U.S. patent application number 10/663722 was filed with the patent office on 2006-07-20 for multivalent immunogenic composition containing rsv subunit composition and influenza virus preparation.
Invention is credited to David Burt, George A. Cates, Michel H. Klein, Suryaprakash Sambhara.
Application Number | 20060159700 10/663722 |
Document ID | / |
Family ID | 22796441 |
Filed Date | 2006-07-20 |
United States Patent
Application |
20060159700 |
Kind Code |
A1 |
Cates; George A. ; et
al. |
July 20, 2006 |
Multivalent immunogenic composition containing RSV subunit
composition and influenza virus preparation
Abstract
Immunogenic compositions for administration to adults
particularly to the elderly, to protect them against disease caused
by infection by respiratory syncytial virus and influenza virus
comprise an immunoeffective amount of a mixture of purified fusion
(F) protein, attachment (G) protein and matrix (M) protein of RSV
and an immunoeffective amount of a non-virulent influenza virus
preparation. The components of the composition when formulated as a
vaccine for in vivo administration do not impair the immunogenicity
of each other. The immunogenic composition may also contain an
adjuvant.
Inventors: |
Cates; George A.; (Richmond
Hill, CA) ; Sambhara; Suryaprakash; (Atlanta, GA)
; Burt; David; (Dollard Des Ormeaux, CA) ; Klein;
Michel H.; (Willowdale, CA) |
Correspondence
Address: |
SIM & MCBURNEY
330 UNIVERSITY AVENUE
6TH FLOOR
TORONTO
ON
M5G 1R7
CA
|
Family ID: |
22796441 |
Appl. No.: |
10/663722 |
Filed: |
September 17, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09213770 |
Dec 17, 1998 |
|
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10663722 |
Sep 17, 2003 |
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Current U.S.
Class: |
424/202.1 |
Current CPC
Class: |
A61P 31/16 20180101;
A61K 2039/543 20130101; C12N 2760/18534 20130101; A61K 2039/55555
20130101; C12N 2760/16134 20130101; A61K 2039/70 20130101; A61K
2039/5254 20130101; A61K 39/145 20130101; A61P 31/12 20180101; A61K
2039/5252 20130101; A61K 39/155 20130101; A61K 39/12 20130101 |
Class at
Publication: |
424/202.1 |
International
Class: |
A61K 39/295 20060101
A61K039/295 |
Claims
1. A multivalent immunogenic composition for conferring protection
in a host against disease caused by infection by respiratory
syncytial virus (RSV) and influenza virus, which comprises: (a) an
immunoeffective amount of a mixture of purified fusion (F) protein,
attachment (G) protein and matrix (M) protein of RSV, and (b) an
immunoeffective amount of a non-virulent influenza virus
preparation.
2. The immunogenic composition of claim 1 formulated as a vaccine
for in vivo administration to the host wherein the individual
components (a) and (b) of the composition are formulated such that
the immunogenicity of the individual components (a) and (b) is not
impaired.
3. The immunogenic composition of claim 2 further comprising an
adjuvant.
4. The immunogenic composition of claim 3 wherein said adjuvant
imparts an enhanced immune response to RSV when prepared to the
mixture (a) formulated with the adjuvant in the absence of the
non-virulent influenza virus preparation.
5. The immunogenic composition of claim 3 wherein the adjuvant is
poly-di(carboxylatophenoxy)-phosphazene (PCPP).
6. The immunogenic composition of claim 1 wherein said mixture (a)
is present in an amount of about 10 to about 200 .mu.g and (b) is
present in an amount of about 1 to about 100 .mu.g, in a single
dose.
7. The immunogenic composition of claim 1 wherein said fusion (F)
protein comprises multimeric fusion (F) proteins.
8. The immunogenic composition of claim 7 wherein, when analyzed
under non-reducing conditions, said multimeric fusion (F) protein
includes heterodimers of molecular weight approximately 70 kDa and
dimeric and trimeric forms.
9. The immunogenic composition of claim 1 wherein, when analyzed
under non-reducing conditions, said attachment (G) protein
comprises G protein of molecular weight approximately 95 kDa and G
protein of molecular weight approximately 55 kDa and oligomeric G
protein.
10. The immunogenic composition of claim 1 wherein, when analyzed
by SDS-PAGE under non-reducing conditions, said matrix (M) protein
comprises M protein of molecular weight approximately 28 to 34
kDa.
11. The immunogenic composition of claim 1 wherein, when analyzed
by reduced SDS-PAGE analysis, said fusion (F) protein comprises an
F.sub.1 subunit of molecular weight approximately 48 kDa and an
F.sub.2 subunit of molecular weight approximately 23 kDa, said
attachment (G) protein comprises a G protein of molecular weight
approximately 95 kDa and a G protein of molecular weight
approximately 55 kDa, and said matrix (M) protein comprises an M
protein of approximately 31 kDa.
12. The immunogenic composition of claim 1 wherein said F, G and M
proteins are present in mixture (a) in the relative proportions of:
F from about 35 to about 70 wt % G from about 5 to about 30 wt % M
from about 10 to about 40 wt %
13. The immunogenic composition of claim 12 wherein, when analyzed
by SDS-PAGE under reducing conditions and silver stained, the ratio
of F.sub.1 subunit of molecular weight approximately 48 kDa to
F.sub.2 subunit of molecular weight approximately 23 kDa is between
1:1 to about 2:1 as determined by scanning densitometry.
14. The immunogenic composition of claim 13 wherein said mixture is
at least about 75% pure.
15. The immunogenic composition of claim 1 wherein said RSV
proteins in said mixture are from one or both of subtypes RSV A and
RSV B.
16. The immunogenic composition of claim 1 wherein said
non-virulent influenza virus preparation comprises a plurality of
different non-virulent influenza virus strains.
