U.S. patent application number 09/797385 was filed with the patent office on 2002-05-16 for non-iga fc binding forms of the group b streptococcal beta antigens.
Invention is credited to Blake, Milan S., Tai, Joseph Y..
Application Number | 20020058042 09/797385 |
Document ID | / |
Family ID | 21821978 |
Filed Date | 2002-05-16 |
United States Patent
Application |
20020058042 |
Kind Code |
A1 |
Tai, Joseph Y. ; et
al. |
May 16, 2002 |
Non-IgA fc binding forms of the Group B streptococcal beta
antigens
Abstract
The invention relates to a mutant C.beta. protein comprising the
amino acid sequence A-X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5
X.sub.6 X.sub.7 X .sub.8 X.sub.9 X.sub.10 X.sub.11 X.sub.12-B,
wherein A comprises amino acids 1-164 of the sequence shown in FIG.
1 (SEQ ID NO: 2), B represents a sequence starting from amino acid
177 and terminating at an amino acid between residue 1094 and 1127,
inclusive, of the sequence shown in FIG. 1 (SEQ ID NO: 2), and
X.sub.1-X.sub.12 are each selected independently from the group
consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, a bond, and
the wild type amino acid found at the corresponding position of the
sequence shown in FIG. 1 (SEQ ID NO: 2), wherein said amino acid
positions are numbered from the first amino acid of the native
amino acid sequence encoding said protein, with the proviso that at
least one of X.sub.1 through X.sub.12, inclusive, is other than the
wild type amino acid, and wherein the LPXTG motif may be missing
from the mutant C.beta. protein. The invention also relates to a
polynucleotide molecule encoding a mutant C.beta. protein, as well
as vectors comprising such polynucleotide molecules, and host cells
transformed therewith. The invention also relates to a conjugate
comprising the mutant C.beta. protein covalently conjugated to a
capsular polysaccharide. The invention also relates to a vaccine
comprising at least one mutant C.beta. protein of the invention and
a pharmaceutically acceptable carrier. The invention also relates
to a method of inducing an immune response in an animal, comprising
administering the vaccine of the invention to an animal in a
therapeutically effective amount.
Inventors: |
Tai, Joseph Y.; (Fort
Washington, PA) ; Blake, Milan S.; (Fulton,
MD) |
Correspondence
Address: |
Baxter Healthcare Corporation
P.O. Box 15210
Irvine
CA
92614
US
|
Family ID: |
21821978 |
Appl. No.: |
09/797385 |
Filed: |
March 1, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09797385 |
Mar 1, 2001 |
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08923992 |
Sep 5, 1997 |
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6280738 |
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60024707 |
Sep 6, 1996 |
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Current U.S.
Class: |
424/185.1 ;
435/320.1; 435/325; 435/6.16; 435/69.1; 530/324; 536/23.1 |
Current CPC
Class: |
A61K 39/00 20130101;
C07K 14/315 20130101; Y10S 424/831 20130101; A61P 31/04 20180101;
Y10S 530/825 20130101 |
Class at
Publication: |
424/185.1 ;
530/324; 536/23.1; 435/6; 435/69.1; 435/320.1; 435/325 |
International
Class: |
A61K 039/00; C07H
021/04; C12Q 001/68; C07K 014/435; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. A polynucleotide molecule comprising a nucleotide sequence that
encodes a mutant C.beta. protein comprising the amino acid sequence
A-X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7 X.sub.8
X.sub.9 X.sub.10 X.sub.11 X.sub.12-B, wherein A comprises amino
acids 1-164 of the sequence shown in FIG. 1 (SEQ ID NO: 2), B
represents a sequence starting from amino acid 177 and terminating
at an amino acid between residue 1094 and 1127, inclusive, of the
sequence shown in FIG. 1 (SEQ ID NO: 2), and X.sub.1-X.sub.12 are
each selected independently from the group consisting of Ala, Val,
Leu, Ile, Pro, Met, Phe, Trp, a bond, and the wild type amino acid
found at the corresponding position of the sequence shown in FIG. 1
(SEQ ID NO: 2), wherein said amino acid positions are numbered from
the first amino acid of the native amino acid sequence encoding
said protein, with the proviso that at least one of X.sub.1 through
X.sub.12, inclusive, is other than the wild type amino acid, and
wherein the LPXTG motif may be missing from the mutant C.beta.
protein.
2. The polynucleotide molecule of claim 1, wherein X.sub.1,
X.sub.5, X.sub.7, X.sub.8, X.sub.10, X.sub.11, and X.sub.12 are
each selected independently from the group consisting of Ala, Val,
Leu, Ile, Pro, Met, Phe, Trp, a bond, and the wild type amino acid
found at the corresponding position of the sequence shown in FIG. 1
(SEQ ID NO: 2), with the proviso that at least one of X.sub.1,
X.sub.5, X.sub.7, X.sub.8, X.sub.10, X.sub.11, and X.sub.12 is
other than the wild type amino acid.
3. The polynucleotide molecule of claim 1, wherein X.sub.1 and
X.sub.11 are selected from the group consisting of Ala, Val, Leu,
Ile, Pro, Met, Phe, Trp, and a bond.
4. The polynucleotide molecule of claim 1, wherein X.sub.1 and
X.sub.11 are Pro.
5. The polynucleotide molecule of claim 1, wherein X.sub.7 and
X.sub.12 are selected from the group consisting of Ala, Val, Leu,
Ile, Pro, Met, Phe, Trp, and a bond.
6. The polynucleotide molecule of claim 1, wherein X.sub.7 and
X.sub.12 are Ala.
7. The polynucleotide molecule of claim 1, wherein X.sub.5,
X.sub.7, X.sub.8, X.sub.10, X.sub.11 and X.sub.12 are each a
bond.
8. A mutant C.beta. protein comprising the amino acid sequence
A-X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7 X.sub.8
X.sub.9 X.sub.10 X.sub.11 X.sub.12-B, wherein A comprises amino
acids 1-164 of the sequence shown in FIG. 1 (SEQ ID NO: 2), B
represents a sequence starting from amino acid 177 and terminating
at an amino acid between residue 1094 and 1127, inclusive, of the
sequence shown in FIG. 1 (SEQ ID NO: 2), and X.sub.1-X.sub.12 are
each selected independently from the group consisting of Ala, Val,
Leu, Ile, Pro, Met, Phe, Trp, a bond, and the wild type amino acid
found at the corresponding position of the sequence shown in FIG. 1
(SEQ ID NO: 2), wherein said amino acid positions are numbered from
the first amino acid of the native amino acid sequence encoding
said protein, with the proviso that at least one of X.sub.1 through
X.sub.12, inclusive, is other than the wild type amino acid, and
wherein the LPXTG motif may be missing from the mutant C.beta.
protein.
9. The protein of claim 8, wherein X.sub.1, X.sub.5, X.sub.7,
X.sub.8, X.sub.10, X.sub.11, and X.sub.12 are each selected from
the group consisting of Ala, Val, Leu, Ile, Pro, Met, Phe, Trp, a
bond, and the wild type amino acid found at the corresponding
position of the sequence shown in FIG. 1 (SEQ ID NO: 2), with the
proviso that at least one of X.sub.1, X.sub.5, X.sub.7, X.sub.8,
X.sub.10, X.sub.11, and X.sub.12 is other than the wild type amino
acid.
10. The protein of claim 8, wherein X.sub.1 and X.sub.11 are
selected from the group consisting of Ala, Val, Leu, Ile, Pro, Met,
Phe, Trp, and a bond.
11. The protein of claim 8, wherein X.sub.1 and X.sub.11 are
Pro.
12. The protein of claim 8, wherein X.sub.7 and X.sub.12 are
selected from the group consisting of Ala, Val, Leu, Ile, Pro, Met,
Phe, Trp, and a bond.
13. The protein of claim 8, wherein X.sub.7 and X.sub.12 are
Ala.
14. The protein of claim 8, wherein X.sub.5, X.sub.7, X.sub.8,
X.sub.10, X.sub.11 and X.sub.12 are each a bond.
15. The mutant C.beta. protein of claim 8, wherein the hydrophobic
amino acid residues 1108-1116 are replaced by non-hydrophobic amino
acids.
16. The mutant C.beta. protein of claim 8, wherein at least one of
amino acid residues 521-541, inclusive, of C.beta. is either (a)
deleted or (b)) altered, so that the protein is not cleaved in this
region when produced in E. coli.
17. The mutant C.beta. protein of claim 16, wherein at least one of
amino acid residues 533-541, inclusive, of C.beta. is either (a)
deleted or (b) altered.
18. The mutant C.beta. protein of claim 16, wherein at least one of
amino acid residues 537-538 of C.beta. is either (a) deleted or (b)
altered.
19. A polysaccharide-protein conjugate comprising the mutant
C.beta. protein of claim 8 and a streptococcal capsular
polysaccharide.
20. A vector comprising the polynucleotide molecule of claim 1.
21. A host cell transformed with the vector of claim 20.
22. A vaccine comprising at least one mutant C.beta. protein of
claim 8, together with a pharmaceutically acceptable carrier.
23. The vaccine of claim 22, wherein said mutant C.beta. protein is
conjugated to a polysaccharide.
24. The vaccine of claim 23, wherein said polysaccharide to which
said mutant C.beta. protein is conjugated is selected from the
group consisting of Group B streptococcal capsular polysaccharide
types Ia, II, III and V.
25. A combination vaccine comprising at least two C.beta.
protein-polysaccharide conjugates selected from the group
consisting of C.beta.-Ia, C.beta.-II, C.beta.-III and C.beta.-V,
together with a pharmaceutically acceptable carrier, wherein the
C.beta. portion of each conjugate is the mutant C.beta. of claim
8.
26. The vaccine of claim 25, said vaccine comprising C.beta.-Ia,
C.beta.-II, C.beta.-III and C.beta.-V, together with a
pharmaceutically acceptable carrier.
