U.S. patent application number 10/134687 was filed with the patent office on 2003-02-20 for group b streptococcus vaccine.
This patent application is currently assigned to The Brigham and Women's Hospital Inc.. Invention is credited to Kasper, Dennis L., Madoff, Lawrence C., Michel, James L..
Application Number | 20030035805 10/134687 |
Document ID | / |
Family ID | 21905019 |
Filed Date | 2003-02-20 |
United States Patent
Application |
20030035805 |
Kind Code |
A1 |
Michel, James L. ; et
al. |
February 20, 2003 |
Group B Streptococcus vaccine
Abstract
The invention concerns a vaccine capable of protecting a
recipient from infection caused group B Streptococcus. The vaccine
comprises polysaccharide-protein moieties or protein moieties
without a polysaccharide. The vaccine can contain, inter alia, (a)
a group B Streptoccus polysaccharide conjugated to (b) either the
N-terminal region of the epsilon antigen, a fragment thereof or
their functional derivatives such that the vaccine retains the
ability to elicit protective antibodies against group B
Streptoccus. The vaccine may contain only one type of such
polysaccharide-protein unit or may contain a mixture of more than
one type of unit. Alternatively, the vaccine may contain antigens
from different species of Group B Streptococcus. Additionally, the
invention concerns a passive vaccine obtained following
immunization with either the capsular polysaccharide-protein
conjugate or the non-conjugated protein.
Inventors: |
Michel, James L.; (Chestnut
Hill, MA) ; Madoff, Lawrence C.; (Brooklin, MA)
; Kasper, Dennis L.; (Newton Centre, MA) |
Correspondence
Address: |
STERNE, KESSLER, GOLDSTEIN & FOX PLLC
1100 NEW YORK AVENUE, N.W., SUITE 600
WASHINGTON
DC
20005-3934
US
|
Assignee: |
The Brigham and Women's Hospital
Inc.
|
Family ID: |
21905019 |
Appl. No.: |
10/134687 |
Filed: |
April 30, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10134687 |
Apr 30, 2002 |
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09040181 |
Mar 18, 1998 |
|
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6426074 |
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60039353 |
Mar 19, 1997 |
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Current U.S.
Class: |
424/184.1 |
Current CPC
Class: |
A61K 39/00 20130101;
C07K 14/315 20130101 |
Class at
Publication: |
424/184.1 |
International
Class: |
A61K 039/00; A61K
039/38 |
Goverment Interests
[0002] Part of the work performed during development of the
invention utilized U.S. Government funds. Therefore, the U.S.
Government may have certain rights in this invention.
Claims
What is claimed is:
1. A conjugate vaccine capable of conferring host immunity to an
infection by group B Streptococcus, said vaccine comprising (a) a
group B Streptococcus capsular polysaccharide conjugated to (b) the
N-terminal region of the epsilon antigen or a fragment thereof,
wherein said N-terminal region or fragment thereof is capable of
eliciting protective antibodies against said group B Streptococcus,
and wherein said conjugate vaccine is substantially free of
streptococcal proteins other than said C protein epsilon antigen,
and both said capsular polysaccharide and said C protein contribute
to the development of said protective host immunity.
2. A conjugate vaccine capable of conferring host immunity to an
infection by group B Streptococcus, said vaccine comprising (a) a
group B Streptococcus capsular polysaccharide that elicits
antibodies to group B Streptococcus; conjugated to (b) the
N-terminal region of the epsilon antigen or fragment thereof, and
further comprising said capsular polysaccharide conjugated to a
C-protein selected from the group B Streptococcus C proteins
consisting of at least one of an alpha antigen, a beta antigen,
fragments of said alpha antigen and fragments of said beta antigen,
wherein said N-terminal region or fragment thereof is capable of
eliciting protective antibodies against said group B Streptococcus
and both said capsular polysaccharide and said C proteins
contribute to the development of said protective host immunity.
3. A passive vaccine that confers passive host immunity against an
infection by a C protein epsilon antigen positive group B
Streptococcus wherein said passive vaccine comprises a composition
containing a therapeutically effective amount of antibodies raised
against a conjugate vaccine, said conjugate vaccine comprising (a)
a capsular polysaccharide that elicits antibodies to group B
Streptococcus; conjugated to (b) the N-terminal region of the
epsilon antigen or fragment thereof, and wherein said N-terminal
region or fragment thereof is capable of eliciting protective
antibodies against said group B Streptococcus, and wherein said
conjugate vaccine is substantially free of streptococcal proteins
other than said C protein epsilon antigen, and both said capsular
polysaccharide and said C protein contributes to the development of
said protective host immunity.
4. A passive vaccine that confers passive host immunity to an
infection by group B Streptococcus wherein said passive vaccine
comprises a composition containing a therapeutically effective
amount of antibodies raised against a conjugate vaccine, said
conjugate vaccine comprising: (a) a group B Streptococcus capsular
polysaccharide that elicits antibodies to Group B Streptococcus:
conjugated to (b) the N-terminal region of the epsilon antigen or
fragment thereof, and further comprising said capsular
polysaccharide conjugated to a C-protein selected from the group B
Streptococcus C proteins selected from the group consisting of at
least one of an alpha antigen, a beta antigen, fragments of said
alpha antigen and fragments of said beta antigen, wherein said
N-terminal region or fragment thereof is capable of eliciting
protective antibodies against said group B Streptococcus and both
said capsular polysaccharide and C proteins contribute to the
development of said protective host immunity.
5. A method for preventing or attenuating an infection caused by a
group B Streptococcus comprising administering to an individual, an
effective amount of the conjugate vaccine of any one of claims 1 or
2.
6. A method for preventing or attenuating an infection caused by a
group B Streptococcus comprising administering to a female an
effective amount of the conjugate vaccine of any one of claims 1 or
2, said vaccine capable of conferring immunity to said infection to
an unborn offspring of said female.
7. A method for preventing or attenuating an infection caused by a
group B Streptococcus comprising administering to an individual an
effective amount of an antisera elicited from the exposure of a
second individual to the conjugate vaccine of any one of claims 1
or 2.
8. The conjugate vaccine of any one of claims 1-4 wherein said
capsular polysaccharide is type-specific.
9. The conjugate vaccine of any one of claims 1-4 wherein said
capsular polysaccharide is group-specific.
10. The vaccine of any one of claims 1-4 wherein a functional
equivalent or a derivative of the N-terminal region of the epsilon
antigen is used in the conjugate vaccine.
11. The vaccine of any one of claims 1-4 or 12-15, further
comprising a pharmacologically acceptable solution.
12. The conjugate vaccine of any one of claims 1-4 or 12-15,
wherein said epsilon antigen from more than one strain of Group B
Streptococcus is used in said vaccine.
13. A vaccine capable of conferring host immunity to an infection
by group B Streptococcus, said vaccine comprising the N-terminal
region of the epsilon antigen or a fragment thereof, wherein said
N-terminal region or fragment thereof is capable of eliciting
protective antibodies against said group B Streptococcus.
14. A vaccine capable of conferring host immunity to an infection
by group B Streptococcus, said vaccine comprising the N-terminal
region of the epsilon antigen or fragment thereof, and further
comprising a C-protein selected from the group B Streptococcus C
proteins consisting of at least one of an alpha antigen, a beta
antigen, fragments of said alpha antigen and fragments of said beta
antigen, wherein said N-terminal region or fragment thereof is
capable of eliciting protective antibodies against said group B
Streptococcus.
15. A passive vaccine that confers passive host immunity against an
infection by a C protein epsilon antigen positive group B
Streptococcus wherein said passive vaccine comprises a composition
containing a therapeutically effective amount of antibodies raised
against a first vaccine comprising the N-terminal region of the
epsilon antigen or fragment thereof, and wherein said N-terminal
region or fragment thereof is capable of eliciting protective
antibodies against said group B Streptococcus and said first
vaccine is substantially free of streptococcal proteins other than
said C protein epsilon antigen.
16. A passive vaccine that confers passive host immunity to an
infection by group B Streptococcus wherein said passive vaccine
comprises a composition containing a therapeutically effective
amount of antibodies raised against a first vaccine, said first
vaccine comprising the N-terminal region of the epsilon antigen or
fragment thereof, and further comprising group B Streptococcus C
protein selected from the group consisting of at least one of an
alpha antigen, a beta antigen, or fragments of said alpha antigen
and fragments of said beta antigen, wherein said N-terminal region
or fragment thereof is capable of eliciting protective
antibodies.
17. The plasmid pJMS36 comprising the DNA of FIG. 6A encoding the
N-terminal region of the epsilon antigen.
Description
[0001] This application claims the benefit of the filing date of
U.S. Provisional Application No. 60/039,353, filed Mar. 19, 1997
and incorporates said application herein by reference.
FIELD OF THE INVENTION
[0003] The invention relates to the fields of microbiology and
vaccine technology, and concerns the development of vaccines
capable of conferring immunity to infection by group B
Streptococcus.
BACKGROUND OF THE INVENTION
[0004] Streptococcus agalactiae (GBS) is the leading cause of
neonatal sepsis and early onset meningitis in infants in the United
States, causing over 2,000 deaths each year. Thus GBS is an
important disease pathogen. GBS is also responsible for more than
50,000 cases of maternal postpartum endometritis (Baker, C., In:
Infectious Diseases of the Fetus and Newborn Infant. Remington J.,
et al. eds. Philadelphia: W. B. Saunders, (1990), pp. 742-811).
Recently, GBS has increasingly been seen to cause serious
infections in nonpregnant adults, primarily among the
immunocompromised, elderly individuals and diabetics. Invasive
infection has been diagnosed in 4.4 per 100,000 nonpregnant adults,
with a mortality rate of 21% (Farley, M. M., et al., N. Engl. J Med
328(25):1807-1811 (1993)). This prospective surveillance study
documented an annual incidence of invasive GBS disease to be 9.2
cases per 100,000 population. The incidence of invasive GBS
infections in adults is higher than the incidence of infections
caused by many other important pathogens, including the
meningococci. Although GBS is sensitive to antibiotics, the rapid
onset of the disease in neonates and infants also leads to high
morbidity (50%) and mortality (20%) (Baker, C., In: Infectious
Diseases of the Fetus and Newborn Infant. Remington J., et al. eds.
Philadelphia: W. B. Saunders, (1990), pp. 742-811; Michel, J. L.,
Infectious Disease Practice 13:1-12 (1990)). Therefore, there is a
need to develop vaccines capable of conferring immunity to
infection by GBS.
[0005] The pathogenic streptococci express a number of
surface-associated, opsonic, and protective polysaccharides and
protein antigens (Kehoe, M. A., Vaccine 9:797-806 (1991);
Lachenauer, C. S. & L. C. Madoff, Infect. Immun. 64:4255-4260
(1996)). The type-specific capsular polysaccharide by itself is not
very immunogenic; however, antibodies to conjugates of the capsular
polysaccharides and protein antigens elicit protection in animal
models of GBS infections (Kasper, D., et al., in Vaccines 94, E.
Norrby (ed.), Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., (1994), pp. 113-117; Weis, J. H. in Current Protocols
in Molecular Biology, Vol. 1, F. M., Ausubel, et al., (eds.), John
Wiley & Sons, Inc., New York, (1994), pp. 6.2.1-6.2.3.;
Paoletti, L. C., et al., Infect. Immun. 62:3236-3243 (1994);
Wessels, M. R., et al., Infect. Immun. 61:4760-4766 (1993)).
[0006] GBS also expresses a family of protective and
well-characterized protein antigens called C proteins (alpha and
beta) and R protein (Rib), also known as R4 (Heden, L. et al., Eur.
J Immunol. 21:1481-1490 (1991); Jerlstrom, P. G., et al., Mol.
Microbiol. 5:843-849 (1991); Larsson, C., et al., Infect. Immun.
64:3518-3523 (1996); Michel, J. L., et al., in Genetics and
Molecular Biology of Streptococci, Lactococci, and Enterococci, G.
M. Dunny, et al., (eds.), American Society for Microbiology,
Washington, D.C., (1991), pp. 214-218; Michel, J. L., et al.,
Infect. Immun. 59:2023-2028 (1991); Michel, J. L., et al., Proc.
Natl. Acad Sci. USA. 89:10060-10064(1992);
St.ang.lhammar-Carlemalm, M., et al., J. Exp. Med. 177:1593-1603
(1993); Wastfelt, M., et al., J Biol. Chem. 271:18892-18897
(1996)). Recently an additional C-protein, the epsilon antigen was
discovered (DuBois, N. B. Genetic and phenotypic properties of the
surface proteins of group B Streptococcus and the identification of
a new protein, Bachelor of Arts thesis in Biology, Harvard College,
(1995)
[0007] The C proteins (alpha and beta, and epsilon) are
surface-associated proteins on GBS carrying immunogenic epitopes
that elicit protective antibodies. Antibodies raised against
partially purified C proteins in rabbits were originally shown by
Lancefield et al. to provide passive protection in mice against
challenge with C protein-positive strains; C protein antibodies did
not protect against C protein-negative strains (Lancefield, R. C.,
et al., J. Exp. Med. 142(1):165-179 (1975)). Strains bearing C
proteins resist phagocytosis and inhibit intracellular killing
(Payne, N. R, et al., J. Infec. Dis. 151:672-681 (1985)).
[0008] The C proteins have been divided into two species
(Russel-Jones et al., J. Exp. Med. 160:1476-1484; 1984) that are
independently expressed and antigenically distinct, and have been
defined biochemically and immunologically.
[0009] The alpha antigen is trypsin-resistant and has been shown to
increase resistance to opsonophagocytic killing (Madoffet al.,
Infect. Immun 59:2638-2644,1991)), whereas the beta antigen is
trypsin-sensitive, and binds preferentially to human serum IgA
(Russel-Jones, G. J., J. Exp. Med. 160:1467-1475 (1984)) by a
non-immune mechanism. Additionally, there is the epsilon antigen
which is believed to be a member of the alpha antigen family. The
specific biological roles of these proteins in virulence are not
known.
[0010] The sequence of the alpha C-protein gene (bca) of GBS
reveals four distinct domains: a signal sequence, an N-terminal
region, a tandem repeat region, and a C-terminal anchor region
(Michel, J.L., et al., Proc. Natl. Acad. Sci. USA. 89:10060-10064
(1992)). Presumably, the epsilon antigen gene (bce) has similar,
though not necessarily identical domains. Identification and
characterization of protective epitopes within the domains of the
alpha, beta and epsilon C proteins will help determine the
immunological properties of these regions. These protective
epitopes could be used to develop a C-protein-capsular
polysaccharide conjugate vaccines to protect against a broad range
of GBS strains.
[0011] The alpha antigen gene, bca, was previously cloned from the
prototype Ia/C(.alpha./.beta.) strain A909. The antibodies raised
to the cloned gene product were protective in an animal model
(Michel, J. L. et al., Inf. Immun. 59:2023-2028 (1991)). In
addition, the nucleotide sequence was determined and the derived
animo acid sequence analyzed (Michel, J. L., et al., Proc. Natl.
Acad. Sci. USA. 89:10060-10064 (1992)). The nucleotide sequence of
alpha antigen revealed an open reading frame of 3,060 nucleotides
encoding a precursor protein of 108.7 kilodaltons (kDa). The gene
is composed of four distinct regions. Cleavage of aputative signal
sequence of 56 amino acid yields amature protein of 104.1 kDa The
20.4 kDa N-terminal region shows no homology to previously
described protein sequences and is followed by a series of nine
tandem repeating units that make up 74% of the mature protein. The
repeating units are identical, and each consists of 82 amino acids
with a molecular mass of 8.7 kDa, which is encoded by 246
nucleotides. The C-terminal region of the alpha antigen contains a
membrane anchor domain motif that is shared by a number of
gram-positive surface-associated proteins (Michel, J. L., et al.,
Proc. Natl. Acad. Sci. USA 89(21):10060-10065 (1992)) including the
group A streptococcal M proteins and the IgG-binding proteins of
Staphylococcus (Fischetti, V., et al., Mol. Microbiol. 4:1603-1605,
(1990); Wren, B. W., Mol Microbiol 5(4):797-803 (1991)). Immunoblot
analysis of the native alpha antigen probed with either antisra to
the cloned alpha antigen or an alpha antigen specific monoclonal
antibody, 4G8, which binds to the repeat region, yields a regularly
spaced ladder pattern of heterogeneous peptides. The bands are
spaced at 8-kDa intervals, corresponding to the coding region
defined by a single repeating subunit in the gene (Michel, J. L.,
et al., Proc. Natl. Acad. Sci. USA 89(21):10060-10065 (1992)). This
correlation suggests that the repeat region is responsible for the
laddered peptide heterogeneity of the alpha antigen.
