in stable health. Subjects were randomized in parallel in a
1:1:1:1:2 ratio to 1 of 5 groups to receive 2 single intramuscular
(IM) injections of study vaccine 12 months apart (Table 1).
Safety Results
[0116] An interim analysis at 28 days after first vaccination was
performed. Subjects that received 5.times.10.sup.10 vp
Ad26.RSV.preF on Day 1, were pooled together (Group 1 and 2), the
same for subjects that received 1.times.10.sup.11 vp Ad26.RSV.preF
(Group 3 and 4). The primary interim analysis post first dose of
all 72 subjects demonstrates that this vaccine is well tolerated at
both dose levels. The median duration of solicited adverse events
(AEs) typically ranged from 1 to 3.5 days. There was no serious AE
related (SAE) to the vaccine, and no AEs led to withdrawal from the
study. There was no apparent difference in reactogenicity between
the two vaccine doses used. The interim data did not reveal an
effect of dose on the safety.
[0117] In conclusion, overall solicited and unsolicited AEs
reported post-vaccination were mild in the majority of subjects,
transient in nature, and resolved without sequelae. Based on the
above, it was concluded that both doses of Ad26.RSV.preF were safe
and well-tolerated by participants of both dosage groups.
Immunogenicity Assays
Pre-F ELISA
[0118] Total IgG levels against the pre-F stabilized conformation
(based on RSV A2 F, Genbank ACO83301.1) were evaluated. The pre-F
protein was biotinylated and sequentially captured by streptavidin
coated plates on 96-well microtiter plates. Serially diluted test
samples were incubated and pre-F-specific antibodies were detected
with anti-human IgG antibodies conjugated with horseradish
peroxidase followed by a chemiluminescent reaction. The IC.sub.50
was reported as the binding titer.
Post-F ELISA
[0119] Total IgG levels against the post-F protein (RSV A2 F,
Genbank ACO83301.1) were evaluated. The post-F protein was coated
on 96-well microtiter plates. Serially diluted test samples were
incubated and post-F-specific antibodies were detected with
anti-human IgG antibodies conjugated with horseradish peroxidase
followed by a chemiluminescent reaction. The IC.sub.50 was reported
as the binding titer.
RSV Neutralization Assay A2 Strain (A2-FFL) or B Strain
(A2-BAGdup-O36634.1F-FFL)
[0120] The functionality of the vaccine-induced antibody responses
was investigated by the determination of virus neutralizing
antibodies (VNA) in a virus neutralization assay using A549 cells
and RSV A2 virus which expresses luciferase (Luc) (RSV A2 FFL or
RSV B FFL). Neutralizing antibodies were measured in A549 cells as
a function of reduction in firefly Luc reporter gene expression
after a single round of infection with RSV A2-FFL (or B Gdup-FFL).
A fixed amount of RSV A2 FFL (or B Gdup-FFL) was mixed with a
serially diluted clinical serum sample. After 1 hour incubation,
A549 cells were added to the mixture. RSV A2-FFL (or B Gdup FFL)
infection or inhibition was measured at 20-21 hours by the Luc
reporter gene expression system. IC.sub.50 was reported as the
neutralization titer.
ELISpot
[0121] Frozen PBMCs were analyzed by IFN.gamma. ELISpot. PBMCs were
stimulated with peptide pools matched to the preF protein vaccine
insert (SEQ ID NO: 1). The number of SFC/10.sup.6 stimulated PBMCs,
after subtraction of mock stimulated PBMCs was reported.
CD4+ and CD8+ T-Cell Responses (ICS)
[0122] The induction or boosting of CD4 and CD8 T-cell subsets
expressing IFN.gamma., IL-2 and TNF.alpha. was determined by ICS
after RSV F-protein peptide stimulation. Total cytokine responses
were reported as the percentage of CD4+ and CD8+ T cells that
produce at least IFN.gamma., IL-2 and/or TNF.alpha.. For the
Th1/Th2 balance, the CD4 cells expressing either IFN.gamma. (Th1)
or IL-4 (Th2) was reported . The total responding (log.sub.10
scale) CD4+ or CD8+ T cells, expressing IFN.gamma., IL-2,
TNF.alpha. as a single cytokine or in any combination thereof are
added and reported
Immunogenicity Results
[0123] The humoral immunogenicity analysis included the following
primary immuno assays (secondary objective): VNA to the RSV A2
strain and binding ELISA to pre-fusion and post-fusion F protein.
In addition, a VNA to an RSV B strain was performed. Cellular
immunogenicity analysis included IFN.gamma. ELISPOT assay. The
results are shown in Tables 2 -7.