27. A method of inducing an immune response in a mammal, comprising
administering a vaccine comprising at least one mutant C.beta.
protein comprising the amino acid sequence A-X.sub.1 X.sub.2
X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7 X.sub.8 X.sub.9 X.sub.10
X.sub.11 X.sub.12-B, wherein A comprises amino acids 1-164 of the
sequence shown in FIG. 1 (SEQ ID NO: 2), B represents a sequence
starting from amino acid 177 and terminating at an amino acid
between residue 1094 and 1127, inclusive, of the sequence shown in
FIG. 1 (SEQ ID NO: 2), and X.sub.1-X.sub.12 are each selected
independently from the group consisting of Ala, Val, Leu, Ile, Pro,
Met, Phe, Trp, a bond, and the wild type amino acid found at the
corresponding position of the sequence shown in FIG. 1 (SEQ ID NO:
2), wherein said amino acid positions are numbered from the first
amino acid of the native amino acid sequence encoding said protein,
with the proviso that at least one of X.sub.1 through X.sub.12,
inclusive, is other than the wild type amino acid, and wherein the
LPXTG motif may be missing from the mutant C.beta. protein,
together with a pharmaceutically acceptable carrier, in an amount
sufficient to induce an immune response in a mammal.
28. The method of claim 27, wherein said mutant C.beta. protein is
conjugated to a streptococcal capsular polysaccharide.
29. The method of claim 28, wherein said mammal is a human.
Description
BACKGROUND OF THE INVENTION
[0001] 1. Field of the Invention
[0002] The present invention concerns the construction of a protein
having a reduced or eliminated ability to bind human IgA, but that
retains the immunological properties useful for formulating a
conjugate vaccine against Group B streptococci.
[0003] 2. Related Art
[0004] Streptococci are a large and varied set of gram-positive
bacteria which have been ordered into several groups based on the
antigenicity and structure of their cell wall polysaccharide
(Lancefield, R. C., J. Exp. Med. 57: 571-595 (1933); Lancefield, R.
C., Proc. Soc. Exp. Biol. and Med. 38: 473-478 (1938)). Two of
these groups have been associated with serious human infections.
Those that have been classified into Group A streptococci are the
bacteria that people are most familiar and are the organisms which
cause "strep throat." Organisms of Group A streptococci also are
associated with the more serious infections of rheumatic fever,
streptococcal impetigo, and sepsis.
[0005] Group B streptococci were not known as a human pathogen in
standard medical textbooks until the early 1970's. Since that time,
studies have shown that Group B streptococci are an important
perinatal pathogen in both the United States as well as the
developing countries (Smith, A. L. and J. Haas, Infections of the
Central Nervous System, Raven Press, Ltd., New York. (1991) p.
313-333). Systemic Group B streptococcal infections during the
first two months of life affect approximately three out of every
1000 births (Dillon, H. C., Jr., et al., J. Pediat. 110: 31-36
(1987)), resulting in 11,000 cases annually in the United States.
These infections cause symptoms of congenital pneumonia, sepsis,
and meningitis. A substantial number of these infants die or have
permanent neurological sequelae. Furthermore, these Group B
streptococcal infections may be implicated in the high
pregnancy-related morbidity which occurs in nearly 50,000 women
annually. Others who are at risk from Group B streptococcal
infections include those who either congenitally,
chemotherapeutically, or by other means, have an altered immune
response.
[0006] Group B streptococci can be further classified into several
different types based on the bacteria's capsular polysaccharide.
The most pathogenically important of these different types are
streptococci having types Ia, Ib, II, or III capsular
polysaccharides. Group B streptococci of these four types represent
over 90% of all reported cases. The structure of each of these
various polysaccharide types has been elucidated and characterized
(Jennings, H. J., et al., Biochemistry 22: 1258-1263 (1983);
Jennings, H. J., et al., Can. J. Biochem. 58: 112-120 (1980);
Jennings, H. J., et al., Proc. Nat. Acad. Sci. USA. 77: 2931-2935
(1980); Jennings, H. J., et al., J. Biol. Chem. 258: 1793-1798
(1983); Wessels, M. R., et al., J. Biol. Chem. 262: 8262-8267
(1987)). As is found with many other human bacterial pathogens, it
has been ascertained that the capsular polysaccharides of Group B
streptococci, when used as vaccines, provide very effective,
efficacious protection against infections with these bacteria. This
was first noted by Lancefield (Lancefield, R. C., et al., J. Exp.
Med. 142: 165-179 (1975)) and more recently in the numerous studies
of Kasper and coworkers (Baker, C. J., et al., N. Engl. J. Med.
319: 1180-1185 (1988); Baltimore, R. S., et al., J. Infect. Dis.
140: 81-86 (1979); Kasper, D. L., et al., J. Exp. Med. 149: 327-339
(1979); Madoff, L. C., et al., J. Clin. Invest. 94: 286-292 (1994);
Marques, M. B., et al., Infect. Immun. 62: 1593-1599 (1994);
Wessels, M. R., et al., J. Clin. Invest. 86: 1428-1433 (1990);
Wessels, M. R., et al., Infect. Immun. 61: 4760-4766 (1993); Wyle,
S. A., et al., J. Infect. Dis. 126: 514-522 (1972)). However, much
like many other capsular polysaccharide vaccines (Anderson, P., et
al., J. Clin. Invest. 51: 39-44 (1972); Gold, R., et al., J. Clin.
Invest. 56: 1536-1547 (1975); Gold, R., et al., J. Infect. Dis.
136S: S31-S35 (1977); Gold, R. M., et al., J. Infect. Dis. 138:
731-735 (1978); Mkel, P. R. H., et al., J. Infect. Dis. 136: S43-50
(1977); Peltola, A., et al., Pediatrics 60: 730-737 (1977);
Peltola, H., et al. N. Engl. J. Med. 297: 686-691 (1977)), vaccines
formulated from pure type Ia, Ib, II, and III capsular
carbohydrates are relatively poor immunogens and have very little
efficacy in children under the age of 18 months (Baker, C. J. and
D. L. Kasper. Rev. Inf. Dis. 7: 458-467 (1985); Baker, C. J., et
al., N. Engl. J. Med. 319: 1180-1185 (1988); Baker, C. J., et al.,
New Engl. J. Med. 322: 1857-1860 (1990)). These pure
polysaccharides are classified as T cell independent antigens
because they induce a similar immunological response in animals
devoid of T lymphocytes (Howard, J. G., et al., Cell. Immunol. 2:
614-626 (1971)). It is thought that these polysaccharides do not
evoke a secondary booster response because they do not interact
with T cells, and therefore fail to provoke a subsequent "helper
response" via the secretion of various cytokines. For this reason,
each consecutive administration of the polysaccharide as a vaccine
results in the release of a constant amount of antibodies, while a
T cell dependent antigen would elicit an ever increasing
concentration of antibodies each time it was administered.
[0007] Goebel and Avery found in 1931 that by covalently linking a
pure polysaccharide to a protein that they could evoke an immune
response to the polysaccharide which could not be accomplished
using the polysaccharide alone (Avery, O. T. and W. F. Goebel, J.
Exp. Med. 54: 437-447 (1931); Goebel, W. F. and O. T. Avery, J.
Exp. Med. 54: 431-436 (1931)). These observations initiated and
formed the basis of the current conjugate vaccine technology.
Numerous studies have followed and show that when polysaccharides
are coupled to proteins prior to their administration as vaccines,
the immune response to the polysaccharides changes from a T
independent response to a T dependent response (see (Dick, W. E.,
Jr. and M. Beurret, Glycoconjugates of bacterial carbohydrate
antigens In: Contributions to Microbiology and Immunology. Cruse et
al., eds., (1989) p.48-114; Jennings, H. J. and R. K. Sood,
Neoglycoconjugates: Preparation and Applications. Y. C. Lee and R.
T. Lee, eds., Academic Press, New York. (1994) p. 325-371; Robbins,
J. B. and R. Schneerson, J. Infect. Dis. 161: 821-832 (1990)) for
reviews). Currently, most of these polysaccharide-protein conjugate
vaccines are formulated with well known proteins such as tetanus
toxoid and diphtheria toxoid or mutants thereof. These proteins
were originally used because they were already licensed for human
use and were well characterized. However, as more and more
polysaccharides were coupled to these proteins and used as
vaccines, interference between the various vaccines which used the
same protein became apparent. For example, if several different
polysaccharides were linked to tetanus toxoid and given
sequentially, the immune response to the first administered
polysaccharide conjugate would be much larger than the last. If,
however, each of the polysaccharides were coupled to a different
protein and administered sequentially, the immune response to each
of the polysaccharides would be the same. Carrier suppression is
the term used to describe this observed phenomenon. One approach to
overcome this problem is to match the protein and polysaccharide so
that they are derived from the same organism.
[0008] Among the various antigens used to classify and subgroup
Group B streptococci, one was a protein known as the Ibc antigen.
This protein antigen was first described by Wilkinson and Eagon in
1971 (Wilkinson, H. W. and R. G. Eagon, Infect. Immun. 4: 596-604
(1971)) and was known to be made up of two distinct proteins
designated as alpha and beta. Later, the Ibc antigen was shown to
be effective when used as a vaccine antigen in a mouse model of
infection by Lancefield and co-workers (Lancefield, R. C., et al.,
J. Exp. Med. 142: 165-179 (1975)). The isolation, purification and
functional characterization of the beta antigen (C.beta.) protein
of Group B streptococci was accomplished by Russell-Jones, et al.