17. The immunogenic composition of claim 16 wherein said
non-virulent influenza virus preparation is an inactivated
influenza virus preparation.
18. A method of immunizing a human host against disease caused by
infection by respiratory syncytial virus (RSV) and influenza virus,
which comprises administering to the host an immunoeffective amount
of the immunogenic composition of claim 1.
19. The method of claim 18 wherein said immunogenic composition is
formulated as a vaccine for in vivo administration to the host
wherein the individual components (a) and (b) of the composition
are formulated such that the immunogenicity of the individual
components (a) and (b) is not impaired.
20. The method of claim 19 wherein said host is a human host of at
least 18 years of age.
Description
FIELD OF INVENTION
[0001] This invention relates to multivalent immunogenic
composition, particularly for administration to adults.
BACKGROUND TO THE INVENTION
[0002] Human respiratory syncytial virus is the main cause of lower
respiratory tract infections among infants and young children
(refs. 1 to 3--a list of references appears at the end of the
disclosure and each of the references in the list is incorporated
herein by reference thereto). Globally, 65 million infections occur
every year resulting in 160,000 deaths (ref. 4). In the USA alone
100,000 children may require hospitalization for pneumonia and
bronchiolitis caused by RS virus in a single year (refs. 5, 6).
Providing inpatient and ambulatory care for children with RS virus
infections costs in excess of $340 million annually in the USA
(ref. 7). Severe lower respiratory tract disease due to RS virus
infection predominantly occurs in infants two to six months of age
(ref. 8). Approximately 4,000 infants in the USA die each year from
complications arising from severe respiratory tract disease caused
by infection with RS virus and Parainfluenza type 3 virus (PIV-3).
The World Health Organization (WHO) and the National Institute of
Allergy and Infectious Disease (NIAID) vaccine advisory committees
have ranked RS virus second only to HIV for vaccine
development.
[0003] RSV infection in adults was initially considered a
significant problem only in certain high-risk populations, such as
the institutionalized elderly. However, evidence has been
accumulating that the infection occurs frequently in previously
healthy adults (ref. 9).
[0004] RSV infections in the elderly usually represent reinfections
in those who have had many prior episodes. These infections have
been reported to cause altered airway resistance and exacerbration
of chronic obstructive lung disease.
[0005] In adults over 60 years old, RSV usually causes mild nasal
congestion and may also result in fewer, anorexia, pneumonia,
brochitis and deaths (ref. 10).
[0006] The structure and composition of RSV has been elucidated and
is described in detail in the textbook "Fields Virology", Fields,
B. N. et al. Raven Press, N.Y. (1996), in particular, Chapter 44,
pp 1313-1351 "Respiratory Syncytial Virus" by Collins, P.,
McIntosh, K., and Chanock, R. M. (ref. 11).
[0007] The two major protective antigens of RSV are the envelope
fusion (F) and attachment (G) glycoproteins (ref. 12). The F
protein is synthesized as an about 68 kDa precursor molecule
(F.sub.0) which is proteolytically cleaved into disulfide-linked
F.sub.1 (about 48 kDa) and F.sub.2 (about 20 kDa) polypeptide
fragments (ref. 13). The G protein (about 33 kDa) is heavily
O-glycosylated giving rise to a glycoprotein of apparent molecular
weight of about 90 kDa (ref. 14). Two broad subtypes of RS virus
have been defined A and B (ref. 15). The major antigenic
differences between these subtypes are found in the G glycoprotein
while the F glycoprotein is more conserved (refs. 7, 16).
[0008] In addition to the antibody response generated by the F and
G glycoproteins, human cytotoxic T cells produced by RSV infection
have been shown to recognize the RSV F protein, matrix protein M,
nucleoprotein N, small hydrophobic protein SH, and the
nonstructural protein lb (ref. 17).
[0009] A safe and effective RSV vaccine is not available and is
urgently needed. Approaches to the development of RS virus vaccines
have included inactivation of the virus with formalin (ref. 18),
isolation of cold-adapted and/or temperature-sensitive mutant
viruses (ref. 19) and purified F or G glycoproteins (refs. 20, 21,
22). Clinical trial results have shown that both live attenuated
and formalin-inactivated vaccines failed to adequately protect
vaccines against RS virus infection (refs. 23 to 25). Problems
encountered with attenuated cold-adapted and/or
temperature-sensitive RS virus mutants administered intranasally
included clinical morbidity, genetic instability and
overattenuation (refs. 26 to 28). A live RS virus vaccine
administered subcutaneously also was not efficacious (ref. 29).
Inactivated RS viral vaccines have typically been prepared using
formaldehyde as the inactivating agent. Murphy et al. (ref. 30)
have reported data on the immune response in infants and children
immunized with formalin-inactivated RS virus. Infants (2 to 6
months of age) developed a high titre of antibodies to the F
glycoprotein but had a poor response to the G protein. Older
individuals (7 to 40 months of age) developed titres of F and G
antibodies comparable to those in children who were infected with
RS virus. However, both infants and children developed a lower
level of neutralizing antibodies than did individuals of comparable
age with natural RS virus infections. The unbalanced immune
response, with high titres of antibodies to the main immunogenic RS
virus proteins F (fusion) and G (attachment) proteins but a low
neutralizing antibody titre, may be in part due to alterations of
important epitopes in the F and G glycoproteins by the formalin
treatment. Furthermore, some infants who received the
formalin-inactivated RS virus vaccine developed a more serious
lower respiratory tract disease following subsequent exposure to
natural RS virus than did non-immunized individuals (refs. 24, 25).
The formalin-inactivated RS virus vaccines, therefore, have been
deemed unacceptable for human use.
