U.S. patent application number 10/523114 was filed with the patent office on 2006-02-16 for vaccine composition comprising transferrin binding protein and hsf from gram negative bacteria.
Invention is credited to Francois-Xavier Jacques Berthet, Ralph Biemans, Philippe DEnoel, Christiane Feron, Carine Goraj, Jan Poolman, Vincent Weynants.
Application Number | 20060034854 10/523114 |
Document ID | / |
Family ID | 31722002 |
Filed Date | 2006-02-16 |
United States Patent
Application |
20060034854 |
Kind Code |
A1 |
Berthet; Francois-Xavier Jacques ;
et al. |
February 16, 2006 |
Vaccine composition comprising transferrin binding protein and hsf
from gram negative bacteria
Abstract
The present invention relates to immunogenic compositions and
vaccines for the prevention or treatment of Gram negative bacterial
infection. Immunogenic compositions of the invention comprise
transferrin binding protein and Hsf, and the combination of these
two antigens have been shown to act synergistically to produce
antibodies with high activity in a serum bactericidal assay. This
combination of antigens is useful for use in vaccines against
Neisseria meningitidis, Neisseria gonorrhoeae, Moraxella
catarrhalis and Haemophilus influenzae.
Inventors: |
Berthet; Francois-Xavier
Jacques; (Rixensart, BE) ; Biemans; Ralph;
(Rixensart, BE) ; DEnoel; Philippe; (Rixensart,
BE) ; Feron; Christiane; (Rixensart, BE) ;
Goraj; Carine; (Rixensart, BE) ; Poolman; Jan;
(Rixensart, BE) ; Weynants; Vincent; (Rixensart,
BE) |
Correspondence
Address: |
SMITHKLINE BEECHAM CORPORATION;CORPORATE INTELLECTUAL PROPERTY-US, UW2220
P. O. BOX 1539
KING OF PRUSSIA
PA
19406-0939
US
|
Family ID: |
31722002 |
Appl. No.: |
10/523114 |
Filed: |
July 31, 2003 |
PCT Filed: |
July 31, 2003 |
PCT NO: |
PCT/EP03/08567 |
371 Date: |
August 2, 2005 |
Current U.S.
Class: |
424/184.1 ;
424/190.1; 530/350 |
Current CPC
Class: |
A61K 39/102 20130101;
A61K 39/1045 20130101; A61P 37/02 20180101; A61K 2039/55577
20130101; A61P 11/00 20180101; A61K 2039/70 20130101; Y02A 50/396
20180101; C07K 14/22 20130101; A61K 2039/521 20130101; A61P 37/04
20180101; A61P 31/00 20180101; Y02A 50/30 20180101; A61K 2039/55516
20130101; A61P 37/00 20180101; A61P 31/04 20180101; A61K 2039/55572
20130101; A61K 39/095 20130101; A61P 43/00 20180101; A61K
2039/55505 20130101 |
Class at
Publication: |
424/184.1 ;
424/190.1; 530/350 |
International
Class: |
A61K 39/02 20060101
A61K039/02; A61K 39/00 20060101 A61K039/00; A61K 39/38 20060101
A61K039/38; C07K 14/22 20060101 C07K014/22 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 2, 2002 |
GB |
0218037.0 |
Aug 2, 2002 |
GB |
0218036.2 |
Aug 2, 2002 |
GB |
0218035.2 |
Aug 2, 2002 |
GB |
0218051.1 |
Aug 30, 2002 |
GB |
0220197.8 |
Aug 30, 2002 |
GB |
0220199.4 |
Nov 1, 2002 |
GB |
0225524.8 |
Nov 1, 2002 |
GB |
0225531.3 |
Dec 24, 2002 |
GB |
0230164.6 |
Dec 24, 2002 |
GB |
0230168.7 |
Dec 24, 2002 |
GB |
0230170.3 |
Mar 5, 2003 |
GB |
0305028.3 |
Claims
1. An immunogenic composition comprising an isolated transferrin
binding protein (Tbp) or antigenic fragment thereof and an isolated
Hsf like protein or antigenic fragment thereof from the same or
different Gram negative bacteria.
2. The immunogenic composition of claim 1 in which the transferrin
binding protein or fragment thereof and Hsf like protein or
fragment thereof are from Neisseria.
3. The immunogenic composition of claim 1 in which the transferrin
binding protein or fragment thereof is derived from N.
meningitidis.
4. The immunogenic composition of claim 1 in which the Hsf like
protein or fragment thereof is derived from N. meningitidis.
5. The immunogenic composition of claim 1 in which the transferrin
binding protein or fragment thereof is derived from N. meningitidis
serogroup B.
6. The immunogenic composition of claim 1 in which the Hsf like
protein or fragment thereof is derived from N. meningitidis
serogroup B.
7. The immunogenic composition of claim 1 in which the transferrin
binding protein or fragment thereof is derived from N.
gonorrhoeae.
8. The immunogenic composition of claim 1 in which the Hsf like
protein or antigenic fragment thereof is derived from N.
gonorrhoeae.
9. The immunogenic composition of claim 1 in which the transferrin
binding protein or antigenic fragment thereof is derived from
Moraxella catarrhalis.
10. The immunogenic composition of claim 1 in which the Hsf like
protein or antigenic fragment thereof is derived from Moraxella
catarrhalis.
11. The immunogenic composition of claim 1 in which the transferrin
binding protein or antigenic fragment thereof is derived from
Haemophilus influenzae.
12. The immunogenic composition of claim 1 in which the Hsf like
protein or antigenic fragment thereof is derived from Haemophilus
influenzae.
13. The immunogenic composition of claim 1 in which the transferrin
binding protein is TbpA or an antigenic fragment thereof.
14. The immunogenic composition of claim 13 comprising high
molecular weight form TbpA or low molecular weight form TbpA or
both high molecular weight form TbpA and low molecular weight form
TbpA.
15. The immunogenic composition of claim 1 in which the Hsf like
protein is Hsf or an antigenic fragment thereof.
16. The immunogenic composition of claim 1 comprising antigenic
fragments of Tbp and/or Hsf like protein capable of generating a
protective response against Neisserial, Moraxella catarrhalis or
Haemophilus influenzae infection.
17. The immunogenic composition of claim 16 comprising antigenic
fragments of TbpA and/or Hsf.
18. The immunogenic composition of claim 1 comprising a fusion
protein of Tbp and Hsf like protein or antigenic fragments
thereof.
19. The immunogenic composition of claim 18 comprising a fusion
protein comprising TbpA and Hsf or antigenic fragments thereof
capable of generating a protective response against Neisserial
infection.
20. An isolated immunogenic composition comprising an outer
membrane vesicle preparation derived from Gram negative bacteria,
in which expression of both transferrin binding protein and Hsf
like protein are at least 1.5 fold higher than naturally occurring
in the unmodified Gram negative bacteria.
21. The immunogenic composition of claim 20 in which the expression
of transferrin binding protein is upregulated by growth under iron
limitation conditions.
22. The immunogenic composition of claim 20 in which at least a
part of the outer membrane vesicle preparation is derived from
Neisseria.
23. The immunogenic composition of claim 20 in which at least a
part of the outer membrane vesicle preparation is derived from
Neisseria meningitidis.
24. The immunogenic composition of claim 20-in which at least a
part of the outer membrane vesicle preparation is derived from
Neisseria meningitidis serogroup B.
25. The immunogenic composition of claim 20 in which at least a
part of the outer membrane vesicle preparation is derived from
Neisseria gonorrhoeae.
26. The immunogenic composition of claim 20 wherein a host cell
from which the outer membrane vesicle preparation is derived has
been engineered so as to down-regulate the expression of one or
more of LgtB and LgtE.
27. The immunogenic composition of claim 20 wherein a host cell
from which the outer membrane vesicle preparation is derived is
unable to synthesise capsular polysaccharides and has preferably
been engineered so as to down-regulate the expression of and
preferably to delete one or more of siaD, ctrA, ctrB, ctrC, ctrD,
synA (equivalent to synX and siaA), synB (equivalent to siaB and
synC (equivalent to siaC).
28. The immunogenic composition of claim 20 wherein a host cell
from which the outer membrane vesicle preparation is derived has
been engineered so as to down-regulate the expression of and
preferably delete one or more of OpC, OpA and PorA.
29. The immunogenic composition of claim 20 wherein a host cell
from which the outer membrane vesicle preparation is derived has
been engineered so as to down-regulate the expression of FrpB.
30. The immunogenic composition of claim 20 wherein a host cell
from which the outer membrane vesicle preparation is derived has
been engineered so as to down-regulate the expression of msbB or
HtrB.
31. The immunogenic composition of claim 20 wherein the outer
membrane vesicle preparation contains LPS which is conjugated to an
outer membrane protein (OMP).
32. The immunogenic composition of claim 31 wherein LPS is
conjugated (preferably intra-bleb ) to OMP in situ in the outer
membrane vesicle preparation.
33. The immunogenic composition of claim 20 in which at least a
part of the outer membrane vesicle preparation is derived from
Moraxella catarrhalis.
34. The immunogenic composition of claim 20 in which at least a
part of the outer membrane vesicle preparation is derived from
Haemophilus influenzae.
35. The immunogenic composition of claim 20 comprising an outer
membrane vesicle preparation isolated from two or more strains of
Gram negative bacteria.
36. The immunogenic composition of claim 35 in which transferrin
binding protein and Hsf like protein are upregulated on different
vesicles originating from different bacterial strains or on the
same vesicles originating from the same bacterial strain.
37. The immunogenic preparation of claim 20 comprising an outer
membrane vesicle preparation in which enhanced transferrin binding
protein expression is derived from a polynucleic acid introduced
into the Gram negative bacteria.
38. The immunogenic composition of claim 20 comprising an outer
membrane vesicle preparation in which enhanced Hsf like protein
expression is derived from a polynucleic acid introduced into the
Gram negative bacteria.
39. The immunogenic composition of claim 20 comprising an outer
membrane vesicle preparation in which enhanced transferrin binding
protein and Hsf like protein expression is derived from a
polynucleic acid encoding both proteins which was introduced into
the Gram negative bacteria.
40. The immunogenic composition of claim 20 in which a bacterial
strain has been genetically engineered so as to introduce a
stronger promoter sequence upstream of a gene encoding transferrin
binding protein.
41. The immunogenic composition of claim 20 in which a bacterial
strain has been genetically engineered so as to introduce a
stronger promoter sequence upstream of a gene encoding Hsf like
protein.
42. The immunogenic composition of claim 20 in which a bacterial
strain has been genetically engineered so as to introduce a
stronger promoter sequence upstream of genes encoding transferrin
binding protein and Hsf like protein.
43. The immunogenic composition of claim 20 in which the
transferrin binding protein is TbpA which is high molecular weight
TbpA, low molecular weight TbpA or both high molecular weight TbpA
and low molecular weight TbpA from N. meningitidis.
44. The immunogenic composition of claim 20 in which the Hsf like
protein is Hsf from Neisseria meningitidis.
45. The immunogenic composition of claim 1 further comprising plain
or conjugated bacterial capsular polysaccharide or
oligosaccharide.
46. The immunogenic composition of claim 1 comprising two or more
bacterial capsular polysaccharides or oligosaccharides conjugated
to transferrin binding protein or Hsf like proteins or both.
47. The immunogenic composition of claim 45 wherein the capsular
polysaccharide or oligosaccharide is derived from one or more
bacteria selected from the group consisting of Neisseria
meningitidis serogroup A, Neisseria meningitidis serogroup C,
Neisseria meningitidis serogroup Y, Neisseria meningitidis
serogroup W-135, Haemophilus influenzae b, Streptococcus
pneumoniae, Group A Streptococci, Group B Streptococci,
Staphylococcus aureus and Staphylococcus epidermidis.
48. An immunogenic composition comprising one or more
polynucleotide(s) encoding a transferrin binding protein or
antigenic fragment thereof and a Hsf like proteinor antigenic
fragment thereof whose expression is driven by a eukaryotic
promoter.
49. The immunogenic composition of claim 48 wherein TbpA and Hsf of
Neisseria are encoded.
50. The immunogenic composition of claim 48 wherein TbpA and Hsf of
Neisseria meningitidis are encoded.
51. The immunogenic composition of claim 1 comprising an
adjuvant.
52. The immunogenic composition of claim 51 comprising aluminium
salts.
53. The immunogenic composition of claim 51 comprising 3D-MPL.
54. The immunogenic composition of claim 51 comprising an adjuvant
containing CpG.
55. A vaccine comprising the immunogenic composition of claim 1 and
a pharmaceutically acceptable excipient.
56. A method for treatment or prevention of Gram negative bacterial
disease comprising administering a protective dose or an effective
amount of the vaccine of claim 55.
57. The method of claim 56 in which Neisserial infection is
prevented or treated.
58. A use of the vaccine of claim 55 in the preparation of a
medicament for treatment or prevention of Gram negative bacterial
infection.
59. The use of claim 58 in the preparation of a medicament for
treatment or prevention of Neisserial infection.
60. A genetically engineered Gram negative bacterial strain from
which the outer membrane vesicles within the immunogenic
composition of claim 20 can be derived.
61. A method of making the immunogenic composition of claim 1
comprising a step of mixing together isolated transferrin binding
protein and isolated Hsf like protein or antigenic fragments
thereof.
62. A method of making the immunogenic composition of claim 20
comprising a step of isolating outer membrane vesicles from a Gram
negative bacterial culture.
63. The method of claim 62 wherein the step of isolating outer
membrane vesicles involves extraction with 0-0.5%, 0.02-0.4%,
0.04-0.3%, 0.06-0.2%, 0.08-0.15% or preferably 0.1% detergent.
64. A method of making the immunogenic composition of claim 47
comprising the step of conjugating bacterial capsular
polysaccharides or oligosaccharides to transferrin binding protein
and/or Hsf like protein.
65. A method of making the vaccine of claim 55 comprising a step of
combining the immunogenic composition with a pharmaceutically
acceptable excipient.
66. A method of preparing an immune globulin for use in prevention
or treatment of Neisserial infection comprising the steps of
immunising a recipient with the vaccine of claim 55 and isolating
immune globulin from the recipient.
67. An immune globulin preparation obtainable from the method of
claim 66.
68. A pharmaceutical preparation comprising the immune globulin
preparation of claim 67 and a pharmaceutically acceptable
excipient.
69. A pharmaceutical preparation comprising monoclonal antibodies
against TbpA and Hsf of Neisseria meningitidis and a
pharmaceutically acceptable excipient.
70. A method for treatment or prevention of Gram negative bacterial
infection comprising a step of administering to the patient an
effective amount of the pharmaceutical preparation of claim 68.
71. A use of the pharmaceutical preparation of claim 68 in the
manufacture of a medicament for the treatment or prevention of Gram
negative bacterial disease.
Description
TECHNICAL FIELD
[0001] The present invention relates to the field of Gram-negative
bacterial immunogenic compositions and vaccines, their manufacture
and the use of such compositions in medicine. More particularly, it
relates to vaccine compositions comprising both transferrin binding
protein and Hsf. The presence of both these antigens leads to the
production of higher levels of bactericidal antibodies.
BACKGROUND
[0002] Gram negative bacteria are the causative agents for a number
of human pathologies and there is a need for effective vaccines to
be developed against many of these bacteria. In particular
Bordetella pertussis, Borrelia burgdorferi, Brucella melitensis,
Brucella ovis, Chlamydia psittaci, Chlamydia trachomatis,
Esherichia coli, Haemophilus influenzae, Legionella pneumophila,
Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas
aeruginosa and Yersinia enterocolitica are Gram negative bacteria
which cause pathologies which could be treated by vaccination.