(Russell-Jones, G. J. and E. C. Gotschlich, J. Exp. Med. 160:
1476-1484 (1984); Russell-Jones, G. J., et al., J. Exp. Med. 160:
1467-1475 (1984)) [see U.S. Pat. No. 4,757,134]. They could
demonstrate that one of the properties of the C.beta. protein was
to bind specifically to human IgA immunoglobulin. The binding site
on the IgA molecule was localized to the Fc portion of the heavy
chain of this immunoglobulin. They further showed that the C.beta.
protein consisted of a single polypeptide having an estimated
molecular weight of 130,000 daltons. The gene responsible for the
expression of the C.beta. protein was cloned (Cleat, P. H. and K.
N. Timmis, Infect. Immun. 55: 1151-1155 (1987)) and sequenced
(Jerlstrom, P. G., et al., Molec. Microbiol. 5: 843-849 (1991)) by
a group led by Timmis. His later study demonstrated that the IgA
binding activity could be assigned to a 746 bp DNA fragment of the
gene defined by a leading BglII restriction endonuclease cleavage
site and ending with a HpaI restriction endonuclease cleavage
site.
[0009] As stated previously, the 1975 Lancefield study showed that
the Ibc antigen was an effective vaccine antigen in a mouse model
of Group B streptococcal infection (Lancefield, R. C., et al., J.
Exp. Med. 142: 165-179 (1975)). It was not clear at the time
whether the alpha or beta protein component of the Ibc antigen was
responsible for this protection. Madoff et al., began to shed light
on this question and demonstrated that the purified C.beta. protein
used as a vaccine could protect infant mice from experimental
infection with Group B streptococci expressing this protein
(Madoff, L. C., et al., Infect. Immun. 60: 4989-4994 (1992)).
Madoff et al., then went on to show that when they coupled a Type
III streptococcal capsular polysaccharide to the C.beta. protein,
producing a conjugate vaccine, this vaccine would protect infant
mice against infection with either a Type III Group B streptococci
(expressing no C.beta.) or a Type Ib Group B streptococci
(expressing C.beta. but lacking a Type III capsular polysaccharide)
(Madoff, L. C., et al., J. Clin. Invest. 94: 286-292 (1994)). Thus,
such a C.beta. protein conjugate vaccine served several functions:
the polysaccharide elicited protective antibodies to the
polysaccharide capsule and the C.beta. protein evoked protective
antibodies to the protein as well as modified the immune response
to the polysaccharide from a T independent response to a T
dependent response.
[0010] This polysaccharide-C.beta. protein conjugate strategy works
well in mice. But clearly, the goal is to protect humans against
Group B streptococcal infections. The only caveat with using the
same strategy in humans is that the C.beta. protein binds human IgA
immunoglobulins non-specifically (C.beta. does not bind mouse IgA).
This human IgA binding activity of C.beta. could diminish the
efficacy of a polysaccharide-C.beta. protein conjugate vaccine for
humans, as antigens bound to IgA can be cleared from the system so
rapidly that an antigen-specific antibody response is not produced.
Furthermore, potentially protective epitopes on the C.beta. protein
could be hidden when the human IgA binds to the C.beta. molecule.
Thus, it would be advantageous to obtain a mutant C.beta. protein
which lacks the IgA binding capacity but retains as much of the
native structure as possible.
[0011] With this goal in mind, several groups have attempted to
determine the IgA binding region of the C.beta. protein. Jerlstrom
et al. (Molec. Microbiol. 5: 843-849 (1991)) used experiments
wherein subfragments of the C.beta. protein were expressed as
fusion proteins to identify two regions of the C.beta. protein
capable of binding IgA. These experiments localized the IgA binding
domains to a 747 bp BglII-HpaI fragment and a 1461 bp HpaI-HindIII
fragment of the C.beta. protein. Furthermore, International Patent
Application No. PCT/US/06111 describes the isolation of a C.beta.
protein bearing a deletion of a region that binds IgA.
SUMMARY OF THE INVENTION
[0012] The invention relates to a mutant C.beta. protein, wherein
the IgA binding by the C.beta. protein is reduced or eliminated,
while the antigenicity of the protein when administered either
alone or as part of a polysaccharide-protein conjugate is
substantially retained.
[0013] In particular, the invention relates to a mutant C.beta.
protein comprising the amino acid sequence A-X.sub.1 X.sub.2
X.sub.3 X.sub.4 X.sub.5 X.sub.6 X.sub.7 X.sub.8 X.sub.9 X.sub.10
X.sub.11 X.sub.12-B, wherein A comprises amino acids 1-164 of the
sequence shown in FIG. 1 (SEQ ID NO: 2), B represents a sequence
starting from amino acid 177 and terminating at an amino acid
between residue 1094 and 1127, inclusive, of the sequence shown in
FIG. 1 (SEQ ID NO: 2), and X.sub.1-X.sub.12 are each selected
independently from the group consisting of Ala, Val, Leu, Ile, Pro,
Met, Phe, Trp, a bond, and the wild type amino acid found at the
corresponding position of the sequence shown in FIG. 1 (SEQ ID NO:
2), wherein said amino acid positions are numbered from the first
amino acid of the native amino acid sequence encoding said protein,
with the proviso that at least one of X.sub.1 through X.sub.12,
inclusive, is other than the wild type amino acid.
[0014] The invention also relates to a polynucleotide molecule
encoding a mutant C.beta. protein, as well as vectors comprising
such polynucleotide molecules, and host cells transformed
therewith.
[0015] The invention also relates to a conjugate comprising the
mutant C.beta. protein covalently conjugated to a capsular
polysaccharide.
[0016] The invention also relates to a vaccine comprising the
mutant C.beta. protein of the invention and a pharmaceutically
acceptable carrier.
[0017] The invention also relates to a method of inducing an immune
response in an animal, comprising administering the vaccine of the
invention to an animal in an effective amount.
BRIEF DESCRIPTION OF THE FIGURES
[0018] FIG. 1 shows the DNA sequence and deduced amino acid
sequence of wild type C.beta.1 (Jerlstrom, P. G., et al., Molec.
Microbiol. 5: 843-849 (1991)). The BglII and PstI sites shown in
FIGS. 2, 3 and 4 are identified.
[0019] FIG. 2 is a map of the region of the C.beta. gene which
encodes the IgA binding site of the C.beta. protein; 2 amino acid
substitutions are indicated, generating mutant dgb2 (see Table
1).
[0020] FIG. 3 is a map of the region of the C.beta. gene which
encodes the IgA binding site of the C.beta. protein; 2 amino acid
substitutions are indicated, generating mutant nv34qp (see Table
1).
[0021] FIG. 4 is a map of the region of the C.beta. gene which
encodes the IgA binding site of the C.beta. protein; 6 amino acids
have been deleted from this region in the mutant protein,
generating mutant dgb1 (see Table 1).
[0022] FIG. 5 is a graph showing the competitive inhibition of
ELISA reactivity by C.beta. proteins.
[0023] FIGS. 6A, 6B and 6C show the complete DNA sequence of the
gene encoding C.beta. mutant dgb2 (see Table 1), as well as the
deduced amino acid sequence of this mutant. The mutations are
underlined.
[0024] FIGS. 7A, 7B and 7C show the complete DNA sequence of the
gene encoding C.beta. mutant nv34qp (see Table 1), as well as the
deduced amino acid sequence of this mutant. The mutations are
underlined.
[0025] FIGS. 8A, 8B and 8C show the complete DNA sequence of the
gene encoding C.beta. mutant dgb1 (see Table 1), as well as the
deduced amino acid sequence of this mutant.
[0026] FIGS. 9A, 9B and 9C show the complete DNA sequence of the
gene encoding C.beta. mutant pnv231 (see Table 1), as well as the
deduced amino acid sequence of this mutant. The mutations are
underlined.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0027] The invention relates to a mutant C.beta. protein of the
group B streptococcal (GBS) beta antigen, wherein IgA binding by
the C.beta. protein is reduced or eliminated and wherein at least a
majority of the antigenicity of the protein is retained.
[0028] It has been discovered that mutation of a region of the
C.beta. protein located between about amino acid residues 163 and
176 of the wildtype C.beta. sequence shown in FIG. 1 (SEQ ID NO: 2)
results in a C.beta. protein which has reduced or eliminated IgA
binding properties, but which retains enough of its tertiary
structure to maintain the majority of its antigenicity (see
Examples 4 and 5).
[0029] As the region of the C.beta. polypeptide has been found
which is responsible for IgA binding, and as it has been
demonstrated in the Examples below that amino acid substitutions or
deletions in this region reduce or eliminate IgA binding while
maintaining antigenicity of the protein, those of ordinary skill in
the art will understand how to alter the amino acid sequence of the
C.beta. polypeptide so as to achieve the objects of the invention.
Appropriate amino acid substitutions which eliminate IgA binding
will include replacement of one or more residues with an amino acid
having different properties. For example, a strongly hydrophilic
amino acid can be replaced with a strongly hydrophobic amino acid.
Amino acids which can be grouped together include the aliphatic
amino acids Ala, Val, Leu and Ile; the hydroxyl residues Ser and
Thr, the acidic residues Asp and Glu, the amide residues Asn and
Gln, the basic residues Lys and Arg and the aromatic residues Phe
and Tyr. Thus, those of ordinary skill in the art will understand
how to determine suitable amino acid substitutions or deletions in
the region between about residues 163 and 176 in the C.beta.
protein in order to reduce or eliminate IgA binding.
[0030] Further guidance concerning which amino acid changes are
likely to have a significant deleterious effect on a function can
be found in Bowie, J. U., et al., "Deciphering the Message in
Protein Sequences: Tolerance to Amino Acid Substitutions," Science
247: 1306-1310 (1990).