[0010] Evidence of an aberrant immune response also was seen in
cotton rats immunized with formalin-inactivated RS virus (ref. 31).
Furthermore, evaluation of RS virus formalin-inactivated vaccine in
cotton rats also showed that upon live virus challenge, immunized
animals developed enhanced pulmonary histopathology (ref. 32).
[0011] The mechanism of disease potentiation caused by
formalin-inactivated RS virus vaccine preparations remains to be
defined but is a major obstacle in the development of an effective
RS virus vaccine. The potentiation may be partly due to the action
of formalin on the F and G glycoproteins. Additionally, a non-RS
virus specific mechanism of disease potentiation has been
suggested, in which an immunological response to contaminating
cellular or serum components present in the vaccine preparation
could contribute, in part, to the exacerbated disease (ref. 33).
Indeed, mice and cotton rats vaccinated with a lysate of HEp-2
cells and challenged with RS virus grown on HEp-2 cells developed a
heightened pulmonary inflammatory response.
[0012] Furthermore, RS virus glycoproteins purified by
immunoaffinity chromatography using elution at acid pH were
immunogenic and protective but also induced immunopotentiation in
cotton rats (refs. 31, 34).
[0013] Influenza virus infection is one of the most common causes
of respiratory tract diseases. Typically, the disease results in a
high fever, usually 100.degree. F. to 103.degree. F. in adults,
often higher in children, and respiratory symptoms, such as sore
throat, running of stuffy nose, as well as headache, muscle aches
and extreme fatigue. In a typical year, influenza is associated
with about 20,000 deaths in the US, and many more hospitalizations
(CDC).
[0014] Influenza viruses are divided into three types, designated
A, B and C. Types A and B are responsible for epidemics that occur
almost every winter. Influenza viruses continually change over time
by mutation, which is termed antigenic drift.
[0015] Influenza A viruses are classified into sub-types on the
basis of two surface antigens, hemagglutinin (H) and neuraminidase
(N). Three subtypes of the hemagglutinin (H1, H2, H3) and two
sub-types of neuraminidase (N1, N2) are recognized among influenza
A viruses that have caused widespread human diseases. Immunity to
these antigens, reduces the likelihood of infections and lessens
the severity of the disease if infection occurs.
[0016] As a result of antigenic drift, major epidemics of
respiratory disease caused by new variants of influenza continue to
occur. Thus, the antigenic characteristics of the circulating
strains provide the basis for selecting the virus strains included
in each year's vaccine.
[0017] Although there are many actual and potential benefits of
vaccines that combine antigens to confer protection against
multiple pathogens, these combinations may have a detrimental
effect on the immunogenicity of the individual components.
[0018] As described above, RSV and influenza virus infections are
prevalent in the adult population and particularly the elderly and
it would be desirable to confer protection against such infection
by the administration of a single vaccine composition. However, any
potential detrimental effect of combining immunogen suitable for
conferring protection against both RSV and influenza virus in a
single formulation are unknown.
SUMMARY OF THE INVENTION
[0019] The inventors have surprisingly found that combining a
mixture of RSV proteins with non-virulent influenza virus in a
vaccine formulation provides an immune response which is
substantially the same as the response obtained by administration
of the components individually. Accordingly, there is no observed
detrimental effect on the immunogenicity of the individual
components by combining them in a single formulation. The inventors
have also surprisingly found that, in the presence of the
non-virulent influenza virus, an enhanced immune response to the
mixture of RSV proteins can be obtained by formulating the
immunogenic composition with an adjuvant.
[0020] Accordingly, in one aspect of the present invention, there
is provided a multivalent immunogenic composition for conferring
protection in a host against disease caused by infection by
respiratory syncytial virus (RSV) and influenza virus, which
comprises (a) an immunoeffective amount of a mixture of purified
fusion (F) protein, attachment (G) protein and matrix (M) protein
of RSV, and (b) an immunoeffective amount of a non-virulent
influenza virus preparation. The immunogenic composition preferably
is formulated as a vaccine for in vivo administration to the host,
particularly an adult human host (at least 18 years of age),
wherein the individual components (a) and (b) of the composition
are formulated such that the immunogenicity of the individual
comprises (a) and (b) is not impaired.
[0021] The immunogenic compositions of the invention may be
formulated as microparticles, capsules, ISCOMs or liposomes. The
immunogenic compositions may further comprise at least one other
immunogenic or immunostimulating material, which may be at least
one adjuvant or at least one immunomodulator, such as cytokines
including IL-2.
[0022] The immunogenic composition provided herein may further
comprise an adjuvant, particularly an adjuvant S which imparts an
enhanced immune to RSV when compared to the RSV mixture formulated
with the adjuvant in the absence of the non-virulent influenza
virus preparation.
[0023] The at least one adjuvant may be selected from the group
consisting of aluminum phosphate, aluminum hydroxide, QS21, Quil A
or derivatives or components thereof, calcium phosphate, calcium
hydroxide, zinc hydroxide, a glycolipid analog, an octodecyl ester
of an amino acid, a muramyl dipeptide, polyphosphazene, a
lipoprotein, ISCOM matrix, DC-Chol, DDA, and other adjuvants and
bacterial toxins, components and derivatives thereof as, for
example, described in U.S. Ser. No. 08/258,228 filed Jun. 10, 1994,
assigned to the assignee hereof and the disclosure of which is
incorporated herein by reference thereto (WO 95/34323). Under
particular circumstances, adjuvants that induce a Th1 response are
desirable.
[0024] Preferably, the adjuvant in the polyphosphazene,
poly-di(carboxylatophenoxy)-phosphazene (PCPP).
[0025] The immunogenic composition of the invention may be
formulated in single dosage form, wherein the mixture of RSV
proteins is present in an amount of about 10 to about 200 .mu.g,
preferably about 50 to about 100 .mu.g, and the non-virulent
influenza virus preparation is present in an amount of about 1 to
about 100 .mu.g, preferably about 10 to about 75 .mu.g.