[0124] As expected, RSV F specific humoral and cellular responses
were detected in all subjects at baseline (day 1), reflecting a
history of natural exposure to RSV throughout life (Tables
2-5).
[0125] Table 2 includes the results of VNA responses against RSV A2
strain, presented in descriptive statistics of the actual values
and fold increase from baseline with corresponding 95% Confidence
interval (CI) The geometric mean titer (GMT) fold rise for RSV A2
neutralizing antibodies after 1 dose of Ad26.RSV.preF at
5.times.10.sup.10 or 1.times.10.sup.11 vp was 2.5 and 3.2,
respectively.
[0126] The results of VNA responses against RSV B strain are shown
in Table 3, which also includes descriptive statistics of the
actual values and fold increase from baseline with corresponding
95% CI.
The GMT fold rise for RSV B neutralizing antibodies after 1 dose of
Ad26.RSV.preF at 5.times.10.sup.10 or 1.times.10.sup.11 vp was 3.1
and 3.4, respectively. Thus, both RSV A2 and RSV B neutralizing
antibodies increased after vaccination at day 28 post vaccination.
Ad26.RSV preF at a dose of 1.times.10.sup.11 vp resulted in higher
neutralizing antibody levels compared to the dose of
5.times.10.sup.10 vp.
[0127] Pre-F binding ELISA assessed the binding of antibodies to
the pre-fusion conformation of the RSV F protein, while post-F
binding ELISA assessed the binding of antibodies to the post-fusion
conformation of the RSV F protein. Descriptive statistics of the
actual values and fold increase from baseline with corresponding
95% CI of the results are presented in Table 2 for both pre-F
protein binding antibody responses (ELISA) and post-F protein
binding antibody responses (ELISA). Total preF IgG binding antibody
showed GMT fold rises of 2.2 (5.times.10.sup.10 vp) and 2.9
(1.times.10.sup.11 vp), while total postF IgG binding antibody
showed GMT fold increases of 1.9 (5.times.10.sup.10 vp) or 2.1
(1.times.10.sup.11 vp) fold. Thus, both the level of binding
antibodies to F protein in the pre-fusion conformation (preF
antibodies) and of post F antibodies increased after one vaccine
dose. The results of the preF binding ELISA followed the same
pattern as the VNA assay. The highest levels of preF binding
antibodies was observed in the group receiving Ad26.RSV.preF at a
dose of 1.times.10.sup.11 vp. No dose response was observed on the
induction of postF antibodies using 5.times.10.sup.10 vp or
1.times.10.sup.11 vp Ad26.RSV.preF dose.
[0128] T-cell responses induced by Ad26.RSV.preF are measured by
IFN.gamma. ELISpot, in which PBMC are restimulated with the preF
protein peptides. Descriptive statistics of the actual values of
RSV-F specific T cell response (IFN.gamma.) before and after
vaccination are presented in Table 4. Median cellular responses in
these elderly subjects as measured by IFN.gamma. ELISPOT increased
from 103 and 95 at baseline to 325 and 305 SFU/10.sup.6 PBMC
(5.times.10.sup.10 and 1.times.10.sup.11 vp, respectively). The
total responding CD4+ T cells, expressing IFN.gamma., IL-2,
TNF.alpha. as a single cytokine or in any combination thereof are
added and presented in Table 5. An increase in the CD4+ T-cell
response was observed at 28 days after after vaccination with
A26.RSV.preF: the median total cytokine response was 0.03% and
0.03% at day 1 compared to 0.13% and 0.12% at day 28 post
immunization (5.times.10.sup.10 and 1.times.10.sup.11 vp,
respectively). The total responding CD8+ T cells, expressing
IFN.gamma., IL-2, TNF.alpha. as a single cytokine or in any
combination thereof are also presented in Table 5 , the median
total cytokine response was 0.07% and 0.04% at day 1 compared to
0.15% and 0.06% at day 28 post immunization (5.times.10.sup.10 and
1.times.10.sup.11 vp, respectively).
[0129] As shown in Table 6, administration of Ad26.preF changed the
ratio between antibodies binding to pre-fusion F (preF antibodies)
and antibodies binding to post-fusion F (postF antibodies). A
geometric mean increase in preF/postF ratio in the Ad26.preF
vaccinated subjects was seen from 1.5 and 1.2 at baseline to 1.7 to
1.8 (5.times.10.sup.10 and 1.times.10.sup.11 vp, respectively).The
administration of Ad26.RSV.preF thus clearly favoured a higher rise
of preF binding antibodies.