[0031] Thus, in particular, the invention relates to a mutant group
B streptococcal (GBS) beta antigen, C.beta., comprising the amino
acid sequence A-X.sub.1 X.sub.2 X.sub.3 X.sub.4 X.sub.5 X.sub.6
X.sub.7 X.sub.8 X.sub.9 X.sub.10 X.sub.11 X.sub.12-B, wherein A
comprises amino acids 1-164 of the sequence shown in FIG. 1 (SEQ ID
NO: 2), B represents a sequence starting from amino acid 177 and
terminating at an amino acid between residue 1094 and 1127,
inclusive, of the sequence shown in FIG. 1 (SEQ ID NO: 2), and
X.sub.1-X.sub.12 are each selected independently from the group
consisting of Ala, Arg, Asp, Val, Leu, Ile, Pro, Met, Phe, Trp, a
bond, and the wild type amino acid found at the corresponding
position of the sequence shown in FIG. 1 (SEQ ID NO: 2), wherein
said amino acid positions are numbered from the first amino acid of
the native amino acid sequence encoding said protein, with the
proviso that at least one of X.sub.1 through X.sub.12, inclusive,
is other than the wild type amino acid. In a particularly preferred
mutant C.beta. protein, amino acids X.sub.7 and X.sub.12 are Ala
(SEQ ID NO: 3). In another preferred mutant, amino acids X.sub.4
and X.sub.11 are Pro (SEQ ID NO: 4). In another preferred mutant,
amino acid X.sub.7 is Thr and amino acid X.sub.12 is Leu (SEQ ID
NO: 5). In a more preferred mutant, amino acids X.sub.5, X.sub.7,
X.sub.8, X.sub.10, X.sub.11 and X.sub.12 are each replaced with a
bond (SEQ ID NO: 6).
[0032] As the C.beta. protein is, in its wild type state, membrane
bound, it is possible to improve purification of the
above-mentioned C.beta. mutants by eliminating the hydrophobic
residues of the transmembrane domain of the C.beta. protein (the
transmembrane domain corresponds to residues 1095-1127 of the
sequence shown in FIG. 1 (SEQ ID NO: 2)). This can be accomplished
by substitution of non-hydrophobic residues for the hydrophobic
residues (residues 1108-1116 of the sequence shown in FIG. 1 (SEQ
ID NO: 2)) or by deletion of the hydrophobic residues. While
purification of membrane-bound C.beta. requires the use of
detergent, a mutant C.beta. which lacks the hydrophobic membrane
spanning region can be purified without using detergent. Thus, the
invention also relates to a mutant C.beta. wherein the nine
hydrophobic residues making up the transmembrane domain are deleted
or replaced by non-hydrophobic amino acids.
[0033] It has been discovered that the IgA-binding ability of
C.beta. may require dimerization of C.beta.. Thus, even where the
IgA-binding region of C.beta. is not mutated as described above,
mutation of the region of C.beta. which is believed to be required
for dimerization can result in a form of C.beta. that cannot bind
IgA. Deletion of a portion of C.beta. from residue 729 to the
C-terminus of the sequence shown in FIG. 1 (SEQ ID NO: 2)
eliminates dimerization of C.beta.. The results of experiments
supporting this finding may be found in Table 1. Several fragments
of C.beta. were inserted into each of two different vectors. Where
sequences shown in the table are preceded or followed by an outward
facing bracket, this indicates that the C.beta. sequence does not
extend further on that end of the fragment., i.e. that the
nucleotide sequence inserted into the vector encodes only those
amino acids shown, and no more of the C.beta. sequence. Where
sequences shown in the table are preceded or followed by ellipses,
this indicates that the remainder of the C.beta. sequence at that
end of the fragment is also included in the vector. Nucleotide
sequences encoding the peptides shown in the upper part of the
table were inserted into either the vector pTOPE or the vector
pET17b. Both of these vectors allow expression of inserted
fragments from the T7 promoter, and both produce fusion proteins
containing a fragment of the .phi.10 capsid protein N terminal to
the amino acid sequence encoded by the insert. However, while
pET17b encodes only 8 amino acids of the .phi.10 protein, pTOPE
encodes a 288 amino acid fragment of the .phi.10 protein.
[0034] As shown in Table 1, certain fragments of C.beta. produced
from pET17b exhibit reduced IgA-binding, while the same fragment
produced by pTOPE is capable of binding IgA. The fragments tested
lack the region of C.beta. predicted to be involved in
dimerization, but do not contain any mutations in the putative IgA
binding domain (note that the C.beta. fragments inserted into
vector pET24b, shown at the bottom of Table 1, contain the putative
dimerization region but nonetheless exhibit reduced IgA binding due
to mutations in the IgA binding domain, as described above). It is
postulated that these C.beta. fragments bind DNA when produced from
pTOPE because the 288 amino acid fragment of the .phi.10 protein
allows dimerization of the C.beta. fragment. This may be due to the
fact that the .phi.10 capsid protein normally forms oligomers; the
region responsible for oligomerization may thus allow dimerization
of the inserted C.beta. fragments, and thus IgA-binding. Thus, the
invention also relates to a mutant C.beta. protein having a
mutation in the dimerization domain of C.beta., wherein the mutant
C.beta. protein is incapable of binding IgA. Of course, in the
interest of producing a non-IgA binding C.beta. protein retaining
as much of the antigenicity of the wild type C.beta. protein as
possible, dimerization of C.beta. should not be interrupted.
[0035] It has also been discovered that production of C.beta.
protein from E. coli can be problematic because the protein is
cleaved at a specific region, presumably by an E. coli signal
peptidase. This cleavage results in a truncated protein, which
obviously is not ideal for a vaccine, as it lacks many antigenic
epitopes of the wildtype C.beta. protein. The cleavage site has
been predicted by sequence analysis and by matrix assisted laser
desorption initiated time of flight (MALDI-TOF) mass spectrometry
(von Heijne, Nucleic Acids Res. 14: 4683-4690 (1986)). The cleavage
site is between amino acid residues 538 and 539 (after alanine and
before glutamine) of the amino acid sequence shown in FIG. 1 (SEQ
ID NO: 2). The signal peptidase recognition site is located within
a 20 amino acid stretch located between residues 521 and 541 of the
amino acid sequence shown in FIG. 1 (SEQ ID NO: 2). Therefore, by
deleting this region, the C.beta. protein or a non-IgA binding
mutant thereof can successfully be produced in E. coli.
Furthermore, as signal peptidases have very strict sequence
specificity, alteration of the signal peptidase recognition
sequence, including even a single, conservative amino acid
substitution in this region, may eliminate cleavage of C.beta. by
E. coli. The recognition sequence required for cleavage by this
signal peptidase is believed to be GluLeuIleLysSerAlaGlnGlnGlu (SEQ
ID NO: 1), corresponding to amino acid residues 533-541 of the
sequence shown in FIG. 1 (SEQ ID NO: 2). Alteration of either the
serine or the alanine residue of this sequence by either deletion
or non-conservative substitution is expected to eliminate cleavage
by the signal peptidase. Of course, ideally, the mutagenesis of
C.beta. will be kept to a minimum so as to retain the tertiary
structure of the wildtype antigen for the purposes of eliciting an
immunogenic response.
[0036] Thus, the invention also relates to a mutant C.beta. protein
of the group B streptococcal (GBS) beta antigen, wherein IgA
binding by the C.beta. protein is reduced or eliminated by any of
the mutations described above, and wherein at least one of amino
acid residues 521-541 of the amino acid sequence shown in FIG. 1
(SEQ ID NO: 2) is either (a) deleted or (b) altered, so that the
protein is not cleaved in this region when C.beta. is produced in
E. coli. In a preferred embodiment, at least one of amino acid
residues 533-541 of the sequence shown in FIG. 1 (SEQ ID NO: 2) is
either (a) deleted or (b) altered. In a more preferred embodiment,
at least one of amino acid residues 537 and 538 is either (a)
deleted or (b) altered. Of course, one of ordinary skill will be
able to determine other suitable amino acid substitutions by
routine experimentation, and by reference to the article by von
Heijne (Nucleic Acids Res. 14: 4683-4690 (1986)).
[0037] The invention also relates to polynucleotide molecules
encoding the mutant proteins of the invention, vectors comprising
those polynucleotide molecules, and host cells transformed
therewith.
[0038] The invention also relates to the expression of novel mutant
C.beta. polypeptides, wherein IgA binding by the C.beta. protein is
reduced or eliminated, in a cellular host.
[0039] Prokaryotic hosts that may be used for cloning and
expressing the polypeptides of the invention are well known in the
art. Vectors which replicate in such host cells are also well
known.
[0040] Preferred prokaryotic hosts include, but are not limited to,
bacteria of the genus Escherichia, Bacillus, Streptomyces,
Pseudomonas, Salmonella, Serratia, Xanthomonas, etc. Two such
prokaryotic hosts are E. coli DH10B and DH5.alpha.F'IQ (available
from LTI, Gaithersburg, Md.). The most preferred host for cloning
and expressing the polypeptides of the invention is E. coli BL21
(Novagen, Wis.), which is lysogenic for DE3 phage.
[0041] The present invention also relates to vectors which include
the isolated DNA molecules of the present invention, host cells
which are genetically engineered with the recombinant vectors, and
the production of the polypeptides of the invention by recombinant
techniques.
[0042] Host cells can be genetically engineered to incorporate
nucleic acid molecules and express polypeptides of the present
invention. For instance, recombinant constructs may be introduced
into host cells using well known techniques of infection,
transduction, transfection, and transformation. The polynucleotides
may be introduced alone or with other polynucleotides. Such other
polynucleotides may be introduced independently, co-introduced or
introduced joined to the polynucleotides of the invention.
[0043] Thus, for instance, the polynucleotides may be joined to a
vector containing a selectable marker for propagation in a host.
The vector construct may be introduced into host cells by the
aforementioned techniques. Generally, a plasmid vector is
introduced as DNA in a precipitate, such as a calcium phosphate
precipitate, or in a complex with a charged lipid. Electroporation
also may be used to introduce polynucleotides into a host. If the
vector is a virus, it may be packaged in vitro or introduced into a
packaging cell and the packaged virus may be transduced into cells.