[0003] Neisseria gonorrhoeae is the etiologic agent of gonorrhea,
one of the most frequently reported sexually transmitted diseases
in the world with an estimated annual incidence of 62 million cases
(Gerbase et al 1998 Lancet 351; (Suppl 3) 2-4). The clinical
manifestations of gonorrhea include inflammation of the mucus
membranes of the urogenital tract, throat or rectum and neonatal
eye infections. Ascending gonococcal infections in women can lead
to infertility, ectopic pregnancy, chronic pelvic inflammatory
disease and tubo-ovarian abscess formation. Septicemia, arthritis,
endocarditis and menigitis are associated with complicated
gonorrhea.
[0004] The high number of gonococcal strains with resistance to
antibiotics contributes to increased morbidity and complications
associated with gonorrhea. An attractive alternative to treatment
of gonorrhea with antibiotics would be its prevention using
vaccination. No vaccine currently exists for N. gonorrhoeae
infections.
[0005] Neisseria meningitidis is an important pathogen,
particularly in children and young adults. Septicemia and
meningitis are the most life-threatening forms of invasive
meningococcal disease (IMD). This disease has become a worldwide
health problem because of its high morbidity and mortality.
[0006] Thirteen N. meningitidis serogroups have been identified
based on antigenic differences in the capsular polysaccharides, the
most common being A, B and C which are responsible for 90% of
disease worldwide. Serogroup B is the most common cause of
meningococcal disease in Europe, USA and several countries in Latin
America.
[0007] Vaccines based on the capsular polysaccharide of serogroups
A, C, W and Y have been developed and have been shown to control
outbreaks of meningococcal disease (Peltola et al 1985 Pediatrics
76; 91-96). However serogroup B is poorly immunogenic and induces
only a transient antibody response of a predominantly IgM isotype
(Ala'Aldeen D and Cartwright K 1996, J. Infect. 33; 153-157). There
is therefore no broadly effective vaccine currently available
against the serogroup B meningococcus which is responsible for the
majority of disease in most temperate countries. This is
particularly problematic since the incidence of serotype B disease
is increasing in Europe, Australia and America, mostly in children
under 5. The development of a vaccine against serogroup B
meningococcus presents particular difficulties because the
polysaccharide capsule is poorly immunogenic owing to its
immunologic similarity to human neural cell adhesion molecule.
Strategies for vaccine production have therefore concentrated on
the surface exposed structures of the meningococcal outer membrane
but have been hampered by the marked variation in these antigens
among strains.
[0008] Further developments have led to the introduction of
vaccines made up of outer membrane vesicles which will contain a
number of proteins that make up the normal content of the bacterial
membrane. One of these is the VA-MENGOC-BC .RTM. Cuban vaccine
against N. meningitidis serogroups B and C (Rodriguez et al 1999
Mem Inst. Oswaldo Cruz, Rio de Janeiro 94; 433-440). This vaccine
was designed to combat an invasive meningococcal disease outbreak
in Cuba which had not been eliminated by a vaccination programme
using a capsular polysaccharide AC vaccine. The prevailing
serogroups were B and C and the VA-MENGOC-BC .RTM. vaccine was
successful at controlling the outbreak with an estimated vaccine
efficiency of 83% against serogroup B strains of N. meningitidis
(Sierra et al 1990 In Neisseria, Walter Gruyter, Berlin, m. Atchman
et al (eds) p 129-134, Sierra et al 1991, NIPH Ann 14; 195-210).
This vaccine was effective against a specific outbreak, however the
immune response elicited would not protect against other strains of
N. meningitidis.
[0009] Subsequent efficacy studies conducted in Latin America
during epidemics caused by homologous and heterologous serogroup B
meningococcal strains have shown some efficacy in older children
and adults but its effectiveness was significantly lower in younger
children who are at greatest risk of infection (Milagres et al
1994, Infect. Immun. 62; 4419-4424). It is questionable how
effective such a vaccine would be in countries with multistrain
endemic disease such as the UK. Studies of immunogenicity against
heterologous strains have demonstrated only limited cross-reactive
serum bactericidal activity, especially in infants (Tappero et al
1999, JAMA 281; 1520-1527).
[0010] A second outer membrane vesicle vaccine was developed in
Norway using a serotype B isolate typical of those prevalent in
Scandinavia (Fredriksen et al 1991, NIPH Ann, 14; 67-80). This
vaccine was tested in clinical trials and found to have a
protective efficacy after 29 months of 57% (Bjune et al 1991,
Lancet, 338; 1093-1096).
[0011] However, the use of outer membrane vesicles in vaccines is
associated with some problems. For instance, the OMV contain toxic
lipopolysaccharides and they may contain immunodominant antigens
which are either strain specific or are expressed variably. Several
processes have been described which could be used to overcome some
of the problems of outer membrane vesicle preparation vaccines.
WO01/09350 describes processes that address some of these problems
for instance by reducing toxicity and modifying the antigens
present on the outer membrane vesicles.
[0012] There are diverse problems with the anti-meningococcal
vaccines currently available. The protein based outer membrane
vaccines tend to be specific and effective against only a few
strains. The polysaccharide vaccines are also suboptimal since they
tend to elicit poor and short immune responses, particularly
against serogroup B (Lepow et al 1986; Peltola 1998, Pediatrics 76;
91-96).
[0013] Neisseria infections represent a considerable health care
problem for which no vaccines are available in the case of N.
gonorrhoeae or vaccines with limitations on their efficacy and
ability to protect against heterologous strains are available in
the case of N. meningitidis. Clearly there is a need to develop
superior vaccines against Neisserial infections that will improve
on the efficacy of currently available vaccines and allow for
protection against a wider range of strains.
DESCRIPTION OF FIGURES
[0014] FIG. 1.--A Coomassie stained gel showing expression levels
of Hsf, TbpA and NspA in outer membrane vesicle preparations
derived from different N. meningitidis stains. Lane 1--molecular
weight markers; lane 2--outer membrane vesicles prepared from
strain H44/76 in which capsular polysaccharides were downregulated;
lane 3--outer membrane vesicles prepared from strain H44/76 in
which capsular polysaccharides and PorA were downregulated; lane
4--outer membrane vesicles prepared from strain H44/76 in which
capsular polysaccharides and PorA were downregulated and NspA was
upregulated; lane 5--outer membrane vesicles prepared from strain
H44/76 in which capsular polysaccharides and PorA were
downregulated and Hsf was upregulated; lane 6--outer membrane
vesicles prepared from strain H44/76 in which capsular
polysaccharides and PorA were downregulated and TbpA was
upregulated; lane 7--outer membrane vesicles prepared from strain
H44/76 in which capsular polysaccharides and PorA were
downregulated and TbpA and Hsf were upregulated; lane 8--outer
membrane vesicles prepared from strain H44/76 in which capsular
polysaccharides and PorA were downregulated and TbpA and NspA were
upregulated.
DETAILED DESCRIPTION
[0015] The present invention discloses a combination of antigens
which when combined in an immunogenic composition or vaccine, can
induce higher titres of bactericidal antibodies than that induced
by the antigens when administered separately. Preferably the
combination of antigens leads to synergistically higher titres of
bactericidal antibodies. As bactericidal antibodies closely reflect
the efficacy of vaccine candidates, the combination of Tbp and Hsf
in vaccines will produce highly effective vaccines. An additional
advantage of the invention will be that the combination of the two
antigens, Tbp and Hsf, will also enable protection against a wider
range of strains.
[0016] The invention relates to an immunogenic composition
comprising transferrin binding protein and Hsf like protein or
antigenic fragments thereof. These proteins are either isolated or
preferably purified to at least 30%, 40%, more preferably 50%, 60%,
70%, 80%, 90%, 95% or 99% pure or enriched in a mixture with other
antigens. Transferrin binding protein and Hsf like protein may be
isolated or derived from the same or different Gram negative
bacterial strains.
[0017] Isolated means isolated from the protein's natural
environment by the hand of man. Purified means purified to at least
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% pure before the
antigen is combined with other components of the immunogenic
composition of the invention.
[0018] Derived from means that the gene encoding the protein is
derived from or the protein is purified from a particular bacterial
strain. Therefore derived from includes recombinant proteins
produced in a separate expression system if the gene encoding the
protein was derived from the named bacteria.
[0019] When combined, Tbp and Hsf have been shown to interact
advantageously and preferably synergistically to elicit an immune
response that is higher in terms of bactericidal activity (for
example as measured by serum bactericidal assay or SBA) and
preferably higher than the additive response elicited by the
antigens individually, more preferably by a factor of at least 1.1,
1.2, 1.5, two, three, four, five, six, seven, eight, nine, most
preferably by a factor of at least ten. The addition of both Tbp
and Hsf to a vaccine will have considerable advantages over
currently available vaccines in eliciting a strong bactericidal
immune response and allowing protection against multiple
strains.
[0020] One embodiment of the invention is an immunogenic
composition comprising both transferrin binding protein and Hsf
like protein. An immunogenic composition is a composition
comprising at least one antigen which is capable of generating an
immune response when administered to a host. Tbp and Hsf like
protein can be derived from any strain of Gram negative bacteria
including Moraxella catarrhalis, Haemophilus influenzae,
Bordetella, Neisseria (including Neisseria meningitidis which could
be serogroup A, B, C, W135 or Y and Neisseria gonorrhoeae) or any
of those Gram negative bacteria hereinbefore described. The
invention covers immunogenic compositions in which Tbp and Hsf like
protein are derived from either the same or different strains of
Gram negative bacteria.
Transferrin Binding Proteins
[0021] Transferrin binding protein (Tbp) is a protein or protein
complex on the outer membrane of Gram negative bacteria, which
binds transferrin. Some proteins in this family will form a
beta-barrel anchored in the outer membrane. Structurally, the
transferrin binding protein may contain an intracellular N-terminal
domain with a TonB box and plug domain, multiple transmembrane beta
strands linked by short intracellular and longer extracellular
loops. Other examples are lipoproteins which interact to form a
complex with the integral membrane protein. Examples of this family
of proteins are TbpA and TbpB. The term Tbp encompasses either of
these proteins individually or in combination, and a complex formed
from TbpA and TbpB. Preferably at least TbpA is present in the
immunogenic compositions of the invention.
[0022] Two families of TbpB have been distinguished, having a high
molecular weight and a low molecular weight respectively. High and
low molecular weight forms of TbpB (WO93/06861; EP586266) associate
with different families of TbpA (WO93/06861; EP586266; WO92/03467;
U.S. Pat. No. 5,912,336) which are distinguishable on the basis of
homology. Despite being of the same molecular weight, TbpA are
known as the high molecular weight and low molecular weight
families because of their association with the high or low
molecular weight form of TbpB (Rokbi et al FEMS Microbiol. Lett.
100; 51, 1993). TbpA and TbpB are known to be expressed in a
variety of bacteria including N. meningitidis (WO93/06861;
EP586266; WO92/03467; U.S. Pat. No. 5,912,336), N. gonorrhoeae
(WO92/03467; U.S. Pat. No. 5,912,336), H. influenzae (Gray-Owen et
al Infect. Immun. 1995; 63:1201-1210, Schryvers J. Med. Microbiol.
1989; 29: 121-130; WO95/13370; WO96/40929), A. pleuropneumoniae, M.
cararrhalis (Mathers et al FEMS Immunol. Med. Microbiol. 1997; 19:
231; Chen et al Vaccine 1999; 18: 109; WO97/13785; WO99/52947) and
P. haemolytica (Cornelissen et al Infection and Immunity 68; 4725,
2000). TbpA and TbpB have also been referred to as Tbp1 (NMB 0461)
and Tbp2 (NMB 0460) respectively (Cornelissen et al Infection and
Immunity 65; 822, 1997).
[0023] As used herein, Tbp denotes the transferrin binding protein
from Gram negative bacteria, including Moraxella catarrhalis and
Haemophilus influenzae, preferably Neisseria, more preferably N.
meningitidis or N. gonorrheoea and most preferably N. meningitidis
of serotype B. Tbp encompasses both TbpA and TbpB and the high
molecular weight and low molecular weight forms of TbpA and TbpB.
Tbp encompasses individual proteins described above and complexes
of the proteins and any other proteins or complexes thereof capable
of binding transferrin.
[0024] Although Tbp can refer to either the high or low molecular
forms of TbpA or TbpB, it is preferred that both high molecular
weight and low molecular weight forms of TbpA and/or TbpB are
present in the immunogenic compositions of the invention. Most
preferably, high molecular weight and low molecular weight TbpA is
present.
[0025] It is also thought that instead of, or in addition to, Tbps,
other iron acquisition proteins may be included in the immunogenic
compositions of the invention. Iron acquisition proteins of
Moraxella catarrhalis include TbpA, TbpB, Ton-B dependent receptor,
CopB (Sethi et al Infect. Immun. 1997; 65: 3666-3671), HasR, OmpB1
and LbpB (Du et al Infect. Immun. 1998; 66:3656-3665; Mathers et al
FEMS Immunol. Med. Microbiol. 1997; 19: 231-236; Chen et al Vaccine
1999; 18: 109-118). Iron acquisition proteins of Haemophilus
influenzae include TbpB, HasR, TonB-dependent receptor,
hemoglobin-binding protein, HhuA, HgpA, HgbA, HgbB and HgbC (Cope
et al Infect. Immun. 2000; 68: 4092-4101; Maciver et al Infect.
Immun. 1996; 64:3703-3712; Jin et al Infect. Immun. 1996;
64:3134-3141; Morton et al J. Gen. Microbiol. 1990; 136:927-933;
Schryvers J. Med. Microbiol. 1989; 29: 121-130). Iron aquisition
proteins from Neisseria meningitidis include Tbp1 (NMB 0461), Tbp2
(NMB 0460), FbpA (NMB 0634), FbpB, BfrA (NMB 1207), BfrB (NMB
1206), LbpA (NMB 1540), LbpB (NMB 1541), Lipo28 also known as
GNA2132 (NMB 2132), Sibp (NMB 1882), Ton B dependent receptors (NMB
0964 and NMB 0293) and HmbR (Tettelin et al Science 287; 1809-1815
2000).
[0026] Tbp proteins included in the immunogenic compositions of the
invention are proteins sharing homology with TbpA and TbpB from N.
meningitidis as described in WO93/06861 and EP586266; preferably
sharing over 40%, 45%, 50%, 60%, 70%, more preferably over 80% or
90%, most preferably over 95%, 96%, 97%, 98%, 99% identity with the
amino acid sequence of TbpA and TbpB as described in WO93/06861 and
EP586266.
[0027] Tbp contains several distinct regions. For example, in the
case of TbpA from N. meningitidis strain H44/76, the amino terminal
186 amino acids form an internal globular domain, 22 beta strands
span the membrane, forming a beta barrel structure. These are
linked by short intracellular loops and larger extracellular loops.
Extracellular loops 2, 3 and 5 have the highest degree of sequence
variability and loop 5 is surface exposed. Loops 5 and 4 are
involved in ligand binding, and are preferred TbpA fragments for
inclusion in the immunogenic compositions of the present
invention.
[0028] In addition to genetic upregulation techniques described
herein, transferrin binding proteins may also be upregulated in
Gram negative bacteria when grown under iron limitation conditions
as described below. In immunogenic compositions of the invention in
which transferrin binding protein is upregulated in an outer
membrane vesicle, upregulation is preferably achieved by growth of
the host strain under iron limitation conditions. This process will
also result in the upregulation of variable iron-regulated
proteins, particularly FrpB in Neisserial stains and heme/hemopexin
utilisation protein C, HgpA and HgpB in Haemophilus influenzae,
which may become immunodominant. It is therefore advantageous to
downregulate the expression of (and preferably delete the genes
encoding) such proteins as described below, to ensure that the
immunogenic composition of the invention elicits an immune response
against antigens present in a wide range of strains.