[0026] The fusion (F) protein may comprise multimeric fusion (F)
proteins, which may include, when analyzed under non-reducing
conditions, heterodimers of molecular weight approximately 70 kDa
and dimeric and trimeric forms.
[0027] The attachment (G) protein may comprise, when analyzed under
non-reducing conditions, oligomeric G protein, G protein of
molecular weight approximately 95 kDa and G protein of molecular
weight approximately 55 kDa.
[0028] The matrix (M) protein may comprise, when analyzed under
non-reducing conditions, protein of molecular weight approximately
28 to 34 kDa.
[0029] The RSV protein mixture employed herein, when analyzed by
reduced SDS-PAGE analysis, may comprise the fusion (F) protein
comprising an F.sub.1 subunit of molecular weight approximately 48
kDa and an F.sub.2 subunit of about 23 kDa, the attachment (G)
protein comprising a G protein of molecular weight approximately 95
kDa and a G protein of molecular weight approximately 55 kDa, and
the matrix (M) protein comprising an M protein of approximately 31
kDa.
[0030] The RSV protein mixture employed in the invention may
comprise the F, G and M proteins in the relative proportions of:
[0031] F about 35 to about 70 wt % [0032] G about 5 to about 30 wt
% [0033] M about 10 to about 40 wt % When analyzed by SDS-PAGE
under reducing conditions and densitometric scanning following
silver staining, the ratio of F.sub.1 subunit of molecular weight
approximately 48 kDa to F.sub.2 subunit of molecular weight
approximately 23 kDa in this mixture may be approximately between
1:1 and 2:1. The mixture of F, G and M proteins may have a purity
of at least about 75%, preferably at least about 85%.
[0034] The mixture employed herein in accordance with this aspect
of the invention, having regard to the method of isolation employed
herein as described below, is devoid of monoclonal antibodies and
devoid of lentil lectin and concanavalin A.
[0035] The RSV proteins provided in the mixture of proteins
employed herein generally are substantially non-denatured by the
mild conditions of preparation and may comprise RSV proteins from
one or both of subtypes RSV A and RSV B.
[0036] The composition and manner of preparation of the mixture of
RSV protein is fully described in U.S. patent application Ser. No.
08/679,060, filed Jul. 12, 1996, and in published PCT Application
WO 98/02457, the disclosures of which are incorporated herein by
reference. As described therein, the mixture of RSV protein may be
obtained by coisolating and copurifying the mixture from the virus.
RSV cells are grown in a culture medium and separated from the
culture medium. The F, G and M proteins are solubilized from the
separated virus and the solubilized RSV protein are coisolated and
copurified. Such coisolation and copurification may be effected by
loading the solubilized protein outer an ion-exchange matrix,
preferably a calcium phosphate matrix, specifically a
hydroxyapatite matrix, and selectively relating the F, G and M
protein from the ion-exchange matrix. The grown virus may first be
worked with urea to remove contaminants without substantially
removing F, G and M protein.
[0037] The non-virulent influenza preparation employed herein
usually comprises a plurality of different non-virulent influenza
virus strain. Conventionally influenza virus vaccines are
formulated annually based on the strains prevalent and extent
during the provisions flu season and may comprise two, three or
more different strains. Such influenza virus preparation may be
rendered non-virulent in any convenient manner, such as by
inactivation with any convenient inactivating agent, such as
formaldehyde.
[0038] In a further aspect of the present invention, there is
provided a method of immunizing a human host against disease caused
by infection by respiratory syncytial virus (RSV) and influenza
virus, which comprises administering to the host an immunoeffective
amount of the immunogenic composition provided herein.
[0039] The immunogenic composition preferably is formulated as a
vaccine for in vivo administration to the host wherein the
individual components (a) and (b) of the composition are formulated
such that the immunogenicity of the individual components (a) and
(b) is not impaired. The formulation provided herein enables the
elderly to be protected by such immunization.
[0040] The present invention provides, in an additional aspect
thereof, a method of producing a vaccine for protection against
disease caused by respiratory syncytial virus (RSV) infection and
by influenza virus infection, comprising administering the
immunogenic composition provided herein to a test host to determine
the amount of and frequency of administration thereof to confer
protection against disease caused by RSV and by influenza virus;
and formulating the immunogenic composition in a form suitable for
administration to a treated host in accordance with the determined
amount and frequency of administration. The treated host may be a
human.
[0041] Advantages of the present invention include the provision of
a single vaccine formulation which permits immunization of the
elderly against disease caused by infection by RSV and influenza
virus in a single immunization regimen.
BRIEF DESCRIPTION OF DRAWINGS
[0042] In each of the Figures, a common legend is used for
identification of with immunogens used in the experiments for which
the data is presented in the Figures as follows:
[0043] (a) phosphate buffered saline (PBS); (b) 200 .mu.g PCPP
adjuvant; (c) 1.5.times.10.sup.6 pfu live RSV; (d) 200 to 400 HA
units live influenza; (e) 5 .mu.g Fluzone.RTM. vaccine with PCPP
adjuvant; (f) 1 .mu.g RSV vaccine with PCPP adjuvant; (g) 5 .mu.g
Fluzone.RTM. vaccine plus 1 .mu.g RSV vaccine with PCPP adjuvant;
(h) 5 .mu.g Fluzone.RTM. vaccine plus 1 .mu.g RSV vaccine with no
adjuvant; (i) 5 .mu.g Fluzone.RTM. vaccine with no adjuvant; (j) 1
.mu.g RSV vaccine with no adjuvant.