A wide variety of techniques suitable for making polynucleotides
and for introducing polynucleotides into cells in accordance with
this aspect of the invention are well known and routine to those of
skill in the art. Such techniques are reviewed at length in
Sambrook et al. cited above, which is illustrative of the many
laboratory manuals that detail these techniques.
[0044] In accordance with this aspect of the invention the vector
may be, for example, a plasmid vector, a single or double-stranded
phage vector, a single or double-stranded RNA or DNA viral vector.
Such vectors may be introduced into cells as polynucleotides,
preferably DNA, by well known techniques for introducing DNA and
RNA into cells. The vectors, in the case of phage and viral vectors
also may be and preferably are introduced into cells as packaged or
encapsulated virus by well known techniques for infection and
transduction. Viral vectors may be replication competent or
replication defective. In the latter case viral propagation
generally will occur only in complementing host cells.
[0045] Preferred among vectors, in certain respects, are those for
expression of polynucleotides and polypeptides of the present
invention. Generally, such vectors comprise cis-acting control
regions effective for expression in a host operatively linked to
the polynucleotide to be expressed. Appropriate transacting factors
either are supplied by the host, supplied by a complementing vector
or supplied by the vector itself upon introduction into the
host.
[0046] In certain preferred embodiments in this regard, the vectors
provide for specific expression. Such specific expression may be
inducible expression or expression only in certain types of cells
or both inducible and cell-specific. Particularly preferred among
inducible vectors are vectors that can be induced for expression by
environmental factors that are easy to manipulate, such as
temperature and nutrient additives. A variety of vectors suitable
to this aspect of the invention, including constitutive and
inducible expression vectors for use in prokaryotic and eukaryotic
hosts, are well known and employed routinely by those of skill in
the art (see U.S. Pat. No. 5,464,758).
[0047] The engineered host cells can be cultured in conventional
nutrient media, which may be modified as appropriate for, inter
alia, activating promoters, selecting transformants or amplifying
genes. Culture conditions, such as temperature, pH and the like,
previously used with the host cell selected for expression
generally will be suitable for expression of polypeptides of the
present invention as will be apparent to those of skill in the
art.
[0048] A great variety of expression vectors can be used to express
a polypeptide of the invention. Such vectors include chromosomal,
episomal and virus-derived vectors e.g., vectors derived from
bacterial plasmids, from bacteriophage, from yeast episomes, from
yeast chromosomal elements, from viruses such as baculoviruses,
papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl
pox viruses, pseudorabies viruses and retroviruses, and vectors
derived from combinations thereof, such as those derived from
plasmid and bacteriophage genetic elements, such as cosmids and
phagemids, all may be used for expression in accordance with this
aspect of the present invention. Generally, any vector suitable to
maintain or propagate, polynucleotides, or to express a
polypeptide, in a host may be used for expression in this
regard.
[0049] The appropriate DNA molecule may be inserted into the vector
by any of a variety of well-known and routine techniques. In
general, a DNA molecule for expression is joined to an expression
vector by cleaving the DNA sequence and the expression vector with
one or more restriction endonucleases and then joining the
restriction fragments together using T4 DNA ligase. Procedures for
restriction and ligation that can be used to this end are well
known and routine to those of skill in the art. Suitable procedures
in this regard, and for constructing expression vectors using
alternative techniques, which also are well known and routine to
those skill, are set forth in great detail in Sambrook et al. cited
above.
[0050] The DNA molecule inserted in the expression vector is
operatively linked to appropriate expression control sequence(s),
including, for instance, a promoter to direct mRNA transcription.
Representatives of such promoters include the phage lambda PL
promoter, the E. coli lac, trp and tac promoters, the SV40 early
and late promoters and promoters of retroviral LTRs, to name just a
few of the well-known promoters. It will be understood that
numerous promoters not mentioned are suitable for use in this
aspect of the invention are well known and readily may be employed
by those of skill in the art in the manner illustrated by the
discussion and the examples herein.
[0051] In general, expression constructs will contain sites for
transcription initiation and termination, and, in the transcribed
region, a ribosome binding site for translation. The coding portion
of the mature transcripts expressed by the constructs will include
a translation initiating AUG at the beginning and a termination
codon appropriately positioned at the end of the polypeptide to be
translated.
[0052] In addition, the constructs may contain control regions that
regulate as well as engender expression. Generally, in accordance
with many commonly practiced procedures, such regions will operate
by controlling transcription, such as repressor binding sites and
enhancers, among others.
[0053] Vectors for propagation and expression generally will
include selectable markers. Such markers also may be suitable for
amplification or the vectors may contain additional markers for
this purpose. In this regard, the expression vectors preferably
contain one or more selectable marker genes to provide a phenotypic
trait for selection of transformed host cells. Preferred markers
include dihydrofolate reductase or neomycin resistance for
eukaryotic cell culture, and tetracycline or ampicillin resistance
genes for culturing E. coli and other bacteria.
[0054] The vector containing the appropriate DNA sequence as
described elsewhere herein, as well as an appropriate promoter, and
other appropriate control sequences, may be introduced into an
appropriate host using a variety of well known techniques suitable
to expression therein of a desired polypeptide. Representative
examples of appropriate hosts include bacterial cells, such as E.
coli, Streptomyces and Salmonella typhimurium cells. Hosts for of a
great variety of expression constructs are well known, and those of
skill will be enabled by the present disclosure readily to select a
host for expressing a polypeptides in accordance with this aspect
of the present invention.
[0055] More particularly, the present invention also includes
recombinant constructs, such as expression constructs, comprising
one or more of the sequences described above. The constructs
comprise a vector, such as a plasmid or viral vector, into which
such a sequence of the invention has been inserted. The sequence
may be inserted in a forward or reverse orientation. In certain
preferred embodiments in this regard, the construct further
comprises regulatory sequences, including, for example, a promoter,
operably linked to the sequence. Large numbers of suitable vectors
and promoters are known to those of skill in the art, and there are
many commercially available vectors suitable for use in the present
invention.
[0056] As the invention concerns the construction of a protein
having a reduced or eliminated ability to bind human IgA, the
invention thus relates to using in vitro mutagenesis methods to
generate the mutant C.beta. proteins of the invention. A number of
in vitro mutagenesis methods are well known to those of skill in
the art; several are provided here as examples.
[0057] One such method introduces deletions or insertions into a
polynucleotide molecule inserted into a plasmid by either partially
or completely digesting the plasmid with an appropriate restriction
enzyme, and then ligating the ends to again generate a plasmid.
Very short deletions can be made by first cutting a plasmid at a
restriction site, and then subjecting the linear DNA to controlled
nuclease digestion to remove small groups of bases at each end.
Precise insertions may also be made by ligating double stranded
oligonucleotide linkers to a plasmid cut at a single restriction
site.
[0058] Chemical methods can also be used to introduce mutations to
a single stranded polynucleotide molecule. For example, single base
pair changes at cytosine residues can be created using chemicals
such as bisulfite, which deaminates cytosine to uracil, thus
converting GC base pairs to AT base pairs.
[0059] Preferably, oligonucleotide directed mutagenesis will be
used so that all possible classes of base pair changes at any
determined site along a DNA molecule can be made. In general, this
technique involves annealing a oligonucleotide complementary
(except for one or more mismatches) to a single stranded nucleotide
sequence of interest. The mismatched oligonucleotide is then
extended by DNA polymerase, generating a double stranded DNA
molecule which contains the desired change in sequence on one
strand. The changes in sequence can of course result in the
deletion, substitution, or insertion of an amino acid if the change
is made in the coding region of a gene. The double stranded
polynucleotide can then be inserted into an appropriate expression
vector, and a mutant polypeptide can thus be produced. The
above-described oligonucleotide directed mutagenesis can of course
be carried out via PCR. An example of such a system is the
Ex-Site.TM. PCR site-directed mutagenesis technique (Stratagene,
Calif.) used in Example 4.
[0060] Using the Ex-Site.TM. PCR site-directed mutagenesis
technique, several different oligonucleotides were made to induce
different changes in the DNA sequence in the region of interest. In
one particular example, overlapping primers were obtained, wherein
both primers contained the sequence required to change lysine to
alanine at amino acids 170 and 175 in the sequence shown in FIG. 1
(SEQ ID NO: 2) (see FIG. 2 and Table 1). The forward primer,
designated C.beta. 613, had the sequence (SEQ ID NO: 6) 5'-GTT GAA
GCA ATG GCA GAG CAA GCG GGA ATC ACA AAT GAA G-3' and the reverse
primer, designated C.beta. 642R had the sequence (SEQ ID NO: 7)
5'-GAT TCC CGC TTG CTC TGC CAT TGC TTC AAC TTG ACT TTT TTG-3' (the
substitutions are noted in BOLD). These oligonucleotides were
combined with pNV222 template, which consists of the C.beta. gene
inserted into the pSP76 vector. PCR was performed, and the products
were ligated and introduced into E. coli strain DH5.alpha., thus
generating clones containing the mutant C.beta. gene.
[0061] The following vectors, which are commercially available, may
be used in the practice of the invention. Among vectors preferred
for use in bacteria are pQE70, pQE60 and pQE-9, available from
Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors,
pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; ptrc99a,
pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia; pUC18,
pUC19 and pPROEX-1, available from LTI, and pTOPE, pET17b, and
pET24a (Novagen, Madison, Wis.). These vectors are listed solely by
way of illustration of the many commercially available and well
known vectors that are available to those of skill in the art for
use in accordance with this aspect of the present invention. It
will be appreciated that any other plasmid or vector suitable for,
for example, introduction, maintenance, propagation or expression
of a polynucleotide or polypeptide of the invention in a host may
be used in this aspect of the invention.