Hsf Like Proteins
[0029] Hsf like proteins are autotransporter proteins sharing
homology with Hsf of N. meningitidis with the sequences found in
WO99/31132; preferably sharing over 40%, 50%, 60%, 70%, more
preferably over 80%, most preferably over 90%, most preferably over
95%, 96%, 97%, 98%, 99% identity with an Hsf amino acid sequence
found in WO99/31132 (preferably SEQ ID NO 2, 4, 6 or 8). Hsf like
proteins are surface exposed proteins and are thought to function
as adhesins. These proteins form a multimeric complex and are
expressed during infection and colonisation.
[0030] Hsf-like proteins are found in many Gram negative bacteria
including Neisseria meningitidis, Neisseria gonorrheoea,
Haemophilus influenzae, Moraxella catarrhalis and Escherichia coli.
Examples of Hsf-like proteins found in Neisseria meningitidis
include Hsf (also kown as NhhA-NMB 0992) (WO99/31132), Aida-1 like
protein (Peak et al 2000, FEMS Imm. Med. Microbiol. 28; 329), IgA
protease, Ssh-2, Hap (WO99/55873), NadA (J. Exp Med. 2002 195;
1445), UspA2 and Tsh. Examples of Hsf-like proteins in Moraxella
catarrhalis include Hsf, UspA1 (WO93/03761), UspA2 (WO93/03761),
outer membrane esterase and YtfN. Examples of Hsf-like proteins in
Haemophilus influenzae include Hia/Hsf (St Geme et al J. Bacteriol.
2000 182: 6005-6013), Hap, IgA1 protease, HMW1, HMW2 (Barenkamp et
al Infect. Immun. 1992 60; 1302-1313), YadA, YadAc and YtfN
(Hendrixson et al Mol Cell 1998; 2:941-850; St Geme et al Mol
Microbiol. 1994; 14:217-233; Grass and St Geme Infect. Immunol.
2001; 69; 307-314; St Geme and Cutter J. Bacteriology 2000; 182;
6005-6013). Examples of Hsf-like proteins in Escherichia coli
include Hsf, Hia, and Hap.
[0031] Hsf has a structure that is common to autotransporter
proteins. For example, Hsf from N. meningitidis strain H44/76
consists of a head region at the amino terminus of the protein
(amino acids 52-479) that is surface exposed and contains variable
regions (amino acids 52-106, 121-124, 191-210 and 230-234), a neck
region (amino acids 480-509), a hydrophobic alpha-helix region
(amino acids 518-529) and an anchoring domain in which four
transmembrane strands span the outer membrane (amino acids
539-591).
[0032] Hsf may refer to the full length polypeptide including the
signal sequence that consists of amino acids 1-51. The invention
also encompasses Hsf with the signal sequence removed so that the
polypeptide would consist of the mature form of Hsf. Other
preferred forms of Hsf may be truncated so as to delete variable
regions of the protein disclosed in WO01/55182. Preferred variants
would include the deletion of one, two, three, four, or five
variable regions as defined in WO01/55182. Preferred variants would
delete residues from between amino acid sequence 52 through to 237
or would delete amino acids 54 to 237, more preferably deleting
residues between amino acid 52 through to 133 or amino acids 55 to
133. It is understood that truncated variants may include or
exclude the signal sequence from amino acids 1 to 51 of Hsf. The
above sequence and those described below can be truncated or
extended by 1, 2, 3, 4, 5, 7, 10, or 15 amino acids at either or
both N and C termini.
[0033] Where Hsf is used in a subunit vaccine, it is preferred that
a portion of the soluble passenger domain is used; for instance the
complete domain of amino acids 52 to 479, most preferably a
conserved portion thereof for instance amino acids 134 to 479.
[0034] Although full length Tbp and/or Hsf like protein (in
particular TbpA and Hsf) is preferably used, or natural variants
thereof, or such full length sequences lacking no more than 60
amino acids from the N and/or C termini, antigenic fragments of Tbp
and/or Hsf like proteins are also included in the immunogenic
composition of the invention. These are fragments containing at
least 10 amino acids, preferably 20 amino acids, more preferably 30
amino acids, more preferably 40 amino acids or most preferably 50
amino acids, taken contiguously from the amino acid sequence of Tbp
and Hsf like protein, preferably TbpA and Hsf. In addition,
antigenic fragments denotes fragments that are immunologically
reactive with antibodies generated against the N. meningitidis Tbp
or Hsf like protein, preerably TbpA or Hsf or with antibodies
generated by infection of a mammalian host with N. meningitidis.
Antigenic fragments also includes fragments that elicit an immune
response that is specific against Tbp or Hsf like protein,
preferably TbpA or Hsf of Gram negative bacteria from which they
are derived. Preferably it is protective against infection from the
Bacterium from which it is derived, preferably Neisserial
infection, more preferably it is protective against N. meningitidis
infection, most preferably it is protective against N. meningitidis
serogroup B infection.
[0035] Preferred fragments of TbpA include the extracellular loops
of TbpA. Using the sequence of TbpA from N. meningitidis strain
H44/76, these loops correspond to amino acids 200-202 for loop 1,
amino acids 226-303 for loop 2, amino acids 348-395 for loop 3,
amino acids 438-471 for loop 4, amino acids 512-576 for loop 5,
amino acids 609-625 for loop 6, amino acids 661-671 for loop 7,
amino acids 707-723 for loop 8, amino acids 769-790 for loop 9,
amino acids 814-844 for loop 10 and amino acids 872-903 for loop
11. The corresponding sequences, after sequence alignment, in other
Tbp proteins would also constitute preferred fragments. Most
preferred fragments would include amino acid sequences comprising
loop 2, loop 3, loop 4 or loop 5 of Tbp.
[0036] Although the preferred fragments of Tbp or TbpA proteins
described above relate to N. meningitidis, one skilled in the art
would readily be able to find the equivalent peptides in Tbp or
TbpA proteins from all the above Gram negative strains on the basis
of sequence homology, which are also fragments of the
invention.
[0037] Preferred fragments of Hsf include the entire head region of
Hsf, preferably containing amino acids 52-473 of Hsf. Additional
preferred fragments of Hsf include surface exposed regions of the
head including amino acids 52-62, 76-93, 116-134, 147-157, 157-175,
199-211, 230-252, 252-270, 284-306, 328-338, 362-391, 408-418,
430-440 and 469-479. Most preferred fragments are 134-591 for use
in a OMV preparation of the invention and 134-479 for use in a
subunit composition of the invention.
[0038] Although the preferred fragments of Hsf like or Hsf proteins
described above relate to N. meningitidis, one skilled in the art
would readily be able to find the equivalent peptides in Hsf like
or Hsf proteins from all the above Gram negative strains on the
basis of sequence homology, which are also fragments of the
invention.
[0039] Also included in the invention are fusion proteins of Tbp
and Hsf-like protein, preferably TbpA and Hsf. These may combine
both Tbp and Hsf like protein, preferably TbpA and Hsf, or
fragments thereof combined in the same polypeptide. Alternatively,
the invention also includes individual fusion proteins of Tbp and
Hsf like protein, preferably TbpA and/or Hsf, or fragments thereof,
provided that both Tbp and Hsf like protein, preferably TbpA and
Hsf, or fragments thereof are present in the composition of the
invention. TbpA or Hsf could for example form a fusion protein with
.beta.-galactosidase, glutathione-S-transferase, green fluorescent
proteins (GFP), epitope tags such as FLAG, myc tag, poly histidine,
or viral/bacterial surface proteins such as influenza virus
haemagglutinin, tetaunus toxoid, diphtheria toxoid or CRM197.
[0040] Isolated transferrin binding proteins which could be
introduced into an immunogenic composition are well known in the
art (WO0025811). They may be expressed in a bacterial host,
extracted using detergent (for instance 2% Elugent) and purified by
affinity chromatography or using standard column chromatography
techniques well known to the art (Oakhill et al Biochem J. 2002
364; 613-6). Similarly, the isolation of Hsf could be achieved
using techniques well known in the art. Recombinant Hsf could be
expressed in E. coli or other bacterial strains. The protein could
be purified using affinity chromatography. This would be a routine
procedure if a tag were introduced into the Hsf sequence.
[0041] The terms `comprising`, `comprise` and `comprises` herein
are intended by the inventors to be optionally substitutable with
the terms `consisting of`, `consist of` and `consists of`,
respectively, in every instance.
Vaccine Combinations
[0042] The invention relates to combinations of antigens including
Tbp and Hsf-like protein, which are effective at eliciting a high
bactericidal activity against Gram negative bacteria. Antigenic
compositions of the invention may comprise antigens in addition to
Tbp and Hsf. They may comprise other protein antigens from Gram
negative bacteria, preferably Neisseria and more preferably from N.
meningitidis.
N. meningitidis
[0043] For N. meningitidis, the immunogenic compositions of the
invention preferably comprise Hsf and TbpA. In a OMV preparation,
it is preferred that Hsf and TbpA are upregulated in the N.
meningitidis strain from which the OMV is derived. TbpA may be
present as either the high or low molecular weight form and
preferably both high and low molecular weight forms are
represented. Hsf is preferably present in OMVs as a membrane
integrated truncate preferably amino acids 134-591. Hsf may also be
present as a subunit vaccine preferably as a passenger domain
(amino acid 52-479) most preferably as a passenger domain truncate
of amino acids 134-479.
[0044] Further antigens may be added to the above compositions (or
upregulated if presented in a OMV), for example, NspA (WO96/29412),
Hap (PCT/EP99/02766), PorA, PorB, OMP85 (also known as D15)
(WO00/23595), PilQ (PCT/EP99/03603), PldA (PCT/EP99/06718), FrpB
(WO96/31618 see SEQ ID NO:38), FrpA (NMB 0585) or FrpC or a
conserved portion in commen to both of at least 30, 50, 100, 500,
750 amino acids (WO92/01460), LbpA and/or LbpB (PCT/EP98/05117;
Schryvers et al Med. Microbiol. 1999 32: 1117), FhaB (WO98/02547
SEQ ID NO:38 [nucleotides 3083-9025]), HasR (PCT/EP99/05989),
lipo02 (PCT/EP99/08315), MltA (WO99/57280) (NMB0033) and ctrA
(PCT/EP00/00135).
[0045] Preferred combinations of antigens in an immunogenic
composition of the invention include combinations comprising Tbp
and Hsf-like protein and FhaB; Tbp and Hsf-like protein and PilQ;
Tbp and Hsf-like protein and NspA; Tbp and Hsf-like protein and
FrpC; more preferably comprising Tbp and Hsf-like protein and Hap;
Tbp and Hsf-like protein and FrpA/C; Tbp and Hsf-like protein and
LbpB; Tbp and Hsf-like protein and D15. Most preferably, D15 would
be incorporated as part of an outer membrane vesicle
preparation.
Moraxella catarrhalis Antigens
[0046] One or more of the following proteins from Moraxella
catarrhalis are preferred for incorporation into the immunogenic
composition of the invention (preferably where the TbpA and Hsf
like proteins are derived from Moraxella catarrhalis): OMP106 (WO
97/41731 & WO 96/34960), HasR (PCT/EP99/03824), PilQ
(PCT/EP99/03823), OMP85 (PCT/EP00/01468), lipo06 (GB 9917977.2),
lipo10 (GB 9918208.1), lipo11 (GB 9918302.2), lipo18 (GB
9918038.2), P6 (PCT/EP99/03038), ompCD, CopB (Helminen M E, et al
(1993) Infect. Immun. 61:2003-2010), D15 (PCT/EP99/03822), Omp1A1
(PCT/EP99/06781), Hly3 (PCT/EP99/03257), LbpA and LbpB (WO
98/55606), TbpA and TbpB (WO 97/13785 & WO 97/32980), OmpE,
UspA1 and UspA2 (WO 93/03761), and Omp21.
Haemophilus influenzae Antigens
[0047] One or more of the following proteins from Haemophilus
influenzae are preferred for inclusion in a immunogenic composition
of the invention (preferably where the TbpA and Hsf like proteins
are derived from Haemophilus influenzae): D15 (WO 94/12641), P6 (EP
281673), TbpA, TbpB, P2, P5 (WO 94/26304), OMP26 (WO 97/01638),
HMW1, HMW2, HMW3, HMW4, Hia, Hsf, Hap, Hin47, and Hif.
[0048] A further aspect of the invention are vaccine combinations
comprising the antigenic composition of the invention with other
antigens which are advantageously used against certain disease
states including those associated with viral or Gram positive
bacteria.
[0049] In one preferred combination, the antigenic compositions
comprising Tbp and Hsf-like protein of the invention are formulated
with 1, 2, 3 or preferably all 4 of the following meningococcal
capsular polysaccharides or oligosaccharides which may be plain or
conjugated to a protein carrier: A, C, Y or W-135. Such a vaccine
containing TbpA and Hsf from N. meningitidis may be advantageously
used as a global meningococcus vaccine. Preferably conjugated
meningococcal capsular polysaccharide C, C and Y or A and C are
included.
[0050] In a further preferred embodiment, the antigenic
compositions comprising TbpA and Hsf of the invention, preferably
formulated with 1, 2, 3 or all 4 of the plain or conjugated
meningococcal capsular polysaccharides (or oligosaccharides) A, C,
Y or W-135 as described above, are formulated with a conjugated H.
influenzae b capsular polysaccharide or oligosaccharide, and/or one
or more plain or conjugated pneumococcal capsular polysaccharides
or oligosaccharides. Optionally, the vaccine may also comprise one
or more protein antigens that can protect a host against
Streptococcus pneumoniae infection. Such a vaccine may be
advantageously used as a meningitis/streptococcus pneumonia
vaccine.
[0051] In a still further preferred embodiment, the immunogenic
composition comprising Tbp and Hsf-like protein of the invention is
formulated with capsular polysaccharides derived from one or more
of Neisseria meningitidis, Haemophilus influenzae b, Streptococcus
pneumoniae, Group A Streptococci, Group B Streptococci,
Staphylococcus aureus or Staphylococcus epidermidis. In a preferred
embodiment, the immunogenic composition would comprise capsular
polysaccharides derived from one or more of serogroups A, C, W-135
and Y of Neisseria meningitidis. A further preferred embodiment
would comprise capsular polysaccharides derived from Streptococcus
pneumoniae. The pneumococcal capsular polysaccharide antigens are
preferably selected from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N,
9V, 10A, 11A, 12F, 14, 15B, 17F, 18C, 19A, 19F, 20, 22F, 23F and
33F (most preferably from serotypes 1, 3, 4, 5, 6B, 7F, 9V, 14,
18C, 19F and 23F). A further preferred embodiment would contain the
PRP capsular polysaccharides of Haemophilus influenzae. A further
preferred embodiment would contain the Type 5, Type 8 or 336
capsular polysaccharides of Staphylococcus aureus. A further
preferred embodiment would contain the Type I, Type II or Type III
capsular polysaccharides of Staphylococcus epidermidis. A further
preferred embodiment would contain the Type Ia, Type Ic, Type II or
Type III capsular polysaccharides of Group B streptocoocus. A
further preferred embodiment would contain the capsular
polysaccharides of Group A streptococcus, preferably further
comprising at least one M protein and more preferably multiple
types of M protein.