[0044] FIG. 1 shows the anti-RSV F immunoglobulin titres in mice
immunized with each of the immunogens;
[0045] FIG. 2 shows the RSV plaque reduction titres in mice
immunized with each of the immunogens;
[0046] FIG. 3 shows the RSV titres in the lungs of mice immunized
with each of the immunogens and then challenged with live RSV;
[0047] FIG. 4 shows the anti-A/Johannesburg influenza
immunoglobulin titres in mice immunized with each of the
immunogens;
[0048] FIG. 5 shows the anti-A/Texas influenza immunoglobulin
titres in mice immunized with each of the immunogens;
[0049] FIG. 6 shows the anti-B/Harbin influenza immunoglobulin
titres in mice immunized with each of the immunogens;
[0050] FIG. 7 shows the anti-A/Johannesburg influenza
hemagglutination inhibition titres in mice immunized with each of
the immunogens;
[0051] FIG. 8 shows the anti-A/Texas influenza hemagglutination
inhibition titres in mice immunized with each of the immunogens;
and
[0052] FIG. 9 shows the anti-B/Harbin influenza hemagglutination
inhibition titres in mice immunized with each of the
immunogens.
GENERAL DESCRIPTION OF INVENTION
[0053] The mixture of F, G and M proteins of RSV used herein may be
coisolated and copurified from RS virus. As described in the
aforesaid U.S. application Ser. No. 08/679,060 and WO 98/02457, the
virus is grown on a vaccine quality cell line, such as VERO cells
and human diploid cells, such as MRC5 and WI38, and the grown virus
is harvested. The fermentation may be effected in the presence of
fetal bovine serum (FBS) and trypsin.
[0054] The viral harvest is filtered and then concentrated,
typically using tangential flow ultrafiltration with a membrane of
desired molecular weight cut-off, and diafiltered. The virus
harvest concentrate may be centrifuged and the supernatant
discarded. The pellet following centrifugation may first be washed
with a buffer containing urea to remove soluble contaminants while
leaving the F, G and M proteins substantially unaffected, and then
recentrifuged. The pellet from the centrifugation then is detergent
extracted to solubilize the F, G and M proteins from the pellet.
Such detergent extraction may be effected by resuspending the
pellet to the original harvest concentrate volume in an extraction
buffer containing a detergent, such as a non-ionic detergent,
including TRITON.RTM. X-100, a non-ionic detergent which is
octadienyl phenol (ethylene glycol).sub.10. Other detergents
include octylglucoside and Mega detergents.
[0055] Following centrifugation to remove non-soluble proteins, the
F, G and M protein extract is purified by chromatographic
procedures. The extract may first be applied to an ion exchange
chromatography matrix to permit binding of the F, G and M proteins
to the matrix while impurities are permitted to flow through the
column. The ion-exchange chromatography matrix may be any desired
chromatography material, particularly a calcium phosphate matrix,
specifically hydroxyapatite, although other materials, such as DEAE
and TMAE and others, may be used.
[0056] The bound F, G and M proteins then are coeluted from the
column by a suitable eluant. The resulting copurified F, G and M
proteins may be further processed to increase the purity
thereof.
[0057] The purified F, G and M proteins employed herein may be in
the form of homo and hetero oligomers including F:G heterodimers
and including dimers, tetramers and higher species. The RSV protein
preparations prepared following this procedure demonstrated no
evidence of any adventitious agent, hemadsorbing agent or live
virus.
[0058] The influenza virus vaccine utilized herein is a sterile
suspension prepared from influenza virus propagated in chicken
embryos. The virus containing allantoic fluids, are harvested and
inactivated with formaldehyde. The virus is then concentrated and
purified in a linear sucrose density gradient solution, using a
continuous flow centrifuge. The virus is then chemically disrupted
using Glyco p-, Isooctylphenyl Ether (Triton.RTM. X-100) producing
a split-antigen. The split-antigen is then further purified by
chemical means and suspended in sodium phosphate-buffered isotonic
sodium chloride solution. Gelatin (0.05%) is then added as a
stabilizer and thimerosol (1:10,000) is added as a
preservative.
[0059] The commercial vaccine (Fluzone.RTM.) as used herein was
obtained from Connaught Laboratories, Swiftwater, Pa.
[0060] As set forth in detail in the Examples below, various
combinations of RSV-A subunit vaccine and trivalent influenza
vaccine were prepared with or without PCPP adjuvant and were tested
for immunogenicity in comparison to several controls. In the
immunization studies, details of which are provided below, Balb/c
mice were immunized with two injections of immunogen given three
weeks apart. Bleeds were collected to monitor the immune response
and, at the end of the study, the mice were challenged with either
influenza or RSV to determine whether protection was obtained.
[0061] Details of the results obtained are set forth in the
Examples below and in FIGS. 1 to 9. It was found that neither the
RSV nor influenza antigen interfered or impaired the immunogenicity
of the other, both in adjuvanted and unadjuvanted form. In
addition, when adjuvanted the combination of the RSV and influenza
immunogen produced an enhanced immune response to RSV in comparison
to the absence of the influenza immunogens.
[0062] It is clearly apparent to one skilled in the art, that the
various embodiments of the present invention have many applications
in the fields of vaccination, diagnosis and treatment of
respiratory syncytial virus and influenza virus infections, and the
generation of immunological agents. A further non-limiting
discussion of such issue is further presented below.
1. Vaccine Preparation and Use
[0063] Immunogenic compositions, suitable to be used as vaccines,
may be prepared from mixtures comprising immunogenic F, G and M
proteins of RSV along with a non-virulent influenza virus
preparation. The immunogenic composition elicits an immune response
which produces antibodies, including anti-RSV antibodies including
anti-F, anti-G and anti-M antibodies as well as anti-influenza
antibodies to each of the strains present in the formulation. Such
antibodies may be viral neutralizing and/or anti-fusion
antibodies.