[0062] Promoter regions can be selected from any desired gene using
vectors that contain a reporter transcription unit lacking a
promoter region, such as a chloramphenicol acetyl transferase
("CAT") transcription unit, downstream of restriction site or sites
for introducing a candidate promoter fragment; i.e., a fragment
that may contain a promoter. As is well known, introduction into
the vector of a promoter-containing fragment at the restriction
site upstream of the CAT gene engenders production of CAT activity,
which can be detected by standard CAT assays. Vectors suitable to
this end are well known and readily available. Two such vectors are
pKK232-8 and pCM7. Thus, promoters for expression of
polynucleotides of the present invention include not only well
known and readily available promoters, but also promoters that
readily may be obtained by the foregoing technique, using a
reporter gene.
[0063] Among known bacterial promoters suitable for expression of
polynucleotides and polypeptides in accordance with the present
invention are the E. coli lacI and lacZ and promoters, the T3 and
T7 promoters, the gpt promoter, the lambda PR, PL promoters and the
trp promoter.
[0064] Selection of appropriate vectors and promoters for
expression in a host cell is a well known procedure and the
requisite techniques for expression vector construction,
introduction of the vector into the host and expression in the host
are routine skills in the art.
[0065] The present invention also relates to host cells containing
the constructs discussed above. The host cell can be a prokaryotic
cell, such as a bacterial cell.
[0066] Constructs in host cells can be used in a conventional
manner to produce the gene product encoded by the recombinant
sequence. Alternatively, the polypeptides of the invention can be
synthetically produced by conventional peptide synthesizers.
[0067] Following transformation of a suitable host strain and
growth of the host strain to an appropriate cell density, where the
selected promoter is inducible, it is induced by appropriate means
(e.g., temperature shift or exposure to chemical inducer) and cells
are cultured for an additional period.
[0068] Cells typically are then harvested by centrifugation,
disrupted by physical or chemical means, and the resulting crude
extract retained for further purification.
[0069] Microbial cells employed in expression of proteins can be
disrupted by any convenient method, including freeze-thaw cycling,
sonication, mechanical disruption, or use of cell lysing agents;
such methods are well know to those skilled in the art.
[0070] The invention also relates to a vaccine comprising a mutant
C.beta. protein, wherein IgA binding by the C.beta. protein is
reduced or eliminated as described herein, together with a
pharmaceutically acceptable carrier. In a preferred embodiment, the
protein is conjugated to a polysaccharide.
[0071] The conjugates of the invention may be formed by reacting
the reducing end groups of the polysaccharide to primary amino
groups (that is, lysine residues) of the C.beta. protein by
reductive amination. The polysaccharide may be conjugated to any or
all of the primary amino groups of the protein. The reducing groups
may be formed by selective hydrolysis or specific oxidative
cleavage, or a combination of both. Preferably, the C.beta. protein
is conjugated to the polysaccharide by the method of Jennings et
al., U.S. Pat. No. 4,356,170, which involves controlled oxidation
of the polysaccharide with periodate followed by reductive
amination with the C.beta. protein of the invention.
[0072] In a preferred embodiment, the polysaccharide is one of the
Group B streptococcal capsular polysaccharides selected from types
Ia, II, III and V. See Baker, C. J. and D. L. Kasper. Rev. Inf.
Dis. 7: 458-467 (1985); Baker, C. J., et al., N. Engl. J. Med. 319:
1180-1185 (1988); Baker, C. J., et al., New Engl. J. Med. 322:
1857-1860 (1990). The vaccine may also be a combination vaccine
comprising one or more of the C.beta. protein-polysaccharide
conjugates selected from the group consisting of C.beta. conjugated
to Group B capsular polysaccharide type Ia (C.beta.-Ia); C.beta.
conjugated to Group B capsular polysaccharide type II (C.beta.-II);
C.beta. conjugated to Group B capsular polysaccharide type III
(C.beta.-III); and C.beta. conjugated to Group B capsular
polysaccharide type V (C.beta.-V). Most preferably, the vaccine is
a combination vaccine comprising C.beta.-Ia, C.beta.-II,
C.beta.-III and C.beta.-V. Such a combination vaccine will elicit
antibodies to Group B streptoccoci of Types Ia, II, III, V, and Ib
(as Type Ib Group B streptococci also express C.beta.).
Furthermore, the immune response to the polysaccharides of the
combination vaccine will be a T dependent response.
[0073] The vaccine of the present invention comprises one or more
of the C.beta. protein vaccines or conjugate vaccines in amounts
effective depending on the route of administration. Although
subcutaneous or intramuscular routes of administration are
preferred, the vaccine of the present invention can also be
administered by an intraperitoneal or intravenous route. One
skilled in the art will appreciate that the amounts to be
administered for any particular treatment protocol can be readily
determined without undue experimentation. With respect to each
conjugate, suitable amounts are expected to fall within the range
of 2 micrograms of the protein per kg body weight to 100 micrograms
per kg body weight. In a preferred embodiment, the vaccine
comprises about 2 .mu.g of the C.beta. protein or an equivalent
amount of the protein-polysaccharide conjugate. In another
preferred embodiment, the vaccine comprises about 5 .mu.g of the
C.beta. protein or an equivalent amount of the
protein-polysaccharide conjugate.
[0074] The vaccine of the present invention may be employed in such
forms as capsules, liquid solutions, suspensions or elixirs for
oral administration, or sterile liquid forms such as solutions or
suspensions. Any inert carrier is preferably used, such as saline,
phosphate-buffered saline, or any such carrier in which the non-IgA
Fc binding group B streptococcal C.beta. protein or conjugate
vaccine have suitable solubility properties. The vaccines may be in
the form of single dose preparations or in multi-dose flasks which
can be used for mass vaccination programs. Reference is made to
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., Osol (ed.) (1980); and New Trends and Developments in
Vaccines, Voller et al. (eds.), University Park Press, Baltimore,
Md. (1978), for methods of preparing and using vaccines.
[0075] The vaccines of the present invention may further comprise
adjuvants which enhance production of C.beta.-specific antibodies.
Such adjuvants include, but are not limited to, various oil
formulations such as Freund's complete adjuvant (CFA), stearyl
tyrosine (ST, see U.S. Pat. No. 4,258,029), the dipeptide known as
MDP, saponin (see U.S. Pat. No. 5,057,540), aluminum hydroxide, and
lymphatic cytokine.
[0076] Freund's adjuvant is an emulsion of mineral oil and water
which is mixed with the immunogenic substance. Although Freund's
adjuvant is powerful, it is usually not administered to humans.
Instead, the adjuvant alum (aluminum hydroxide) or ST may be used
for administration to a human. The C.beta. protein vaccine or a
conjugate vaccine thereof may be absorbed onto the aluminum
hydroxide from which it is slowly released after injection. The
vaccine may also be encapsulated within liposomes according to
Fullerton, U.S. Pat. No. 4,235,877.
[0077] In another preferred embodiment, the present invention
relates to a method of inducing an immune response in an animal
comprising administering to the animal the vaccine of the
invention, produced according to methods described, in an amount
effective to induce an immune response.
[0078] 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.
EXAMPLES
Example 1
Cloning and Expression of the Gene Encoding C.beta.
[0079] To locate the IgA binding site on the C.beta. protein, two
oligonucleotides were synthesized. The first oligonucleotide, oligo
1, corresponds to the 5' end of the mature protein, and has the
sequence (SEQ ID NO: 8) 5'-AAGGATCCAAGTGAGCTTGTAAAGGACGAT-3', which
includes a BamHI site. The second oligonucleotide falls just short
of the 3' end of the gene, and has the sequence (SEQ ID NO: 9)
5'-AAAACTCGAGTTTCTTCCGTTGTT- GATGTA-3', and includes a XhoI site.
The oligonucleotide for the 3' end of the gene was chosen to
eliminate the LPXTG motif found in most gram positive cell wall
proteins. This sequence motif has been shown to be involved in the
processing of these cell wall proteins and is the part of these
proteins which eventually becomes covalently bound to peptidoglycan
(Navarre, W. W. and O. Schneewind, Molec. Microbiol. 14: 115-121
(1994); Schneewind, O., et al., Science 268: 103-106 (1995)). Using
chromosomal DNA from Strain A909 Group B streptococci containing
the gene for the C.beta. protein as a template, and standard PCR
procedures, a product of approximately 3.2 kb was produced as
observed when electrophoresed on a 1% agarose gel. The PCR product
containing the C.beta. protein gene was cleaved with the
endonuclease restriction enzymes BamHI and XhoI. This BamHI-XhoI
DNA fragment contained the sequence for the entire C.beta. protein
except for the last 33 amino acids at the carboxyl terminus,
including the putative IgA binding site. The DNA fragment was then
ligated into the appropriately restricted T7 expression plasmid
pET17b (Novagen Inc., Madison Wis.) using a standard T4 ligase
procedure. The plasmid was then transformed into the E. coli strain
BL21(DE3) using the manufacturer's suggested protocols (Novagen
Inc.). E. coli cells containing the plasmid were selected on LB
plates containing 50 .mu.g/ml carbenicillin. These plates were
incubated overnight at 37.degree. C. The transformant colonies were
carefully lifted onto nitrocellulose filters saturated with IPTG.
After 30 min, the bacteria were lysed by placing the filters into a
chloroform vapor chamber for 15 min at room temperature.
[0080] After the filters were removed from the chamber, they were
placed, colony-side up, onto a Whatman 3MM filter which had been
previously saturated with 20 mM Tris-HCl, pH 7.9, 6 M urea, and 0.5
M NaCl. After 15 min, the filters were washed three times in PBS
and incubated for 1 hr with purified human IgA in PBS-Tween. The
filters were then rewashed in the PBS-Tween and developed by
standard procedures (Blake, M. S., et al., Analyt. Biochem. 136:
175-17 (1984)) using a goat antihuman IgA-alkaline phosphatase
conjugate (Cappel Research Products, West Chester, Pa.). Several
colonies demonstrating high IgA binding activity were selected and
grown overnight in 1 ml LB broth containing carbenicillin at
30.degree. C. These cultures were then diluted 1 to 100 with fresh
LB-carbenicillin broth and incubated at 30.degree. C. for an
addition 6 hr. Expression was then induced by the addition of IPTG
and the culture continued for an addition 2 hr at 30.degree. C. The
cells were collected by centrifugation, resuspended in water and
subjected to several freeze-thaw cycles. The cells were once again
collected by centrifugation and the supernatants saved for
examination of their IgA binding activity.