[0052] Preferred pneumococcal proteins antigens are those
pneumococcal proteins which are exposed on the outer surface of the
pneumococcus (capable of being recognised by a host's immune system
during at least part of the life cycle of the pneumococcus), or are
proteins which are secreted or released by the pneumococcus. Most
preferably, the protein is a toxin, adhesin, 2-component signal
tranducer, or lipoprotein of Streptococcus pneumoniae, or fragments
thereof. Particularly preferred proteins include, but are not
limited to: pneumolysin (preferably detoxified by chemical
treatment or mutation) [Mitchell et al. Nucleic Acids Res. 1990
Jul. 11; 18(13): 4010 "Comparison of pneumolysin genes and proteins
from Streptococcus pneumoniae types 1 and 2.", Mitchell et al.
Biochim Biophys Acta 1989 Jan. 23; 1007(1): 67-72 "Expression of
the pneumolysin gene in Escherichia coli: rapid purification and
biological properties.", WO 96/05859 (A. Cyanamid), WO 90/06951
(Paton et al), WO 99/03884 (NAVA)]; PspA and transmembrane deletion
variants thereof (U.S. Pat. No. 5,804,193--Briles et al.); PspC and
transmembrane deletion variants thereof (WO 97/09994--Briles et
al); PsaA and transmembrane deletion variants thereof (Berry &
Paton, Infect Immun 1996 December; 64(12):5255-62 "Sequence
heterogeneity of PsaA, a 37-kilodalton putative adhesin essential
for virulence of Streptococcus pneumoniae"); pneumococcal choline
binding proteins and transmembrane deletion variants thereof; CbpA
and transmembrane deletion variants thereof (WO 97/41151; WO
99/51266); Glyceraldehyde-3-phosphate--dehydrogenase (Infect.
Immun. 1996 64:3544); HSP70 (WO 96/40928); PcpA (Sanchez-Beato et
al. FEMS Microbiol Lett 1998, 164:207-14); M like protein, (EP
0837130) and adhesin 18627, (EP 0834568). Further preferred
pneumococcal protein antigens are those disclosed in WO 98/18931,
particularly those selected in WO 98/18930 and PCT/US99/30390.
[0053] The vaccine may also optionally comprise antigens providing
protection against one or more of Diphtheria, tetanus and
Bordetella pertussis infections. The pertussis component may be
killed whole cell B. pertussis (Pw) or acellular pertussis (Pa)
which comprises at least one antigen (and preferably all three)
from PT, FHA and 69 kDa pertactin. Typically, the antigens
providing protection against Diphtheria and tetanus would be
Diphtheria toxoid and tetanus toxoid. The toxoids may chemically
inactivated toxins or toxins inactivated by the introduction of
point mutations.
[0054] The vaccine may also optionally comprise one or more
antigens that can protect a host against non-typeable Haemophilus
influenzae, RSV and/or one or more antigens that can protect a host
against influenza virus. Such a vaccine may be advantageously used
as a global otitis media vaccine.
[0055] Preferred non-typeable H. influenzae protein antigens
include Fimbrin protein (U.S. Pat. No. 5,766,608) and fusions
comprising peptides therefrom (eg LB1 Fusion) (U.S. Pat. No.
5,843,464--Ohio State Research Foundation), OMP26, P6, protein D,
TbpA, TbpB, Hia, Hmw1, Hmw2, Hap, and D15.
[0056] Preferred influenza virus antigens include whole, live or
inactivated virus, split influenza virus, grown in eggs or MDCK
cells, or Vero cells or whole flu virosomes (as described by R.
Gluck, Vaccine, 1992, 10, 915-920) or purified or recombinant
proteins thereof, such as HA, NP, NA, or M proteins, or
combinations thereof.
[0057] Preferred RSV (Respiratory Syncytial Virus) antigens include
the F glycoprotein, the G glycoprotein, the HN protein, the M
protein or derivatives thereof.
[0058] It should be appreciated that antigenic compositions of the
invention may comprise one or more capsular polysaccharide from a
single species of bacteria. Antigenic compositions may also
comprise capsular polysaccharides derived from one or more species
of bacteria.
[0059] Such capsular polysaccharides may be unconjugated or
conjugated to a carrier protein such as tetatus toxoid, tetanus
toxoid fragment C, diphtheria toxoid, CRM197, pneumolysin, Protein
D (U.S. Pat. No. 6,342,224), TbpA or Hsf. One embodiment of the
invention would contain separate capsular polysaccharides
conjugated to TbpA and Hsf.
[0060] The polysaccharide conjugate may be prepared by any known
coupling technique. For example the polysaccharide can be coupled
via a thioether linkage. This conjugation method relies on
activation of the polysaccharide with 1-cyano-4-dimethylamino
pyridinium tetrafluoroborate (CDAP) to form a cyanate ester. The
activated polysaccharide may thus be coupled directly or via a
spacer group to an amino group on the carrier protein. Preferably,
the cyanate ester is coupled with hexane diamine and the
amino-derivatised polysaccharide is conjugated to the carrier
protein using heteroligation chemistry involving the formation of
the thioether linkage. Such conjugates are described in PCT
published application WO93/15760 Uniformed Services University.
[0061] The conjugates can also be prepared by direct reductive
amination methods as described in U.S. Pat. No. 4,365,170
(Jennings) and U.S. Pat. No. 4,673,574 (Anderson). Other methods
are described in EP-0-161-188, EP-208375 and EP-0-477508.
[0062] A further method involves the coupling of a cyanogen bromide
activated polysaccharide derivatised with adipic acid hydrazide
(ADH) to the protein carrier by Carbodiimide condensation (Chu C.
et al Infect. Immunity, 1983 245 256).
Antigenic Compositions Comprising Outer Membrane Vesicles
[0063] A preferred aspect of the present invention is the
upregulation, or overexpression, of Tbp and Hsf in an OMV. Gram
negative bacteria are separated from the external medium by two
successive layers of membrane structures, the cytoplasmic membrane
and the outer membrane. The outer membrane of Gram-negative
bacteria is dynamic and depending on environmental conditions can
undergo drastic morphological transformations. Among these
manifestations, the formation of outer membrane vesicles or blebs
has been studied and documented in many Gram-negative bacteria
(Zhou et al 1998). Among these, a non-exhaustive list of bacterial
pathogens reported to produce blebs include: Bordetella pertussis,
Borrelia burgdorferi, Brucella melitensis, Brucella ovis, Chlamydia
psittaci, Chlamydia trachomatis, Esherichia coli, Haemophilus
influenzae, Legionella pneumophila, Neisseria gonorrhoeae,
Neisseria meningitidis, Pseudomonas aeruginosa and Yersinia
enterocolitica. Although the biochemical mechanism responsible for
the production of OMV/blebs is not fully understood, these outer
membrane vesicles have been extensively studied as they represent a
powerful methodology in order to isolate outer-membrane protein
preparations in their native conformation. In that context, the use
of outer-membrane preparations is of particular interest to develop
vaccines against Neisseria, Moraxella catarrhalis, Haemophilus
influenzae, Pseudomonas aeruginosa and Chlamydia. Moreover, outer
membrane blebs combine multiple proteinaceaous and
non-proteinaceous antigens that are likely to confer extended
protection against intra-species variants.
[0064] The outer membrane vesicles of the invention will have Tbp
and Hsf-like protein (preferably TbpA and Hsf) upregulated. This is
optionally achieved by having Hsf-like protein and Tbp upregulated
in outer membrane vesicles derived from a single Gram negative
bacterial, preferably Neisserial strain. Hsf-like protein and Tbp
may also be upregulated separately in outer membrane vesicles
derived from different Gram negative bacterial strains, preferably
Neisserial strains. In a preferred embodiment, the different
strains of Neisseria in which Tbp and Hsf-like protein, more
preferably TbpA and Hsf are upregulated will be a L2 and L3 or L3
and L2, repectively immunotype of N. meningitidis.
[0065] The manufacture of bleb preparations from Neisserial strains
may be achieved by any of the methods well known to a skilled
person. Preferably the methods disclosed in EP 301992, U.S. Pat.
No. 5,597,572, EP 11243 or U.S. Pat. No. 4,271,147, Frederikson et
al. (NIPH Annals [1991], 14:67-80), Zollinger et al. (J. Clin.
Invest. [1979], 63:836-848), Saunders et al. (Infect. Immun.
[1999], 67:113-119), Drabick et al. (Vaccine [2000], 18:160-172) or
WO 01/09350 (Example 8) are used. In general, OMVs are extracted
with a detergent, preferably deoxycholate, and nucleic acids are
optionally removed enzymatically. Purification is achieved by
ultracentrifugation optionally followed by size exclusion
chromatography. If 2 or more different blebs of the invention are
included, they may be combined in a single container to form a
multivalent preparation of the invention (although a preparation is
also considered multivalent if the different blebs of the invention
are separate compositions in separate containers which are
administered at the same time [the same visit to a practitioner] to
a host). OMV preparations are usually sterilised by filtration
through a 0.2 .mu.m filter, and are preferably stored in a sucrose
solution (e.g. 3%) which is known to stabilise the bleb
preparations.
[0066] Upregulation of Tbp and Hsf-like protein within outer
membrane vesicle preparations may be achieved by insertion of an
extra copy of a gene into the Gram negative bacteria from which the
OMV preparation is derived. Alternatively, the promoter of a gene
can be exchanged for a stronger promoter in the bacterial strain
from which the OMV preparation is derived. Such techniques are
described in WO01/09350. Upregulation of a protein will lead to a
higher level of protein being present in OMV compared to the level
of protein present in OMV derived from unmodified N. meningitidis
(for instance strain H44/76). Preferably the level will be at least
1.2, 1.5, 2, 3, 4, 5, 7, 10 or 20 times higher.
[0067] Where LPS is intended to be an additional antigen in the
OMV, a protocol using a low concentration of extracting detergent
(for example deoxycholate or DOC) may preferably be used in the OMV
preparation method so as to preserve high levels of bound LPS
whilst removing particularly toxic, poorly bound LPS. The
concentration of DOC used is preferably 0-0.5% DOC, more preferably
0.02%-0.4%, 0.03%-0.3%, 0.04%-0.2%, 0.05%-0.15%, 0.05%-0.2% DOC,
most preferably around or exactly 0.1% DOC.
[0068] "Stronger promoter sequence" refers to a regulatory control
element that increases transcription for a gene encoding antigen of
interest.
[0069] "Upregulating expression" refers to any means to enhance the
expression of an antigen of interest, relative to that of the
non-modified (i.e., naturally occurring) bleb. It is understood
that the amount of `upregulation` will vary depending on the
particular antigen of interest but will not exceed an amount that
will disrupt the membrane integrity of the bleb. Upregulation of an
antigen refers to expression that is at least 10% higher than that
of the non-modified bleb. Preferably it is at least 50% higher.
More preferably it is at least 100% (2 fold) higher. Most
preferably, it is at least 3, 4, 5, 7, 10, 20 fold higher.
Preferably the level of expression is assessed when blebs have been
derived from bacteria grown in iron limited conditions (for
instance in the presence of an iron chelator).
[0070] Alternatively or additionally, upregulating expression may
refer to rendering expression non-conditional on metabolic or
nutritional changes, particularly in the case of TbpA, TbpB, LbpA
and LbpB.
[0071] Again for the purpose of clarity, the terms `engineering a
bacterial strain to produce less of said antigen` or down
regulation refers to any means to reduce the expression of an
antigen (or the expression of a functional gene product) of
interest, relative to that of the non-modified (i.e., naturally
occurring bleb), preferably by deletion, such that expression is at
least 10% lower than that of the non-modified bleb. Preferably it
is at least 50% lower and most preferably completely absent. If the
down regulated protein is an enzyme or a functional protein, the
downregulation may be achieved by introducing one or more mutations
resulting in a 10%, 20%, 50%, 80% or preferably a 100% reduction in
enzymatic or functional activity.
[0072] The engineering steps required to modulate the expression of
Neisserial proteins can be carried out in a variety of ways known
to the skilled person. For instance, sequences (e.g. promoters or
open reading frames) can be inserted, and promoters/genes can be
disrupted by the technique of transposon insertion. For instance,
for upregulating a gene's expression, a strong promoter could be
inserted via a transposon up to 2 kb upstream of the gene's
initiation codon (more preferably 200-600 bp upstream, most
preferably approximately 400 bp upstream). Point mutation or
deletion may also be used (particularly for down-regulating
expression of a gene).
[0073] Such methods, however, may be quite unstable or uncertain,
and therefore it is preferred that the engineering step is
performed via a homologous recombination event. Preferably, the
event takes place between a sequence (a recombinogenic region) of
at least 30 nucleotides on the bacterial chromosome, and a sequence
(a second recombinogenic region) of at least 30 nucleotides on a
vector transformed within the strain. Preferably the regions are
40-1000 nucleotides, more preferably 100-800 nucleotides, most
preferably 500 nucleotides). These recombinogenic regions should be
sufficiently similar that they are capable of hybridising to one
another under highly stringent conditions.
[0074] Methods used to carry out the genetic modification events
herein described (such as the upregulation or downregulation of
genes by recombination events and the introduction of further gene
sequences into a Neisserial genome) are described in WO01/09350.
Typical strong promoters that may be integrated in Neisseria are
porA, porB, lgtF, Opa, p110, lst, and hpuAB. PorA and PorB are
preferred as constitutive, strong promoters. It has been
established that the PorB promoter activity is contained in a
fragment corresponding to nucleotides -1 to -250 upstream of the
initation codon of porB.
Upregulation of Expression of Iron Acquisition Proteins by Growth
under Iron Limitation Conditions
[0075] The upregulation of transferrin binding protein in an outer
membrane vesicle preparation of the invention is preferably
achieved by isolating outer membrane vesicles from a parental
strain of Gram negative bacteria grown under iron limitation
conditions. A low concentration of iron in the medium will result
in increased expression of proteins involved in iron acquisition
including TbpA and TbpB. The expression of these proteins is
thereby upregulated without the need for recombinantly modifying
the gene involved, for instance by inserting a stronger promoter or
inserting an additional copy of the gene. The invention would also
encompass upregulation of transferrin binding protein by growth in
iron limitation medium where the gene has also been recombinantly
modified.
[0076] Iron limitation is achieved by the addition of an iron
chelator to the culture medium. Suitable iron chelators include
2,2-Dipyridil, EDDHA (ethylenediamine-di(o-hydroxyphenylacetic
acid) and Desferal (deferoxamine mesylate, Sigma). Desferal is the
preferred iron chelator and is added to the culture medium at a
concentration of between 10 and 100 .mu.M, preferably 25-75 .mu.M,
more preferably 50-70 .mu.M, most preferably at 60 .mu.M. The iron
content of medium comes primarily from the yeast extract and soy
peptone constituents and the amount present may vary between
batches. Therefore different concentrations of Desferal may be
optimal to achieve upregulation of iron acquisition proteins in
different batches of medium. The skilled artisan should easily be
able to determine the optimal concentration. In basic terms, enough
iron chelator should be added to the medium to upregulate the
expression of the desired iron-regulated protein, but not so much
so as to adversely affect the growth of the bacteria.
[0077] Preferably, upregulation of transferrin binding protein by
growth under iron limited conditions is combined with recombinant
upregulation of Hsf like protein so that the outer membrane vesicle
of the invention is achieved.