[0012] It was reported that the size of the largest alpha antigen
expressed by a given strain varies widely, from 54 to >200 kDa
Opsonophagocytic killing in the presence of 4G8 antibodies
correlated directly with increasing molecular mass of the alpha
antigen and with the quantity of the alpha antigen expressed on the
surface of GBS. GBS strains bearing the alpha antigen are resistant
to killing by polymorphonuclear leukocytes in the absence of alpha
antigen specific antibody. However, this resistance is not
dependent on the overall size of the antigen expressed by a given
strain. While a given strain produces an alpha antigen with a
consistent size distribution, occasional colonies are isolated
expressing smaller protein sizes; this phenomenon has also been
observed in strain pairs from mothers and infants (Hervas, J. A. et
al., Clin. Infect. Dis. 16:714-718 (1993)) In recent work, it was
found that immune mice infected with GBS bearing the alpha antigen
yielded strains with either smaller or absent alpha antigen
expression (Beseth, B. D., A genetic analysis of phenotypic
diversity of the C protein alpha antigen of group B Streptococcus,
Bachelor of Arts thesis in Biology, Harvard College, (1992);
Madoff, L. C., et al., Proc. Natl. Acad. Sci. USA.
93:4131-4136(1996)).
[0013] To explore the molecular basis for the smaller size of alpha
antigen seen in different strains of GBS, a panel of GBS isolates
was examined and the size of the alpha antigen was compared with
the size and composition of the alpha antigen gene . In doing so,
it was discovered that the alpha antigen gene family was, in fact,
composed of at least two different but related proteins. The new
class of proteins has been named epsilon. The N-terminal region of
the epsilon gene (bce), has been cloned and the nucleotide sequence
analyzed. The molecular basis for size variation among alpha and
epsilon bearing strains of GBS, and their potential for multiple
intramolecular regions of antigenic variability, is potentially
important both in understanding mechanisms of pathogenesis of GBS
and in the development of a conjugate vaccine against GBS.
[0014] Development of an effective C-protein-based conjugate
vaccine is assisted by a better understanding of the immunogenic
and protection inducing effect of the alpha and epsilon antigens,
particularly since the alpha antigen appears to undergo antigenic
variation in isolates from neonates and their mothers (Hervas, J.
A., et al., Clin. Infect. Dis. 16:714-718 (1993); Madoff, L. C., et
al., Proc. Natl. Acad. Sci. USA. 93:4131-4136 (1996)). Deletions in
the number of tandem repeats within the bca (Gravekamp, C., et al.,
Infect. Immun. 64:3576-3583 (1996) and the bce gene may give rise
to antigenically variable polypeptides due to conformational
epitopes that vary as a function of the number of repeats of the
bce gene.
[0015] If an effective conjugate GBS vaccine is to be developed,
protective epitopes that are conserved in the parental stains and
their deletion mutants need to be identified. It has been observed
that the bca gene was deleted in the neonatal isolates in the
tandem repeat region but not in the N- and C-termini of the bca
gene (Beseth, B. D., A genetic analysis of phenotypic diversity of
the C protein alpha antigen of group B Streptococcus, Bachelor of
Arts thesis in Biology, Harvard College, (1992); Madoff, L. C., et
al., Proc. Natl. Acad Sci. USA. 93:4131-4136 (1996)). Therefore,
conserved epitopes are likely to be localized to the N- and
C-terminal regions. The N-terminus of the alpha and epsilon C
protein is a likely location for protective epitopes of these
C-proteins that are conserved in spontaneous deletions and
wild-type strains. However, the C-terminus of the alpha C protein
may not contain protective epitopes, since it is thought to be
involved in the antigen's attachment to the cell-wall peptidoglycan
(Michel, J. L., et al., Proc. Natl. Acad. Sci. USA. 89:10060-10064
(1992); Navarre, W. W. & Schneewind, O., Mol. Microbiol.
14-115-121 (1994); Schneewind, O., et al., EMBO J 12:4803-4811
(1993)).
SUMMARY OF THE INVENTION
[0016] The present invention concerns the development of a
conjugate vaccine to group B Streptococcus (i.e. Streptococcus
agalactiae) that utilizes the N-terminal region of the epsilon
antigen or fragments thereof.
[0017] This novel conjugate vaccine should have the advantages both
of eliciting T-cell dependent protection via the adjuvant action of
the carrier protein and also providing additional protective
epitopes that are present on the group B streptococcal protein
(Insel, R. A, et al., New Eng. J. Med. (Editorial) 319(18):
1219-1220 (1988); Baker, C. J, et al., Rev. of Infec. Dis.
7:458-467 (1985)).
[0018] The advantage to use of the N-terminal region of the epsilon
antigen in the vaccine is that the N-terminal is a likely location
for protective epitopes of the epsilon antigen that are conserved
in spontaneous deletions and wild-type strains. Further, the
N-terminal region of the C-proteins may be more genetically stable
than other regions of the molecule. The repeat regions of other
C-proteins are known to contain antigenic epitotpes but also
exhibit antigenic variability. (Klinge, et al., Infect. Immun.
(April 1997, In press)).
[0019] In detail, the invention provides a conjugate vaccine
capable of conferring host immunity to an infection by group B
Streptococcus, the vaccine comprising (a) a polysaccharide
conjugated to (b) a protein; wherein both the polysaccharide and
the protein are characteristic molecules of the group B
Streptococcus, and wherein the protein is a derivative of the C
protein epsilon antigen N-terminal region that retains the ability
to elicit protective antibodies against the group B
Streptococcus.
[0020] The conjugate vaccine of the invention, can include the
N-terminal region of the epsilon antigen or fragment thereof as the
antigen in the vaccine plus the alpha and/or beta antigen or
fragments thereof in combination with the epsilon antigen. These
conjugate vaccines may also be used to obtain the passive vaccines
of the invention.
[0021] The vaccine of the invention may also comprise the
N-terminal region of the epsilon antigen or fragments thereof as
the antigen in the vaccine either by itself or with the alpha
and/or beta antigen or fragments thereof in combination with the
epsilon antigen. Such fragments or combinations of fragments may be
used in the vaccine of the invention in a non-conjugated form, i.e.
not conjugated to a polysaccharide. These vaccines may also be used
to obtain the passive vaccines of the invention.
[0022] The invention also concerns a method for preventing or
attenuating an infection caused by a group B Streptococcus which
comprises administering to an individual, suspected of being at
risk for such an infection, an effective amount of the conjugate
vaccine of the invention, such that it provides host immunity
against the infection.
[0023] The invention further concerns a method for preventing or
attenuating infection caused by a group B Streptococcus which
comprises administering to a pregnant female an effective amount of
a conjugate vaccine of the invention, such that it provides
immunity to the infection to an unborn offspring of the female.
[0024] The invention also provides a method for preventing or
attenuating an infection caused by a group B Streptococcus which
comprises administering to an individual suspected of being at risk
for such an infection an effective amount of an antisera elicited
from the exposure of a second individual to a conjugate vaccine of
the invention, such that it provides host Immunity to the
infection.
[0025] The invention also provides for the use of the N-terminal
region of the epsilon antigen as an immunogenic composition and for
use of such a composition in diagnostic procedures.
[0026] The invention also provides for a plasmid (ATCC Accession
No. 98365, deposited Mar. 18, 1997 at The American Type Culture
Collection, Rockville, Md.) containing the N-terminal region of the
epsilon antigen. The plasmid is referred to as pJMS36.
BRIEF DESCRIPTION OF THE FIGURES
[0027] FIGS. 1A-1C. Map of the C protein alpha antigen structural
gene and nucleotide probes. The map of the alpha C-protein (FIG.
1A) indicates the signal sequence, the amino terminus, the nine
repeating subunits, and the carboxyl terminal anchor region. The
nucleotide residues are indicated above the map and the amino acid
residues of the mature protein below. Restriction sites (FIG. 1B)
within the gene and the flanking regions are indicated. The
nucleotide probes (FIG. 1C) used for genetic analysis are shown
below the map. The amino terminus probe is a PCR product, the
repeat probe is digested with StyI, and the whole gene probe is
digested with HindIII.
[0028] FIG. 2A-2C. (FIG. 2A) Correlation of the molecular mass of
expressed alpha antigen with the size of its structural gene. The
maximal sizes of the alpha antigen from 18 strains of GBS were
determined by western blot and compared with the sizes of the
structural gene defined by DraI and detected on genomic Southern
blots. The correlation coefficient for this relationship is 0.91.
(FIG. 2B) Correlation of alpha antigen gene size with repeat region
size. The size of the structural gene (DraI) on Southern blots was
compared with the size of the repeat region defined by BsaBI
genomic Southern blots. The correlation coefficient for this
relationship is 0.96. (FIG. 2C) Divergence of two populations of
GBS alpha antigens. Southern blot mapping of the structural gene,
bca, in 18 clinical isolates revealed two populations of alpha
antigen-positive strains. The open boxes are alpha and the closed
boxes are epsilon. The correlation coefficients for both alpha and
epsilon are 0.99, and by multiple regression modeling, the
difference in slope and intercept between these data sets is
significant (p<0.001).
[0029] FIG. 3A-3B. Immunological analysis of the C protein alpha
and epsilon antigens. 4G8 (anti-repeat) antiserum binds to both
alpha and epsilon (FIG. 3A). Epsilon, however, displays a
ladder-pattern that is out of phase with respect to alpha. The
antiserum to the amino terminus of alpha fails to bind epsilon on
immunoblot (FIG. 3B), indicating the region of difference between
the two antigens. Lanes 1, 3, 5, 7, and 9 (72, 144, A909, Carson,
and DK 13) are alpha-positive and lanes 2, 4, 6, 8, and 10 (GH7,
DK8, 515, DK 4-1, and Robinson) are epsilon-positive. Lane 11 is C
protein-negative. Molecular weight markers are to the left and
calculated molecular weights to the right of samples.
[0030] FIG. 4. Genetic comparison of C protein alpha and epsilon
antigens. Southern blot analysis of the alpha and epsilon antigens
revealed homology in the repeat regions, but divergence in the
amino terminus. Blot A was probed with he alpha-repeat probe, and
blot B was probed with he alpha-amino terminus probe. Samples 1 and
2 (A909 and H36B) are alpha-positive, samples 3 and 4 (515 and
Davis) are epsilon-positive, and sample 5 (18RS21) is C
protein-negative. Molecular weight markers are to the left and
calculated molecular weights to the right of the samples.
[0031] FIG. 5. Cloning strategy for the amino terminus of the C
protein epsilon antigen. The amino terminus of the epsilon antigen
was cloned using a PCR product as shown. The template for PCR was
genomic DNA from the epsilon-positive strain, 515.
[0032] FIG. 6A-6B. Nucleotide and amino acid sequences of the
epsilon antigen. The nucleotide sequence (FIG. 6A) of the cloned
amino terminus of epsilon was determined and translated to give the
amino acid sequence (FIG. 6B). Structural features of the gene are
indicated, as well as the -10-promoter consensus sequence and the
ribosomal binding site (RBS).
[0033] FIG. 7. Divergence of the alpha and epsilon antigen amino
terminal The nucleotide sequence of epsilon is compared to alpha,
showing homology in the upstream flanking DNA and the signal
sequence. The amino termini, however, are distinct. Similar results
are seen when the amino acid sequences are compared.
[0034] FIG. 8. Distribution of sizes of the repetitive surface
antigens alpha and epsilon. 24 strains of GBS expressing the alpha
or the epsilon antigen were plotted according to the size of
peptide expressed. The chart shows a distribution around 120 kD
which corresponds to the size of peptide from the prototype nine
repeat peptide.
[0035] FIG. 9. Restriction maps of bca gene subclones, defining the
location of monoclonal antibody 4G8 binding within the alpha
C-protein. pJMS23-1 is the bca gene clone. pJMS23-9 was used to
develop nested deletions of the bca gene (Michel, J. L., et al.,
Proc. Natl. Acad. Sci. 89:10060-10064 (1992)). pSKOF1-13 was
derived from pJMS23-9 and contains part of the repeat region.
pDEK14 encodes the alpha C-protein N-terminus and pDEK15 encodes
the alpha C-protein C-terminus. The restriction endonuclease sites
are A, AluI; N, NsiI; H, HindIII; and E, EcoRI. Promoters include
Sp6 and T7, and His represents a six-residue histidine tag of
vector pET24a.
[0036] FIG. 10. Expression of the alpha C-protein N- and C-termini.
As seen in Coomassie-stained 15% polyacrylamide gel of extracts of
E. coli strain BL21(DE3) containing pET24a (lane 1), pDEK14 (lane
2), and pDEK15 (lane 3) after induction with IPTG. Arrows indicate
a 23-kDa peptide that corresponds to the N-terminal fiagment and a
10-kDa peptide that corresponds to the C-terminal fragment.
[0037] FIG. 11. Detection of epitopes located in the repeat region
of the alpha C-protein by monoclonal antibody 4G8. Shown are
western blot of extracts of GBS strains A909 (positive control,
lane 1) and 090 (negative control, lane 2); .E coli stain DH5a
containing pJMS23-1 (positive control, lane 3), pGEM-7Zf(-)
(negative control, lane 4); pSKOFI-13 (alpha C-protein repeat
region, lane 5); and E. coli strain BL21(DE3) containing pET24a
(negative control, lane 6), pDEK14 (alpha C-protein N-terminus,
lane 7), and pDEK15 (alpha C-protein C-terminus, lane 8). Arrow
indicates 40-kDa band that corresponds to the expressed gene
product from pSKOF1-13.
[0038] FIG. 12. ELISA inhibition. Relative binding affinities of
monoclonal antibody 4G8 to purified alpha C proteins with 1, 2, 9,
and 16 repeats are shown. Plates were coated with 9-repeat alpha C
protein (0.125 .mu.g/ml). 4G8 was used as the primary antibody
(dilution 1:8,000), and alkaline phosphatase-conjugated antibody to
rabbit IgG Fc (1:2,000) was used as the secondary antibody.
Two-fold dilutions were made of 1-, 2-, 9-, and 16-repeat alpha C
protein, and used as inhibiting antigen (starting dilution 5
.mu.g/ml).
[0039] FIG. 13A-13B. FIG. 13A. Induction of expression and
purification of the alpha C-protein N-terminal peptide. Lanes 1 and
2 show Coomassie-stained gels of extracts of E. coli containing
pDEK14 before and after induction with IPTG, respectively. Lane 3
shows a 10-.mu.g sample of the eluate from a lysate of E. coli
containing pDEK14 after Ni.sup.2+ affinity column chromatography.
FIG. 13B. Detection of native alpha C-protein by antibodies to the
gene product of pDEK14. Western blots are shown of extracts of GBS
strains 090 (negative control, lane 1) and A909 (lane 2) probed
with antibodies raised to the alpha C-protein N-terminus.
[0040] FIG. 14. Opsonophagocytosis Assay. Results of an
opsonophagocytosis assay used to determine whether alpha C-protein
N-terminal antibodies were opsonic. Negative controls included the
presence of heat-killed complement (c-), the absence of PMN (PMN-),
and preimmunization sera (pre-bleed). Polyclonal antibodies to the
alpha C-protein (anti-alpha). Polyclonal antibodies (anti-alpha)
served as the positive control. Opsonization is expressed as
log-kill: the log number of GBS at start of the assay minus the log
number of GBS after 1 hr of incubation.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0041] Significance and Clinical Perspective
[0042] Maternal immunoprophylaxis with a vaccine to group B
Streptococcus has been proposed as a potential route for protecting
against infection both in the mother and in the young infant
through the peripartum transfer of antibodies (Baker, C. J. et al.,
New Eng. J. Med. (Editorial) 314(26):1702-1704 (1986); Baker, C. J.
et al., New Eng. J. Med. 319:1180 (1988); Baker, C. J. et al., J.
Infect. Dis. 7:458 (1985)). As is the case with other encapsulated
bacteria, susceptibility to infection correlates with the absence
of type-specific antibody (Kasper, D. L., et al., J. Clin. Invest.
72:260-269 (1983), Kasper, D. L., et al., Antibiot. Chemother.
35:90-100 (1985)). The lack of opsonically active type-specific
anti-capsular antibodies to group B Streptococcus is a risk factor
for the development of disease following colonization with group B
Streptococcus (Kasper, D. L. et al., J. Infec. Dis. 153:407415
(1986)).
[0043] One approach has been to vaccinate with purified
type-specific capsular polysaccharides. Methods of producing such
vaccines, and the use of such vaccines to immunize against group B
Streptococcus are disclosed by Kasper, D. L. (U.S. Pat. No.