[0064] Immunogenic compositions including vaccines may be prepared
as injectables, as liquid solutions, suspensions or emulsions. The
active immunogenic ingredients may be mixed with pharmaceutically
acceptable excipients which are compatible therewith. Such
excipients may include water, saline, dextrose, glycerol, ethanol,
and combinations thereof. The immunogenic compositions and vaccines
may further contain auxiliary substances, such as wetting or
emulsifying agents, pH buffering agents, or adjuvants to enhance
the effectiveness thereof. Immunogenic compositions and vaccines
may be administered parenterally, by injection subcutaneous,
intradermal or intramuscularly injection. Alternatively, the
immunogenic compositions formulated according to the present
invention, may be formulated and delivered in a manner to evoke an
immune response at mucosal surfaces. Thus, the immunogenic
composition may be administered to mucosal surfaces by, for
example, the nasal or oral (intragastric) routes. Alternatively,
other modes of administration including suppositories and oral
formulations may be desirable. For suppositories, binders and
carriers may include, for example, polyalkalene glycols or
triglycerides. Such suppositories may be formed from mixtures
containing the active immunogenic ingredient(s) in the range of
about 0.5 to about 10%, preferably about 1 to 2%. Oral formulations
may include normally employed carriers such as, pharmaceutical
grades of saccharine, cellulose and magnesium carbonate. These
compositions can take the form of solutions, suspensions, tablets,
pills, capsules, sustained release formulations or powders and
contain about 1 to 95% of the active ingredients, preferably about
20 to about 75%.
[0065] The immunogenic preparations and vaccines are administered
in a manner compatible with the dosage formulation, and in such
amount as will be therapeutically effective, immunogenic and
protective. The quantity to be administered depends on the subject
to be treated, including, for example, the capacity of the
individual's immune system to synthesize antibodies, and, if
needed, to produce a cell-mediated immune response. Precise amounts
of active ingredients required to be administered depend on the
judgment of the practitioner. However, suitable dosage ranges are
readily determinable by one skilled in the art and may be of the
order of micrograms to milligrams of the active ingredient(s) per
vaccination. Suitable regimes for initial administration and
booster doses are also variable, but may include an initial
administration followed by subsequent booster administrations. The
dosage may also depend on the route of administration and will vary
according to the size of the host.
[0066] The concentration of the active ingredients in an
immunogenic composition according to the invention is in general
about 1 to 95%. A vaccine which contains antigenic material of only
one pathogen is a monovalent vaccine.
[0067] Immunogenicity can be significantly improved if the antigens
are co-administered with adjuvants. Adjuvants enhance the
immunogenicity of an antigen but are not necessarily immunogenic
themselves. Adjuvants may act by retaining the antigen locally near
the site of administration to produce a depot effect facilitating a
slow, sustained release of antigen to cells of the immune system.
Adjuvants can also attract cells of the immune system to an antigen
depot and stimulate such cells to elicit immune responses.
[0068] Immunostimulatory agents or adjuvants have been used for
many years to improve the host immune responses to, for example,
vaccines. Intrinsic adjuvants, such as lipopolysaccharides,
normally are the components of the killed or attenuated bacteria
used as vaccines. Extrinsic adjuvants are immunomodulators which
are formulated to enhance the host immune responses. Thus,
adjuvants have been identified that enhance the immune response to
antigens delivered parenterally. Some of these adjuvants are toxic,
however, and can cause undesirable side-effects, making them
unsuitable for use in humans and many animals. Indeed, only
aluminum hydroxide and aluminum phosphate (collectively commonly
referred to as alum) are routinely used as adjuvants in human and
veterinary vaccines. The efficacy of alum in increasing antibody
responses to diphtheria and tetanus toxoids is well established.
While the usefulness of alum is well established for some
applications, it has limitations. For example, alum is ineffective
for influenza vaccination and usually does not elicit a cell
mediated immune response. The antibodies elicited by
alum-adjuvanted antigens are mainly of the IgG1 isotype in the
mouse, which may not be optimal for protection by some vaccinal
agents.
[0069] A wide range of extrinsic adjuvants can provoke potent
immune responses to antigens. These include saponins complexed to
membrane protein antigens (immune stimulating complexes), pluronic
polymers with mineral oil, killed mycobacteria in mineral oil,
Freund's incomplete adjuvant, bacterial products, such as muramyl
dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A,
and liposomes.
[0070] To efficiently induce humoral immune responses (HIR) and
cell-mediated immunity (CMI), immunogens are often emulsified in
adjuvants. Many adjuvants are toxic, inducing granulomas, acute and
chronic inflammations (Freund's complete adjuvant, FCA), cytolysis
(saponins and Pluronic polymers) and pyrogenicity, arthritis and
anterior uveitis (LPS and MDP). Although FCA is an excellent
adjuvant and widely used in research, it is not licensed for use in
human or veterinary vaccines because of its toxicity.
EXAMPLES
[0071] The above disclosure generally describes the present
invention. A more complete understanding can be obtained by
reference to the following specific Examples. These Examples are
described solely for purposes of illustration and are not intended
to limit the scope of the invention. Changes in form and
substitution of equivalents are contemplated as circumstances may
suggest or render expedient. Although specific terms have been
employed herein, such terms are intended in a descriptive sense and
not for purposes of limitation.
[0072] Methods of determining tissue culture infectious dose.sub.50
(TCID.sub.50/mL), plaque and neutralization titres, not explicitly
described in this disclosure are amply reported in the scientific
literature and well within the scope of those skilled in the art.
Protein concentrations were determined by the bicinchoninic acid
(BCA) method as described in the Pierce Manual (23220, 23225;
Pierce Chemical company, U.S.A.), incorporated herein by
reference.