[0130] The geometric mean preF/VNA ratio in the Ad26.preF
vaccinated subjects was similar for both groups (5.times.10.sup.10
and 1.times.10.sup.11 vp): 0.8 at baseline and 0.7 at 28 days after
vaccination (Table 7), while geometric mean postF/VNA ratio in the
Ad26.preF vaccinated subjects was 0.5 and 0.6 at baseline and 0.4
and 0.4 (5.times.10.sup.10 and 1.times.10.sup.11 vp, respectively)
at day 29 (Table 8).
[0131] In previously conducted clinical trials VAC18192RSV1001
(ClinicalTrials.gov Identifier: NCT02440035) and VAC18192RSV1003
(ClinicalTrials.gov Identifier: NCT02561871) with the prototype
Ad26.RSV FA2 RSV expressing the wild type RSV F from RSV A2,
healthy subjects of 18-50 years of age received Ad26.RSV.FA2 at
5.times.10.sup.10 vp dose. Both humoral and cellular immune
responses were increased 28 days after receiving Ad26.RSV.FA2. The
geometric mean fold rise for RSV neutralizing antibodies was 2.0
and 1.9 for RSV A2 and RSV B respectively. The mean fold rise of
total postF IgG binding antibody showed geometric mean increases of
3.0 fold, while the geometric mean fold rise of preF IgG binding
showed a 2.4 fold rise. Median cellular responses in these subjects
as measured by IFN.gamma. ELISPOT increased from 76 to 290
SFU/10.sup.6 PBMC (data not shown). In these clinical trials with
Ad26.RSV.FA2 an increase in preF/postF ratio was not observed. More
strikingly, the geometric mean preF/post F ratio was 1.4 at
baseline and decreased to 1.1 at 28 days after vaccination. The
geometric mean preF/VNA ratio was 0.6 at baseline and 0.7 at 28
days after vaccination; while the geometric mean postF/VNA ratio
was 0.4 at baseline and increased slightly to 0.6 at 28 days after
vaccination (Tables 10-12).
[0132] In conclusion, according to the present invention it was
shown that both humoral and cellular immune responses were strongly
increased 28 days after the first vaccine dose for both dose levels
of Ad26.RSV.preF (i.e. recombinant adenovirus encoding the RSV F
protein in the pre-fusion conformation, in particular the RSV F
protein of SEQ ID NO: 1). The highest humoral responses were
observed with 1.times.10.sup.11 vp dose, which was still safe. The
boost in humoral immune response was preferably directed against
the pre-F epitopes, as illustrated by a shift in the preF/postF
binding antibody ratio from baseline. One dose of Ad26.RSV.preF was
safe and well tolerated in this phase 1 study and boosted
favourable humoral and cellular responses in RSV experienced older
adults, even at a higher dose of 1.times.10.sup.11 vp of
adenovirus.
TABLE-US-00001 TABLE 1 Study design Group N Day 1 Day 365.sup.a 1
12 Ad26.RSV.preF Ad26.RSV.preF (5 .times. 10.sup.10 vp) (5 .times.
10.sup.10 vp) 2 12 Ad26.RSV.preF Placebo (5 .times. 10.sup.10 vp) 3
12 Ad26.RSV.preF Ad26.RSV.preF (1 .times. 10.sup.11 vp) (1 .times.
10.sup.11 vp) 4 12 Ad26.RSV.preF Placebo (1 .times. 10.sup.11 vp) 5
24 Placebo Placebo .sup.aFor operational reasons, the second
vaccination may occur between 12-14 months after the first
vaccination