4,207,414 and U.S. Reissue Pat. No. RE31672, and U.S. Pat. Nos.
4,324,887, 4,356,263, 4,367,221, 4,367,222, and 4,367,223), by
Carlo, D. J. (U.S. Pat. No. 4,413,057, European Patent Publication
38,265), and by Yavordios, D. et al. European Patent Publication
71,515), all of which references are incorporated herein by
reference.
[0044] Although the polysaccharide capsule of group B Streptococcus
is well characterized and has been shown to play a role in both
virulence and immunity (Kasper, D. L. J. Infect. Dis. 153:407
(1986)), these capsular components have been found to vary in their
immunogenicity depending both on the specific capsular type and on
factors in the host's immune system (Baker, C. J, et@, Rev. of
Infec. Dis. 7:458467 (1985)). A clinical trial evaluating a
capsular polysaccharide vaccine of group B Streptococcus showed an
overall response rate of 63% and indicated that such a vaccine was
not optimally immunogenic (Baker C. J, et al., New Eng. J. Med.
319(18):1180-1185 (1988)).
[0045] Differences in immunogenicity have also been observed with
the capsular polysaccharides of other bacteria. For example, the
vaccine against the type C meningococcal capsule is highly active
while the group B meningococcal polysaccharide vaccine is not
immunogenic (Kasper, D. L. et al., J. Infec. Dis. 153:407415
(1986)). T-cell independent functions of the host's immune system
are often required for mounting an antibody response to
polysaccharide antigens. The lack of a T-cell independent response
to polysaccharide antigens may be responsible for the low levels of
antibody against group B Streptococcus present in mothers whose
children subsequently develop an infection with group B
Streptococcus. In addition, children prior to 18 or 24 months of
age have a poorly developed immune response to T-cell independent
antigens.
[0046] Determinants of Virulence and Immunity in Group B
Streptococcus
[0047] There are at least nine serotypes of group B Streptococcus
that share a common group specific polysaccharide antigen. However,
antibody of the group antigen is not protective in animal models.
Lancefield originally classified group B Streptococcus into four
serotypes (Ia, Ib, II and III) using precipitin techniques. The
composition and structure of the unique type-specific capsular
polysaccharides for each of the serotypes was subsequently
determined (Jennings, H. J, et al., Biochem. 22:1258-1264 (1983),
Kasper, D. L. et al., J. Infec. Dis. 153:407-415 (1986), Wessels,
M. R, et al., Trans. Assoc. Amer. Phys. 98:384-391 (1985)).
Wilkinson defined a fifth serotype, Ic, by the identification of a
protein antigen (originally called the Ibc protein) present on all
strains of serotype Ib and some strains with the type Ia capsule
(Wilkinson, H. W, et al., J. Bacteriol. 97:629-634 (1969),
Wilkinson, H. W, et al., Infec. and Immun. 4:596-604 (1971)). This
protein was later found to vary in prevalence between the different
serotypes of group B Streptococcus but was absent in serotype Ia
(Johnson, D. R, et al., J. Clin. Microbiol. 19:506-510 (1984)).
[0048] The nomenclature, however, has been changed to classify the
serotypes of group B Streptococcus solely by the capsular
type-specific polysaccharides, with a fifth capsular type having
been described (type IV) (Pritchard, D. G, et al., Rev. Infec. Dis.
10(8):5367-5371 (1988)) as well as a type V and VII (Lachaenauer et
al., Inf. Immun. 64:42554260, (1996)).Therefore, the typing of
group B Streptococcus strains is no longer based on the antigenic
Ibc protein, which is now called the C protein. The type Ic strain
is reclassified as serotype Ia on the basis of its capsular
polysaccharide composition, with the additional information that it
also carries the C protein.
[0049] Immunological, epidemiological and genetic data suggest that
the type-specific capsule plays an important role in immunity to
group B Streptococcus infections. The composition and structure of
the type-specific capsular polysaccharides and their role in
virulence and immunity have been the subjects of intensive
investigation (Ferrieri, P. et al., Infec. Immun. 27:1023-1032
(1980), Krause, R. M, et al., J. Exp. Med. 142:165-179 (1975),
Levy, N. J, et al., J. Infec. Dis. 149:851-860 (1984), Wagner, B,
et al., J. Gen. Microbiol. 118:95-105 (1980), Wessels, M. R, et
al., Trans. Assoc. Amer. Phys. 98:384-391 (1985)).
[0050] Controversy has existed regarding the structural arrangement
of the type-specific and group B streptococcal polysaccharides on
the cell surface, on the immunologically important determinants
with in the type-specific polysaccharide, and on the mechanisms of
capsule determined virulence of group B Streptococcus (Kasper, D.
L. et al., J. Infec. Dis. 153:407415 (1986)). To study the role of
the capsule in virulence, Rubens et al. used transposon mutagenesis
to create an isogeneic strain of type m group B Streptococcus that
is unencapsulated (Rubens, C. E, et al., Proc. Natl. Acad. Sci. USA
84:7208-7212 (1987)). They demonstrated that the loss of capsule
expression results in significant loss of virulence in a neonatal
rat model. However, the virulence of clinical isolates with similar
capsular composition varies widely. This suggests that other
bacterial virulence factors, in addition to capsule, play a role in
the pathogenesis of group B Streptococcus.
[0051] A number of proteins and other bacterial products have been
described in group B Streptococcus whose roles in virulence and
immunity have not been established, CAMP (Christine Atks-Much
Peterson) factor, pigment (probably carotenoid), R antigen, X
antigen, anti-phagocytic factors and poorly defined "pulmonary
toxins" (Ferrieri, P, et al., J. Exp. Med. 151:56-68 (1980);
Ferrieri, P. et al., Rev. Inf. Dis. 10(2):1004-1071 (1988); Hill,
H. R. et al., Sexually Transmitted Diseases, McGraw-Hill, pp.
397-407). The C proteins are discussed below.
[0052] Isogeneic strains of group B Streptococcus lacking hemolysin
show no decrease in virulence in the neonatal rat model (Weiser, J.
N, et al., Infec. and Immun. 55:2314-2316 (1987)). Both hemolysin
and neuraminidase are not always present in clinical isolates
associated with infection. The CAMP factor is an extracellular
protein of group B Streptococcus with a molecule weight of 23,500
daltons that in the presence of staphylococcal beta-toxin (a
sphingomyelinase) leads to the lysis of erythrocyte membranes. The
gene for the CAMP factor in group B Streptococcus was recently
cloned and expressed in E. coli (Schneewind, O, et al., Infec. and
Immun. 56:2174-2179 (1988)). The role, if any, of the CAMP factor,
X and R antigens, and other factors listed above in the
pathogenesis of group B Streptococcus is not disclosed in the prior
art (Febrenbach, F. J, et al., In: Bacterial Protein Toxins, Gustav
Fischer Verlag, Stuttgart (1988); Hill, H. R. et al., Sexually
Transmitted Diseases, McGraw-Hill, NY, pp. 397407 (1984)).
[0053] The C protein(s) are a group of a cell surface associated
protein antigens of group B Streptococcus that were originally
extracted from group B Streptococcus by Wilkinson et al.
(Wilkinson, H. W, et al., J. Bacteriol. 97:629-634 (1969),
Wilkinson, H. W, et al., Infec. and Immun. 4:596-604 (1971)). They
used hot hydrochloric acid (HCl) to extract the cell wall and
trichloroacetic acid (TCA) to precipitate protein antigens. Two
antigenically distinct populations of C proteins have been
described: (1) A group of proteins that are sensitive to
degradation by pepsin but not by trypsin, and called either TR
(trypsin resistant) or alpha (a). (2) Another group of group B
Streptococcus proteins that are sensitive to degradation by both
pepsin and trypsin, and called TS (trypsin sensitive) or beta (p)
(Bevanger, L, et al., Acta Path. Microbiol Scand Sect. B. 87:51-54
(1979), Bevanger, L, et al., Acta Path. Microbiol. Scand. Sect. B.
89:205-209 (1981), Bevanger, L. et al., Acta Path. Microbiol.
Scand. Sect. B. 91:231-234 (1983), Bevanger, L. et al., Acta Path.
Microbiol. Scand. Sect. B. 93:113-119 (1985), Bevanger, L, et al.,
Acta Path. Microbiol Immunol . Scand . Sect. B. 93 :121-124 (1985),
Johnson, D. R, et al., J. Clin. Microbiol. 19:506-510 (1984),
Russell-Jones, G. J, et al., J. Exp. Med. 160:1476-1484
(1984)).
[0054] In 1975, Lancefield et aL used mouse protection studies with
antisera raised in rabbits to define the C proteins functionally
for their ability to confer protective immunity against group B
Streptococcus strains carrying similar protein antigens
(Lancefield, R. C, et al., J. Exp. Med. 142:165-179 (1975)).
Numerous investigators have obtained crude preparations of
antigenic proteins from group B Streptococcus, that have been
called C proteins, by chemical exaction from the cell wall using
either HCl or detergents (Bevanger, L, et al., Acta Path.
Microbiol. Scand. Sect. B. 89:205-209 (1981), Bevanger, L. et al.,
Acta Path. Microbiol. Scand. Sect. B. 93:113-119 (1985),
Russell-Jones, G. J, et al., J. Exp. Med. 160:1476-1484 (1984),
Valtonen, M. V, et al., Microb. Path. 1:191-204 (1986), Wilkinson,
H. W, et al., Infec. and Immun. 4.596-604(1971)). The reported
sizes for these antigens have varied between 10 and 190
kilodaltons, and a single protein species has not been isolated or
characterized (Ferrieri, P. et al., Rev. Inf. Dis. 10(2):1004-1071
(1988)).
[0055] By screening with protective antisera, C proteins can be
detected in about 60% of clinical isolates of group B
Streptococcus, and are found in all serotypes but with differing
frequencies (Johnson, D. R, et al., J. Clin. Microbiol. 19:506-510
(1984)). Individual group B Streptococcus isolates may have both
the TR and TS antigens, or only one, or neither of these antigens.
Except for the ability of the partially purified antigens to elicit
protective immunity, the role of these antigens in pathogenesis has
not been studied in vitro. In vivo studies with group B
Streptococcus strains that carry C proteins provides some evidence
that the C proteins may be responsible for resistance to
opsonization (Payne, N. R, et al., J. Infec. Dis. 151:672681
(1985)), and the C proteins may inhibit the intracellular killing
of group B Streptococcus following phagocytosis (Payne, N. R, et
al., Infect. and Immun. 55:1243-1251 (1987)). It has been shown
that type II strains of group B Streptococcus carrying the C
proteins are more virulent in the neonatal rat sepsis model
(Ferrieri, P, et al., Infect. Immun. 27:1023-1032 (1980), Ferrieri,
P. et al., Rev. Inf. Dis. 10(2): 1004-1071 (1988)). Since there is
no genetic data on the C proteins, isogeneic strains lacking the C
proteins have not previously been studied. There is evidence that
one of the TS, or .beta., C proteins binds to IgA (Russell-Jones,
G. J, et al., J. Exp. Med. 160:1476-1484 (1984)). The role, if any,
that the binding of IgA by the C proteins has on virulence is,
however, not disclosed.
[0056] In 1986, Valtonen et al. isolated group B Streptococcus
proteins from culture supernatants that elicit protection in the
mouse model (Valtonen, M. V, et al., Microb. Path. 1:191-204
(1986)). They identified, and partially purified, a trypsin
resistant group B Streptococcus protein with a molecular weight of
14,000 daltons. Antisera raised to this protein in rabbits
protected mice against subsequent challenge with type Ib group B
Streptococcus (89% protection). This protein is, by Lancefield's
definition, a C protein. However, when antisera raised against this
protein were used to immunoprecipitate extracts of group B
Streptococcus antigens, a number of higher molecular weight
proteins were found to be reactive. This suggested that the 14,000
m.w. protein may represent a common epitope of several group B
Streptococcus proteins, or that it is a degradation product found
in the supernatants of group B Streptococcus cultures. The
diversity in the sizes in C proteins isolated from both the
bacterial cells and supernatants suggests that the C proteins may
represent a gene family, and maintain antigenic diversity as a
mechanism for protection against the immune system.
[0057] The range of reported molecular weights and difficulties
encountered in purifying individual C proteins are similar to the
problems that many investigators have faced in isolating the M
protein of group A Streptococcus (Dale, J. B, et al., Infec. and
Immun. 46(1):267-269 (1984), Fischetti, V. A, et al., J. Exp. Med.
144:32-53 (1976), Fischetti, V. A, et al., J. Exp. Med.
146:1108-1123 (1977)). The gene for the M protein has now been
cloned and sequenced, and found to contain a number of repeated DNA
sequences (Hollingshead, S. K, et al., J. Biol. Chem. 261:1677-1686
(1986), Scott, J. R, et al., Proc. Natl. Acad. Sci USA 82:1822-1826
(1986), Scott, J. R, et al., Infec. and Immun. 52:609-612 (1986)).
These repeated sequences may be responsible for
post-transcriptional processing that results in a diversity in the
size of M proteins that are produced. The mechanism by which this
occurs is not understood. The range of molecular weights described
for the C proteins of group B Streptococcus might result from a
similar process.
[0058] Cleat et al. attempted to clone the C proteins by using two
preparations of antisera to group B Streptococcus obtained from
Bevanger (.alpha. and .beta.) to screen a library of group B
Streptococcus DNA in E. coli (Bevanger, L. et al., Acta Path.
Microbiol. Immunol. Scand. Sect. B. 93:113-119 (1985), Cleat, P. H,
et al., Infec. and Immun. 55(5):1151-1155 (1987), which references
are incorporated herein by reference). These investigators
described two clones that produce proteins that bind to
antistreptococcal antibodies. However, they failed to determine
whether either of the cloned proteins had the ability to elicit
protective antibody, or whether the prevalence of these genes
correlated the with group B Streptococcus strains known to carry
the C proteins. The role of the cloned gene sequences in the
virulence of group B Streptococcus was not investigated. Since the
C proteins are defined by their ability to elicit protective
antibodies, this work failed to provide evidence that either of the
clones encodes a C protein.
[0059] The Conjugated Vaccine of the Present Invention
[0060] Embodiments of the present invention surmount the
above-discussed deficiencies of prior vaccines to group B
Streptococcus through the development of a conjugate vaccine in
which the capsular polysaccharides are covalently linked to a
protein backbone. This approach supports the development of a
T-cell dependent antibody response to the capsular polysaccharide
antigens and circumvents the T-cell independent requirements for
antibody production (Baker, C. J, et al., Rev. of Infec. Dis.
7:458467 (1985), Kasper, D. L. et al., J. Infec. Dis. 153:407415
(1986), Kasper, D., et al., Vaccines 94, pgs. 113-117, Cold Spring
Harbor Laboratory Press (1994)) which references are incorporated
herein by reference).
[0061] In a conjugate vaccine, an antigenic molecule, such as the
capsular polysaccharides of group B Streptococcus (discussed
above), is covalently linked to a "carrier" protein or polypeptide.
The linkage serves to increase the antigenicity of the conjugated
molecule. Methods for forming conjugate vaccines from an antigenic
molecule and a "carrier" protein or polypeptide are known in the
art (Jacob, C. O, et al., Eur. J. Immunol. 16:1057-1062 (1986);
Parker, J. M. R. et al., In: Modern Approaches to Vaccines,
Chanock, R. M. et al., eds, pp. 133-138, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1983); Zurawski, V. R, et
al., J. Immunol. 121:122-129 (1978); Klipstein, F. A, et al.,
Infect. Immun. 37:550-557 (1982); Bessler, W. G, Immunobiol.
170:239-244 (1985); Posnett, D. N, et al., J. Biol. Chem.
263:1719-1725 (1988); Ghose, A. C, et al., Molec. Imumol.
25:223-230 (1988); all of which references are incorporated herein
by reference).
[0062] A prototype model for conjugate vaccines was developed
against Hemophilus influenzae (Anderson, P, Infec. and Immun.
39:223-238 (1983); Chu, C, et al., Infect. Immun. 40:245-256
(1983); Lepow, M, Pediat. Infect. Dis. J. 6:804-807 (1987), which
references are incorporated herein by reference), and this model
may be employed in constructing the novel vaccines of the present
invention. Additional methods for producing such a conjugate
vaccine are disclosed by Anderson, P. W, et al., European Patent
Publication 245,045; Anderson, P. W, et al., U.S. Pat. Nos.
4,673,574 and 4,761,283; Frank, R. et al., U.S. Pat. No. 4,789,735;
European Patent Publication No. 206,852; Gordon, L. K, U.S. Pat.