Down Regulation/Removal of Variable and Non-Protective
Immunodominant Antigens
[0078] Many surface antigens are variable among bacterial strains
and as a consequence are protective only against a limited set of
closely related strains. An aspect of this invention covers outer
membrane vesicles comprising Tbp and Hsf like protein, preferably
TbpA and Hsf in which the expression of other proteins is reduced,
or, preferably, gene(s) encoding variable surface protein(s) are
deleted. Such deletion results in a bacterial strain producing
blebs which, when administered in a vaccine, have a stronger
potential for cross-reactivity against various strains due to a
higher influence exerted by conserved proteins (retained on the
outer membranes) on the vaccinee's immune system. Examples of such
variable antigens include: for Neisseria--pili (PilC) which
undergoes antigenic variations, PorA, Opa, OpC, PilC, PorB, TbpB,
FrpB; for H. influenzae--P2, P5, pilin, IgA1-protease; and for
Moraxella--OMP106.
[0079] Other types of gene that could be down-regulated or switched
off are genes which, in vivo, can easily be switched on (expressed)
or off by the bacterium. As outer membrane proteins encoded by such
genes are not always present on the bacteria, the presence of such
proteins in the bleb preparations can also be detrimental to the
effectiveness of the vaccine for the reasons stated above. A
preferred example to down-regulate or delete is Neisseria Opc
protein. Anti-Opc immunity induced by an Opc containing bleb
vaccine would only have limited protective capacity as the
infecting organism could easily become Opc.sup.-. H. influenzae
HgpA and HgpB are other examples of such proteins.
[0080] For example, these variable or non-protective genes may be
down-regulated in expression, or terminally switched off. This has
the advantage of concentrating the immune system on better antigens
that are present in low amounts on the outer surface of blebs.
Methods for Downregulation of Expression are Disclosed in
WO01/09350.
[0081] By down regulation of an immunodominant outer membrane
protein is it meant that levels of expression are decreased and
preferably switched off or that mutations and/or deletions of
surface exposed immunodominant loops render the outer membrane
protein less immunodominant. By down regulation of a protein with
enzymatic function it is meant that the level of expression of the
protein is decreased or preferably switched off or can mean that
the expression of functional enzyme is reduced or preferably
eliminated.
[0082] Preferred meningococcal strains of bacteria to use in making
immunogenic compositions of the invention have downregulation,
preferably deletion of 1, 2 or 3 of PorA, OpA and Opc. Preferably
PorA and Opa; PorA and OpC; OpA and OpC; PorA and Opa and OpC are
downregulated.
[0083] Four different Opa genes are known to exist in the
meningococcal genome (Aho et al. 1991 Mol. Microbiol. 5:1429-37),
therefore where Opa is said to be downregulated in expression it is
meant that preferably 1, 2, 3 or (preferably) all 4 genes present
in meningococcus are so downregulated. Such downregulation may be
performed genetically as described in WO 01/09350 or by seeking
readily-found, natural, stable meningococcal strains that have no
or low expression from the Opa loci. Such strains can be found
using the technique described in Poolman et al (1985 J. Med. Micro.
19:203-209) where cells that are Opa have a different phenotype to
cells expressing Opa which can be seen looking at the appearance of
the cells on plates or under a microscope. Once found, the strain
can be shown to be stably Opa.sup.- by performing a Western blot on
cell contents after a fermentation run to establish the lack of
Opa.
[0084] Where upregulation of transferrin binding protein in the
outer membrane vesicles of the invention is achieved by growth
under iron limitation conditions, variable iron-regulated proteins
may also be upregulated. These include FrpB in Neisseria
meningitidis and Neisseria gonorrhoeae (Microbiology 142;
3269-3274, (1996); J. Bacteriol. 181; 2895-2901 (1999)), and
heme/hemopexin utilisation protein C (J. Bacteriol. 177; 2644-2653
(1995)) and HgpA, HgpB and HgpC (Infect. Immun. 66; 4733-4741
(1998), Infect. Immun. 67; 2729-2739 (1999), Microbiology 145;
905-914 (1999)) in Haemophilus influenzae. The inventors have found
that it is advantageous to downregulate expression of at least the
variable portions of such proteins when iron limitation is used to
upregulate transferrin binding protein expression. This is achieved
either by using the processes described in WO01/09350 or by
deleting the variable part(s) of the protein. This will ensure that
the immune response elicited by the immunogenic composition is
directed towards antigens that are present in a wide range of
strains. Down regulation of FrpB is preferably combined with down
regulation of PorA and OpA; PorA and OpC; OpA and OpC; PorA and OpA
and OpC in the bleb immunogenic compositions of the invention
derived from Gram negative bacterial strains, preferably Moraxella
catarrhalis, Haemophilus influenzae or Neisserial (more preferably
N. meningitidis) strains.
[0085] In an alternative embodiment of the invention, FrpB is
downregulated in outer membrane vesicles which have been prepared
from Gram negative bacterial strains, preferably Moraxella
catarrhalis, Haemophilus influenzae or Neisserial (more preferably
N. meningitidis) strains, not necessarily grown under iron
limitation conditions.
Detoxification of LPS
[0086] The OMVs in the immunogenic composition of the invention may
be detoxifed via methods for detoxification of LPS which are
disclosed in WO01/09350. In particular, methods for detoxification
of LPS involve the downregulation, preferably deletion of htrB
and/or msbB enzymes which are disclosed in WO 01/09350. Deletion
mutants of these genes are characterised phenotypically by the
msbB- mutant LPS losing one secondary acyl chain compared to wild
type and the htrB- mutants LPS losing 2 (or both) secondary acyl
chains. Such methods are preferably combined with methods of OMV
extraction involving low levels of DOC, preferably 0-0.3% DOC, more
preferably 0.05-0.2% DOC, most preferably around 0.1% DOC.
[0087] Further methods of LPS detoxification include adding to the
bleb preparations a non-toxic peptide functional equivalent of
polymyxin B [a molecule with high affinity to Lipid A] (preferably
SAEP 2) (see WO 93/14115, WO 95/03327, Velucchi et al (1997) J
Endotoxin Res 4: 1-12, and EP 976402 for further details of
non-toxic peptide functional equivalents of polymyxin
B--particularly the use of the peptide SAEP 2 (of sequence
KTKCKFLKKC where the 2 cysteines form a disulphide bridge)).
Cross-Reactive Polysaccharides
[0088] The isolation of bacterial outer-membrane blebs from
encapsulated Gram-negative bacteria often results in the
co-purification of capsular polysaccharide. In some cases, this
"contaminant" material may prove useful since polysaccharide may
enhance the immune response conferred by other bleb components. In
other cases however, the presence of contaminating polysaccharide
material in bacterial bleb preparations may prove detrimental to
the use of the blebs in a vaccine. For instance, it has been shown
at least in the case of N. meningitidis that the serogroup B
capsular polysaccharide does not confer protective immunity and is
susceptible to induce an adverse auto-immune response in humans.
Consequently, outer membrane vesicles of the invention may be
isolated from a bacterial strain for bleb production, which has
been engineered such that it is free of capsular polysaccharide.
The blebs will then be suitable for use in humans. A particularly
preferred example of such a bleb preparation is one from N.
meningitidis serogroup B devoid of capsular polysaccharide. In
general, isolation of outer membrane vesicles should be from Gram
negative bacterial strains that cannot synthesise capsular
pooysaccharides, particularly where the strain is a msbB- mutant
described above.
[0089] This may be achieved by using modified bleb production
strains in which the genes necessary for capsular biosynthesis
and/or export have been impaired. Inactivation of the gene coding
for capsular polysaccharide biosynthesis or export can be achieved
by mutating (point mutation, deletion or insertion) either the
control region, the coding region or both (preferably using the
homologous recombination techniques described above), or by any
other way of decreasing the enzymatic function of such genes.
Moreover, inactivation of capsular biosynthesis genes may also be
achieved by antisense over-expression or transposon mutagenesis. A
preferred method is the deletion of some or all of the Neisseria
meningitidis capsular polysaccharide (cps) genes required for
polysaccharide biosynthesis and export. For this purpose, the
replacement plasmid pMF121 (described in Frosh et al. 1990, Mol.
Microbiol. 4:1215-1218) can be used to deliver a mutation deleting
the cpsCAD (+galE) gene cluster.
[0090] Where the above immunogenic compositions of the invention
are derived from a meningococcus B strain, it is further preferred
that the capsular polysaccharide (which also contains human-like
saccharide structures) is also removed. Although many genes could
be switched off to achieve this, the inventors have advantageously
shown that it is preferred that the bleb production strain has been
genetically engineered to permanently downregulate the expression
of functional gene product from the siaD gene (i.e. downregulating
.alpha.-2-8 polysialyltransferase activity), preferably by
switching the gene off, most preferably by deleting all or part of
the promoter and/or open-reading frame of the gene. Such an
inactivation is described in WO 01/09350. The siaD (also known as
synD) mutation is the most advantageous of many mutations that can
result in removing the human-similar epitope from the capsular
polysaccharide, because it one of the only mutations that has no
effect on the biosynthesis of the protective epitopes of LOS, thus
being advantageous in a process which aims at ultimately using LOS
as a protective antigen, and has a minimal effect on the growth of
the bacterium. A preferred aspect of the invention is therefore a
bleb immunogenic preparation as described above which is derived
from an lgtE.sup.- siaD.sup.-, an lgtA.sup.- siaD.sup.- or,
preferably, an lgtB.sup.- siaD.sup.- meningococcus B mutant strain.
The strain itself is a further aspect of the invention.
[0091] Although siaD.sup.- mutation is preferable for the above
reasons, other mutations which switch off neisserial (preferably
meningococcus B) capsular polysaccharide synthesis may be used.
Thus bleb production strain can be genetically engineered to
permanently downregulate the expression of functional gene product
from one or more of the following genes: ctrA, ctrB, ctrC, ctrD,
synA (equivalent to synX and siaA), synB (equivalent to siaB) or
synC (equivalent to siaC) genes, preferably by switching the gene
off, most preferably by deleting all or part of the promoter and/or
open-reading frame of the gene. The lgtE.sup.- mutation may be
combined with one or more of these mutations. Preferably the
lgtB.sup.- mutation is combined with one or more of these
mutations. A further aspect of the invention is therefore a bleb
immunogenic preparation as described above which is derived from
such a combined mutant strain of meningococcus B. The strain itself
is a further aspect of the invention.
[0092] Heterogeneity within the oligosaccharide moiety of the LPS
generates structural and antigenic diversity among different
neisserial strains (Griffiss et al. Inf. Immun. 1987; 55:
1792-1800). This has been used to subdivide meningococcal strains
into 12 immunotypes (Scholtan et al. J Med Microbiol 1994,
41:236-243). Immunotypes L3, L7, & L9 are immunologically
identical and are structurally similar (or even the same) and have
therefore been designated L3,7,9 (or, for the purposes of this
specification, generically as "L3"). Meningococcal LPS L3,7,9 (L3),
L2 and L5 can be modified by sialylation, or by the addition of
cytidine 5'-monophosphate-N-acetylneuraminic acid. Although L2, L4
and L6 LPS are distinguishable immunologically, they are
structurally similar and where L2 is mentioned herein, either L4 or
L6 may be optionally substituted within the scope of the invention.
See M. P. Jennings et al, Microbiology 1999, 145, 3013-3021 and Mol
Microbiol 2002, 43:931-43 for further illustration of LPS structure
and heterogeneity.
[0093] The safety of antibodies raised to L3 or L2 LPS has been
questioned, due to the presence of a structure similar to the
lacto-N-neotetraose oligosaccharide group
(Gal.beta.1-4GlcNAc.beta.1-3Gal.beta.1-4Glc.beta.1-) present in
human glycosphingolipids. Even if a large number of people has been
safely vaccinated with deoxycholate extracted vesicle vaccines
containing residual amount of L3 LPS (G. Bjune et al, Lancet
(1991), 338, 1093-1096; GVG. Sierra et al, NIPH ann (1991), 14,
195-210), the deletion of the terminal part of the LOS saccharidic
is advantageous in preventing any cross-reaction with structures
present at the surface of human tissues. In a preferred embodiment,
inactivation of the lgtB gene results in an intermediate LPS
structure in which the terminal galactose residue and the sialic
acid are absent (the mutation leaves a
4GlcNAc.beta.1-3Gal.beta.1-4Glc.beta.1- structure in L2 and L3
LOS). Such intermediates could be obtained in an L3 and an L2 LPS
strain. An alternative and less preferred (short) version of the
LPS can be obtained by turning off the lgtE gene. A further
alternative and less preferred version of the LPS can be obtained
by turning off the lgtA gene. If such an lgtA.sup.- mutation is
selected it is preferred to also turn off lgtC expression to
prevent the non-immunogenic L1 immunotype being formed.
[0094] LgtB.sup.- mutants are most preferred as the inventors have
found that this is the optimal truncation for resolving the safety
issue whilst still retaining an LPS protective oligosaccharide
epitope that can still induce a bactericidal antibody response.
[0095] Therefore, immunogenic compositions of the invention further
containing L2 or L3 preparations (whether purified or in an
isolated bleb) or meningococcal bleb preparations in general are
advantageously derived from a Neisserial strain (preferably
meningococcal) that has been genetic engineered to permanently
downregulate the expression of functional gene product from the
lgtB, lgtA or lgtE gene, preferably by switching the gene off, most
preferably by deleting all or part of the promoter and/or
open-reading frame of the gene.
[0096] A Neisserial locus containing various lgt genes, including
lgtB and lgtE, and its sequence is known in the art (see M. P.
Jennings et al, Microbiology 1999, 145, 3013-3021 and references
cited therein, and J. Exp. Med. 180:2181-2190 [1994]).
[0097] In bleb preparations, particularly in preparations extracted
with low DOC concentrations LPS may be used as an antigen in the
immunogenic composition of the invention. It is however
advantageous to downregulate/delete/inactivate enzymatic function
of either the lgtE, lgtA (particularly in combination with lgtC),
or, preferably, lgtB genes/gene products in order to remove human
like lacto-N-neotetraose structures. The Neisserial locus (and
sequence thereof) comprising the lgt genes for the biosynthesis of
LPS oligosaccharide structure is known in the art (Jennings et al
Microbiology 1999 145; 3013-3021 and references cited therein, and
J. Exp. Med. 180:2181-2190 [1994]). Downregulation/deletion of lgtB
(or functional gene product) is preferred since it leaves the LPS
protective epitope intact.
[0098] In N. meningitidis serogroup B bleb preparations of the
invention, the downregulation/deletion of both siaD and lgtB is
preferred, (although a combination of lgtB.sup.- with any of
ctrA.sup.-, ctrB.sup.-, ctrC.sup.-, ctrD.sup.-, synA.sup.-
(equivalent to synX.sup.- and siaA.sup.-), synB.sup.- (equivalent
to siaB.sup.-) or synC.sup.- (equivalent to siaC.sup.-) in a
meningococcus B bleb production strain may also be used) leading to
a bleb preparation with optimal safety and LPS protective epitope
retention.
[0099] Immunogenic composition of the invention may comprise at
least, one, two, three, four or five different outer membrane
vesicle preparations. Where two or more OMV preparations are
included, at least one antigen of the invention is upregulated in
each OMV. Such OMV preparations may be derived from Neisserial
strains of the same species and serogroup or preferably from
Neisserial strains of different class, serogroup, serotype,
subserotype or immunotype. For example, an immunogenic composition
may comprise one or more outer membrane vesicle preparation(s)
which contains LPS of immunotype L2 and one or more outer membrane
vesicle preparation which contains LPS of immunotype L3. L2 or L3
OMV preparations are preferably derived from a stable strain which
has minimal phase variability in the LPS oligosaccharide synthesis
gene locus.