TABLE-US-00002 TABLE 2 Descriptive Statistics of the Humoral
immunogenicity Assays; Full Analysis Set Ad26.RSV.preF
Ad26.RSV.preF (5 .times. 10.sup.10 vp) (1 .times. 10.sup.11 vp)
Placebo Humoral: ELISA preF Baseline 24 24 23 DAY 1 N 24 24 23
Geometric mean (95% CI) 334 (255; 438) 407 (293; 566) 269 (212;
341) Post-dose 1 23 24 23 DAY 29 N 23 24 23 Geometric mean (95% CI)
709 (519; 967) 1193 (897; 1588) 255 (203; 321) Geometric mean
increase (95% CI) 2.2 (1.7; 2.8) 2.9 (2.2; 3.9) 0.9 (0.9; 1.0)
Humoral: ELISA postF Baseline 24 24 23 DAY 1 N 24 24 23 Geometric
mean (95% CI) 219 (172; 279) 326 (228; 466) 174 (132; 231)
Post-dose 1 23 24 23 DAY 29 N 23 24 23 Geometric mean (95% CI) 407
(305; 543) 680 (509; 908) 170 (129; 224) Geometric mean increase
(95% CI) 1.9 (1.5; 2.3) 2.1 (1.8; 2.4) 1.0 (0.9; 1.0) Humoral: RSV
A2 strain Baseline 24 24 23 DAY 1 N 24 24 23 Geometric mean (95%
CI) 432 (328; 569) 512 (345; 759) 402 (280; 578) Post-dose 1 23 24
23 DAY 29 N 23 24 23 Geometric mean (95% CI) 1031 (839; 1267) 1617
(1126; 2323) 431 (299; 622) Geometric mean increase (95% CI) 2.5
(1.9; 3.2) 3.2 (2.3; 4.2) 1.1 (1.0; 1.1) N: number of subjects with
data
TABLE-US-00003 TABLE 3 Titers of Neutralizing Antibodies to RSV B
strain: Descriptive Statistics of the Actual Values and Fold
Increase from Baseline; Full Analysis Set Ad26.RSV.preF
Ad26.RSV.preF (5 .times. 10.sup.10 vp) (1 .times. 10.sup.11 vp)
Placebo Baseline 24 24 23 DAY 1 N 24 24 23 Geometric mean (95% CI)
1787 (1252; 2551) 2710 (1678; 4376) 1632 (1065; 2502) Post-dose 1
23 24 23 DAY 29 N 23 24 23 Geometric mean (95% CI) 5383 (3770;
7686) 9302 (6273; 13795) 1556 (1073; 2257) Geometric mean increase
(95% CI) 3.1 (2.1; 4.7) 3.4 (2.3; 5) 1 (0.8; 1.1) N: number of
subjects with data
TABLE-US-00004 TABLE 4 ELISpot: Descriptive Statistics of the
Actual Values; Full Analysis Set Ad26.RSV.preF Ad26.RSV.preF (5
.times. 10.sup.10 vp) (1 .times. 10.sup.11 vp) Placebo Baseline 23
21 21 DAY 1 N 23 21 21 Median (Q1; Q3) 103 (42; 175) 95 (37; 245)
114 (42; 169) Post-dose 1 23 21 22 DAY 29 N 23 21 22 Median (Q1;
Q3) 325 (249; 478) 305 (172; 492) 94 (61; 223) N: number of
subjects with data
TABLE-US-00005 TABLE 5 Total Cytokine Response (ICS): Descriptive
Statistics of the Actual Values; Full Analysis Set Ad26.RSV.preF
Ad26.RSV.preF (5 .times. 10.sup.10 vp) (1 .times. 10.sup.11 vp)
Placebo CD4 Baseline 20 20 22 DAY 1 N 20 20 22 Median (Q1; Q3) 0.03
(<0.02; 0.07) 0.03 (<0.02; 0.10) 0.02 (<0.02; 0.06)
Post-dose 1 19 19 20 DAY 29 N 19 19 20 Median (Q1; Q3) 0.13 (0.08;
0.16) 0.12 (0.05; 0.18) <0.02 (<0.02; 0.04) CD8 Baseline 20
20 22 DAY 1 N 20 20 22 Median (Q1; Q3) 0.07 (0.02; 0.20) 0.04
(<0.02; 0.12) 0.09 (<0.02; 0.31) Post-dose 1 19 19 20 DAY 29
N 19 19 20 Median (Q1; Q3) 0.15 (0.08; 0.29) 0.06 (0.03; 0.20) 0.10
(0.03; 0.66) N: number of subjects with data
TABLE-US-00006 TABLE 6 Ratio Antibody Response RSV Pre F-protein
versus Post F-protein over Time; Full Analysis Set Ad26.RSV.preF
Ad26.RSV.preF (5 .times. 10.sup.10 vp) (1 .times. 10.sup.11 vp)
Placebo Baseline 24 24 23 DAY 1 N 24 24 23 Geometric mean 1.5 (1.3;