No. 4,619,828; Beachey, E. H, U.S. Pat. No. 4,284,537; Kuo et al.,
U.S. Pat. No. 5,192,540; Cryz et al., U.S. Pat. No. 5,370,872;
Kasper et. al., U.S. Pat. No. 5,302,386 and Jennings et al., U.S.
Pat. No. 5,576,002 all of which references are incorporated herein
by reference.
[0063] The protein backbones for conjugate vaccines such as the
Hemophilus influenzae vaccine have utilized proteins that do not
share antigenic properties with the target organism from which the
bacterial capsular polysaccharides were obtained (Ward, J. et al.,
In: Vaccines, Plotkin, S. A, et al., eds, Saunders, Philadelphia,
page 300 (1988). A conjugate vaccine of Group B Streptococcus type
III polysaccharride and tetanus toxoid used in baboons has been
shown to induce type III-specific immunoglobulin G with opsonic
activity. (Paoletti et al., Inf. Immun. 64:677-679 (1996)).
Recently a conjugate vaccine has also been used in human trials
(Kasper D., et al., 98:2308-2314, (1996)).
[0064] In contrast, the conjugate vaccine of the present invention
employs immunogenic proteins, e.g. the amino terminal region of the
epsilon antigen, of group B Streptococcus as the backbone for a
conjugate vaccine. Alternatively the conjugate vaccine may employ
the N-terminal region of the epsilon antigen or fragments thereof
and in addition, other C-proteins such as the alpha and/or beta
antigen. Such an approach is believed to lead to more effective
vaccines (Insel, R. A, et al., New Eng. J. Med. (Editorial)
319(18):1219-1220 (1988)).
[0065] The isolation and characterization of the N-terminal region
of the epsilon antigen may allow optimization of both the adjuvant
and antigenic properties of the polypeptide backbone/carrier of the
conjugate vaccine.
[0066] Genetic Studies of the C Proteins
[0067] The present invention thus concerns the cloning of
N-terminal region of the epsilon C protein of group B
Streptococcus, its role in virulence and immunity, and its ability
to serve as an immunogen for a conjugate vaccine against group B
Streptococcus. Discussion of the N-terminal region of the epsilon
antigen refers to the DNA and amino acid sequence of FIGS. 6A-6B
and fragments thereof.
[0068] Despite the extensive literature available on cloning in
many groups of streptococci, only limited genetic manipulations
have been accomplished in group B Streptococcus (Macrina, F. L,
Ann. Rev. Microbiol. 38:193-219 (1984), Wanger, A. R, et al.,
Infec. and Immun. 55:1170-1175 (1987)). The most widely used
technique in group B Streptococcus has been the development of
Tn916 and its use in transposon mutagenesis (Rubens, C. E, et al.,
Proc. Natl. Acad. Sci. USA 84:7208-7212 (1987), Wanger, A. R, et
al., Res. Vet. Sci. 38:202-208 (1985)). However, since it would
appear that there is more than one gene for the C proteins and the
protective antisera bind to several proteins, screening for the C
protein genes by transposon mutagenesis is impractical.
[0069] The present invention accomplishes the cloning of the
N-terminal region of the epsilon antigen. A specific protocol for
cloning the epsilon amino terminal region is provided in Example 7.
Additionally, the protocol-used to obtain expression of the
N-terminal region of the alpha antigen (as described in Example 8)
should be equally applicable for expressing the epsilon antigen
N-terminal region. Of course, other methods known to those of skill
in the art of cloning and expressing recombinant DNA may also be
considered.
[0070] For example, as in PCT Application WO94/10317 it may be
desirable to employ a cloning vector that could be rapidly screened
for expression of proteins which bind to naturally elicited
antibodies to group B Streptococcus. Since such antibodies are
heterologous polyclonal antibodies and not monoclonal antibodies,
it is necessary that a vector be employed which could be easily
screened through many positive clones to identify genes of
interest.
[0071] A number of techniques are available for screening clones
for the expression of antigens that bind to a specific antisera
(Aruffo, A, et al., Proc. Natl. Acad. Sci. USA 84:8573-8577
(1987)). The most widely used system, .lambda.gt11, was developed
by Young and Davis (Huynh, T. V. et al., In: DNA Cloning, A
Practical Approach, Vol. 1 (Glover, D. M, Ed.) IRL Press,
Washington pp. 49-78 (1985); Wong, W. W, et al., J. Immunol.
Methods. 82:303-313 (1985), which references are incorporated
herein by reference). This technique allows for the rapid screening
of clones expressed in the lysogenic phage whose products are
released by phage lysis. Commonly faced problems with this system
include the requirement for subcloning DNA fragments into plasmid
vectors for detailed endonuclease restriction mapping, preparing
probes and DNA sequencing. In addition, the preparation of DNA from
phage stocks is cumbersome and limits the number of potentially
positive clones that can be studied efficiently. Finally, the
preparation of crude protein extracts from cloned genes is
problematic in phage vector hosts.
[0072] To circumvent these problems, a plasmid vector was developed
for screening cloned bacterial chromosomal DNA for the expression
of proteins involved in virulence and/or immunity. The vector is
prepared by modifying the commonly used plasmid cloning vector,
pUC12 (Messing, J, et al., Gene 19:269-276 (1982); Norrander, J, et
al., Gene 26:101-106 (1983); Vieira, J, et al., Gene 19:259-268
(1982); which references are incorporated herein by reference).
[0073] Using this system, plasmid clones can be easily manipulated,
mapped with restriction endonucleases and their DNA inserts
sequences, probes prepared and gene products studied without the
necessity for subcloning. pUC12 is a 2.73 kilobase (kb) high copy
number plasmid that carries a Co1E1 origin of replication,
ampicillin resistance and a polylinker in the lacZ gene (Ausubel,
F. M, et al., Current Topics in Molecular Biology; Greene Publ.
Assn./Wiley Interscience, NY (1987) which reference is incorporated
herein by reference).
[0074] Several modifications are made in the polylinker of pUC12
(Aruffo, A, et al., Proc. Natl. Acad. Sci. USA 84:8573-8577 (1987)
which reference is incorporated herein by reference). The overall
plan in altering pUC12 is to modify the polylinker to present
identical but non-cohesive BstXI sites for cloning, to add a
"stuffer" fragment to allow for easy separation of the linear host
plasmid, and to provide for expression from the lac promoter in all
three translational reading frames.
[0075] In order to provide a site for the insertion of foreign DNA
with a high efficiency and to minimize the possibility for
self-ligation of the plasmid, inverted, non-cohesive BstXI ends are
added to the polylinker. pUC12 is first cut with BamHI (Step 1) and
the plasmid is mixed with two synthetic oligonucleotide adaptors
that are partially complementary: a 15-mer (GATCCATTGTGCTGG) and an
1-mer (GTAACACGACC) (Step 2). When the adaptors are ligated into
pUC12, two new BstI sites are created but the original BamHI sites
are also restored (Step 3). The plasmid is then treated with
polynucleotide kinase and ligated to form a closed circular plasmid
(Step 4). When this plasmid is treated with BstE, the resulting
ends are identical and not cohesive (both have GTGT overhangs)
(Step 5).
[0076] A second modification in the polylinker is done to allow for
the purification of the linear plasmid for cloning without
contamination from partially cut plasmid that can self-ligate. A
blunt end, 365 base pair (bp), FnuD2 fragment is obtained from the
plasmid pCDM. This cassette or "stuffer" fragment, which does not
contain a BstXI site, is blunt end ligated to two synthetic
oligonucleotides that are partially complementary: a 12-mer
(ACACGAGATTTC) and an 8-mer (CTCTAAAG) (Step 6). The resulting
fragment with adaptors has 4 bp overhangs (ACAC) that are
complementary to the ends of the modified pUC12 plasmid shown in
Step 5. The modified pUC12 plasmid is ligated to the PCDM insert
with adaptors; the resulting construct is pUX12.inverted,
non-cohesive BstXI ends is added to the polylinker. As shown in
FIG. 1, pUC12 was first cut with BamHI (Step 1) and the plasmid was
mixed with two synthetic oligonucleotide adaptors that are
partially complementary: a 15-mer (GATCCATTGTGCTGG) and an 11-mer
(GTAACACGACC) (Step 2). When the adaptors are ligated into pUC12,
two new BstI sites are created but the original BamHI sites are
also restored (Step 3). The plasmid was then treated with
polynucleotide kinase and ligated to form a closed circular plasmid
(Step 4). When this plasmid is treated with BstXI, the resulting
ends are identical and not cohesive (both have GTGT overhangs)
(Step 5).
[0077] Another modification in the polylinker can be done to allow
for the purification of the linear plasmid for cloning without
contamination from partially cut plasmid that can self-ligate.
Again, a blunt end, 365 base pair (bp), FnuD2 fragment is obtained
from the plasmid pCDM. This cassette or "stuffer" fragment, which
also does not contain a BstXI site, is blunt end ligated to two
synthetic oligonucleotides that are partially complementary: a
12-mer (ACACGAGATTTC) and an 8-mer (CTCTAAAG) (Step 6). The
resulting fragment with adaptors has 4 bp overhangs (ACAC) that are
complementary to the ends of the modified pUC12 plasmid shown in
Step 5. The modified pUC12 plasmid was ligated to the PCDM insert
with adaptors; the resulting construct, named pUX12, is shown in
FIG. 2. The pUX12 plasmid can also be formed by recombinant methods
(or by homologous recombination), using plasmid pUC12.
[0078] Since pUX12 is to be used as an expression vector, it is
preferable to further modify the polylinker such that it will
contain all three potential reading frames for the lac promoter.
These changes allow for the correct translational reading frame for
cloned gene fragments with a frequency of one in six. For example,
a cloned fragment can insert in the vector in one of two
orientations and one of three reading frames. To construct a +1
reading frame, the pUX12 plasmid is cut with the restriction enzyme
EcoRI which cleaves at a unique site in the polylinker. The single
stranded 5' sticky ends are filled in using the 5'-3' polymerase
activity of T4 DNA polymerase, and the two blunt ends ligated. This
results in the loss of the EcoRI site, and the creation of a new
XmnI site The above construction can be confirmed by demonstrating
the loss of the EcoRI site and confirming the presence of a new
XmnI site in the polylinker. In addition, double stranded DNA
sequencing on the +1 modified pUX12 plasmid is performed using
standard sequencing primers (Ausubel, F. M, et al., Current Topics
in Molecular Biology; Greene Publ. Assn./Wiley Interscience, NY
(1987)). The DNA sequence should show the addition of 4 base pairs
to the polylinker and confirmed the modification of pUX12 to a +1
reading frame. This plasmid is called pUX12+1.
[0079] In order to construct a -1 reading frame, the pUX12 vector
is cut with the restriction enzyme SacI which cuts at a unique site
in the polylinker of pUX12. The single stranded 3' sticky ends are
cut back to blunt ends using the 3'-5' exonuclease activity of T4
polymerase, and the resulting blunt ends ligated.
[0080] The resulting sequence should eliminate the SacI site while
resulting in a new FnuD2 site. However, restriction mapping of the
pUX12-1 plasmids has shown that while the SacI site is absent,
there is no FnuD2 site present. In addition, the SmaI/XmaI sites on
the polylinker were no longer present. Several potential pUX12-1
constructs were sequenced from mini-prep, double-stranded DNA. Of
the six modified plasmids sequenced, one was found with ten
nucleotides absent, thereby creating a -1 reading frame. This
suggests that the T4 DNA polymerase has additional exonuclease
activity and cuts back additional double stranded portions of the
polylinker. Nevertheless, the resulting plasmid had a -1 reading
frame. The plasmid was named pUX12-1.
[0081] The use of the pUX12 vectors in the cloning of antigenic
proteins of group B Streptococcus has been discussed in detail in
PCT Application WO 94/10317. In brief, DNA derived from group B
Streptococcus, or complementary to such DNA is introduced into the
pUX12, pUX12+1 or pUX12-1 vectors and transformed into Escherichia
coli. The cloned DNA is expressed in E. coli and the cellular
lysate is tested to determine whether it contains any protein
capable of binding to antisera to group B Streptococcus.
[0082] There are a number of potentially interesting modifications
of pUX12 that could increase its utility. For example, the lac
promoter could be replaced by another promoter, the origin of
replication could be modified to produce a lower copy number vector
and the drug resistance marker could be changed.
[0083] Any vector capable of providing the desired genetic
information to the desired host cell may be used to provide genetic
sequences encoding the epsilon antigen derivatives of the invention
to a host cell. For example, in addition to plasmids, such vectors
include linear DNA, cosmids, transposons, and phage. A potential
problem with cloning streptococcal proteins is exemplified by
cloning of the repeat region of the alpha C-protein which has been
found to be unstable in some instances. Secondly, problems have
also been encountered in expressing certain streptococcal
proteins.
[0084] The host cell is not limited to E. coli. Any bacterial or
yeast (such as S. cerevisiae) host that is capable of expressing
the derivatives of the invention may be used as an appropriate
host. For example, B. subtilis and the group B Streptococcus may be
used as hosts. Methods for cloning into such hosts are known. For
example, for Gram-positive hosts, see Harwood, C. R., et al., eds.,
"Molecular Biological Methods for Bacillus," Wiley-Interscience,
New York, 1991) for a description of culture methods, genetic
analysis plasmids, gene cloning techniques, the use of transposons,
phage, and integrational vectors for mutagenesis and the
construction of gene fusions, and methods of measuring gene
expression. Appropriate hosts are available from stock centers such
as the American Type Culture Collection (Rockville, Md., USA) and
the Bacillus Genetic Stock Center (Ohio State Univ., Columbus,
Ohio, USA).
[0085] Alternatively, the N-terminal region of the epsilon antigen
may be expressed or clones using a yeast host or shuttle vector,
e.g. S. cerevisiae yeast plasmids including those containing the
2-micron circle or their derivatives. Such plasmids are well known
in the art (Botstein, D. et al., Miami Wntr. Symp. 19:265-274
(1982); Broach J. R. in: Molecular Biology of the Yeast
Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. pp.445470 (1981); Broach J.
R., Cell 28.203-204 (1982); Bollon D. P. et al., J. Clin. Hematl.
Oncol. 10:3948 (1980); Maniatis, T. In: Cell Biology: A
Comprehensive Treatise, Vol. 3, Gene Expression, Academic Press,
N.Y., pp 563-608 (1980); Heslot et al. eds., Molecular Biology and
Genetic Engineering of Yeasts, CRC Press, Boca Raton, (1991)) and
are commercially available.
[0086] One of skill in the art may also obtain the N-terminal
region of the epsilon antigen or fragments thereof, by chemical
synthesis of the amino acid sequence or a fragment of the sequence
in FIG. 6B. An N-terminal region fragment can include inter alia,
the sequence of FIG. 6B without the signal peptide, as well as
those fragments that would be thought to be antigenic by those of
skill in the art.
[0087] Recently, workers in the area of chemical synthesis have
indicated that it should be possible to routinely construct large
synthetic proteins (>200 amino acids in length). (Muir et al.,
Curr. Opin. Biotech. 4:420427, 1993). Several reports of synthesis
of proteins of at least 90-99 amino acids have also appeared.
(Schnolzer et al., Science 256:221-225, 1992; Hojo et-al, Bull.
Chem. Soc. Jpn. 65:3055-3063, 1992). Thus, chemical synthesis
techniques should be a viable option for preparing the N-terminal
region of the epsilon antigen or fragments thereof. In fact a 90
amino acid protein from Bacillus stearothermophilus has already
been synthesized using protected peptide fragments (Hojo et al.,
65:3055-3063, 1992). Further, the use of TASP (template assembled
synthetic proteins) should be helpful in this regard. (Eggleston et
al., Macromolecular Symposia 101:397-404 (1996)).
[0088] The present invention concerns a vaccine comprising a
polysaccharide (such as the capsular polysaccharide) which is
characteristic of the group B Streptococcus conjugated to a protein
which is also characteristic of the group B Streptococcus. The
"polysaccharide" and "protein" of such a conjugated vaccine may be
identical to a molecule which is characteristic of the group B
Streptococcus, or they may be functional derivatives of such
molecules.
[0089] For the purposes of the present invention, a group B
Streptococcus polysaccharide is any group B-specific or
type-specific polysaccharide. Preferably, such polysaccharide is
one which, when introduced into a mammal (either animal or human)
elicits antibodies which are capable of reacting with group B
Streptococcus may be employed. Examples of the preferred
polysaccharides of the present invention include the capsular
polysaccharide of the group B Streptococcus, or their equivalents.
For the purposes of the present invention, any protein which when
introduced into a mammal (either animal or human) either elicits
antibodies which are capable of reacting a protein expressed by
group B Streptococcus, or which increases the immunogenicity of a
polysaccharide to elicit antibodies to a polysaccharide of the
group B Streptococcus may be employed. Examples of the preferred
proteins of the present invention include the C proteins of the
group B Streptococcus, or their equivalents.