Preferred Neisserial Bleb Preparations
[0100] In addition to Hsf and Tbp, one or more of the following
genes (encoding protective antigens) are preferred for upregulation
when carried out on a Neisserial strain, including gonococcus, and
meningococcus (particularly N. meningitidis B): NspA (WO 96/29412),
Hap (PCT/EP99/02766), PorA, PorB (NMB 2039), OMP85 (WO 00/23595),
PilQ (PCT/EP99/03603), PldA (PCT/EP99/06718), FrpB (WO 96/31618),
FrpA/FrpC (WO 92/01460), LbpA/LbpB (PCT/EP98/05117), FhaB (WO
98/02547), HasR (PCT/EP99/05989), lipo02 (PCT/EP99/08315), MltA (WO
99/57280), MafA (NMB 0652), MafB (NMB 0643), Omp26 (NMB 0181),
adhesin NMB 0315, adhesin NMB 0995, adhesin NMB 1119, P2086 (NMB
0399), Lipo28 (NMB 2132), NM-ADPRT (NMB 1343), VapD (NMB 1753) and
ctrA (PCT/EP00/00135). They are also preferred as genes which may
be heterologously introduced into other Gram-negative bacteria.
[0101] One or more of the following genes are preferred for
downregulation: PorA, PorB, PilC, LbpA, LbpB, Opa, Opc, htrB, msbB
and lpxK.
[0102] One or more of the following genes are preferred for
upregulation: pmrA, pmrB, pmrE, and pmrF.
[0103] Preferred repressive control sequences to be modified are:
the fur operator region (particularly for either or both of the
TbpB or LbpB genes); and the DtxR operator region.
[0104] One or more of the following genes are preferred for
downregulation: galE, siaA, siaB, siaC, siaD, ctrA, ctrB, ctrC, and
ctrD.
[0105] Immunogenic compositions of the invention may also comprise
OMV/bleb preparations derived from Gram negative bacteria including
Pseudomonas aeruginosa, Moraxella catarrhalis and Haemophilys
influenzae b.
Preferred Pseudomonas aeruginosa Bleb Preparations
[0106] In addition to Hsf and Tbp, one or more of the following
genes (encoding protective antigens) are preferred for
upregulation: PcrV, OprF, OprI. They are also preferred as genes
which may be heterologously introduced into other Gram-negative
bacteria.
Preferred Moraxella catarrhalis Bleb Preparations
[0107] In addition to Hsf and Tbp, one or more of the following
genes (encoding protective antigens) are preferred for
upregulation: OMP106 (WO 97/41731 & WO 96/34960), HasR
(PCT/EP99/03824), PilQ (PCT/EP99/03823), OMP85 (PCT/EP00/01468),
lipo06 (GB 9917977.2), lipo10 (GB 9918208.1), lipo11 (GB
9918302.2), lipo18 (GB 9918038.2), P6 (PCT/EP99/03038), ompCD, CopB
(Helminen M E, et al (1993) Infect. Immun. 61:2003-2010), D15
(PCT/EP99/03822), Omp1A1 (PCT/EP99/06781), Hly3 (PCT/EP99/03257),
LbpA and LbpB (WO 98/55606), TbpA and TbpB (WO 97/13785, WO95/13370
& WO 97/32980), OmpE, UspA1 and UspA2 (WO 93/03761), and Omp21.
They are also preferred as genes which may be heterologously
introduced into other Gram-negative bacteria.
[0108] One or more of the following genes are preferred for
downregulation: CopB, OMP106, OmpB1, LbpA, and LbpB.
[0109] One or more of the following genes are preferred for
downregulation: htrB, msbB and lpxK.
[0110] One or more of the following genes are preferred for
upregulation: pmrA, pmrB, pmrE, and pmrF.
Preferred Haemophilus influenzae Bleb Preparations
[0111] In addition to Hsf and Tbp, one or more of the following
genes (encoding protective antigens) are preferred for
upregulation: D15 (WO 94/12641, WO95/12641), P6 (EP 281673), P2, P5
(WO 94/26304), OMP26 (WO 97/01638), HMW1, HMW2, HMW3, HMW4, Hia,
Hap, Hin47, and Hif (all genes in this operon should be upregulated
in order to upregulate pilin). They are also preferred as genes
which may be heterologously introduced into other Gram-negative
bacteria.
[0112] One or more of the following genes are preferred for
downregulation: P2, P5, Hif, IgA1-protease, HgpA, HgpB, HMW1, HMW2,
Hxu, htrB, msbB and lpxK.
[0113] One or more of the following genes are preferred for
upregulation: pmrA, pmrB, pmrE, and pmrF.
[0114] Preferably the immunogenic compositions or vaccines of the
invention do not consist of and/or comprise the particular
combinations of SEQ IDs listed in the table spanning from page 3,
line 18 to page 52, line 2 of WO 00/71725 and/or any individual
combination described in the examples 1-11 of WO 00/71725.
[0115] Preferably in addition or alternatively any individualised
combinations disclosed in WO 01/52885 are not claimed in this
invention.
Vaccine Formulations
[0116] A preferred embodiment of the invention is the formulation
of the immunogenic composition of the invention in a vaccine which
may also comprise a pharmaceutically acceptable excipient or
carrier.
[0117] The manufacture of outer membrane vesicle preparations from
any of the aforementioned modified strains may be achieved by any
of the methods well known to a skilled person. Preferably the
methods disclosed in EP 301992, U.S. Pat. No. 5,597,572, EP 11243
or U.S. Pat. No. 4,271,147 are used. Most preferably, the method
described in WO 01/09350 is used.
[0118] Vaccine preparation is generally described in Vaccine Design
("The subunit and adjuvant approach" (eds Powell M. F. & Newman
M. J.) (1995) Plenum Press New York).
[0119] The antigenic compositions of the present invention may be
adjuvanted in the vaccine formulation of the invention. Suitable
adjuvants include an aluminium salt such as aluminum hydroxide gel
(alum) or aluminium phosphate, but may also be a salt of calcium
(particularly calcium carbonate), iron or zinc, or may be an
insoluble suspension of acylated tyrosine, or acylated sugars,
cationically or anionically derivatised polysaccharides, or
polyphosphazenes.
[0120] Suitable Th1 adjuvant systems that may be used include,
Monophosphoryl lipid A, particularly 3-de-O-acylated monophosphoryl
lipid A, and a combination of monophosphoryl lipid A, preferably
3-de-O-acylated monophosphoryl lipid A (3D-MPL) together with an
aluminium salt. An enhanced system involves the combination of a
monophosphoryl lipid A and a saponin derivative particularly the
combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a
less reactogenic composition where the QS21 is quenched with
cholesterol as disclosed in WO96/33739. A particularly potent
adjuvant formulation involving QS21 3D-MPL and tocopherol in an oil
in water emulsion is described in WO95/17210 and is a preferred
formulation.
[0121] The vaccine may comprise a saponin, more preferably QS21. It
may also comprise an oil in water emulsion and tocopherol.
Unmethylated CpG containing oligo nucleotides (WO 96/02555) are
also preferential inducers of a TH1 response and are suitable for
use in the present invention.
[0122] The vaccine preparation of the present invention may be used
to protect or treat a mammal susceptible to infection, by means of
administering said vaccine via systemic or mucosal route. These
administrations may include injection via the intramuscular,
intraperitoneal, intradermal or subcutaneous routes; or via mucosal
administration to the oral/alimentary, respiratory, genitourinary
tracts. Thus one aspect of the present invention is a method of
immunizing a human host against a disease caused by infection of a
gram-negative bacteria, which method comprises administering to the
host an immunoprotective dose of the bleb preparation of the
present invention.
[0123] The amount of antigen in each vaccine dose is selected as an
amount which induces an immunoprotective response without
significant, adverse side effects in typical vaccinees. Such amount
will vary depending upon which specific immunogen is employed and
how it is presented. Generally, it is expected that each dose will
comprise 1-100 .mu.g of protein antigen, preferably 5-50 .mu.g, and
most typically in the range 5-25 .mu.g.
[0124] An optimal amount for a particular vaccine can be
ascertained by standard studies involving observation of
appropriate immune responses in subjects. Following an initial
vaccination, subjects may receive one or several booster
immunisations adequately spaced.
Polynucleotides of the Invention
[0125] "Polynucleotide" generally refers to any polyribonucleotide
or polydeoxribonucleotide, which may be unmodified RNA or DNA or
modified RNA or DNA. "Polynucleotides" include, without limitation
single- and double-stranded DNA, DNA that is a mixture of single-
and double-stranded regions, single- and double-stranded RNA, and
RNA that is mixture of single- and double-stranded regions, hybrid
molecules comprising DNA and RNA that may be single-stranded or,
more typically, double-stranded or a mixture of single- and
double-stranded regions. In addition, "polynucleotide" refers to
triple-stranded regions comprising RNA or DNA or both RNA and DNA.
The term polynucleotide also includes DNAs or RNAs containing one
or more modified bases and DNAs or RNAs with backbones modified for
stability or for other reasons. "Modified" bases include, for
example, tritylated bases and unusual bases such as inosine. A
variety of modifications has been made to DNA and RNA; thus,
"polynucleotide" embraces chemically, enzymatically or
metabolically modified forms of polynucleotides as typically found
in nature, as well as the chemical forms of DNA and RNA
characteristic of viruses and cells. "Polynucleotide" also embraces
relatively short polynucleotides, often referred to as
oligonucleotides.
[0126] Another aspect of the invention relates to an
immunological/vaccine formulation which comprises one or more
polynucleotide(s) encoding Tbp and Hsf-like protein, particularly
those which correspond to protein combinations of the invention.
Such techniques are known in the art, see for example Wolff et al.,
Science, (1990) 247: 1465-8.
[0127] The expression of Tbp and Hsf-like protein, preferably TbpA
and Hsf in such a polynucleotide would be under the control of a
eukaryotic promoter, capable of driving expression within a
mammalian cell. The polynucleotide may additionally comprise
sequence encoding other antigens. Examples of such eukaryotic
promoters include promoters from viruses using mammalian cells as
host including adenovirus promoters, retroviral promoters.
Alternatively, mammalian promoters could be used to drive
expression of TbpA an Hsf-like protein.
Antibodies and Passive Immunisation
[0128] Another aspect of the invention is the use of an immunogenic
composition comprising TbpA and Hsf-like protein to generate immune
globulin which can be used to treat or prevent infection by Gram
negative bacteria or preferably Neisseria, more preferably
Neisseria meningitidis and most preferably Neisseria meningitidis
serogroup B.
[0129] Inocula for polyclonal antibody production are typically
prepared by dispersing the antigenic composition in a
physiologically tolerable diluent such as saline or other adjuvants
suitable for human use to form an aqueous composition. An
immunostimulatory amount of inoculum is administered to a mammal
and the inoculated mammal is then maintained for a time sufficient
for the antigenic composition to induce protective antibodies.
[0130] The antibodies can be isolated to the extent desired by well
known techniques such as affinity chromatography.
[0131] Antibodies can include antiserum preparations from a variety
of commonly used animals e.g. goats, primates, donkeys, swine,
horses, guinea pigs, rats or man. The animals are bled and serum
recovered.
[0132] An immune globulin produced in accordance with the present
invention can include whole antibodies, antibody fragments or
subfragments. Antibodies can be whole immunoglobulins of any class
e.g. IgG, IgM, IgA, IgD or IgE, chimeric antibodies or hybrid
antibodies with dual specificity to Tbp and Hsf. They may also be
fragments e.g. F(ab')2, Fab', Fab, Fv and the like including hybrid
fragments. An immune globulin also includes natural, synthetic or
genetically engineered proteins that acts like an antibody be
binding to specific antigens to form a complex.
[0133] A vaccine of the present invention can be administered to a
recipient who then acts as a source of immune globulin, produced in
response to challenge from the specific vaccine. A subject thus
treated would donate plasma from which hyperimmune globulin would
be obtained via conventional plasma fractionation methodology. The
hyperimmune globulin would be administered to another subject in
order to impart resistance against or treat Neisserial infection.
Hyperimmune globulins of the invention are particularly useful for
treatment or prevention of Neisserial disease in infants, immune
compromised individuals or where treatment is required and there is
no time for the individual to produce antibodies in response to
vaccination. An additional aspect of the invention is a
pharmaceutical composition comprising monoclonal antibodies
reactive against TbpA and Hsf which could be used to treat or
presvent infection by Gram negative bacteria or preferably
Neisseria, more preferably Neisseria meningitidis and most
preferably Neisseria meningitidis serogroup B.
[0134] Such pharmaceutical compositions comprise monoclonal
antibodies that can be whole immunoglobulins of any class e.g. IgG,
IgM, IgA, IgD or IgE, chimeric antibodies or hybrid antibodies with
dual specificity to Tbp and Hsf-like protein. They may also be
fragments e.g. F(ab')2, Fab', Fab, Fv and the like including hybrid
fragments.
[0135] Methods of making monoclonal antibodies are well known in
the art and can include the fusion of splenocytes with myeloma
cells (Kohler and Milstein 1975 Nature 256; 495; Antibodies--a
laboratory manual Harlow and Lane 1988). Alternatively, monoclonal
Fv fragments can be obtained by screening a suitable phage display
library (Vaughan T J et al 1998 Nature Biotechnology 16; 535).
Monoclonal antibodies may also be humanised or part-humanised using
techniques that are well-known in the art.
Serum Bactericidal Assay
[0136] The serum bactericidal assay is the preferred method to
assess the synergistic relationship between antigens when combined
in an immunogenic composition
[0137] Such a synergistic response may be characterised by the SBA
elicited by the combination of antigens being at least 50%, two
times, three times, preferably four times, five times, six times,
seven times, eight times, nine times and most preferably ten times
higher than the SBA elicited by each antigen separately. Preferably
SBA is measured against a homologous strain from which the antigens
are derived and preferably also against a panel of heterologous
strains. (See below for a representative panel for instance BZ10
(B:2b:P1.2) belonging to the A-4 cluster; B16B6 (B:2a:P1.2)
belonging to the ET-37 complex; and H44/76 (B:15:P1.7,16)). SBA is
the most commonly agreed immunological marker to estimate the
efficacy of a meningococcal vaccine (Perkins et al. J Infect Dis.
1998, 177:683-691). Satisfactory SBA can be acertained by any known
method. SBA can be carried out using sera obtained from animal
models (see examples 6-9), or from human subjects.
[0138] A further preferred method of conducting SBA with human sera
is the following. A blood sample is taken prior to the first
vaccination, two months after the second vaccination and one month
after the third vaccination (three vaccinations in one year being a
typical human primary vaccination schedule administered at, for
instance, 0, 2 and 4 months, or 0, 1 and 6 months). Such human
primary vaccination schedules can be carried out on infants under 1
year old (for instance at the same time as Hib vaccinations are
carried out) or 2-4 year olds or adolescents may also be vaccinated
to test SBA with such a primary vaccination schedule. A further
blood sample may be taken 6 to 12 months after primary vaccination
and one month after a booster dose, if applicable.
[0139] SBA will be satisfactory for an antigen or bleb preparation
with homologous bactericidal activity if one month after the third
vaccine dose (of the primary vaccination schedule) (in 2-4 year
olds or adolescents, but preferably in infants in the first year of
life) the percentage of subjects with a four-fold increase in terms
of SBA (antibody dilution) titre (compared with pre-vaccination
titre) against the strain of meningococcus from which the antigens
of the invention were derived is greater than 30%, preferably
greater than 40%, more preferably greater than 50%, and most
preferably greater than 60% of the subjects.
[0140] Of course an antigen or bleb preparation with heterologous
bactericidal activity can also constitute bleb preparation with
homologous bactericidal activity if it can also elicit satisfactory
SBA against the meningococcal strain from which it is derived.