Example 2
Identification of the IgA Binding Domain of C.beta.
[0081] Once certain a stable plasmid producing a recombinant
C.beta. protein had been achieved and that the expressed protein
bound human IgA, a strategy similar to that of the Novatope System
(Novagen, Inc.) was utilized to locate the IgA binding region of
C.beta.. This procedure was performed according to the
manufacturer's instructions. Briefly, the purified plasmid
containing the C.beta. gene was randomly digested with DNase I and
electrophoresed in a 2% low melting point agarose gel. Fragments of
the DNA corresponding to sizes between 100 to 300 base pairs were
excised from the gel, purified, and resuspended in TE buffer. A
single dA was added to the fragments using the recommended reaction
mixture and the fragments ligated into the pETOPET vector which
contained single dT ends. After the standard ligation procedure,
the plasmids were transformed into competent NovaBlue (DE3) cells
(Novagen, Inc.) and plated on LB plates containing 50 .mu.g/ml
carbenicillin. These plates were incubated overnight at 37.degree.
C. The transformant colonies were tested for IgA binding activity
as described in Example 1. Several clones were selected on the
bases of their binding to the IgA. The bacteria from each of these
clones were inoculated separately onto fresh LB plates and retested
for their IgA binding ability as before. Plasmid preparations were
made from each by standard means and sequenced.
[0082] The nucleotide sequences of the cloned C.beta. protein gene
fragments were determined by the dideoxy method using denatured
double-stranded plasmid DNA template as described (Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
N.Y. (1993)). Sequenase II kits (United States Biochemical Corp.,
Cleveland, Ohio) were used in accordance with the manufacturer's
instructions. The smallest fragment of DNA obtained that included
part of the C.beta. gene is shown in FIG. 1. The translation of
this sequence corresponds to amino acid 101 to 230 of the mature
C.beta. protein shown in FIG. 1 (SEQ ID NO: 2). Attempts to further
shorten this DNA fragment failed to give any IgA binding
activity.
Example 3
ELISA Inhibition Assays: Peptide Binding Studies
[0083] Several synthetic peptides were made corresponding to the
amino acid sequence contained within this region of the C.beta.
protein. Peptides were synthesized using NMP t-butoxycarbonyl
chemistry on an ABI 430A peptide synthesizer (Applied Biosystems,
Foster City, Calif.) and were deprotected. Peptides from a sample
of the resin were removed from the resin by treatment with HF in
the presence of anisole (0.degree. C./1 h). Preparative
purification of these peptides were performed using a C18 column
(2.14 ID.times.30 cm) (Dynamax-Rainin, Woburn, Mass.). The peptides
were quantitated by PTC amino acid analysis using Waters Picotag
system (Waters, Milford, Mass.). The synthesized peptides eluted
from the C18 column as a major peak consisting of usually 75-85% of
the total elution profile. The amino acid composition of the
purified peptides were in good agreement with the sequence which
was used to synthesize the peptides. These peptides were used in
ELISA inhibition assays to block the binding of human IgA to the
purified C.beta. protein as follows. Microtiter plates (Nunc-Immuno
Plate IIF, Vangard International, Neptune, N.J.) were sensitized by
adding 0.1 ml per well of purified C.beta. at a concentration of
2.0 .mu.g/ml in 0.1 M Carbonate buffer, pH 9.6 with 0.02% azide.
The plates were incubated overnight at room temperature. The plates
were washed five times with 0.9% NaCl, 0.05% Brij 35, 10 mM sodium
acetate pH 7.0, 0.02% azide. A purified human IgA myeloma protein
was purchased from Cappel Laboratories, was diluted in PBS with
0.5% Brij 35 and added to the plate and incubated for 1 hr at room
temperature. The plates were again washed as before and the
secondary antibody, alkaline phosphatase conjugated goat anti-human
IgA (Tago Inc., Burlingame, Calif.), was diluted in PBS-Brij, added
to the plates and incubated for 1 h at room temperature. The plates
were washed as before and p-nitrophenyl phosphate (Sigma
phosphatase substrate 104) (1 mg/ml) in 0.1 M diethanolamine, 1 mM
MgCl.sub.2, 0.1 mM ZnCl.sub.2, 0.02% azide, pH 9.8 added. The
plates were incubated at 37.degree. C. for 1 h and the absorbance
at 405 nm determined using an Elida-5 microtiter plate reader
(Physica, New York, N.Y.). Control wells lacked either the primary
and/or secondary antibody. This was done to obtain a titer of the
human IgA myeloma protein which would give a half-maximal reading
in the ELISA assay. This titer would be used in the inhibition
ELISA. The microtiter plate were sensitized and washed as before.
Purified synthetic peptides were added and diluted in PBS-Brig. The
dilution of the human IgA myeloma protein which gave the half
maximal reading was then added. The mixture was then incubated for
1 hr at room temperature. The plates were rewashed and the
conjugated second antibody added as stated. The plates were then
processed and read as described. The percentage of inhibition would
be calculated as follows:
[0084] 1-(ELISA value with the peptide added)/(ELISA value without
the peptide added).
[0085] The peptide which inhibited in this ELISA assay contained
the sequence
Asn-His-Gln-Lys-Ser-Gln-Val-Glu-Lys-Met-Ala-Glu-Gln-Lys-Gly (SEQ ID
NO: 10). This suggested that at least part of the IgA binding
domain of the C.beta. was comprised within the region of the
protein containing this sequence.
Example 4
Oligonucleotide Directed Mutagenesis of the Gene Encoding
C.beta.
[0086] In order to confirm the importance of this region in the
C.beta. protein for IgA binding activity and to begin to generate
the mutant proteins that in the end would be used in the vaccine
formulation, a modification of the Ex-Site.TM. PCR site-directed
mutagenesis protocol was employed as developed by Stratagene
(Stratagene, Calif.). The template used was a plasmid called pNV222
which consisted of the C.beta. gene inserted into the pSP76 vector
(Promega, Madison, Wis.). DNA oligonucleotides were synthesized on
an Applied Biosystems model 292 DNA Synthesizer (Foster City,
Calif.). The oligonucleotides were manually cleaved from the column
by treatment with 1.5 ml of ammonium hydroxide for 2 hours with
gentle mixing every 15 minutes. They were deprotected at 55.degree.
C. for 16-18 hours. After deprotection they were dried down and
used directly or purified using oligonucleotide purification
columns (Applied Biosystems, Foster City Calif.). Several different
oligonucleotides were made to induce different changes in the DNA
sequence in the region of interest. An example of which is the
following. The primers, in this particular example, were
overlapping primers, both containing the sequence required to
change lysine to alanine at amino acids 170 and 175 in the sequence
shown in FIG. 1 (SEQ ID NO: 2). The forward primer, designated
C.beta. 613, had the sequence (SEQ ID NO: 6) 5'-GTT GAA GCA ATG GCA
GAG CAA GCG GGA ATC ACA AAT GAA G-3' and the reverse primer,
designated C.beta. 642R had the sequence (SEQ ID NO: 7) 5'-GAT TCC
CGC TTG CTC TGC CAT TGC TTC AAC TTG ACT TTT TTG-3' (the
substitutions are noted in BOLD). The reaction conditions were as
follows: 10 ng pNV222 template, 15 pmol. of each primer, 1 mM of
each dNTP, 1X VENT Polymerase Buffer (20 mM Tris-HCl, pH 7.5; 10 mM
KCl; 10 mM (NH.sub.4).sub.2 SO.sub.4; 2 mM MgSO.sub.4 0.1% (v/v)
Triton.RTM. X-100; 0.1 mg/ml bovine serum albumin (BSA)), 10 units
of Vent Polymerase, and H.sub.2O to 100 .mu.l. The reactions were
prepared with PCR Gem 10 wax beads as per the Hot Start Protocol
(Perkin Elmer, Foster City, Calif.). The reactions were run in a
Perkin Elmer Thermocycler (Perkin Elmer, Foster City, Calif.) under
the following conditions: 1 cycle of 94.degree. C. for 5 minutes;
10 cycles of 94.degree. C. for 30 seconds, 37.degree. C. for 2
minutes, 72.degree. C. for 10 minutes; 30 cycles of 94.degree. C.
for 30 seconds, 55.degree. C. for 2 minutes, 72.degree. C. for 10
minutes; and 1 cycle of 72.degree. C. for 12 minutes. The reaction
was treated with 10 units of DpnI at 37.degree. C. for 30 minutes
to destroy the template DNA, followed by a 60 minute treatment at
72.degree. C. with PfuI polymerase to fill in any remaining
overhangs. The reaction was diluted 1:4.6 in 1 X Vent Buffer plus
0.38 mM dATP. The diluted reaction was ligated for 24 hours at
25.degree. C. and transformed into competent DH5.alpha. cells
(Gibco/BRL, Gaithersburg, Md.). Selected colonies were grown in 3
ml of LB plus kanamycin (50 mg/ml) at 37.degree. C. for 16-18
hours. DNA was prepared using QIAspin.TM. columns (Qiagen,
Chatsworth, Calif.). The clones were analyzed for insert size on
0.8% agarose gels and then sequenced. Selected clones were then
grown in 100 ml LB plus kanamycin (50 mg/ml) at 37.degree. C. for
16-18 hours. DNA was prepared using the Qiagen-tip 100 (Qiagen,
Chatsworth, Calif.). They were then digested with NdeI and PstI and
run on 0.8% agarose gels to separate the mutated region. The 2300
bp fragment was isolated and purified from the gel using the
Gene-Clean Spin Kit.TM. (Bio 101, Vista, Calif.). A clone named
pNV34 which consisted of the expression vector pET 24a (Novagen)
and the native C.beta. gene, was also digested with NdeI and PstI
and run on a 0.8% agarose gel. The large band (6300 bp) containing
the pET vector and the remainder of the C.beta. gene was isolated
and purified from the gel using the Gene-Clean Spin Kit.TM. (Bio
101). These two fragments were ligated at 4.degree. C. for 24 hours
and transformed into competant BL21 (DE3) cells. Selected colonies
were grown in 3 ml of LB plus kanamycin (50 mg/ml) at 37.degree. C.
for 16-18 hours. DNA was prepared using QIAspin.TM. columns
(Qiagen) and the clones were analyzed for insert size on 0.8%
agarose gels.