[0090] Examples of functional derivatives or equivalents of the
peptide antigens include fragments of a natural protein, such as
N-terminal fragment, C-terminal fragment or internal sequence
fragments of the group B Streptococcus C protein epsilon antigen
that retain their ability to elicit protective antibodies against
the group B Streptococcus. The term functional derivatives is also
intended to include variants of a natural protein (such as proteins
having changes in amino acid sequence but that retain the ability
to elicit an immunogenic, virulence or antigenic property as
exhibited by the natural molecule), for example, the variants of
the epsilon antigen with an altered flanking sequence.
[0091] By functional equivalent or derivative is further meant an
amino acid sequence that is not identical to the specific ammo acid
sequence, but rather contains at least some amino acid changes
(deletion, substitutions, inversion, insertions, etc.) that do not
essentially affect the immunogenicity or protective antibody
producing production of the protein as compared to a similar
activity of the specific amino acid sequence, when used for the
desired purpose. Preferably, an "equivalent" or functionally
derivative amino acid sequence contains at least 85-99% homology at
the amino acid level to the specific amino acid sequence, most
preferably 90% and in an especially highly preferable embodiment,
at least 95% homology at the amino acid level.
[0092] The peptide antigen that is conjugated to the polysaccharide
in the vaccine of the invention may be a peptide encoding the
native amino acid sequence of the N-terminal region of the epsilon
antigen of FIG. 6B or it may be a functional derivative or
equivalent of the native amino acid sequence.
[0093] Epsilon antigens from other strains of the group B
Streptococcus may also be prepared and used in a similar manner as
a slight variability in the sequence of the protein, would not
alter the biological properties and their functional ability to
elicit protective antibodies. For example, a group B Streptococcus
epsilon antigen isolated from a different strain of the group B
Streptococcus is intended to be within the scope of the
invention.
[0094] The peptides used in the invention, whether encoding a
native protein or a functional derivative thereof, are conjugated
to a group B Streptococcus carbohydrate moiety by any means that
retains the ability of these proteins to induce protective
antibodies against the group B Streptococcus.
[0095] Heterogeneity in the vaccine may be provided by mixing
specific conjugated species. For example, the vaccine preparation
may contain one or more copies of one of the peptide forms
conjugated to the carbohydrate, or the vaccine preparation may be
prepared to contain more than one form of the above functional
derivatives and/or the native sequence, each conjugated to a
polysaccharide used therein.
[0096] A multivalent vaccine may also be prepared by mixing the
group B-specific conjugates as prepared above with other proteins,
such as diphtheria toxin or tetanus toxin, and/or other
polysaccharides, using techniques known in the art.
[0097] Heterogeneity in the vaccine may also be provided by
utilizing group B Streptococcal preparations from group B
Streptococcal hosts (especially into Streptococcus agalactiae),
that have been transformed with the recombinant constructs such
that the streptococcal host expresses the N-terminal region of the
epsilon antigen protein or a functional derivative thereof. In such
cases, homologous recombination between the genetic sequences
encoding the epsilon antigen will result in spontaneous mutation of
the host, such that a population of hosts is easily generated and
such hosts express a wide range of antigenic epsilon antigen
functional derivatives useful in the vaccines of the invention.
[0098] As used herein, a polysaccharide or protein is
"characteristic" of a bacteria if it is substantially similar in
structure or sequence to a molecule naturally associated with the
bacteria. The term is intended to include both molecules which are
specific to the organism, as well as molecules which, though
present on other organisms, are involved in the virulence or
antigenicity of the bacteria in a human or animal host.
[0099] The vaccine of the present invention may confer resistance
to group B Streptococcus by either passive immunization or active
immunization. In one embodiment of passive immunization, the
vaccine is provided to a host (i.e. a human or mammal) volunteer,
and the elicited antisera is recovered and directly provided to a
recipient suspected of having an infection caused by a group B
Streptococcus.
[0100] The ability to label antibodies, or fragments of antibodies,
with toxin labels provides an additional method for treating group
B Streptococcus infections when this type of passive immunization
is conducted. In this embodiment, antibodies, or fragments of
antibodies which are capable of recognizing the group B
Streptococcus antigens are labeled with toxin molecules prior to
their administration to the patient. When such a toxin derivatized
molecule binds to a group B Streptococcus cell, the toxin moiety
will cause the death of the cell.
[0101] In a second embodiment, the vaccine is provided to a female
(at or prior to pregnancy or parturition), under conditions of time
and amount sufficient to cause the production of antisera which
serve to protect both the female and the fetus or newborn (via
passive incorporation of the antibodies across the placenta).
Passive protection has been shown to result from immunization with
either the alpha or beta C-protein (Michel et al., 59:2023-2028
(1991)). Furthermore, transferance of the protective effect between
mother and unborn offspring has already been show to be successful
following maternal immunization with the beta C-protein or a
polysaccharide-beta antigen conjugate. (Madoff et al. Inf. Immun.
60: 4989-4994, 1992, Madoff et al., J. Clin. Invest. 94:286-292
(1994)).
[0102] The present invention thus concerns and provides a means for
preventing or attenuating infection by group B Streptococcus, or by
organisms which have antigens that can be recognized and bound by
antisera to the polysaccharide and/or protein of the conjugated
vaccine. As used herein, a vaccine is said to prevent or attenuate
a disease if its administration to an individual results either in
the total or partial attenuation (i.e. suppression) of a symptom or
condition of the disease, or in the total or partial immunity of
the individual to the disease.
[0103] The administration of the vaccine (or the antisera which it
elicits) may be for either a "prophylactic" or "therapeutic"
purpose. When provided prophylactically, the compound(s) are
provided in advance of any symptom of group B Streptococcus
infection. The prophylactic administration of the compound(s)
serves to prevent or attenuate any subsequent infection. When
provided therapeutically, the compound(s) is provided upon the
detection of a symptom of actual infection. The therapeutic
administration of the compound(s) serves to attenuate any actual
infection.
[0104] The vaccines of the present invention may, thus, be provided
either prior to the onset of infection (so as to prevent or
attenuate an anticipated infection) or after the initiation of an
actual infection.
[0105] A composition is said to be "pharmacologically acceptable"
if its administration can be tolerated by a recipient patient. Such
an agent is said to be administered in a "therapeutically effective
amount" if the amount administered is physiologically significant.
An agent is physiologically significant if its presence results in
a detectable change in the physiology of a recipient patient.
[0106] As would be understood by one of ordinary skill in the art,
when the vaccine of the present invention is provided to an
individual, it may be in a composition which may contain salts,
buffers, adjuvants, or other substances which are desirable for
improving the efficacy of the composition. Adjuvants are substances
that can be used to specifically augment a specific immune
response. Normally, the adjuvant and the composition are mixed
prior to presentation to the immune system, or presented
separately, but into the same site of the animal being immunized.
Adjuvants can be loosely divided into several groups based upon
their composition. These groups include oil adjuvants (for example,
Freund's complete and incomplete), mineral salts (for example,
AlK(SO.sub.4).sub.2, AlNa(SO.sub.4).sub.2, AlNH.sub.4(SO.sub.4),
silica, kaolin, and carbon), polynucleotides (for example, poly IC
and poly AU acids), and certain natural substances (for example,
wax D from Mycobacterium tuberculosis, as well as substances found
in Corynebacterium parvum, or Bordetella pertussis, and members of
the genus Brucella. Among those substances particularly useful as
adjuvants are the saponins such as, for example, Quil A. (Superfos
A/S, Denmark). Examples of materials suitable for use in vaccine
compositions are provided in Remington's Pharmaceutical Sciences
(Osol, A, Ed, Mack Publishing Co, Easton, Pa., pp. 1324-1341
(1980), which reference is incorporated herein by reference).
[0107] The therapeutic compositions of the present invention can be
administered parenterally by injection, rapid infusion,
nasopharyngeal absorption (intransopharangeally), dermoabsorption,
or orally. The compositions may alternatively be administered
intramuscularly, or intravenously. Compositions for parenteral
administration include sterile aqueous or non-aqueous solutions,
suspensions, and emulsions. Examples of non-aqueous solvents are
propylene glycol, polyethylene glycol, vegetable oils such as olive
oil, and injectable organic esters such as ethyl oleate. Carriers
or occlusive dressings can be used to increase skin permeability
and enhance antigen absorption. Liquid dosage forms for oral
administration may generally comprise a liposome solution
containing the liquid dosage form. Suitable forms for suspending
liposomes include emulsions, suspensions, solutions, syrups, and
elixirs containing inert diluents commonly used in the art, such as
purified water. Besides the inert diluents, such compositions can
also include adjuvants, wetting agents, emulsifying and suspending
agents, or sweetening, flavoring, or perfuming agents.
[0108] Many different techniques exist for the timing of the
immunizations when a multiple administration regimen is utilized.
It is possible to use the compositions of the invention more than
once to increase the levels and diversities of expression of the
immunoglobulin repertoire expressed by the immunized animal.
Typically, if multiple immunizations are given, they will be given
one to two months apart.
[0109] According to the present invention, an "effective amount" of
a therapeutic composition is one which is sufficient to achieve a
desired biological effect. Generally, the dosage needed to provide
an effective amount of the composition will vary depending upon
such factors as the animal's or human's age, condition, sex, and
extent of disease, if any, and other variables which can be
adjusted by one of ordinary skill in the art.
[0110] The antigenic preparations of the invention can be
administered by either single or multiple dosages of an effective
amount. Effective amounts of the compositions of the invention can
vary from 0.01-1,000 .mu.g/ml per dose, more preferably 0.1-500
.mu.g/ml per dose, and most preferably 10-300 .mu.g/ml per
dose.
[0111] Having now generally described the invention, the same will
be more readily understood through reference to the following
examples which are provided by way of illustration, and are not
intended to be limiting of the present invention, unless
specified.
[0112] Bacterial Strains, Plasmids, Transposons and Media.
[0113] 60 clinical isolates and classical lab strains of GBS were
studied, 26 of them from the Channing Laboratory panel of clinical
isolates collected by Dr. Dennis Kasper and his colleagues. Others
were kindly provided by Drs. Carol Baker (Baylor College of
Medicine), Lars Bevanger (Trottenheim, Norway), Henry Blumberg
(CDC, Emory University), Patricia Ferrieri (University of
Minnesota), Emil Gotschlich (from the collection of Rebecca
Lancefield at the Rockefeller University), Gunnar Lindahl
(University of Lund, Sweden), and Graziella Orifici (Instituto
Sanitario, IRIS, Rome Italy). GBS strains were grown on blood-agar
plates supplied by Becton Dickinson or in liquid Todd-Hewitt broth
(1) medium.
[0114] Three strains of E. coli were used to make competent cells
for cloning experiments. Strains DH5-.alpha. (Rec A-) and MC1061
(Rec A+) express a high copy number and strain pCNB expresses the
col E1 replicon as a low copy number plasmid. E. coli were grown on
Lennox L Broth-agar (LB) plates and in liquid LB medium. Antibiotic
selection was achieved with ainpicillin to a final concentration of
100 .mu.g/ml.
EXAMPLE 1
Immunoblotting of GBS Extracts with 4G8 Alpha-Specific Monoclonal
Antibody
[0115] It has been shown previously that different strains of GBS
express different sizes of alpha antigen (Madoff, L. C., et al.,
Infect Immun 59(1):204-210 (1991)). These studies were done using
western immunoblots probing with the a monoclonal antibody, 4G8,
that is known to bind within the repeat region. Strains, like the
prototype Ia/C(.beta.) strain 515, were typed as alpha antigen
negative by classical techniques using polyclonal typing antisera
raised to partially purified C proteins. To characterize this
diversity of size of the alpha antigen on the phenotypic level, 20
different isolates of GBS previously characterized (Madoff, L. C.,
et al., Infect Immun 59(1):204-210 (1991)) and expressing a wide
range of alpha antigen sizes, and four strains of GBS negative for
alpha antigen expression, were chosen from the collection of the
Channing Laboratory and immunoblotted.
[0116] Methods
[0117] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and Western Immunoblots were performed by standard
techniques with a Mighty Small electrophoresis apparatus and a
Transphor electroblotter (Hoefer, San Francisco) (Madoff, L. C., et
al., Infect Immun 59(1):204-210 (1991)).
[0118] Two types of primary antibodies were used for immunoblots
(see Table 1). The first group of antibodies were raised against
individual surface proteins from GBS. Specifically, polyclonal
antibody was raised against the alpha antigen clone pJMS23
(anti-alpha) and the monoclonal antibody 4G8 raised to GBS strain
A909 (Madoff, L. C., et al., Infect Immun 59(1):204-210 (1991))
recognized the C protein alpha antigen. 4G8 is specific to an
epitope in the repetitive region of alpha. Antiserum to the
subclone expressing only the alpha antigen amino terminus was also
used. All antibodies were used at 1:1,000 dilution in PBS/T.
1TABLE 1 Antisera. A table of all antisera used. Anti- body Type
Animal Antigen Comments Source alpha polyclonal rabbit clone
anti-whole alpha Madoff N-term. polyclonal rabbit clone anti-alpha
N-term Michel 4G8 monoclonal mouse A909 anti-alpha repeats Madoff
Beta polyclonal rabbit clone anti-whole beta Madoff Rib polyclonal
rabbit purified anti-whole Rib Lindahl R1 polyclonal rabbit
purified anti-R1 Ferrieri R4 polyclonal rabbit purified anti-R4
Ferrieri 1a polyclonal rabbit O90 anti-type 1a Kasper 1b polyclonal
rabbit H36B anti-type 1b Kasper II polyclonal rabbit 18RS21
anti-type II Kasper III polyclonal rabbit M781 anti-type III Kasper
IV polyclonal rabbit 3139 anti-type IV Kasper V polyclonal rabbit
1169 anti-type V Kasper VI polyclonal rabbit NT6 anti-type VI
Kasper VII polyclonal rabbit 7271 anti-type VII Kasper VIII
polyclonal rabbit JM913 anti-type VIII Kasper
[0119] After being electroblotted onto nitrocellulose sheets,
samples were probed with the 4G8 monoclonal antibody (Madoff, L.
C., et al., Infect Immun 59(1):204-210 (1991)) As expected, 20 of
the isolates were positive for alpha antigen expression. Among
these isolates, the maximum molecular mass of the alpha antigen
varied from 61,000 to 270,000 Da.
[0120] Results
[0121] Protein extracts from these strains displayed the
heterogenous peptide ladder associated with protein alpha (Madoff,
L. C., et al., Infect Immun 59(1):204-210 (1991)). However, two
different phenotypic laddering patterns were observed in this panel
of strains. In 12 of the strains, the peptide ladder fragments were
exactly in line with those of the prototypical alpha antigen
bearing strain A909; this strain was used to clone bca. These
strains were classified as exhibiting the "classic" alpha laddering
pattern. The ladder fragments from strains DK8, DA, 515, DK 4-1,
DK3, RO, and strain isolates GH2A and GH7 were slightly out of
frame with those exhibiting this pattern and were said to exhibit
the epsilon laddering pattern. Although the distance between bands
was the same in both groups, the ladder fragments for epsilon
strains were slightly below those of alpha strains.
[0122] FIG. 3A is a sample immunoblot of 10 of the alpha and
epsilon-positive strains that shows the relative sizes and the
laddering patterns of the alpha and epsilon antigen.
EXAMPLE 2
Determination of Alpha Gene Size with Southern Blotting
[0123] Once the extent of phenotypic diversity of the alpha and
epsilon antigens had been identified in the panel of strains, DNA
blotting was used to characterize gene number and size among
strains. First, it was necessary to determine whether the ladder of
peptide fragments corresponding to the alpha or epsilon antigen
were the product of only one gene. In the M protein of group A
Streptococcus, for example, phenotypic diversity is generated not
only by rearrangement of repeating regions within its gene, but
also by duplications of the M-protein gene itself (Hollinghead et
al, Mol. Gen. Genet 207:196-203, (1987)).
[0124] Methods
[0125] To investigate whether the alpha and epsilon C-protein genes
were present in a single copy within the different strains, genomic
DNA was digested with DraI that cuts outside of the open reading
frame of the alpha antigen gene (see FIG. 2, electrophoresed, and
Southern blotted. The blots were then probed with a 32P-labeled
Styl repeat fragment specific to the repeat pattern of bca.
[0126] Standard procedures were followed from Ausubel et al. for
the preparation of plasmid DNA from E. coli, restriction enzyme
digestion, agarose gel electrophoresis, and Southern blot
hybridization (Ausubel, F. M., et al., Current Protocols in
Molecular Biology, New York: John Wiley & Sons, 1993). Genornic
DNA was isolated by the method of Hull et al. (Infect. Immun.