[0141] SBA will be satisfactory for an antigen or bleb preparation
with heterologous bactericidal activity if one month after the
third vaccine dose (of the primary vaccination schedule) (in 2-4
year olds or adolescents, but preferably in infants in the first
year of life) the percentage of subjects with a four-fold increase
in terms of SBA (antibody dilution) titre (compared with
pre-vaccination titre) against three heterologous strains of
meningococcus is greater than 20%, preferably greater than 30%,
more preferably greater than 35%, and most preferably greater than
40% of the subjects. Such a test is a good indication of whether
the antigen or bleb preparation with heterologous bactericidal
activity can induce cross-bactericidal antibodies against various
meningococcal strains. The three heterologous strains should
preferably have different electrophoretic type (ET)-complex or
multilocus sequence typing (MLST) pattern (see Maiden et al. PNAS
USA 1998, 95:3140-5) to each other and preferably to the strain
from which the antigen or bleb preparation with heterologous
bactericidal activity is made or derived. A skilled person will
readily be able to determine three strains with different
ET-complex which reflect the genetic diversity observed amongst
meningococci, particularly amongst meningococcus type B strains
that are recognised as being the cause of significant disease
burden and/or that represent recognised MenB hyper-virulent
lineages (see Maiden et al. supra). For instance three strains that
could be used are the following: BZ10 (B:2b:P1.2) belonging to the
A-4 cluster; B16B6 (B:2a:P1.2) belonging to the ET-37 complex; and
H44/76 (B:15:P1.7,16) belonging to the ET-5 complex, or any other
strains belonging to the same ET/Cluster. Such strains may be used
for testing an antigen or bleb preparation with heterologous
bactericidal activity made or derived from, for instance,
meningococcal strain CU385 (B:4:P1.15) which belongs to the ET-5
complex. Another sample strain that could be used is from the
Lineage 3 epidemic clone (e.g. NZ124 [B:4:P1.7,4]). Another ET-37
strain is NGP165 (B:2a:P1.2).
[0142] Processes for measuring SBA activity are known in the art.
For instance a method that might be used is described in WO
99/09176 in Example 10C. In general terms, a culture of the strain
to be tested is grown (preferably in conditions of iron
depletion--by addition of an iron chelator such as EDDA to the
growth medium) in the log phase of growth. This can be suspended in
a medium with BSA (such as Hanks medium with 0.3% BSA) in order to
obtain a working cell suspension adjusted to approximately 20000
CFU/ml. A series of reaction mixes can be made mixing a series of
two-fold dilutions of sera to be tested (preferably
heat-inactivated at 56.degree. C. for 30 min) [for example in a 50
.mu.l/well volume] and the 20000 CFU/ml meningococcal strain
suspension to be tested [for example in a 25 .mu.l/well volume].
The reaction vials should be incubated (e.g. 37.degree. C. for 15
minutes) and shaken (e.g. at 210 rpm). The final reaction mixture
[for example in a 100 .mu.l volume] additionally contains a
complement source [such as 25% final volume of pretested baby
rabbit serum], and is incubated as above [e.g. 37.degree. C. for 60
min]. A sterile polystyrene U-bottom 96-well microtiter plate can
be used for this assay. A aliquot [e.g. 10 .mu.l] can be taken from
each well using a multichannel pipette, and dropped onto
Mueller-Hinton agar plates (preferably containing 1% Isovitalex and
1% heat-inactivated Horse Serum) and incubated (for example for 18
hours at 37.degree. C. in 5% CO.sub.2). Preferably, individual
colonies can be counted up to 80 CFU per aliquot. The following
three test samples can be used as controls:
buffer+bacteria+complement; buffer+bacteria+inactivated complement;
serum+bacteria+inactivated complement. SBA titers can be
straightforwardly calculated using a program which processes the
data to give a measurement of the dilution which corresponds to 50%
of cell killing by a regression calculation.
[0143] All references or patent applications cited within this
patent specification are incorporated by reference herein.
METHOD OF INDUSTRIAL APPLICATION OF THE INVENTION
[0144] The examples below are carried our using standard
techniques, which are well known and routine to those of skill in
the art, except where otherwise described in detail. The examples
are illustrative, but do not limit the invention.
EXAMPLE 1
Methods for Constructing Strains of Neisseria meningitidis
Serogroup B Used in Outer Membrane Vesicle Preparations
[0145] WO01/09350 provides detailed methods for preparing outer
membrane vesicles and manipulating the bacterial strains from which
the outer membrane vesicles are derived. Where the outer membrane
vesicles are to retain lipoproteins such as TbpB and or
lipopolysaccharides, methods of isolation with low levels or no
deoxycholate are preferred.
EXAMPLE 2
Up-Regulation of the Hsf Protein Antigen in a Recombinant
Neisseiria meningitidis Serogroup B Strain Lacking Functional cps
Genes but Expressing PorA
[0146] As described in WO01/09350 examples, in certain countries,
the presence of PorA in outer membrane vesicles may be
advantageous, and can strengthen the vaccine efficacy of
recombinant improved blebs. In the following example, we have used
a modified pCMK(+) vector to up-regulate the expression of the Hsf
protein antigen in a strain lacking functional cps genes but
expressing PorA. The original pCMK(+) vector contains a chimeric
porA/lacO promoter repressed in E. coli host expressing lacI.sup.q
but transcriptionally active in Neisseria meningitidis. In the
modified pCMK(+), the native porA promoter was used to drive the
transcription of the hsf gene. The gene coding for Hsf was PCR
amplified using the HSF 01-NdeI and HSF 02-NheI oligonucleotide
primers, presented in the table below. Because of the sequence of
the HSF 01-NdeI primer the Hsf protein expressed will contain two
methionine residues at the 5' end. The conditions used for PCR
amplification were those described by the supplier (HiFi DNA
polymerase, Boehringer Mannheim, GmbH). Thermal cycling was the
following: 25 times (94.degree. C. 1 min., 48.degree. C. 1 min.,
72.degree. C. 3 min.) and 1 time (72.degree. C. 10 min., 4.degree.
C. up to recovery). The corresponding amplicon was subsequently
cloned in the corresponding restriction sites of pCMK(+) delivery
vector. In this recombinant plasmid, designed pCMK(+)-Hsf, we
deleted the lacO present in the chimeric porA/lacO promoter by a
recombinant PCR strategy. The pCMK(+)-Hsf plasmid was used as a
template to PCR amplify 2 separate DNA fragments:
[0147] fragment 1 contains the porA 5' recombinogenic region, the
Kanamycin resistance gene and the porA promoter. Oligonucleotide
primers used, RP1 (SacII) and RP2, are presented in the table
below. RP1 primer is homologous to the sequence just upstream of
the lac operator.
[0148] fragment 2 contains the Shine-Dalgarno sequence from the
porA gene, the hsf gene and the porA 3' recombinogenic region.
Oligonucleotide primers used, RP3 and RP4(ApaI), are presented in
the table below. RP3 primer is homologous to the sequence just
downstream of the lac operator. The 3' end of fragment 1 and the 5'
end of fragment 2 have 48 bases overlapping. 500 ng of each PCR (1
and 2) were used for a final PCR reaction using primers RP1 and
RP4. The final amplicon obtained was subcloned in pSL1180 vector
restricted with SacII and ApaI. The modified plasmid pCMK(+)-Hsf
was purified at a large scale using the QIAGEN maxiprep kit and 2
.mu.g of this material was used to transform a Neisseiria
meningitidis serogroup B strain lacking functional cps genes. In
order to preserve the expression of porA, integration resulting
from a single crossing-over was selected by a combination of PCR
and Western blot screening procedures. Kanamycin resistant clones
testing positive by porA-specific PCR and western blot were stored
at -70.degree. C. as glycerol stocks and used for further studies.
Bacteria (corresponding to about 5.10.sup.8 bacteria) were
re-suspended in 50 .mu.l of PAGE-SDS buffer, frozen (-20.degree.
C.)/boiled (100.degree. C.) three times and then were separated by
PAGE-SDS electrophoresis on a 12.5% gel. The expression of Hsf was
examined in Whole-cell bacterial lysates (WCBL) derived from NmB
[Cps-, PorA+] or NmB [Cps-, PorA+, Hsf+]. Coomassie staining
detected a significant increase in the expression of Hsf (with
respect to the endogenous Hsf level). This result confirms that the
modified pCMK(+)-Hsf vector is functional and can be used
successfully to up-regulate the expression of outer membrane
proteins, without abolishing the production of the major PorA outer
membrane protein antigen.
[0149] Oligonucleotides used in this work TABLE-US-00001
Oligonucleotides Sequence Remark(s) Hsf 01-Nde 5'-GGA ATT CCA TAT
GAT GAA CAA NdeI cloning site AAT ATA CCG C-3' Hsf 02-Nhe 5'-GTA
GCT AGC TAG CTT ACC ACT Nhe I cloning site GAT AAC CGA C-3'
GFP-mut-Asn 5'-AAC TGC AGA ATT AAT ATG AAA AsnI cloning site GGA
GAA GAA CTT TTC-3' Compatible with NdeI GFP-Spe 5'-GAC ATA CTA GU
TAT TTG TAG SpeI cloning site AGC TCA TCC ATG-3' Compatible with
NheI RP1 (SacII) 5'-TCC CCG CGG GCC GTC TGA ATA SacII cloning site
CAT CCC GTC-3' RP2 5'-CAT ATG GGC TTC CTT TTG TAA ATT TGA GGG CAA
ACA CCC GAT ACG TCT TCA-3' RP3 5'-AGA CGT ATC GGG TGT TTG CCC TCA
AAT TTA CAA AAG GAA GCC CAT ATG-3' RP4(ApaI) 5'-GGG TAT TCC GGG CCC
TTC AGA ApaI cloning site CGG CGC AGC AGG-3'
EXAMPLE 3
Up-Regulation of the N. meningitidis Serogroup B tbpA Gene by
Promoter Replacement
[0150] The aim of the experiment was to replace the endogenous
promoter region of the tbpA gene by the strong porA promoter, in
order to up-regulate the production of the TbpA antigen. For that
purpose, a promoter replacement plasmid was constructed using E.
coli cloning methodologies. A DNA region (731 bp) located upstream
from the tbpA coding sequence was discovered in the private Incyte
PathoSeq data base of the Neisseria meningitidis strain ATCC 13090.
This DNA contains the sequence coding for TbpB antigen. The genes
are organized in an operon. The tbpB gene will be deleted and
replaced by the CmR/porA promoter cassette. For that purpose, a DNA
fragment of 3218 bp corresponding to the 509 bp 5' flanking region
of tbpB gene, the 2139 bp tbpB coding sequence, the 87 bp
intergenic sequence and the 483 first nucleotides of tbpA coding
sequence was PCR amplified from Neisseria meningitidis serogroup B
genomic DNA using oligonucleotides BAD16 (5'- GGC CTA GCT AGC CGT
CTG AAG CGA TTA GAG TTT CAA AAT TTA TTC-3') and BAD17 (5'-GGC CAA
GCT TCA GAC GGC GTT CGA CCG AGT TTG AGC CTT TGC-3') containing
uptake sequences and NheI and HindIII restriction sites
(underlined). This PCR fragment was cleaned with a High Pure Kit
(Boerhinger Mannheim, Germany) and directly cloned in a pGemT
vector (Promega, USA). This plasmid was submitted to circle PCR
mutagenesis (Jones & Winistofer (1992)) in order to (i) insert
suitable restriction sites allowing cloning of a CmR/PorA promoter
cassette and (ii) to delete 209 bp of the 5' flanking sequence of
tbpB and the tbpB coding sequence. The circle PCR was performed
using the BAD 18 (5'-TCC CCC GGG AAG ATC TGG ACG AAA AAT CTC AAG
AAA CCG-3') & the BAD 19 (5'-GGA AGA TCT CCG CTC GAG CAA ATT
TAC AAA AGG AAG CCG ATA TGC AAC AGC AAC ATT TGT TCC G -3')
oligonucleotides containing suitable restriction sites XmaI, BglII
and XhoI (underlined). The CmR/PorA promoter cassette was amplified
from the pUC D15/Omp85 plasmid previously described, using primers
BAD21 (5'- GGA AGA TCT CCG CTC GAG ACA TCG GGC AAA CAC CCG-3')
& BAD20 (5'- TCC CCC GGG AGA TCT CAC TAG TAT TAC CCT GTT ATC
CC-3') containing suitable restriction sites XmaI, SpeI, BglII and
XhoI (underlined). This PCR fragment was cloned in the circle PCR
plasmid. This plasmid will be used to transform Neisseria
meningitidis serogroup B [cps-] and [cps- porA-] strains.
Integration by double crossing-over in the upstream region of tbpA
will direct the insertion of the porA promoter directly upstream of
the tbpA ATG.
EXAMPLE 4
Construction of a N. meningitidis Serogroup B Strain Up-Regulated
for the Expression of Two Antigens: TbpA and Hsf
[0151] The aim of the experiment was to up-regulate the expression
of TbpA and Hsf simultaneously in the same N. meningitidis
serogroup B strain. The production of TbpA was up-regulated by
replacing its endogenous promoter region by the strong porA
promoter (promoter replacement). In this context, the tbpB gene,
located upstream of tbpA is deleted, and the TbpB protein no longer
present in the outer-membrane. The expression of Hsf was
up-regulated by insertion (homologous recombination) of a second
copy of the corresponding gene at the porA locus (gene delivery).
Both strains have been described in a separate patent referred to
as WO01/09350. The selection markers used in both strategies
(Cm.sup.R or Kan.sup.R) allowed the combination of both
integrations into the same chromosome.
[0152] Total genomic DNA was extracted from the recombinant Nm.B
cps-/TbpA+/PorA+ strain by the Qiagen Genomic tip 500-G protocol.
Ten .mu.g of DNA was restricted o/n with DraIII restriction enzyme
and used to transform Neisseria meningitidis serogroup B by the
classical transformation protocol. Cells used for transformation
were either recombinant NmB cps-/Hsf+/PorA+ (homologous
recombination by 1 crossing over into the porA locus) or
recombinant NmB cps-/Hsf+/PorA- (Allelic exchange/homologous
recombination by 2 crossing over into the porA locus). They were
plated over-night on GC agar containing 200 .mu.g/ml kanamycine,
diluted to DO.sub.650=0.1 in GC liquid medium 10 mM MgCl.sub.2, and
incubated 6 hours at 37.degree. C. under vigorous agitation with 10
.mu.g of DraIII restricted genomic DNA. Recombinant Neisseria
meningitidis resulting from a double crossing over event (PCR
screening) were selected on GC medium containing 200 .mu.g/ml
kanamycin and 5 .mu.g/ml chloramphenicol and analyzed for TbpA and
Hsf expression in OMV preparations. As represented in FIG. 1, the
production of both TbpA and Hsf was significantly increased in the
OMV prepared from the TbpA/Hsf recombinant NmB strain when compared
to the OMV prepared from the control NmB cps- strains. The level of
over expression of each protein in the dual recombinant is
comparable with the level of expression obtained in the
corresponding single recombinants. The level of over expression of
TbpA and Hsf was comparable in PorA+ and PorA- strains (data not
shown). All together, these data demonstrate that: (i) expression
of TbpA and Hsf can be jointly and concomitantly up-regulated into
N. meningitidis and (ii) recombinant blebs enriched for TbpA and
Hsf can be obtained and used for immunization.
Analysis of Hsf and TbpA Content of Outer Membrane Vesicles
Coommassie Blue Stained SDS-PAGE
[0153] 15 .mu.g of protein in outer membrane vesicle preparations
with up-regulation of Hsf or TbpA or both Hsf and TbpA, were
diluted in a sample buffer containing .beta.-mercaptoethanol and
heated at 95.degree. C. for 10 minutes. The samples were then run
on SDS-PAGE polyacrylamide gel (Novex 4-20% Tris-glycine 1.5 mm
2Dwell SDS Page), stained in Coomassie blue for one hour and
destained in several washes of destain. Results are shown in FIG.