1.8) 1.2 (1; 1.6) 1.5 (1.3; 1.8) (95% CI) Post-dose 1 23 24 23 DAY
29 N 23 24 23 Geometric mean 1.7 (1.5; 2.1) 1.8 (1.4; 2.3) 1.5
(1.3; 1.8) (95% CI) N: number of subjects with data
TABLE-US-00007 TABLE 7 Ratio Antibody Response RSV Pre F-protein
versus VNA A2 over Time; Full Analysis Set Ad26.RSV.preF
Ad26.RSV.preF (5 .times. 10.sup.10 vp) (1 .times. 10.sup.11 vp)
Placebo Baseline 24 24 23 DAY 1 N 24 24 23 Geometric mean 0.8 (0.6;
0.9) 0.8 (0.7; 1.0) 0.7 (0.5; 0.9) (95% CI) Post-dose 1 23 24 23
DAY 29 N 23 24 23 Geometric mean 0.7 (0.5; 0.9) 0.7 (0.6; 0.9) 0.6
(0.4; 0.8) (95% CI) N: number of subjects with data
TABLE-US-00008 TABLE 8 Ratio Antibody Response RSV Post-F-protein
versus VNA A2 over Time; Full Analysis Set Ad26.RSV.preF
Ad26.RSV.preF (5 .times. 10.sup.10 vp) (1 .times. 10.sup.11 vp)
Placebo Baseline 24 24 23 DAY 1 N 24 24 23 Geometric mean 0.5 (0.4;
0.6) 0.6 (0.5; 0.8) 0.7 (0.5; 0.9) (95% CI) Post-dose 1 23 24 23
DAY 29 N 23 24 23 Geometric mean 0.4 (0.3; 0.5) 0.4 (0.3; 0.6) 0.6
(0.4; 0.8) (95% CI) N: number of subjects with data
TABLE-US-00009 TABLE 9 Descriptive Statistics of all Humoral
Assays; Immunogenicity Analysis Set (Study VAC18192RSV1001 +
VAC18192RSV1003) Ad26.RSV.FA2, (5 .times. 10.sup.10 vp) Humoral:
ELISA pre Baseline 35 DAY 1 N 35 Geometric mean (95% CI) 248 (203;
304) Post-Ad.RSV.FA2 dose 35 DAY 29 N 35 Geometric mean (95% CI)
596 (493; 720) Geometric mean increase (95% CI) 2.4 (2.0; 2.9)
Humoral: ELISA post Baseline 35 DAY 1 N 35 Geometric mean (95% CI)
184 (151; 224) Post-Ad.RSV.FA2 dose 35 DAY 29 N 35 Geometric mean
(95% CI) 556 (445; 695) Geometric mean increase (95% CI) 3.0 (2.5;
3.7) Humoral: RSV A2 strain Baseline 35 DAY 1 N 35 Geometric mean
(95% CI) 433 (345; 543) Post-Ad.RSV.FA2 dose 35 DAY 29 N 35
Geometric mean (95% CI) 856 (675; 1085) Geometric mean increase
(95% CI) 2.0 (1.7; 2.4) Humoral: RSV B strain Baseline 35 DAY 1 N
35 Geometric mean (95% CI) 2231 (1851; 2689) Post-Ad.RSV.FA2 dose
23 DAY 29 N 23 Geometric mean (95% CI) 3983 (3206; 4948) Geometric
mean increase (95% CI) 1.9 (1.6; 2.1) N: number of subjects with
data
TABLE-US-00010 TABLE 10 Ratio Antibody Response RSV Pre F-protein
versus Post F-protein over Time; Immunogenicity Analysis Set (Study
VAC18192RSV1001 + VAC18192RSV1003) Ad26.RSV.FA2, (5 .times.
10.sup.10 vp) Baseline 35 DAY 1 N 35 Geometric mean (95% CI) 1.4
(1.2; 1.6) Post-Ad.RSV.FA2 dose 35 DAY 29 N 35 Geometric mean (95%
CI) 1.1 (0.9; 1.2) N: number of subjects with data
TABLE-US-00011 TABLE 11 Ratio Antibody Response RSV Pre F-protein
versus VNA A2 over Time; Immunogenicity Analysis Set (Study
VAC18192RSV1001 + VAC18192RSV1003) Ad26.RSV.FA2, (5 .times.
10.sup.10 vp) Baseline 35 DAY 1 N 35 Geometric mean (95% CI) 0.6
(0.5; 0.7) Post-Ad.RSV.FA2 dose 35 DAY 29 N 35 Geometric mean (95%
CI) 0.7 (0.6; 0.9) N: number of subjects with data
TABLE-US-00012 TABLE 12 Ratio Antibody Response RSV Post-F-protein
versus VNA A2 over Time; Immunogenicity Analysis Set (Study
VAC18192RSV1001 + VAC18192RSV1003) Ad26.RSV.FA2, (5 .times.