[0073] CMRL 1969 and Iscove's Modified Dulbecco's Medium (IMDM)
culture media were used for cell culture and virus growth. The
cells used in this study are vaccine quality African green monkey
kidney cells (VERO lot M6) obtained from Institut Merieux. The RS
viruses used were the RS virus subtype A (Long and A2 strains)
obtained from the American Type culture Collection (ATCC), a recent
subtype A clinical isolate and RSV subtype B clinical isolate from
Baylor College of Medicine.
Example 1
[0074] This Example illustrates the production of RSV on a
mammalian cell line on microcarrier beads in a 150 L controlled
fermenter.
[0075] Vaccine quality African green monkey kidney cells (VERO) at
a concentration of 10.sup.5 cells/mL were added to 60 L of CMRL
1969 medium, pH 7.2 in a 150 L bioreactor containing 360 g of
Cytodex-1 microcarrier beads and stirred for 2 hours. An additional
60 L of CMRL 1969 was added to give a total volume of 120 L. Fetal
bovine serum was added to achieve a final concentration of 3.5%.
Glucose was added to a final concentration of 3 g/L and L-glutamine
was added to a final concentration of 0.6 g/L. Dissolved oxygen
(40%), pH (7.2), agitation (36 rpm), and temperature (37.degree.
C.) were controlled. Cell growth, glucose, lactate, and glutamine
levels were monitored. At day 4, the culture medium was drained
from the fermenter and 100 L of E199 media (no fetal bovine serum)
was added and stirred for 10 minutes. The fermentor was drained and
filled again with 120 L of E199.
[0076] An RSV inoculum of RSV subtype A was added at a multiplicity
of infection (M.O.I.) of 0.001 and the culture was then maintained
for 3 days before one-third to one-half of the medium was drained
and replaced with fresh medium. On day 6 post-infection, the
stirring was stopped and the beads allowed to settle. The viral
culture fluid was drained and filtered through a 20 mm filter
followed by a 3 mm filter prior to further processing.
[0077] The clarified viral harvest was concentrated 75- to 150-fold
using tangential flow ultrafiltration with 300 NMWL membranes and
diafiltered with phosphate buffered saline containing 10% glycerol.
The viral concentrate was stored frozen at -70.degree. C. prior to
further purification.
Example 2
[0078] This Example illustrates the process of purifying RSV
subunit from a viral concentrate of RSV subtype A.
[0079] A solution of 50% polyethylene glycol-8000 was added to an
aliquot of virus concentrate prepared as described in Example 1 to
give a final concentration of 6%. After stirring at room
temperature for one hour, the mixture was centrifuged at 15,000 RPM
for 30 min in a Sorvall SS-34 rotor at 4.degree. C. The viral
pellet was suspended in 1 mM sodium phosphate, pH 6.8, 2 M urea,
0.15 M NaCl, stirred for 1 hour at room temperature, and then
recentrifuged at 15,000 RPM for 30 min. in a Sorvall SS-34 rotor at
4.degree. C. The viral pellet was then suspended in 1 mM sodium
phosphate, pH 6.8, 50 mM NaCl, 1% Triton X-100 and stirred for 30
minutes at room temperature. The insoluble virus core was removed
by centrifugation at 15,000 RPM for 30 min. in a Sorval SS-34 rotor
at 4.degree. C. The soluble protein supernatant was applied to a
column of ceramic hydroxyapatite (type II, Bio-Rad Laboratories)
and the column was then washed with five column volumes of 1 mM
sodium phosphate, pH 6.8, 50 mM NaCl, 0.02% Triton X-100. The RSV
subunit composition from RSV subtype A, containing the F, G and M
proteins, was obtained by eluting the column with 10 column volumes
of 1 mM sodium phosphate, pH 6.8, 400 mM NaCl, 0.02% Triton
X-100.
Example 3
[0080] This Example illustrates the production of influenza
virus.
[0081] The influenza virus vaccine utilized herein is a sterile
suspension prepared from influenza virus propagated in chicken
embryos. The virus containing allantoic fluids, are harvested and
inactivated with formaldehyde. The virus is then concentrated and
purified in a linear sucrose density gradient solution, using a
continuous flow, centrifuge. The virus is then chemically disrupted
using Glyco p-, Isooctylphenyl Ether (Triton.RTM. X-100) producing
a split-antigen. The split-antigen is then further purified by
chemical means and suspended in sodium phosphate-buffered isotonic
sodium chloride solution. Gelatin (0.05%) is then added as a
stabilizer and thimerosol (1:10,000) is added as a
preservative.
[0082] The commercial vaccine (Fluzone.RTM.) as used herein was
obtained from Connaught Laboratories, Swiftwater, Pa.
Example 4
[0083] This Example illustrates the immunization protocol used in
the mice studies.
[0084] Mice were bleed one day prior to the first immunization and
also on days 22 and 28 of the study. Immunizations were done on
days 1 and 22. Both immunizations were administered intramuscularly
in the thigh muscle. Each immunization was done at two injection
sites (both right and left thigh muscles; 0.05 ml/site). The dose
of RSV vaccine was 1 .mu.g total protein and the dose of Fluzone
vaccine was 5 .mu.g total protein per dose. The RSV or Fluzone
vaccines were administered in the presence or absence of adjuvant.
The adjuvant used was poly-di(carboxylatophenoxy)-phosphazene
(PCPP) given at 200 .mu.g/dose. Mice that received live RSV (A2
strain) as the immunogen were given 1.5.times.10.sup.6 pfu/dose
intranasally. Mice that received live influenza virus (A/Taiwan
Strain) as the immunogen were given 200 to 400 HAU/dose
intraperitonally. Virus challenge with either RSV or influenza was
administered intranasally on day 29 using the same dose as given
for the live virus immunized mice. All animals were sacrificed on
day 33. Lungs were removed and frozen immediately in liquid
nitrogen for later determination of virus titre.