33:933-938 (1981)). as modified by Rubens et al. (Rubens, C. E., et
al., Proc. Natl. Acad. Sci USA 84(20):7208-7212 (1987)).
[0127] Four nucleotide probes were used for Southern blots. A probe
specific for the entire 3,060-bp gene of the alpha antigen was
derived from a subclone of the alpha gene (pJMS23-1) digested with
HindIII. A 246-bp probe specific for the repeats of alpha was also
derived from pJMS23-1 by digestion with StyI and purified from an
agarose gel with the GeneClean kit (Bio101, La Jolla, Calif.). A
probe internal to the amino terminus of the alpha gene (without the
signal sequence), was prepared by a polymerase chain reaction (PCR)
with primers designed from the published alpha gene sequence
(Michel, J. L., et al., Proc. Natl. Acad. Sci. USA
89(21):10060-10065 (1992) #2492) (FIG. 1). A probe for the beta
antigen gene was derived from the beta clone pJMS1 by digestion
with BamHI, agarose gel separation of a 3,100 bp fragment, and
purification by Gene Clean (Bio 101).
[0128] Results
[0129] For all 24 strains, only one fragment homologous to the StyI
probe was found. Fragment size differed between strains and was
found to be larger in strains that had larger alpha or epsilon
C-proteins, indicating that changes in the size of the expressed
antigens may be due to differences in the size of the structural
gene. The finding of a single gene copy per strain was confirmed by
Southern blots following digestion with multiple restriction
enzymes and using pulsed field gel electrophoresis (data not
shown).
[0130] Once it had been determined that the alpha antigen gene was
present in a single copy within the strains, the size of the alpha
C-protein gene in each of the strains was compared with the
apparent molecular mass of the alpha antigen observed on
immunoblots. The size of the alpha antigen gene on Southern blot
was found to range from 1.85 to 10.25 kb and was directly
correlated with the size of expressed alpha or epsilon antigens
(FIG. 2). Therefore, the variability among strains in the maximal
size of these antigens is directly related to the size of their
structural genes.
EXAMPLE 3
Localization of Alpha Gene Size Determinants
[0131] To determine how the composition of the alpha antigen gene
was altered as its size changed, Southern blots were made from
BsaBI digests. BsaBI cuts just outside either end of the repeat
region, within the N-terminal and C-terminal of the gene. As seen
in FIG. 2B shows, there is approximately a one to one relationship
between the increasing size of the repeat region and the size of
the alpha antigen gene. Therefore, differences in the size of the
expressed alpha antigen are largely a function of the number of
repeats contained within the alpha gene.
EXAMPLE 4
Possible Variability in the C-Terminal of the Gene
[0132] After it had been shown that the size of the gene could be
predicted based upon the number of repeats that it contained, new
DNA blots were made to more closely investigate the size of the
amino and carboxy ends of the repeat region of the gene among
different strains. Southern blots made from a StyI-NsiI double
digest were used to examine the C-terminal for any variation
between strains. -As seen in FIG. 2, the StyI-NsiI double digestion
cut out a 435 base pair fragment of the C-terminal anchor and
repeat region. When the blots were probed with the HincII-NsiI DNA
fragment, only the 435 bp fragment was seen. On the basis of this
data (not shown), the size of the C-terminal appears to be
conserved between strains.
EXAMPLE 5
Immunologic Analysis of the Epsilon Antigen
[0133] Laddering proteins are of interest because of the antigenic
variability they introduce. Such variability may have a role in
changing the virulence properties and immune targets of the
organism. The relationship between alpha and the cross-reactive
antigen, epsilon was investigated. 4G8 monoclonal antiserum against
the repeating subunits of alpha and a new alpha amino
terminus-specific antiserum permitted analysis of the different
segments of the gene individually.
[0134] FIG. 3 shows the results of immunoblot of GBS strains
expressing either the alpha or epsilon antigen probed with 4G8
antibody and with alpha amino terminus antiserum. Immunoblot probed
with 4G8 antibody showed the cross-reactivity of alpha and epsilon.
This is strong evidence that the two proteins share significant
homology and, in particular, that the repeat regions share
epitopes. However, antiserum raised against the subeloned amino
terminus of alpha failed to bind the epsilon-positive 515 on an
immunoblot and was highly specific for alpha strains including
A909. C protein-negative control strains failed to bind anti-alpha,
4G8, or anti-alpha amino terminus sera. The immunologic data
supports idea that alpha and epsilon share significant homology
within the repeat region, but that the amino termini are
distinct.
EXAMPLE 6
Southern Blot Analysis of the Epsilon Antigen
[0135] Genomic DNA from A909, the prototype alpha-positive strain,
and 515, the prototype epsilon-positive strain, was double digested
with DraI and StyI and probed on a Southern blot with the
alpha-amino terminus probe to determine the homology of the alpha
and epsilon amino termini. The results are shown in FIG. 4. The
amino terminus probe failed, even under low stringency, to
hybridize to genomic DNA from 515, the prototype epsilon-positive
strain. When the same Southern blots were re-probed with the alpha
repeat probe, both alpha- and epsilon-positive strains hybridized,
thus indicating that the repeat region is largely the same. DNA
from non-C protein-bearing strains was run as a negative control
and failed to hybridize with amino terminus or repeat probe.
[0136] This indicated that alpha and epsilon have significantly
different nucleotide sequences in the amino termini.
[0137] A new surface-associated protein, the epsilon antigen, was
discovered and characterized. Genetic and immunologic analysis of
epsilon indicated that the alpha and epsilon antigens shared a high
degree of homology in the signal sequence, the repetitive region,
and the carboxyl terminus, but are significantly different at the
amino terminus by both nucleotide and antibody probe. This
difference explains the cross-reactivity of anti-alpha serum with
epsilon and the distinct laddering pattern of epsilon on
immunoblot. In identifying an area of marked difference between
alpha and epsilon, a simple two-step screening technique to
differentiate alpha and epsilon by either Southern blot or
immunoblot was created. Both alpha and epsilon bind to the whose
gene or repeat probe from alpha, but only alpha binds to the amino
terminus-specific probe from alpha. Until the clone was isolated,
there was no specific probe for epsilon.
[0138] The alpha and epsilon antigens probably arose from a common
evolutionary predecessor through transferral of the amino terminus
from another gene. By incorporating a new surface antigen, GBS
increases its antigenic diversity, and thus, its ability to survive
a variety of immune response conditions. Since they share a high
degree of homology in the repeat region, a recombinational event
was probably recent in the evolutionary history of the organism. It
is possible, however, that the integrity of the repetitive domains
must be maintained for the survival of the organism and is thus
maintained by selection.
EXAMPLE 7
Cloning of Epsilon Amino Terminus
[0139] Having identified the amino terminus as the principal site
of the difference between alpha and epsilon, the unique region of
epsilon was cloned and sequenced so that it could be further
characterized genetically and immunologically. A clone of epsilon
will be useful for assessing the heterogeneity of and patterns of
cross-reactivity with other surface-associated proteins, and will
help elucidate the biological function of these surface-associated
proteins of GBS.
[0140] Methods
[0141] The clone of the epsilon amino terminal region was derived
by PCR from genomic DNA of the epsilon-positive GBS stain 515.
Primers were designed from the published sequence of the alpha
antigen gene (Michel, J. L., et al, Proc. Natl. Acad. Sci. USA
89(21):10060-10065 (1992)) with sites upstream of the signal
sequence and immediately downstrem of the amino terminus. The
primers were engineered with restriction sites for the enzymes
EcoRI (upstream) and BamHI (downstream). The exact primer sequences
are listed in FIG. 5. The PCR reactions were run according to
Ausubel et al. (Ausubel, F. M., et al., Current Protocols in
Molecular Biology, New York: John Wiley & Sons, 1993), with the
exception that 2 .mu.l of each primer was used per reaction at a 50
mM concentration with no additional magnesium. Reactions with no
template DNA and with only one primer were run to control for
contaminants. The PCR was performed with a Perkin-Elmer Cetus
thermal cycler (model 480) and Vent polymerase (New England
Biolabs) with 90 seconds at 94.degree. C. for denaturing, 120
seconds at 52.degree. C. for annealing, 180 seconds at 72.degree.
C. for extension, and 30 cycles.
[0142] The amino terminal region of epsilon was prepared by PCR,
concentrated from six PCR reactions, and digested overnight with
EcoRI and BamHI (Gibco-BRL). The cloning vector, pBluescript
KS-(Stratagene), containing an ampicillin resistance marker, was
also digested overnight with the same enzymes. Both vector and
insert were run on an agarose gel to remove any uncut pieces or
small fragments from the digestion and purified by Gene Clean
(Bio101). A 20-.mu.l ligation reaction with T4 DNA ligase (New
England Biolabs) was incubated overnight at 16.degree. C. with
controls to check for self-ligation (no insert) and controls to
check for uncut vector (no ligase). 1.5 .mu.l from the ligation was
transformed into 50 .mu.l of competent cells prepared from E. coli
strain MC1061 (Ausubel, F. M., et al., Current Protocols in
Molecular Biology, New York: John Wiley & Sons, 1993). The
transformation was performed by electroporation with a Gene Pulser
(Bio-Rad, Hercules, Calif.) with parameters 2.5 mV, 25 .mu.F, and
200 Ohms. LB plates with ampicillin (100 l/ml) were used to screen
for transformants.
[0143] DNA sequencing was performed at the Beth Israel Hospital's
Molecular Medicine Unit sequencing service with the Applied
Biosystems automated sequencer (model 373A). When preparing plasmid
DNA for sequencing, one additional phenol:chloroform:isoamyl
alcohol (25:24:1) extraction and ethanol precipitation was
performed. DNA was resuspended to a final concentration of 500
.mu.g/ml. Sequence data was confirmed by manual dideoxy sequencing
using the Sequenase version 2.0 DNA sequencing kit (United States
Biochemical, Cleveland, Ohio) and 35S alpha-labeled dATP
(Amersham). DNA was run on a 6% polyacrylamide gel. The gel was
dried and autoradiographed on Scientific Imaging Film (Kodak,
Rochester, N.Y.) overnight at -70.degree. C.
[0144] Analysis of sequence data was performed with the Genetics
Computer Group (Version 8.0) sequence analysis software (University
of Wisconsin, Madison, Wis.) through the Massachusetts General
Hospital Department of Molecular Biology's computer system, FRODO.
The homology of the final gene sequence was compared to a national
database for peptide and nucleotide sequences at the National
Center for Biotechnology Information (Bethesda, Md.) using the
Basic Local Alignment Search Tool (BLAST) network service.
[0145] As shown in FIG. 5, the strategy for cloning the amino
terminus of epsilon was based on the hypothesis that the upstream
flanking region and the downstream region (the repeats) shared
enough homology with those of the alpha antigen that PCR primers
designed from the known alpha sequence would amplify the amino
terminus of epsilon.
[0146] To quantitatively determine the homology of the epsilon and
alpha amino termini and to further characterize the unique features
of epsilon, the nucleotide sequence of the clone was determined,
and it is presented in FIG. 6A. Initial sequencing was accomplished
by automated sequencing. The variable reproducibility of the
results and the abundance of ambiguous nucleotides necessitated
confirmation by manual dideoxy sequencing.
[0147] As shown in FIG. 7, the nucleotide and peptide sequences of
epsilon were similar to alpha in the upstream flanking region and
the signal sequence. However, in the amino terminus, the divergence
between the sequences was significant. For the nucleotide sequence
of the entire clone there was 76% homology between epsilon and
alpha. The amino terminus alone, excluding the flanking DNA and the
signal sequence, however, showed only a 65% homology between the
sequence of the epsilon clone and the alpha antigen. The amino acid
sequence of epsilon has 61% identity with alpha, which drops to
only 54% identity if the identical flanking region and signal
sequence are removed. This finding is consistent with findings from
Southern blot and immunoblot analysis of the epsilon antigen. The
amino terminus sequence of epsilon presented here is markedly
different at the nucleotide and the amino acid level from that of
alpha and supports the importance of epsilon as an independent
antigen.
[0148] Nucleotide sequencing the unique region of the epsilon
antigen also permitted a compositional and structural analysis of
the peptide and comparison with the N-terminus of the alpha
antigen. The isoelectric point of the epsilon N-terminus was 9.79,
whereas the isoelectric point for the alpha N-terminus was 5.19.
The markedly different charged state of the two peptides provides
further evidence for how they may present different epitopes on the
surface of the organism. Furthermore, two-dimensional drawings of
protein structure based on Chou-Fasman predictions for peptide
folding indicate several domains which are similar between the
alpha and epsilon N-termini (see FIG. 10). Epsilon, however, has
several distinct domains that are not present in alpha and
represent a significant change in the epitopic structure of the
antigen. The additional domains on epsilon may explain the failure
of alpha N-terminal antiserum to cross-react with epsilon.
[0149] The sequence from the clone was used to generate a
restriction map of the epsilon antigen amino terminus. Knowledge of
the restriction sites will help further characterize the gene and
permit the creation of an epsilon-specific nucleotide probe. A
restriction map of the amino terminal region of epsilon indicates
several changes from alpha. Sites for the restriction enzymes AciI,
BsaAI, BssSI, SexAI, SfcI, and SnaBI are present in the epsilon
clone, but are not present anywhere in the alpha gene.
[0150] To learn about the biological role of the epsilon antigen,
the amino terminus sequence was compared to the database of
nucleotide and peptide sequences at the National Center for
Biotechnology Information using the BLAST network service. As
expected, the clone showed significant homology to the C protein
alpha antigen of GBS. In addition, homology was noted to other
streptococcal protein precursors including those for IgA binding
proteins, elongation factors, and muramidase. However, comparison
of the nucleotide and amino acid sequences for the amino terminus
of epsilon without the flanking region and signal sequence showed
homology only to the alpha antigen. The sequence of epsilon
presented here provides important insights into the relationship of
alpha and epsilon and, more generally, of laddering
surface-associated proteins. By identifying common and variable
features of the laddering proteins their biological role and
function in virulence and immunity may be more clearly
understood.
[0151] A clone of the amino terminus of epsilon was isolated and
sequenced to characterize the unique portion of the gene. The amino
terminus clone of the epsilon gene has shown conclusively that
epsilon and alpha antigen genes differ significantly at the
nucleotide level. The start site and signal sequence of both genes
are similar. However the majority of the amino terminus of epsilon
shares little homology with alpha Subcloning the epsilon amino
terminus clone into an E. coli expression vector with a histidine
tag will allow for the purification of epsilon amino terminus
peptide. This peptide can be used to immunize rabbits and raise
epsilon-specific antiserum that should identify epsilon on
immunoblot. If this antiserum is cross-protective with strains
expressing other surface-associated antigens, it may play an
important role in vaccine development With the sequence presented
here, it will be possible to learn more about the relationship
between laddering surface proteins and about the nature of the
evolutionary split that led to the production of the related, but
mutually exclusive, alpha and epsilon antigens. Comparing common
and distinct features of the alpha and epsilon genes may lead to a
more clear understanding of their role in virulence and immunity.
Only by sequencing the repeat region of epsilon can it be
determined whether there are any differences with the repeat region
in alpha. In addition, a full-length clone can be expressed and
purified to raise epsilon-specific antibodies for protection
studies or to covalently link with type-specific polysaccharide as
a conjugate vaccine.
[0152] The biological role of surface-associated proteins in
virulence and immunity is not well characterized. Discovery of
strains with silent genes for surface antigens and strains with no
surface antigen expression invites further inquiry into regulation
and expression. Studying the role of immunogenic surface proteins
on GBS is important to understanding the pathogenesis of the
organism and may lead to the development of a polyvalent conjugate
vaccine incorporating protein antigens.
[0153] A new surface protein antigen of GBS has been identified,
sequenced, and characterized. This represents a significant
advance, not only in the knowledge of surface protein structure,
but also in the design of biological tools and techniques that may
be used in the future to study surface proteins genetically and
immunologically. Epsilon, has been defined and its relationship to
other related proteins established. This has generated a new
understanding about the role of surface-associated proteins in the
immune response to GBS.
[0154] Discussion
[0155] In analyzing the phenotypic and genotypic diversity of the
C-protein alpha antigen of GBS, it was found that the heterogenous
ladder of peptide fragments comprising the alpha antigen is coded
for by only one gene in the bacterium and that the size of the
largest peptide fragment in the ladder was a consequence of the
size of the alpha antigen gene and in particular, the size of the
repeat region within that gene. Three novel sources of antigenic
diversity in the C-protein alpha antigen were identified
Additionally, the epsilon antigen which is related to the alpha
antigen was identified and cloned that is distinguishable by its
different N-terminal.