10.sup.10 vp) Baseline 35 DAY 1 N 35 Geometric mean (95% CI) 0.4
(0.3; 0.5) Post-Ad.RSV.FA2 dose 35 DAY 29 N 35 Geometric mean (95%
CI) 0.6 (0.5; 0.8) N: number of subjects with data
TABLE-US-00013 Sequence listing Amino acid sequence of the RSV
pre-fusion F protein (mutations compared to RSV A2 strain are bold
and underlined) SEQ ID NO: 1: RSV preF2.2 amino acid sequence:
MELLILKANA ITTILTAVTF CFASGQNITE EFYQSTCSAV SKGYLSALRT GWYTSVITIE
LSNIKEIKCN GTDAKVKLIK QELDKYKNAV TELQLLMQST PATNNRARRE LPRFMNYTLN
NAKKTNVTLS KKRKRRFLGF LLGVGSAIAS GVAVSKVLHL EGEVNKIKSA LLSTNKAVVS
LSNGVSVLTS KVLDLKNYID KQLLPIVNKQ SCSIPNIETV IEFQQKNNRL LEITREFSVN
AGVTTPVSTY MLTNSELLSL INDMPITNDQ KKLMSNNVQI VRQQSYSIMS IIKEEVLAYV
VQLPLYGVID TPCWKLHTSP LCTTNTKEGS NICLTRTDRG WYCDNAGSVS FFPQAETCKV
QSNRVFCDTM NSLTLPSEVN LCNVDIFNPK YDCKIMTSKT DVSSSVITSL GAIVSCYGKT
KCTASNKNRG IIKTFSNGCD YVSNKGVDTV SVGNTLYYVN KQEGKSLYVK GEPIINFYDP
LVFPSNEFDA SISQVNEKIN QSLAFIRKSD ELLHNVNAVK STTNIMITTI IIVIIVILLS
LIAVGLLLYC KARSTPVTLS KDQLSGINNI AFSN SEQ ID NO: 2: codon optimized
nucleic acid encoding the RSV F pre-F2.2 pre-fusion protein
ATGGAGCTGCTGATCCTGAAGGCCAACGCCATCA
CCACCATCCTGACCGCCGTGACCTTCTGCTTCG CCAGCGGCCAGAACATCACCGAGGAGTTCTACC
AGAGCACCTGCAGCGCCGTGAGCAAGGGCTACC TGAGCGCCCTGAGAACCGGCTGGTACACCAGCG
TGATCACCATCGAGCTGAGCAACATCAAGGAGA TCAAGTGCAACGGCACCGACGCCAAGGTGAAGC
TGATCAAGCAGGAGCTGGACAAGTACAAGAACG CCGTGACCGAGCTGCAGCTGCTGATGCAGAGCA
CCCCCGCCACCAACAACAGAGCCAGAAGAGAGC TGCCCAGATTCATGAACTACACCCTGAACAACG
CCAAGAAGACCAACGTGACCCTGAGCAAGAAGA GAAAGAGAAGATTCCTGGGCTTCCTGCTGGGCG
TGGGCAGCGCCATCGCCAGCGGCGTGGCCGTGA GCAAGGTGCTGCACCTGGAGGGCGAGGTGAACA
AGATCAAGAGCGCCCTGCTGAGCACCAACAAGG CCGTGGTGAGCCTGAGCAACGGCGTGAGCGTGC
TGACCAGCAAGGTGCTGGACCTGAAGAACTACA TCGACAAGCAGCTGCTGCCCATCGTGAACAAGC
AGAGCTGCAGCATCCCCAACATCGAGACCGTGA TCGAGTTCCAGCAGAAGAACAACAGACTGCTGG
AGATCACCAGAGAGTTCAGCGTGAACGCCGGCG TGACCACCCCCGTGAGCACCTACATGCTGACCA
ACAGCGAGCTGCTGAGCCTGATCAACGACATGC CCATCACCAACGACCAGAAGAAGCTGATGAGCA
ACAACGTGCAGATCGTGAGACAGCAGAGCTACA GCATCATGAGCATCATCAAGGAGGAGGTGCTGG
CCTACGTGGTGCAGCTGCCCCTGTACGGCGTGA TCGACACCCCCTGCTGGAAGCTGCACACCAGCC
CCCTGTGCACCACCAACACCAAGGAGGGCAGCA ACATCTGCCTGACCAGAACCGACAGAGGCTGGT
ACTGCGACAACGCCGGCAGCGTGAGCTTCTTCC CCCAGGCCGAGACCTGCAAGGTGCAGAGCAACA
GAGTGTTCTGCGACACCATGAACAGCCTGACCC TGCCCAGCGAGGTGAACCTGTGCAACGTGGACA
TCTTCAACCCCAAGTACGACTGCAAGATCATGA CCAGCAAGACCGACGTGAGCAGCAGCGTGATCA
CCAGCCTGGGCGCCATCGTGAGCTGCTACGGCA AGACCAAGTGCACCGCCAGCAACAAGAACAGAG
GCATCATCAAGACCTTCAGCAACGGCTGCGACT ACGTGAGCAACAAGGGCGTGGACACCGTGAGCG
TGGGCAACACCCTGTACTACGTGAACAAGCAGG AGGGCAAGAGCCTGTACGTGAAGGGCGAGCCCA