Example 5
[0085] This Example illustrates the determination of RSV titres in
the lungs of mice.
[0086] Mouse lungs were removed at the time of sacrifice, quick
frozen in liquid nitrogen, and then stored at -70.degree. C. until
assayed for virus titre. To process the lungs they were thawed,
weighed and then homogenized in Dulbecco's Modified Eagles (DME)
tissue culture media containing 10% fetal bovine serum. The
homogenate was centrifuged at 200.times.g for 15 min to remove cell
debris and the supernatant was collected. The supernatant was
assayed for RSV titres using the RSV plaque assay, as described in
Example 6.
[0087] When the mice were challenged with RSV, FIG. 3, again a good
immune response was observed in the combination and adjuvant (lane
g) showing very low titres in the lungs, comparable to the RSV
alone (lane f) or the live RSV (lane c). This also shows the lack
of interference between the influenza component and the RSV
components.
Example 6
[0088] This Example describes the RSV plaque assay.
[0089] Vero cells were grown in CMRL 1969 media plus 10% FBS for
RSV titrations. Test samples were diluted serially in 10-fold steps
and added to 24-well plates containing confluent Vero cells for 1-2
hours. Following adsorption the sample was removed and replaced
with media containing 0.75% methyl cellulose. After 4-5 days
incubation, virus plaques were detected by probing the wells with
monoclonal anti-F antibody. Bound antibody was visualized using
sequential incubation with horse radish peroxidase-conjugated
donkey anti-mouse immunoglobulin and
4-choro-1-napthol/H.sub.2O.sub.2. Plaques were scored manually.
Example 7
[0090] This Example describes the RSV plaque reduction assay.
[0091] Test sera were heat-inactivated at 56.degree. C. for 30
minutes. Samples were diluted in four-fold serial steps and mixed
with an equal volume of RSV A(long strain; 30-70 PFU) in assay
media containing 10% guinea pig complement. After one hour
incubation at 37.degree. C. the mixture was inoculated onto Vero
cells for 1-2 hours. This was followed by an overlay with 0.75%
methyl cellulose and incubation for 4 to 5 days. Virus plaques were
detected as described for the RSV plaque assay in Example 6. The
neutralization titre is expressed as the reciprocal of the dilution
which results in 60% reduction in plaque formation (as determined
by linear interpolation analysis).
[0092] The enhancement of the RSV response is illustrated in FIG. 2
where plaque reduction titres were looked at. The RSV/Flu
combination (lane g) again shows a higher titre than the RSV alone
(lane f).
Example 8
[0093] This Example describes the mouse anti-RSV F antibody
ELISA.
[0094] Anti-F immunoglobulin antibody titres in mouse sera were
measured in an antigen-specific ELISA employing native F protein as
the solid-phase coat. The F protein was purified by immunoaffinity
chromatography using an immobilized anti-F monoclonal antibody.
Wells were coated with F protein, then blocked with 1% BSA in PBS.
Dilutions of test serum samples were added, and after incubation,
the wells were washed again with 1% BSA. The bound F-specific
antibodies were detected with horse radish peroxidase-labeled
antibody specific for mouse IgG (H+L chains), followed after
further washing by tetramethylbenzidine plus hydrogen peroxide
substrate. Colour formation was measured at 450 nm in an automatic
plate reader. The antibody titre is expressed as the reciprocal of
the greatest four-fold dilution at which optical density remains
double that of a negative control.
[0095] As can be seen from FIG. 1, RSV-F IgG antibody response was
observed in the RSV/Flu immunizations (lanes g+h) either with or
without adjuvant. These results are comparable to RSV immunization
alone (lane f) and in fact the combination immunization (lane g)
shows an enhanced RSV response over RSV alone (lane f). This shows
that there was no interference between the influenza component and
the RSV components.
Example 9
[0096] This Example describes the mouse anti-influenzae antibody
ELISA.
[0097] Influenza strain-specific antibody titres in mouse sera were
measured using microitre plates coated with the appropriate
influenza strain (A/Texas, A/Johannesburg, or B/Harbin). Plate
processing and development was done as described for the RSV-F
antibody ELISA in Example 8.
[0098] As can be seen from FIGS. 4, 5, 6, all three strains of
influenzae elicited a good antibody response to influenza virus in
the combination RSV/Flu administration (lane g). This was
comparable to the flu vaccine administered alone (lane e). This
again shows that the combination vaccine did not reduce or
interfere with the immune response to the influenza component.
Example 10
[0099] This Example describes the influenza hemagglutination
inhibition assay.
[0100] Sera samples were heated at 56.degree. C. for 30 minutes to
inactivate complement and then treated with trypsin and potassium
periodate to destroy endogenous inhibitors of hemagglutination.
Serially diluted antisera were tested for their ability to inhibit
the agglutination of chicken red blood cells by four HA units of
influenza virus (A/Texas, A/Johannesburg, or B/Harbin) in a
standard hemagglutination inhibition assay.
[0101] FIGS. 7, 8 and 9 shows that the combination vaccine (lane g)
produced as good haemagglutinin (HA) titres as the Flu vaccine as
its own. Again this illustrates that the RSV component d:d not
interfere with the eliciting of a good influenza immune response,
in this case as measured by HAI.
SUMMARY OF THE DISCLOSURE
[0102] In summary of the disclosure, the present invention provides
a multivalent immunogenic composition comprising an RSV protein
subunit component and a non-virulent influenza virus preparation
wherein the active ingredients do not interfere with the
immunogenicity of the other and which is suitable for
administration to s adults and the elderly. Modifications are
possible with the scope of the invention.
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