[0156] The tandem repeats within the repeat region of the alpha
antigen gene are the primary source of diversity in the C-protein
alpha antigen. For the panel of strains analyzed, the size of the
repeat region in bca varied by as much as 7 kb between strains, and
protein size varied by as much as 200,000 Da. When the biological
function of C-protein alpha antigen becomes more well defined, it
will be possible to better appreciate these differences in terms of
their effects upon biological function.
[0157] The two properties that have been ascribed to the alpha
antigen are the ability to resist phagocytosis and the ability to
elicit protective antibodies S (Madoff, L. C., et al., Infect Immun
59(1):204-210 (1991);Madoff, L. C., et al., Infect Immun
59(1):204-210 (1991); Michel, J. L., et al., Infect Immun
59(6):2023-2028 (1991)). However, there are other biological
functions of the alpha C-protein that have not yet been defined.
One can imagine that a strain encoding an alpha antigen of almost
200,000 Da would be extremely sensitive to killing in the presence
of specific antibody. Clearly there must be some function of the
repeats that makes it beneficial for some strains to include a
large repeat region within their alpha C-proteins. To look for
possible answers to this question, one can first examine the role
that repeating units play in the surface-associated proteins of
other gram-positive cocci. Repeat regions in the M protein of group
A Streptococcus for example are able to rearrange spontaneously,
generating antigenic diversity by changing the peptide sequence and
number of repeats (Hollinghead et al, Mol. Gen. Genet 207:196-203,
(1987)). It has not been shown that changing numbers of repeats
within the alpha antigen can change its protective epitopes, but if
this were possible, it could be a reason for the maintenance of
large numbers of repeats within the gene.
[0158] The unique amino terminus of the epsilon antigen was cloned
and sequenced. The sequencing indicated a large area of divergence
from the alpha antigen. The results have important implications for
understanding the biological role of surface-associated proteins
and for incorporating them into experimental vaccines.
EXAMPLE 8
Expression of the Recombinant Alpha C-Protein N- and C-Terminal
Peptides
[0159] The alpha C-protein monoclonal antibody 4G8 binds a
protective epitope of the alpha C protein (Madoff, L. C., et al.,
Infect. Immun. 59:204-210 (1991)). To map the location of this
epitope, plasmid constructs consisting of the N-terminus, repeat
region, and C-terminus were cloned and expressed in E. coli.
[0160] Methods
[0161] Bacterial strains, plasmids, and media. For this Example as
well as Examples 9-14 the following bacterial strains, plasmids and
media were used. Bacterial strains used in this study included GBS
strains A909 and 090 (Lancefield, R. C., et al., J. Exp. Med.
142:165-179 (1975)) and E. coli strains BL21(DE3) (Grodberg, J.
& Dunn, J. J., J. Bacteriol. 1 70:1245-1253 (1988)), DH5a
(Gibco/BRL, Bethesda, Md.), and NK8032 (kindly provided by Dr.
Nancy Kleckner). Alpha C-protein subclones pJMS23-1 and pJMS23-9
have been described (Michel, J. L., et al., Infect. Immun.
59:2023-2028 (1991); Michel, J. L., et al., Proc. Natl. Acad. Sci.
USA. 89:10060-10064 (1992)), and plasmids pSKOF1-13, pDEK14, and
pDEK15 are described below. Plasmid vectors included pET24a
(Novagen, Madison, Wis.) and pGEM-7Zf(-) (Promega, Madison, Wis.).
GBS strains were grown in Todd-Hewitt broth (Difco, Detroit, Mich.)
and on tryptose soy agar (TSA) with 5% sheep's blood plates (Becton
Dickinson, Woburn, Mass.). E. coli strains were grown in L-broth
(Difco). Antibiotic concentrations were: ampicillin (100 .mu.g/ml)
and kanamycin (50 .mu.g/ml).
[0162] DNA procedures/Sublones of the bca gene. Restriction
endonucleases, ligases, and calf-intestine alkaline-phosphatase
were obtained from New England Biolabs (Beverly, Mass.) and
Boehringer Mannheim (Indianapolis, Ind.). Staggered-end ligations
were carried out at 14.degree. C. (labor, S. in Current Prdtocols
in Molecular Biology, Vol. 1, F. M., Ausubel, et al., (eds.), John
Wiley & Sons, Inc., New York, (1994), pp. 3.14.3-3.14.4).
Plasmids used in this study are shown in FIG. 11. pSKOF1-13,
containing a 1.24-kb insert consisting of tandem repeats, was
developed by digesting pJMS23-9, first with HindIl and NsiI and
then with exonuclease III (Michel, J. L., et al., Proc. Natl. Acad.
Sci. USA. 89:10060-10064 (1992)). pSKOF1-13 encodes 11 amino acids
of the C-terminal partial repeat and the first amino acid from the
C-terminus expressed from the lacZ promoter of the pGEM-7Zf(-)
vector. The gene product expressed from pSKOF1-13 contains the
alpha C-protein repeat region and a single amino acid of the
C-terminus.
[0163] The DNA encoding the alpha C-protein N- and C-termini were
PCR cloned into a pET24a overexpression vector containing a T7
polymerase promoter that facilitates
isopropyl-.sctn.-D-thiogalactosidase (IPTG)-inducible, high-level
expression of the recombinant gene fragment and a C-terminal
six-residue histidine tag that supports purification of the gene
products by Ni.sup.2+ affinity chromatography. Oligonucleotide
preparations for PCR were synthesized at an institutional core
facility with the Expedite Nucleic Acid Synthesis System, Model
8909 (Millipore Corp., Bedford, Mass.). The DNA sequence encoding
the alpha C-protein N-terminus was amplified from the bca gene
subclone pJMS23-1 by PCR with the following oligonucleotide
primers:
2 5'-GTATATGGATCCATAGTTGCTGCATCTACA-3'and
5'-GGGCTGAAGCTTCAATACTAACAATTTCTC-3'.
[0164] The oligonucleotide primers used to amplify the DNA encoding
the alpha C-protein C-terminus are:
3 5'-GTATATGGATCCAAAGCTCAGCAAGTCAAC-3'and
5'-GGGCTGAAGCTTATCCTCTTTTTTCTTAGAAAC-3'.
[0165] Conditions of the amplification were as follows:
denaturation, 3 min at 94.degree. C.; annealing, 2 min at
39.degree. C.; and polymerization, 3 min at 72.degree. C.
Amplification was carried out with a Vent polymerase kit, with 1.5
mM MgCI.sub.2 (New England Biolabs). The BamHI and HindIII
restriction endonuclease sites (underlined) were encoded in the
primers to facilitate cloning into the pET24a vector.
[0166] A 542-bp fragment was amplified with the N-terminal-specific
primers by PCR from pJMS23-1 and ligated into the BamHI- and
HindIII-digested pET24a vector. Plasmid pDEK14 (FIG. 9) contained
such a 542-bp insert and was verified to encode the alpha C-protein
N-terminus by partial nucleotide sequence analysis. With
C-terminal-specific primers, a 144-bp fragment was amplified from
pJMS23-1 and ligated into the pET24a vector . Recombinant clones
were screened by colony-blot hybridization and probed with the
amplified C-terminal PCR product; insert size was determined by
digestion with BamHI and HindIII. Plasmid pDEK15 (FIG. 9) contained
a 135-bp fragment encoding the alpha C-protein C-terminus as
confirmed by nucleotide sequence.
[0167] Colony-blot hybridization for E. coli Colony-blot
hybridization was carried out as described previously (Weis, J. H.
in Current Protocols in Molecular Biology, Vol. 1, F. M., Ausubel,
et al., (eds.), John Wiley & Sons, Inc., New York, (1994), pp.
6.2.1-6.2.3). The amplified PCR fragments were labeled by random
priming reaction with .sup.32P-dCTP (Amersham, Arlington Heights,
Ill.) with use of a Random Priming kit (Boehringer Mannheim). The
labeled probes were separated from unincorporated nucleotides on
NucTrap columns (Stratagene, La Jolla, Calif.).
[0168] FIG. 9 shows the subclones of the bca gene that were used to
map the protective epitope. PAGE was used to characterize the
expressed gene products from the N- and C-terminal clones. Extracts
of strain BL21 (DE3) containing pET24a (negative control), pDEK14
(N-terminal clone), and pDEK15 (C-terminal clone) were prepared
after IPTG induction and electrophoresed on 15% polyacrylamide
gels. FIG. 10 shows a band of 23 kDa (the expected size of the
N-terminal fragment) in the lane corresponding to the extract of E.
coli containing pDEK14 (lane 2). Lane 3 shows a 6.2-kDa band in the
E. coli extract containing pDEK15, which is similar to the expected
size of the recombinant alpha C-protein C-terminal peptide.
EXAMPLE 9
Localization of the Protective Epitope(s) Defined by 4G8
[0169] Western blot analysis was used to determine whether the
epitope bound by 4G8 is locali to the N-terminus, repeat region, or
C-terminus of the alpha C protein.
[0170] Methods
[0171] SDS/polyacrylamide gel electrophoresis (PAGE), western
immunoblotting, and antibodies. Proteins were analyzed on SDSIPAGE
(8% and 15%), with both Coomassie blue staining and western
immunoblots by standard methods (Gallagher, S. A. & Smith, J.
A. in Current Protocols in Molecular Biology, Vol. 2. F. M.
Ausubel, et al., (eds.), John Wiley & Sons, Inc., New York,
(1994), pp. 10.2.1-10.2.21; Sasse, J. in Current Protocols in
Molecular Biology, Vol. 2, F. M., Ausubel, et al., (eds.), John
Wiley & Sons, Inc., New York, (1994), p.-10.6.1-10.6.8) Primary
antibodies and secondary alkaline-phosphatase conjugates. (Organon
Teknika, West Chester, Pa.) were used at a dilution of {fraction
(1/1000)}. Blots were developed with alkaline-phosphatase substrate
buffer (Sigma, St. Louis, Mo.). Antibodies used in this study
include polyclonal alpha C-protein antibodies (Gravekamp, C., et
al., Infect. Immun. 64:3576-3583 (1996)), alpha C-protein
N-terminal antibodies (Kling et al., Inf. Immun. (In press, April
1997)), and the alpha C-protein monoclonal antibody 4G8 (Madoff, L.
C., et a., Infect. Immun. 59:204-210 (1991)).
[0172] FIG. 11 shows a series of bands ranging in size from 27 to
40 kDa in the E. coli extract containing pSKOF1-13 (tandem repeat
region) but not in the extracts of E. coli carrying pDEK14
(N-terminus) or pDEK15 (C-terminus). The top band (40 kDa)
corresponds approximately to the expected size of the recombinant
gene product (45 kDa). These data indicate that 4G8 specifically
detects an epitope in the tandem repeat region of the alpha
antigen. Bands ranging in size from 34- to 95-kDa are found in GBS
strain A909 (as a positive control for alpha C-protein expression)
but not in 090 (negative control). This result is similar to the
previously observed size range of the alpha C-protein bands from
A909 (36 to 116 kDa) (Madoff, L. C., et al., Infect. Immun.
59:204-210 (1991)). In addition, bands ranging from 32 to 105 kDa
are detected in the extract of E. coli containing pJMS23-1. These
bands correspond to the approximate sizes of the recombinant alpha
C protein (40 to 120 kDa) (Michel, J. L., et al., Infect. Immun.
59:2023-2028 (1991)). No bands are detected in the E. coli extracts
carrying pGEM-7Zf(-) or pET24a (negative controls). Thus, 4G8
detects the alpha C protein from both the native antigen (A909) and
the recombinant antigen (pJMS23-1) (Madoff, L. C., et al., Infect.
Immun. 59:204-210 (1991); Michel, J. L., et al., Infect. Immun.
59:2023-2028 (1991)). These data indicate that 4G8 does not bind
either terminus and is specific for the alpha C-protein tandem
repeat region.
EXAMPLE 10
ELISA Inhibition
[0173] ELISA inhibition was used to study the relationship between
the relative affinity of monoclonal antibody 4G8 and the numbers of
repeats expressed by alpha C protein.
[0174] Methods
[0175] Development of alpha C-protein N-terminal specific
antibodies. To obtain alpha C-protein N-terminal-specific
antibodies, purified alpha C-protein N-terminal peptides were
lyophilized and sent to Lampire Biologicals (Malvern, Pa.) for
rabbit immunization: 100 .mu.g of the alpha C-protein N-terminal
peptide was resuspended in 2.5 ml of PBS, emulsified in 2.5 ml of
complete Freund's adjuvant, and injected subcutaneously at 6 sites
on day 1. Booster immunizations given at 21 and 42 days consisted
of solubilized antigen, emulsified with incomplete Freund's
adjuvant. Blood was drawn on days 1 (preimmunization bleed), 21,
and 42 for antibody testing. At day 56, a 50-ml blood sample was
drawn (postimmunization bleed).
[0176] Titers of the mouse monoclonal antibody 4G8 and rabbit
antiserum elicited to the alpha C-protein N-terminal peptide were
measured by ELISA inhibition (Gravekamp, C., et al., Infect. Immun.
64:3576-3583 (1996)).
[0177] FIG. 12 indicates that a 40-, 133-, and 222-fold higher
concentration of 1-repeat antigen was required than of 2-, 9-, and
16-repeat antigens, respectively, to obtain 50% inhibition of
antigen-antibody binding. These data show a high affinity of 4G8
for 2-, 9-, and 16-repeat alpha C protein, but a much lower
affinity for the 1-repeat alpha C protein. These data are similar
to results of other studies of the effects of repeat number on
antibody binding (Gravekamp, C., et al., Infect. Immun.
64:3576-3583 (1996)).
EXAMPLE 11
Purification of the Alpha C-Protein N-Terminal Peptide
[0178] The N-terminal peptide expressed from E. coli was purified
by Ni.sup.2+ affinity chromatography.
[0179] Methods
[0180] Ni.sup.2+-affinity chromatography. The recombinant alpha
C-protein N-terminal peptide was purified with a Ni.sup.2+ affinity
column according to the Novagen pET system manual. Eluted fractions
containing the largest amounts of protein were identified by
Bradford assay (Smith, J. A., in Current Protocols in Molecular
Biology, Vol. .2, F. M. Ausubel, et al., (eds.) John Wiley &
Sons, Inc., New York, (1994), pp. 10.1.1-10.1.3) and desalted with
an Amicon microconcentrator, P-10,000 molecular weight cutoff.
[0181] FIG. 13A shows a Coomassie-stained polyacrylamide gel of the
cell extracts of E. coli (BL21/DE3) containing pDEK14--before and
after induction with IPTG, as well as a sample of the eluate from
the Ni.sup.2+ column (lane 3). A single 22-kDa band is seen in the
eluate from the Ni.sup.2+ column which demonstrates that the
recombinant alpha C-protein N-terminal peptide was successfully
induced, expressed, and purified by Ni.sup.2+ affinity
chromatography.
EXAMPLE 12
Raising Antibodies to the Recombinant Alpha C-Protein N-Terminal
Peptide
[0182] Antibodies were raised in rabbits to the recombinant
Ni.sup.2+ purified N-terminal peptide. Western blot analysis of the
postimmunization serum specifically detected a band of 22 kDa, in
the extract of E. coil carrying pDEK14 after induction with IPTG,
that corresponds to the expected size of the N-terminal peptide
(data not shown). An ELISA was used to quantitate the titer of the
N-terminal antiserum. Titers of antiserum were found to be high, in
excess of a 1/102,400 fold dilution.
[0183] Western blot analysis was used to determine whether
antibodies specific for the alpha C-protein N-terminus can bind the
native protein. FIG. 13B shows a ladder pattern of bands with a
size range from 36 to 116 kDa detected in extracts prepared from
GBS strain A909 but not from strain 090 (negative control) probed
with the postimmunization serum. This ladder pattern corresponds to
the expected size range of the alpha C-protein bands (Madoff, L.
C., et al., Infect. Immun. 59:2638-2644 (1991)). Thus, alpha
C-protein N-terminal antiserum can detect native alpha C
protein.
EXAMPLE 13
Opsonophagocytosis Assay Using Alpha C-Protein N-Terminal-Specific
Antibodies
[0184] An in vitro opsonophagocytosis assay was used to determine
whether alpha C-protein N-terminal antibodies are opsonic for GBS
(Gallagher, S. A. & Smith, J. A. in Current Protocols in
Molecular Biology, Vol. 2. F. M. Ausubel, et al., (eds.), John
Wiley & Sons, Inc., New York, (1994), pp. 10.2.1-10.2.21).