TCATCAACTTCTACGACCCCCTGGTGTTCCCCA GCAACGAGTTCGACGCCAGCATCAGCCAGGTGA
ACGAGAAGATCAACCAGAGCCTGGCCTTCATCA GAAAGAGCGACGAGCTGCTGCACAACGTGAACG
CCGTGAAGAGCACCACCAACATCATGATCACCA CCATCATCATCGTGATCATCGTGATCCTGCTGA
GCCTGATCGCCGTGGGCCTGCTGCTGTACTGCA AGGCCAGAAGCACCCCCGTGACCCTGAGCAAGG
ACCAGCTGAGCGGCATCAACAACATCGCCTTCA GCAACTGA
Sequence CWU 1
1
21574PRTArtificial SequenceRSV preF2.2 amino acid sequence 1Met Glu
Leu Leu Ile Leu Lys Ala Asn Ala Ile Thr Thr Ile Leu Thr1 5 10 15Ala
Val Thr Phe Cys Phe Ala Ser Gly Gln Asn Ile Thr Glu Glu Phe 20 25
30Tyr Gln Ser Thr Cys Ser Ala Val Ser Lys Gly Tyr Leu Ser Ala Leu
35 40 45Arg Thr Gly Trp Tyr Thr Ser Val Ile Thr Ile Glu Leu Ser Asn
Ile 50 55 60Lys Glu Ile Lys Cys Asn Gly Thr Asp Ala Lys Val Lys Leu
Ile Lys65 70 75 80Gln Glu Leu Asp Lys Tyr Lys Asn Ala Val Thr Glu
Leu Gln Leu Leu 85 90 95Met Gln Ser Thr Pro Ala Thr Asn Asn Arg Ala
Arg Arg Glu Leu Pro 100 105 110Arg Phe Met Asn Tyr Thr Leu Asn Asn
Ala Lys Lys Thr Asn Val Thr 115 120 125Leu Ser Lys Lys Arg Lys Arg
Arg Phe Leu Gly Phe Leu Leu Gly Val 130 135 140Gly Ser Ala Ile Ala
Ser Gly Val Ala Val Ser Lys Val Leu His Leu145 150 155 160Glu Gly
Glu Val Asn Lys Ile Lys Ser Ala Leu Leu Ser Thr Asn Lys 165 170
175Ala Val Val Ser Leu Ser Asn Gly Val Ser Val Leu Thr Ser Lys Val
180 185 190Leu Asp Leu Lys Asn Tyr Ile Asp Lys Gln Leu Leu Pro Ile
Val Asn 195 200 205Lys Gln Ser Cys Ser Ile Pro Asn Ile Glu Thr Val
Ile Glu Phe Gln 210 215 220Gln Lys Asn Asn Arg Leu Leu Glu Ile Thr
Arg Glu Phe Ser Val Asn225 230 235 240Ala Gly Val Thr Thr Pro Val
Ser Thr Tyr Met Leu Thr Asn Ser Glu 245 250 255Leu Leu Ser Leu Ile
Asn Asp Met Pro Ile Thr Asn Asp Gln Lys Lys 260 265 270Leu Met Ser
Asn Asn Val Gln Ile Val Arg Gln Gln Ser Tyr Ser Ile 275 280 285Met
Ser Ile Ile Lys Glu Glu Val Leu Ala Tyr Val Val Gln Leu Pro 290 295
300Leu Tyr Gly Val Ile Asp Thr Pro Cys Trp Lys Leu His Thr Ser
Pro305 310 315 320Leu Cys Thr Thr Asn Thr Lys Glu Gly Ser Asn Ile
Cys Leu Thr Arg 325 330 335Thr Asp Arg Gly Trp Tyr Cys Asp Asn Ala
Gly Ser Val Ser Phe Phe 340 345 350Pro Gln Ala Glu Thr Cys Lys Val
Gln Ser Asn Arg Val Phe Cys Asp 355 360 365Thr Met Asn Ser Leu Thr
Leu Pro Ser Glu Val Asn Leu Cys Asn Val 370 375 380Asp Ile Phe Asn
Pro Lys Tyr Asp Cys Lys Ile Met Thr Ser Lys Thr385 390 395 400Asp
Val Ser Ser Ser Val Ile Thr Ser Leu Gly Ala Ile Val Ser Cys 405 410
415Tyr Gly Lys Thr Lys Cys Thr Ala Ser Asn Lys Asn Arg Gly Ile Ile
420 425 430Lys Thr Phe Ser Asn Gly Cys Asp Tyr Val Ser Asn Lys Gly
Val Asp 435 440 445Thr Val Ser Val Gly Asn Thr Leu Tyr Tyr Val Asn
Lys Gln Glu Gly 450 455 460Lys Ser Leu Tyr Val Lys Gly Glu Pro Ile