1, which shows that the level of Hsf and TbpA are considerably
higher in outer membrane vesicle preparations, derived from N.
meningitidis where their level of expression had been enhanced.
EXAMPLE 5
Immunogenicity of OMVs with Upregulation of Hsf and/or TbpA
[0154] Groups of 20 mice were immunised three times with OMV by the
intramuscular route on days 0, 21 and 28. Each innoculation was
made up of 5 .mu.g (protein content) of OMVs formulated on AlPO4
with MPL. The OMVs were derived from N. meningitidis strain H44/76,
engineered so that capsular polysaccharides and PorA were down
regulated. A comparison was made of OMVs in which Hsf, TbpA, both
Hsf and TbpA or neither were upregulated. On day 41, blood samples
were taken for analysis by ELISA or by serum bactericidal
assay.
ELISA to Detect Antibodies against Hsf
[0155] 96 well microplates (Nunc, Maxisorb) were coated overnight
at 4.degree. C. with 100 .mu.l of 1 .mu.g/ml of specific antigen in
PBS. After washing with NaCl 150 mM Tween 20 0.05%, plates were
saturated with 100 .mu.l of PBS-BSA 1% under shaking at room
temperature for 30 minutes. Between each step (performed under
shaking at room temperature during 30 min and with PBS-BSA 0.2% as
diluant buffer), reagents in excess were removed by washing with
NaCl-Tween 20. One hundred micro-liters of diluted serum samples
were added per micro-well. Bound antibodies were recognized by a
biotinylated anti-mouse Ig (Prosan) (1/2000). The antigen-antibody
complex was revealed by incubation with streptavidin-biotinylated
peroxidase conjugate (Amersham) (1/4000).
OrthoPhenileneDiamine/H.sub.2O.sub.2 (4 mg/10 ml citrate buffer
0.1M pH 4.5+5 .mu.l H.sub.2O.sub.2) is used to reveal the assay.
Plates were incubated for 15 min at room temperature in the dark
before stoping the reaction by addition of 50 .mu.l of 1N HCl. The
absorbance was read at 490 nm. TABLE-US-00002 Titre Mid-Point (on
pooled sera) g1, blebs TbpA-HSF, IM 15471 g2, blebs TbpA, IM 15.41
g3, blebs HSF, IM 14508 g4, blebs CPS(-)PorA(-), IM -- g5,
MPL/AIPO4, IM --
[0156] The results shown in the table above, show that high and
equivalent antibody titres against Hsf were raised by immunisation
with OMVs with upregulation of Hsf or both Hsf and TbpA. Virtually
no antibody against Hsf could be detected in sera raised after
inoculation with adjuvant alone or OMV in which neither Hsf nor
TbpA had been upregulated or OMV in which only TbpA had been
upregulated.
EXAMPLE 6
Serum Bactericidal Activity of Antisera Raised against OMVs with
Up-Regulation of Hsf and/or TbpA
[0157] The serum bactericidal activity of antisera from the mice
inoculated with OMVs with upregulation of Hsf, TbpA, both Hsf and
TbpA or without upregulation were compared in assays using either
the homologous strain H44/76 or the heterologous strain Cu385. The
serum bactericidal assay has been shown to show good correlation
with the protection and is therefore a good indication of how
effective a candidate composition will be in eliciting a protective
immune response.
[0158] Neisseria meningitidis serogroup B wild type strains (H44/76
strain=B:15 P1.7,16 L3,7,9 and CU385 strain=B: 4 P1.19,15 L3,7,9)
were cultured overnight on MH+1% Polyvitex+1% horse serum Petri
dishes at 37.degree. C.+5% CO2. They were sub-cultured for 3 hours
in a liquid TSB medium supplemented with 50 .mu.M of Desferal (Iron
chelator) at 37.degree. C. under shaking to reach an optical
density of approximately 0.5 at 470 nm.
[0159] Pooled or individual serum were inactivated for 40 min at
56.degree. C. Serum samples were diluted 1/100 in HBSS-BSA 0.3% and
then serially diluted two fold (8 dilutions) in a volume of 50
.mu.l in round bottom microplates.
[0160] Bacteria, at the appropriate OD, were diluted in HBSS-BSA
0.3% to yield 1.3 10e4 CFU per ml. 37.5 .mu.l of this dilution was
added to the serum dilutions and microplates were incubated for 15
minutes at 37.degree. C. under shaking. Then, 12.5 .mu.l of rabbit
complement were added to each well. After 1 hour of incubation at
37.degree. C. and under shaking, the microplates were placed on ice
to stop the killing.
[0161] Using the tilt method, 20 .mu.l of each well were platted on
MH+1% Polyvitex+1% horse serum Petri dishes and incubated overnight
at 37.degree. C.+CO2. The CFU's were counted and the percent of
killing calculated. The serum bactericidal titer is the last
dilution yielding.gtoreq.50% killing. TABLE-US-00003 H44/76 CU385 %
% OMV GMT responders GMT responders CPS(-) PorA (-) 93 30% 58 5%
CPS(-) PorA (-) Hsf 158 40% 108 20% CPS(-) PorA (-) TbpA 327 60%
147 30% CPS(-) PorA (-) Hsf - TbpA 3355 100% 1174 80%
[0162] Similar results to those shown in the above table were
obtained in two other similar experiments.
[0163] A dramatic increase in the bactericidal titres (GMT) against
the homologous strain and a heterologous strain were seen after
vaccination with OMV in which both Hsf and TbpA were upregulated.
By comparison, bactericidal GMTs measured on mice vaccinated with
Hsf or TbpA upregulated OMVs were similar to those obtained with
mice vaccinated with control OMVs.
[0164] The benefit of double up-regulation was also clearly
observed in the percentage of mice producing a significant level of
bactericidal antibodies (titres greater than 1/100), particularly
in experiments using the heterologous strain.
EXAMPLE 7
Effect of Mixing Anti-Hsf and Anti-TbpA Sera on Bactericidal
Activity
[0165] Groups of 20 mice were immunised three times with OMV by the
intramuscular route on days 0, 21 and 28. Each inoculation was made
up of 5 .mu.g (protein content) of OMVs formulated on AlPO4 with
MPL. The OMVs were derived from N. meningitidis strain H44/76,
engineered so that capsular polysaccharides and PorA were down
regulated. One group of mice was immunised with control OMVs in
which there was no up-regulation of proteins. In a second group,
Hsf expression was up-regulated, in a third group TbpA expression
was up-regulated and in a fourth group, the expression of both Hsf
and TbpA was up-regulated.
[0166] The sera were pooled, either using sera from mice in the
same group or by mixing sera isolated from the group in with Hsf
alone or TbpA alone had been up-regulated. Serum bactericidal
activity was measured for each of the pooled sera and the results
are shown in the table below. TABLE-US-00004 SBA done on pooled
sera from mice immunized with SBA titer TbpA-Hsf blebs 774 TbpA
blebs 200 Hsf blebs 50 CPS(-) PorA(-) blebs 50 Mix anti-TbpA +
anti-Hsf sera 1162
[0167] The results in the above table show that mixing of anti-Hsf
and anti-TbpA antisera resulted in a much higher serum bactericidal
activity than was achieved by either antisera individually. The
synergistic effect seems to be achieved by the presence of
antibodies against both Hsf and TbpA.
EXAMPLE 8
Truncated Hsf Proteins may Combine Synergistically with TbpA
[0168] A series of truncated Hsf constructs were made using
standard molecular biology procedures. These include a construct
that encodes amino acids 1 to 54 which contains the signal sequence
of Hsf and amino acids 134 to 592 of Hsf (Tr1Hsf). A second
truncated Hsf contained amino acids 1-53 of the signal sequence of
Hsf followed by amino acids 238-592 of Hsf (Tr2Hsf). These two
truncated Hsf constructs and full length Hsf were introduced into
N. Meningitidis B strain MC58 siaD-, Opc-, PorA- so that their
expression would be up-regulated and outer membrane vesicles were
produced using the methods described above.
[0169] The outer membrane vesicle preparations were adsorbed onto
Al(OH)3 and injected into mice on days 0, 21 and 28. On day 42, the
mice were bled and sera prepared. The sera were mixed with sera
from mice vaccinated with up-regulated TbpA OMVs and serum
bactericidal assays were performed as described above.
[0170] Results TABLE-US-00005 Serum Bactericidal titres Group
H44/76 CU385 MC58 PorA + siaD+ 25600 25600 MC58 PorA - siaD - Hsf
1530 800 MC58 PorA - siaD - Tr1Hsf 1015 1360 MC58 PorA - siaD -
Tr2Hsf 50 50 Negative control 50 50 TbpA + MC58 PorA + siaD+ 25600
24182 TbpA + MC58 PorA - siaD - Hsf 2595 1438 TbpA + MC58 PorA -
siaD - Tr1Hsf 4383 2891 TbpA + MC58 PorA - siaD - Tr2Hsf 1568 742
TbpA + Negative control 778 532
[0171] The results shown in the above table reveal that the first
truncation (Tr1Hsf) elicits an immune response which is capable of
combining with antisera against TbpA to produce a larger serum
bactericidal activity than when full length Hsf is used. However,
the extent of the truncation is important and the truncation
produced in Tr2 has a deleterious effect compared to the full
length Hsf. The enhanced bactericidal activity of Tr1Hsf was seen
against both the strains used.
EXAMPLE 9
Serum Bactericidal Activity of Antibodies against TbpA, Hsf and a
Third Meningococcal Protein
[0172] N. meningitidis strain H66/76 in which PorA and capsular
polysaccharides were down regulated as described above, was used as
the background strain for up-regulating TbpA and Hsf, LbpB, D15,
PilQ or NspA using the procedure described above. Outer membrane
vesicles were prepared from each strain as described above.
Recombinant FhaB, FrpC, FrpA/C and Hap were made using techniques
well known in the art as described in PCT/EP99/02766, WO92/01460
and WO98/02547.
[0173] The outer membrane vesicle preparations and recombinant
proteins were adsorbed onto Al(OH)3 and injected into mice on days
0, 21 and 28. On day 42, the mice were bled and sera prepared. The
sera against TbpA and Hsf up-regulated OMVs were mixed with sera
from mice vaccinated with up-regulated LbpB, D15, PilQ or NspA OMVs
or recombinant FhaB, FrpC, FrpA/C or Hap and serum bactericidal
assays were performed as described above.
Results
[0174] Results are shown in the table below. In assays using the
homologous H44/76 stain, the addition of antibodies against a third
meningococcal antigen, with the exception of FrpC, did not produce
a serum bactericidal titre higher than that produced using
antibodies against TbpA and Hsf alone.
[0175] However, the addition of antibodies against a third antigen
was advantageous in serum bactericidal assays using a heterologous
strain. Antibodies against D15 (OMP85), Hap, FrpA/C and LbpB were
particularly effective at increasing the serum bactericidal titre
against the CU385 strain. TABLE-US-00006 Serum Bactericidal Titre
Antisera Mix H44/76 CU385 anti-TbpA-Hsf and nonimmune sera 5378
2141 anti-TbpA-Hsf and anti-FhaB 5260 2563 anti-TbpA-Hsf and
anti-Hap 4577 5150 anti-TbpA-Hsf and anti-FrpA/C 5034 4358
anti-TbpA-Hsf and anti-LbpB 5400 4834 anti-TbpA-Hsf and anti-D15
4823 4657 anti-TbpA-Hsf and anti-PilQ 4708 2242 anti-TbpA-Hsf and
anti-NspA 4738 2518 anti-TbpA-Hsf and anti-FrpC 6082 2300
EXAMPLE 10
Effect of FrpB KO in Outer Membrane Vesicles on Their Ability to
Elicit a Bactericidal Immune Response in Homologous and
Heterologous Strains
[0176] Two strains of H44/76 N. meningitidis were used to prepare
outer membrane vesicle preparations as described in WO01/09350,
using a 0.1% DOC extraction so that the LOS content was around 20%.
Strain B1733 is siaD(-), PorA(-), has upregulation of Tr1 Hsf
(example 8) and lgtB is knocked out. Strain B1820 B1733 is siaD(-),
PorA(-), has upregulation of Tr1 Hsf, lgtB is knocked out and FrpB
is also knocked out. Both strains were cultured in media
supplemented with 60 .mu.M Desferal so that iron regulated proteins
such as LbpA/B and TbpA/B are upregulated.
[0177] The bleb preparations were adsorbed onto Al(OH)3 and 5 .mu.g
were injected intramuscularly into groups of 30 mice on day 0 and
day 21. Blood samples were taken on day 28.
[0178] Serum bactericidal assays were carried out on three L3
strains (the homologous wild type strain H44/76 and two
heterologous L3 strains; NZ124 and M97250687), as described in
example 6.
[0179] Results TABLE-US-00007 Blebs used for H44/76 M97250687 NZ124
inoculation GMT SC GMT SC GMT SC B1733 1518 30/30 151 11/30 70 4/29
B1820 781 19/30 1316 24/30 276 19/30
[0180] GMT indicates the geometric mean titre of the sera in the
SBA. [0181] SC indicates the number of mice seroconverting (SBA
titre>1/100).
[0182] The results clearly show that FrpB KO (B1820) blebs induce a
better heterologous cross-bactericidal response than FrpB(+) blebs
(B1733). The SBA titres were higher and a higher proportion of mice
seroconverted in strains M97250687 and NZ124. The results in the
homologous strain was not quite as good when FrpB was deleted.
[0183] These data suggest that FrpB drives the immune response, but
since this outer membrane protein is highly variable, antibodies
against this protein are only able to induce killing of the
homologous strain.
Sequence CWU 1
1
14 1 31 DNA Artificial Sequence Primer 1 ggaattccat atgatgaaca
aaatataccg c 31 2 31 DNA Artificial Sequence Primer 2 gtagctagct
agcttaccac tgataaccga c 31 3 36 DNA Artificial Sequence Primer 3
aactgcagaa ttaatatgaa aggagaagaa cttttc 36 4 33 DNA Artificial
Sequence Primer 4 gacatactag tttatttgta gagctcatcc atg 33 5 30 DNA
Artificial Sequence Primer 5 tccccgcggg ccgtctgaat acatcccgtc 30 6
51 DNA Artificial Sequence Primer 6 catatgggct tccttttgta
aatttgaggg caaacacccg atacgtcttc a 51 7 48 DNA Artificial Sequence
Primer 7 agacgtatcg ggtgtttgcc ctcaaattta caaaaggaag cccatatg 48 8
33 DNA Artificial Sequence Primer 8 gggtattccg ggcccttcag
acggcgcagc agg 33 9 45 DNA Artificial Sequence Primer 9 ggcctagcta
gccgtctgaa gcgattagag tttcaaaatt tattc 45 10 42 DNA Artificial
Sequence Primer 10 ggccaagctt cagacggcgt tcgaccgagt ttgagccttt gc
42 11 39 DNA Artificial Sequence Primer 11 tcccccggga agatctggac
gaaaaatctc aagaaaccg 39 12 64 DNA Artificial Sequence Primer 12
ggaagatctc cgctcgagca aatttacaaa aggaagccga tatgcaacag caacatttgt
60 tccg 64 13 36 DNA Artificial Sequence Primer 13 ggaagatctc
cgctcgagac atcgggcaaa cacccg 36 14 38 DNA Artificial Sequence
Primer 14 tcccccggga gatctcacta gtattaccct gttatccc 38
* * * * *