[0185] Methods
[0186] The opsonophagocytosis assay to determine the functionality
of the alpha C-protein N-terminal-specific antibodies was carried
out as described (Baltimore, R. S., et al., J. Immunol. 118:673-678
(1977)). This assay requires human serum (used as a complement
source), GBS (.about.1.5.times.10.sup.6 CFU), polymorphonucleocytes
(PMNs) (-3.0.times.10.sup.6 cells), and antibodies (final dilution
{fraction (1/100)}) combined in a 500-.sub.--1 volume. -The amount
of opsonophagocytic killing (log-kill) was determined by
subtracting the log of the number of colonies surviving the 1-hr
assay from the log of the number of CFU at the zero time point.
[0187] FIG. 14 shows that the postimmunzation antiserum kills
approximately 0.6-log more GBS than the preimmunization serum; this
degree of killing is comparable to that by antiserum raised to the
whole recombinant molecule (alpha C-protein polyclonal serum). As
expected, no killing is observed when PMNs are not added or when
heat-killed complement is used. These data demonstrate that alpha
C-protein N-terminal-specific antibodies are opsonic for GBS.
EXAMPLE 14
Mouse Protection by Alpha C-Protein N-Terminal-Specific
Antibodies
[0188] A mouse protection study was conducted to determine whether
alpha C-protein N-terminal-specific antibodies can protect neonatal
mice against infection with alpha C-protein-bearing strains of GBS.
Pregnant dams were passively immunized with postimmunization rabbit
antiserum raised to the N-terminal, preimmunization rabbit serum,
(negative control), and rabbit antiserum to the Ia-TT
protein-capsular-polysacchari- de conjugate (positive control)
(Wessels, M. R., et al., Infect. Immun. 61:4760-4766 (1993)).
[0189] Neonatal mouse model. The following is a modification of the
neonatal mouse model described by Rodewald et al. (Rodewald, A. K.,
et al., J. Infect. Dis. 166:635-639 (1992)). CD-1 outbred mice
arrived 17- to 18-days pregnant (Charles River Laboratories). The
second day after arrival mice were divided into three groups with 4
pregnant mice per group. The mice were immunized intraperitoneally
with 0.5 ml of the postimmunization antiserum raised to the
N-terminal polypeptide, preimmunization serum, or antiserum raised
to a protein-polysaccharide conjugate consisting of the Ia capsular
polysaccharide covalently coupled to tetanus toxoid (Ia-T)
(Wastfelt, M., et al., J. Biol. Chem. 271:18892-18897 (1996)).
Newborn mouse pups were challenged with 3.times.10.sup.4 CFU of GBS
strain A909 by intraperitoneal injection. After 48 hours, numbers
of dead and surviving mice were counted.
[0190] Table 2 shows that 69% of the mice immunized with N-terminal
antibodies survived challenge with GBS strain A909, whereas only
15% of those immunized with the preimmunization serum survived.
This protection was significant (P<0.0001, Fisher's exact test).
These results show that the alpha C-protein N-terminal antibodies
are significantly more protective than control serum. Therefore,
the alpha C-protein N-terminus contains a protective epitope.
4TABLE 2 Passive protection of neonatal mice with
N-terminal-specific antibodies to GBS. Number of pups Antiserum
(alive/total) % Survival Ia-TT.sub.a 45/46 98 preimmunzation.sub.b
6/39 15 N-terminal 29/42.sub.c 69 .sub.aPositive control (GBS Ia
polysaccharide conjugated to tetanus toxoid) .sub.bNegative control
.sub.cP < 0.0001 compared with the preimmunization serum by
Fisher's exact test.
[0191] Discussion of Examples 11-14
[0192] The alpha and presumably the epsilon C protein of GBS are
surface-associated proteins that are thought to play a role in the
virulence of and immunity to GBS. The different structural domains
of the alpha C protein, the N-terminus, repeat region, and
C-terminus, may have different biological and immunologic
properties. It is assumed that this is also true for the epsilon
antigen.
[0193] Thus, either in a conjugated or non-conjugated form, the
different structural domains (e.g. the C or N-terminal regions) of
the alpha, beta or epsilon antigens either individually or in
various combinations with each other may be used to make vaccines
against GBS. In order to develop a conjugate vaccine to protect
against alpha C-protein-bearing strains, the opsonic and protective
epitopes of the antigen were initially mapped. Epitopes of the
alpha C protein that are both opsonic and protective have now been
localized to the repeat region and the N-terminus of the alpha
C-protein.
[0194] Clearly, there is a divergence between the N-termini of the
alpha and epsilon C-proteins. Studies of other surface-associated
proteins of group B Streptococcus that have repetitive sequences
(e.g., Rib, epsilon, and type V strains) suggest that there is also
divergence from the alpha antigen in the N-terminal (Beseth, B. D.,
A genetic analysis of phenotypic diversity of the C protein alpha
antigen of group B Streptococcus, Bachelor of Arts thesis in
Biology, Harvard College, (1992); DuBois, N. B. Genetic and
phenotypic properties of the surface proteins of group B
Streptococcus and the identification of a new protein, Bachelor of
Arts thesis in Biology, Harvard College, (1995); Lachenauer, C. S.
& L. C. Madoff, Infect. Immun. 64:4255-4260 (1996); Wstfelt,
M., et al., J. Biol. Chem. 271:18892-18897 (1996)).
[0195] To determine whether the alpha C-protein N-terminus
contained protective epitopes, specific antibodies to the
N-terminus were raised that conferred passive protection against
alpha C-protein-bearing strains in a neonatal mouse model. It is
interesting that specific antibodies to the alpha C-protein
N-terminus conferred 70% passive-protection, whereas the polyclonal
antibodies to the 9-repeat alpha C protein conferred 41% passive
protection (Gravekamp, C., et al., Infect. Immun. 64:3576-3583
(1996)). However, polyclonal antibodies elicited to recombinant 1-
or 2-repeat alpha C proteins give greater than 75% protection
(Gravekamp, C., et al., Infect. Immun. 64:3576-3583 (1996)).
Hypothetically, antibodies directed at the full-length alpha or
epsilon C protein might select for deletions within the repeat
region.
[0196] A mouse model of GBS infection was used to determine whether
alpha C-protein tandem repeat deletion mutants were selected in
vivo (Madoff, L. C., et al., Proc. Natl. Acad. Sci. USA.
93:4131-4136 (1996)). Mice immunized with antibodies to the alpha C
protein were challenged with GBS strains expressing the alpha C
protein. The size of the alpha C protein in strains of GBS isolated
from the spleens of the mice was determined by western blot
analysis. Fifty percent of the recovered GBS strains expressed
truncated forms of the alpha C protein, a result suggesting the
selection of deletion mutants within the repeat region of the bca
gene in the presence of alpha C-protein antibodies. Because these
mutants were isolated at a relatively high frequency, they may have
been protected against opsonophagocytosis by the alpha C-protein
antibodies.
[0197] In an in vitro opsonophagocytosis assay, strains of GBS with
deletions in the bca gene were killed less frequently than parental
strains by antibodies to the full-length alpha C-protein (Madoff,
L. C., et al., Proc. Natl. Acad. Sci. USA. 93:41314136 (1996)).
This apparent lesser susceptibility to opsonization may be
explained by fewer tandem repeat epitopes than are present in
strains with a full-length alpha C-protein. In addition to being
fewer in number, the alpha C-protein protective epitopes may be
conformational, and deletion mutants may express fewer alpha
C-protein epitopes. By ELISA inhibition the affinity for
recombinant alpha C proteins containing a single repeat is reduced
compared with that of recombinant proteins containing larger
numbers of repeats (Gravekamp, C., et al., Infect. Immun.
64:3576-3583 (1996)). These antibodies recognize epitopes expressed
by proteins containing more repeats (9 or 16) but lose their
overall binding affinity for epitopes expressed by proteins
containing fewer repeats (1 or 2).
[0198] These observations support the possibility that the epitopes
of the alpha C-protein tandem repeat region are conformational.
Alternatively, the reduced affinity for the single repeat
recombinant protein can also be explained if the recombinant alpha
C protein is proteolytically processed. Thus, the 1-repeat
recombinant protein may lose repeat-region epitopes due to
proteolytic processing within the single repeat and have a reduced
ability to inhibit antibody binding to these epitopes. In contrast
to epitopes within the repeat region, the N-terminal epitopes are
conserved in both parental and deletion mutant strains of GBS
strain A909, and these mutants are susceptible to
opsonophagocytosis with N-terminal antiserum (Madoff, L. C., et
al., Proc. Natl. Acad. Sci. USA. 93:4131-4136 (1996)). Therefore,
the recombinant alpha and epsilon C-protein N-terminus are prime
candidates for use in a protein-polysaccharide conjugate
vaccine.
[0199] The protective monoclonal antibody 4G8 was localized to the
repeat region of the alpha C protein (Madoff, L. C., et al.,
Infect. Immun. 59:204-210 (1991)). Attempts to further localize the
epitope bound by 4G8 within the repeat region have not been
successful. Our unpublished studies using synthetic peptides
corresponding to overlapping 10-amino acid segments within the
alpha C-protein repeats did not reveal a binding site for 4G8. The
inability to define a peptide binding site for 4G8 on the alpha
C-protein could be explained if the site detected by 4G8 were a
conformational epitope or if the binding site contains
noncontiguous segments from within the repeat region.
[0200] Protective epitopes have been mapped within the closely
related M protein of group A Streptococcus (GAS) (Beachey, E. H.,
et al., J. Exp. Med. 166:647-656 (1987)). In studies assessing
whether protective epitopes were localized to the N-terminal half,
the N-terminus of the M-protein was liberated from the cell wall of
GAS by pepsin cleavage. Antibodies were raised to the isolated
pepsin-cleaved N-terminal fragments (pepM) (Beachey, E. H., et al.,
J. Exp. Med. 150:862-877 (1979)). Such antibodies have been
demonstrated to be opsonic and protective in studies of several M
proteins (Kehoe, M. A., Vaccine 9:797-806 (1991)). Protective
epitopes of the M5, M6, and M24 proteins were mapped to the extreme
N-terminal regions of the mature proteins (Beachey, E. H., et al.,
J. Exp. Med. 166:647-656 (1987)), and a synthetic peptide vaccine
consisting of these epitopes was developed. This vaccine elicited
opsonic antiserum that protected mice against challenge with M5,
M6, and M24 strains of GAS.
[0201] The identification of two protective epitopes of GBS, the
N-terminus and the tandem repeat region, will facilitate the
development of a conjugate vaccine against GBS. One vaccine
strategy is to use the recombinant alpha containing the N-terminus
plus two tandem repeats (Gravekamp, C., et al., Infect. Immun.
64:3576-3583 (1996)). This recombinant protein confers passive
protection in a neonatal mouse protection assay. However, to
increase the efficacy of a vaccine based on the two-repeat protein,
this recombinant antigen can be coupled to several different
N-terminal and repeat regions from other surface-associated
proteins of GBS such as the Rib protein, which is commonly found on
many serotype III strains. By combining multiple, distinct, and
conserved protective epitopes to form a multivalent vaccine, it
should be possible to raise antibodies against the majority of
clinically significant GBS isolates. Similar strategies for
producing vaccines will be used with the N-terminal portion of the
epsilon antigen.
EXAMPLE 15
Mouse Protection by Epsilon C-Protein N-Terminal-Specific
Antibodies
[0202] A mouse protection study is conducted to investigate using
epsilon C-protein N-terminal-specific antibodies (either in a
conjugated or a non-conjugated form) to protect neonatal mice
against infection with alpha and/or epsilon C-protein-bearing
strains of GBS. Pregnant dams are passively immunized with
postimmunization rabbit antiserum raised to the N-terminal region
of the epsilon antigen, preimmunization rabbit serum, (negative
control), and rabbit antiserum to the Ia-TT
protein-capsular-polysaccharide conjugate (positive control)
(Wessels, M. R., et al., Infect. Immun. 61:4760-4766 (1993)).
Antiserum is also raised to a conjugate vaccine comprising (a) a
group B Streptococcus capsular polysaccharide conjugated to (b) the
N-terminal region of the epsilon antigen or a fragment thereof,
where said N-terminal region or fragment is capable of eliciting
protective antibodies against the group B Streptococcus, and where
said conjugate vaccine is substantially free of streptococcal
proteins other than the C-protein epsilon antigen.
[0203] A preparation that is "substantially free" of streptococcal
proteins other than the N-terminal region of the epsilon antigen
should be understood as referring to a preparation wherein the only
streptococcal protein is that of the terminal region of the epsilon
antigen or fragments thereof. Though proteins may be present in the
preparation which are homologous to other streptococcal proteins,
the sample is still said to be substantially free of other
streptococcal proteins as long as the homologous protein contained
in the sample are not expressed from genes obtained from
Streptococcus. Finally, "substantially free" of streptococcal
proteins other than the N-terminal region of the epsilon antigen is
not mean to exclude preparations which might contain trace amounts
of such proteins.
[0204] The neonatal mouse model of Rodewald (Rodewald et al.,
Infect. Immun. 61:4760-4766 (1993) is then used to determine the
protective effect of the antiserum. An animal model for determining
the dose response of humans to conjugate vaccines comprising a
bacterial polysaccharide and a strongly immunogenic carrier protein
is that described in U.S. Pat. No. 5,604,108.
EXAMPLE 16
GBS Type III Polysaccharide-Epsilon Antigen N-Terminal Region
Conjugate Vaccine
[0205] A conjugate vaccine is prepared similar to Madoffet al. (J.
Clin. Invest. 94:286-292 (1994). Oxidized type III polysaccharide
(5.5 mg) is combined with 5 mg. of the N-terminal region of the
epsilon antigen in a phosphate buffered saline at pH 9.0.
Cyanoborohydride is added to the mixture incubated at room
temperature insure complete coupling. The pH of the reaction is
maintained at 9.0-9.5. Progress of the conjugation is monitored by
gel filtration chromatography (Paoletti et al., Trends in Glycosci.
Glycotechnol. 4:269-278, (1992)). the conjugation is considered to
be complete when the magnitude of the protein peak occurring at the
void volume of a Superose 6 column (Pharmacia Fine Chemical,
Piscataway, N.J. remains constant. After completion, the conjugate
vaccine is separated from uncoupled components.
EXAMPLE 17
Passive Immunization
[0206] The invention is also related to a method of passive
immunization, used particularly for infants or compromised adults.
The capsular polysaccharide-N-terminal region epsilon antigen
conjugate or a non-conjugated N-terminal region epsilon antigen is
injected into a human to raise antibodies thereto to a high titer.
The antiserum from the blood of the human is separated and
fractionated to produce a gamma globulin fraction containing the
antibodies, that can be used for passive immunization. This method
of establishing donors for passive immunization is useful because,
although occasional non-immunized individual have very high levels
of type-specific GBS antibody in their sera, it would be necessary
to screen very large populations to select those individuals whose
plasma could be pooled to make sufficiently high titered globulin
fractions to be useful for passive immunizations.
[0207] For passive immunization, pools of human sera from selected
individuals vaccinated with the conjugated vaccine can be
concentrated and fractionated by conventional procedures. This
provides a globulin fraction containing most of the type-specific
antibody and has sufficiently high activity so that the hyperimmune
globulin is effective in small doses of 0.3-1.0 ml, preferably a
about 0.5 ml. The globulin fraction is administered either
intravenously or intramuscularly in a suitable physiologically
acceptable carrier. Such a carrier includes, but is not limited to,
normal saline. The concentration of the globulin in the carrier may
be from 5 to 20% by weight. The hyperimmune globulin can be
administered either to pregnant women prior to deliver, to
neonates, or to immunologically compromised individuals, to provide
passive immunization or therapy.
EXAMPLE 18
Diagnostic Tool
[0208] The DNA obtained by cloning the plasmid pJMS36 may be used
as a diagnostic tool for determining infections with Group B
Streptococcus expressing the epsilon antigen. Such knowledge should
provide those of skill in the art with more rational approaches for
the treatment of GBS infection. Such a tool is a useful as an
adjunct for diagnosis of GBS infection or as a molecular marker of
epidemiological significance.
[0209] After obtaining an appropriate sample for culturing from an
individual in need of diagnosis, the bacterial DNA is extracted and
analyzed by Southern blot analysis to determine whether the GBS
bacteria carries an epsilon antigen. The means for culturing the
sample, extracting and analyzing the DNA from the infected
individual are well-known to those of skill in the art.
[0210] All references cited are incorporated herein by reference.
While the invention has been described in detail and with reference
to specific embodiments thereof, it will be apparent to one skilled
in the art that various changes and modifications could be made
therein without departing from the spirt and scope thereof.
* * * * *