Ile Asn Phe Tyr Asp Pro465 470 475 480Leu Val Phe Pro Ser Asn Glu
Phe Asp Ala Ser Ile Ser Gln Val Asn 485 490 495Glu Lys Ile Asn Gln
Ser Leu Ala Phe Ile Arg Lys Ser Asp Glu Leu 500 505 510Leu His Asn
Val Asn Ala Val Lys Ser Thr Thr Asn Ile Met Ile Thr 515 520 525Thr
Ile Ile Ile Val Ile Ile Val Ile Leu Leu Ser Leu Ile Ala Val 530 535
540Gly Leu Leu Leu Tyr Cys Lys Ala Arg Ser Thr Pro Val Thr Leu
Ser545 550 555 560Lys Asp Gln Leu Ser Gly Ile Asn Asn Ile Ala Phe
Ser Asn 565 57021725DNAArtificial Sequencecodon optimized nucleic
acid encoding the RSV F pre-F2.2 pre-fusion protein 2atggagctgc
tgatcctgaa ggccaacgcc atcaccacca tcctgaccgc cgtgaccttc 60tgcttcgcca
gcggccagaa catcaccgag gagttctacc agagcacctg cagcgccgtg
120agcaagggct acctgagcgc cctgagaacc ggctggtaca ccagcgtgat
caccatcgag 180ctgagcaaca tcaaggagat caagtgcaac ggcaccgacg
ccaaggtgaa gctgatcaag 240caggagctgg acaagtacaa gaacgccgtg
accgagctgc agctgctgat gcagagcacc 300cccgccacca acaacagagc
cagaagagag ctgcccagat tcatgaacta caccctgaac 360aacgccaaga
agaccaacgt gaccctgagc aagaagagaa agagaagatt cctgggcttc
420ctgctgggcg tgggcagcgc catcgccagc ggcgtggccg tgagcaaggt
gctgcacctg 480gagggcgagg tgaacaagat caagagcgcc ctgctgagca
ccaacaaggc cgtggtgagc 540ctgagcaacg gcgtgagcgt gctgaccagc
aaggtgctgg acctgaagaa ctacatcgac 600aagcagctgc tgcccatcgt
gaacaagcag agctgcagca tccccaacat cgagaccgtg 660atcgagttcc
agcagaagaa caacagactg ctggagatca ccagagagtt cagcgtgaac
720gccggcgtga ccacccccgt gagcacctac atgctgacca acagcgagct
gctgagcctg 780atcaacgaca tgcccatcac caacgaccag aagaagctga
tgagcaacaa cgtgcagatc 840gtgagacagc agagctacag catcatgagc
atcatcaagg aggaggtgct ggcctacgtg 900gtgcagctgc ccctgtacgg
cgtgatcgac accccctgct ggaagctgca caccagcccc 960ctgtgcacca
ccaacaccaa ggagggcagc aacatctgcc tgaccagaac cgacagaggc
1020tggtactgcg acaacgccgg cagcgtgagc ttcttccccc aggccgagac
ctgcaaggtg 1080cagagcaaca gagtgttctg cgacaccatg aacagcctga
ccctgcccag cgaggtgaac 1140ctgtgcaacg tggacatctt caaccccaag
tacgactgca agatcatgac cagcaagacc 1200gacgtgagca gcagcgtgat
caccagcctg ggcgccatcg tgagctgcta cggcaagacc 1260aagtgcaccg
ccagcaacaa gaacagaggc atcatcaaga ccttcagcaa cggctgcgac
1320tacgtgagca acaagggcgt ggacaccgtg agcgtgggca acaccctgta
ctacgtgaac 1380aagcaggagg gcaagagcct gtacgtgaag ggcgagccca
tcatcaactt ctacgacccc 1440ctggtgttcc ccagcaacga gttcgacgcc
agcatcagcc aggtgaacga gaagatcaac 1500cagagcctgg ccttcatcag
aaagagcgac gagctgctgc acaacgtgaa cgccgtgaag 1560agcaccacca
acatcatgat caccaccatc atcatcgtga tcatcgtgat cctgctgagc
1620ctgatcgccg tgggcctgct gctgtactgc aaggccagaa gcacccccgt
gaccctgagc 1680aaggaccagc tgagcggcat caacaacatc gccttcagca actga
1725
* * * * *