[0087] Also constructed were clones encoding mutant C.beta.
proteins wherein two glutaminyl residues are replaced by prolinyl
residues (FIG. 3), and wherein a deletion in the C.beta. gene had
occurred resulting in a 6 amino acid deletion in the region of
interest (FIG. 4).
[0088] Clones expressing a C.beta. protein which lacked or had
reduced IgA binding activity but still reacted with the
anti-.beta.ag antiserum were selected (see Example 5) and grown in
100 ml LB plus kanamycin (50 mg/ml) at 37.degree. C. for 16-18
hours. Plasmid DNA from these clones was prepared using Qiagen tip
100 (Qiagen) and the mutated C.beta. gene entirely sequenced.
Example 5
Western Blot and ELISA Analysis of IgA Binding by C.beta.
Mutants
[0089] The proteins encoded by the mutated genes were expressed and
subjected to SDS-PAGE and western blot analysis in order to
determine if mutations in the gene encoding the C.beta. protein
reduced or eliminated IgA binding, while retaining C.beta.
antigenicity. Two western blots were made for each sample and
reacted with either the purified human IgA myeloma protein or
hyperimmune rabbit anti-.beta.ag protein antiserum. The clone
expressing a C.beta. protein wherein lysine is changed to alanine
at amino acids 170 and 175 in the sequence shown in FIG. 1 (SEQ ID
NO: 2) demonstrated almost no IgA binding activity, but the ability
of the protein to react with anti-C.beta. antiserum remained high.
IgA binding activity was also substantially eliminated in the clone
expressing a C.beta. protein wherein two glutaminyl residues are
replaced by prolinyl residues (FIG. 3) and in the clone encoding a
C.beta. protein having a six amino acid deletion (FIG. 4), while
reactivity with the anti-C.beta. antiserum was maintained for both.
The data for the clone having a six amino acid deletion suggested
that the residues responsible for the IgA binding activity of the
C.beta. protein were located within this region of the protein, and
that other possible mutations within this area would effect the IgA
binding activity.
[0090] A competitive inhibition ELISA was used to more precisely
determine the amount of antigenic and/or structure change the
sequence modifications had on the C.beta. protein. Microtiter
plates (Nunc-Immuno Plate IIF, Vangard International, Neptune,
N.J.) were sensitized by adding 0.1 ml per well of purified C.beta.
at a concentration of 2.0 .mu.g/ml in 0.1 M carbonate buffer, pH
9.6 with 0.02% azide. The plates were incubated overnight at room
temperature. The plates were washed five times with 0.9% NaCl,
0.05% Brij 35, 10 mM sodium acetate pH 7.0, 0.02% azide.
Hyperimmune rabbit antiserum to the C.beta. protein was diluted in
PBS with 0.5% Brij 35 and added to the plate and incubated for 1 hr
at room temperature. The plates were again washed as before and the
secondary antibody, alkaline phosphatase conjugated goat
anti-rabbit IgG (Tago Inc., Burlingame, Calif.), was diluted in
PBS-Brij, added to the plates and incubated for 1 h at room
temperature. The plates were washed as before and p-nitrophenyl
phosphate (Sigma Phosphatase Substrate 104) (1 mg/ml) in 0.1 M
diethanolamine, 1 mM MgCl.sub.2, 0.1 mM ZnCl.sub.2, 0.02% azide, pH
9.8, was added. The plates were incubated at 37.degree. C. for 1 h
and the absorbance at 405 nm determined using an Elida-5 microtiter
plate reader (Physica, New York, N.Y.). Control wells lacked either
the primary and/or secondary antibody. This was done to obtain a
titer of the rabbit anti-C.beta. protein which would give a
half-maximal reading in the ELISA assay. This titer would be used
in the inhibition ELISA. The microtiter plate were sensitized and
washed as before. Purified C.beta. protein or mutations of the
C.beta. protein were added and diluted in PBS-Brig. The dilution of
the rabbit anti-C.beta. protein which gave the half maximal reading
was then added. The mixture was then incubated for 1 hr at room
temperature. The plates were rewashed and the conjugated second
antibody added as stated. The plates were then processed and read
as described. The percentage of inhibition would be calculated as
follows:
[0091] 1-(ELISA value with the protein added)/(ELISA value without
the proteins added).
[0092] FIG. 4 shows the results of one of these inhibition ELISA
assays. In this assay the inhibition of the wildtype C.beta.
protein from streptococci is compared with the recombinant C.beta.
protein and the glutaminyl to prolinyl mutants, both expressed in
E. coli. As can be seen from the figure, this assay is sensitive
enough to detect the absence of the membrane spanning region in the
recombinants of the C.beta. proteins. However, when the recombinant
C.beta. protein containing the wildtype sequence is compared to the
substitution mutant lacking IgA binding activity, the antigenic
differences are minimal. This would suggest that such substitution
mutants maintain most of the antigenic character of the C.beta.
protein but lack the unwanted the IgA binding activity.
[0093] Although the foregoing refers to particular preferred
embodiments, it will be understood that the present invention is
not so limited. It will occur to those of ordinary skill in the art
that various modifications may be made to the disclosed embodiments
and that such modifications are intended to be within the scope of
the present invention, which is defined by the following Claims.
All patents and publications cited herein are incorporated by
reference herein in their entirety.
1 % wild- Name Sequence Vector type IgAbs+
]LLHIKQHEEVEKDKKAKQQKTLKQSDTKVDLSNIDKELNHQKSQVEKMAEQKGI-
TNEDKDSMLKKIEDIRKQAQQA pTOPE 100 DKKEDAEVKVREELGKLFSSTKAGLDQEIQ[
dgb6
]DSDALLELENQFNETNRLLHIKQHEEVEKDKKAKQQKTLKQSDTKVDLSNIDKELNHQKSQV-
EKMAEQKGITNEDK 17b DSMLKKIEDIRKQAQQAKKEDAVEVKVREELGKLFSSTKAGLDQEIQ-
EHVKKETSSEENTQKVDEHYANSL[ dgb6p
]DSDALLELENQFNETNRLLHIKQHEEVEKDKKAK-
QQKTLKQSDTKVDLSNIDKELNHQKSQVEKMAEQKGITNEDK pTOPE
DSMLKKIEDIRKQAQQADKKEDAEVKVREELGKLFSSTKAGLDQEIQEHVKKETSSEENQKVDEHYANSL[
dgb7
]VDLSNIDKELNHQKSQVEKMAEQKGITNEDKDSMLKKIEDIRKQAQQADKKEDAEVKVREEL-
GKLFSSTKAGLDQE 17b 0 IQEHVKKETSSEENTQKVDEHYANSLQNLAQKSLE[ dgb7p
]VDLSNIDKELNHQKSQVEKMAEQKGITNEDKDSMLKKIEDIRKQAQQADKKEDAEVKREELGKLFS-
STKAGLDQEI pTOPE 100 QEHVKKETSSEENTQKVDEHYANSLQNLAQKSLE[ dgb8
]VDLSNIDKELNHQKSQVEKMAEQKGITNEDKDSMLKKIEDIRKQAQQDKKEDAEVKVREELGKLFSS-
TKAGLDEIQ 17b 0 EHVKKETSSEENTQKVDEHYANLQNLAQKSLEELDKATTNE[ dgb8p
]VDLSNIDKELHNQKSQVEKMAEQKGITNEDKDSMLKKIEDIRKQAQQADKKEDAEVKVREELGKLF-
SSTKAGLDQE pTOPE 100 IQEHVKKETSSEENTQKVDEHYANSLQNLAQKSLEELDKATTNE[
dgp10 ]VDLSNIDKELNHQKSQVEKMAEQKGITNEDKDSMLKKIEDIRKQAQQADKKEDAEVKV[
17b 10 dgb12 ...VDLSNIDKELNHQKSQVEKMAEQKGITNEDKDSMLKKIEDIRKQAQQADK-
KEDAEVKV[ 17b 20 dgb11
...VDLSNIDKELNHQKSQVEKMAEQKGITNEDKDSMLKKIEDI-
RKQAQQADKKEDAEVKVREELGKLFSSTKAGLDQE 17b 20 IQFHVKKETSSEENTQKVDEHYA-
NSL[ nv34qp
...VDLSNIDKELNHQKSPVEKMAEPKGITNEDKDSMLKKIEDIRKQAQQADKKE-
DAEVKVREELGKLFSSTKAGLDQE 24a 10 IQ... dgb2
...VDLSNIDKELNHQKSQVEAMAEQAGITNEDKSMLKKIEDIRKQAQQADKKEDAEVKVREELGKLFSSTKA-
GLDQE 24a 60 IQ... dgb1 ...VDLNIDKELNHQKSQE(....DELTA....)A-
GITNEDKDSMLKKIEDIRKQAQQADKKEDAEVKVREELGKLFSSTKAGLDQEIQ... 24a 0
pnv231
...VDLSNIDKELNHQKSQVETMAEQLGITNEDKDSMLKKIEDIRKQAQQADKKEDAEVKVREELG-
KLFSSTKAGLDQE 24a IQ...
* * * * *