U.S. patent application number 10/785673 was filed with the patent office on 2005-03-31 for modified immunogenic pneumolysin compositions as vaccines.
Invention is credited to Liang, Shu-Mei, Michon, Francis, Minetti, Conceicao, Polvino-Bodnar, Mary Ellen, Pullen, Jeffrey K., Tai, Joseph Y..
Application Number | 20050070695 10/785673 |
Document ID | / |
Family ID | 26731692 |
Filed Date | 2005-03-31 |
United States Patent
Application |
20050070695 |
Kind Code |
A1 |
Minetti, Conceicao ; et
al. |
March 31, 2005 |
Modified immunogenic pneumolysin compositions as vaccines
Abstract
This invention relates to modified pneumolysin polypeptides that
retain the immunogenic nature of pneumolysin but have reduced or
undetectable hemolytic activity compared to native pneumolysin. The
invention also provides a method for generating novel pneumolysin
variants with these desired characteristic properties. The
invention also provides immunogenic compositions useful as
pharmaceutical compositions including vaccines in which non-toxic,
modified pneumolysin is used to stimulate protective immunity
against Streptococcus pneumoniae. The vaccines may be compositions
in which the modified pneumolysin is conjugated to bacterial
polysaccharides or may be carried on an attenuated viral vector. In
addition, the invention also provides a method of using the
non-toxic, modified pneumolysin toxoid in order to stimulate
antibodies against Streptococcus pneumoniae in a treated individual
which are then isolated and transferred to a second individual,
thereby conferring protection against Streptococcus pneumoniae in
the second individual.
Inventors: |
Minetti, Conceicao; (Silver
Spring, MD) ; Michon, Francis; (Bethesda, MD)
; Pullen, Jeffrey K.; (Columbia, MD) ;
Polvino-Bodnar, Mary Ellen; (Annapolis, MD) ; Liang,
Shu-Mei; (Taipei, TW) ; Tai, Joseph Y.;
(Collegeville, PA) |
Correspondence
Address: |
MORGAN & FINNEGAN, L.L.P.
345 Park Avenue
New York
NY
10154-0053
US
|
Family ID: |
26731692 |
Appl. No.: |
10/785673 |
Filed: |
February 23, 2004 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10785673 |
Feb 23, 2004 |
|
|
|
09120044 |
Jul 21, 1998 |
|
|
|
6764686 |
|
|
|
|
60053306 |
Jul 21, 1997 |
|
|
|
60073456 |
Feb 2, 1998 |
|
|
|
Current U.S.
Class: |
530/395 |
Current CPC
Class: |
A61K 39/092 20130101;
A61K 2039/6037 20130101; Y10S 424/831 20130101; Y10S 530/825
20130101; C07K 14/3156 20130101 |
Class at
Publication: |
530/395 |
International
Class: |
C07K 014/47 |
Claims
1-15. (cancelled)
16. A recombinant nucleic acid molecule encoding a modified type 14
pneumolysin polypeptide comprising one or more amino acid
substitutions in a wild-type pneumolysin polypeptide having the
amino acid sequence of SEQ ID NO:3, wherein said one amino acid
substitution occurs at a position selected from the group
consisting of position 61, 148, and 195, or wherein said more than
one amino acid substitution occurs at positions selected from the
group consisting of 17, 18, 33, 41, 45, 46, 61, 63, 66, 83, 101,
102, 128, 148, 189, 195, 239, 243, 255, and 257, and wherein said
modified pneumolysin polypeptide is soluble, elicits antibodies
which are cross-reactive with wild-type pneumolysin, and has
attenuated hemolytic activity.
17. The recombinant nucleic acid molecule according to claim 16
comprising the pneumolysin nucleic acid sequence of SEQ ID NO: 1,
and wherein said nucleic acid sequence comprises one or more of the
nucleotide substitutions selected from the group consisting of:
A-50.fwdarw.G, G-54.fwdarw.T, T-181.fwdarw.C, A-196.fwdarw.T and
T-302.fwdarw.C; A-122.fwdarw.G, A-514.fwdarw.G, T-583.fwdarw.A and
A-764.fwdarw.G; A-187.fwdarw.T, T-380.fwdarw.A, A-382.fwdarw.C and
T-443.fwdarw.A; T-98.fwdarw.C, T-137.fwdarw.C, T-248.fwdarw.C,
T-717.fwdarw.A and A-770.fwdarw.G; T-134.fwdarw.C, A-305.fwdarw.G,
A-566.fwdarw.G and T-583.fwdarw.G; T-583.fwdarw.G; T-583.fwdarw.A;
T-443.fwdarw.A; and T-181.fwdarw.C.
18. The recombinant nucleic acid molecule of claim 16 as contained
in a vector.
19. A microorganism comprising the nucleic acid molecule of claim
16.
20. The microorganism according to claim 19, wherein the
microorganism is selected from the group consisting of: bacteria,
yeast, mammalian and insect cells.
21. The microorganism according to claim 20, wherein the
microorganism is E. coli.
22-26. (cancelled)
27. A method for killing bacteria comprising contacting said
bacteria with antibodies to an immunogenic molecule comprising
modified pneumolysin comprising one or more amino acid
substitutions in a wild-type pneumolysin polypeptide having the
amino acid sequence of SEQ ID NO:3, wherein said one amino acid
substitution occurs at a position selected from the group
consisting of position 61, 148, and 195, or wherein said more than
one amino acid substitution occurs at positions selected from the
group consisting of 17, 18, 33, 41, 45, 46, 61, 63, 66, 83, 101,
102, 128, 148, 189, 195, 239, 243, 255, and 257, and wherein said
modified pneumolysin polypeptide is soluble, elicits antibodies
which are cross-reactive with wild-type pneumolysin, and has
attenuated hemolytic activity in the presence of complement.
28. The method according to claim 27, wherein the immunogenic
molecule is a polysaccharide-polypeptide conjugate wherein the
polysaccharide is a bacterial capsular polysaccharide.
29. A method for immunization of mammals comprising administering a
vaccine of comprising the modified pneumolysin polypeptide
comprising one or more amino acid substitutions in a wild-type
pneumolysin polypeptide having the amino acid sequence of SEQ ID
NO:3, wherein said one amino acid substitution occurs at a position
selected from the group consisting of position 61, 148, and 195, or
wherein said more than one amino acid substitution occurs at
positions selected from the group consisting of 17, 18, 33, 41, 45,
46, 61, 63, 66, 83, 101, 102, 128, 148, 189, 195, 239, 243, 255,
and 257, and wherein said modified pneumolysin polypeptide is
soluble, elicits antibodies which are cross-reactive with wild-type
pneumolysin, and has attenuated hemolytic activity and a
pharmaceutically acceptable carrier to said mammals.
30. A method for obtaining modified pneumolysin polypeptides,
wherein said modified pneumolysin polypeptides have reduced
hemolytic activity and suitable for eliciting an immunogenetic
response which is cross-reactive with wild-type pneumolysin
comprising the steps of: (a) mutating a nucleic acid molecule
encoding wild-type pneumolysin to produce mutated nucleic acid
molecules encoding modified pneumolysin polypeptides, wherein the
modified pneumolysin polypeptides comprise one or more amino acid
substitutions in a wild-type pneumolysin polypeptide having the
amino acid sequence of SEQ ID NO:3, wherein said one amino acid
substitution occurs at a position selected from the group
consisting of position 61, 148, and 195, or wherein said more than
one amino acid substitution occurs at positions selected from the
group consisting of 17, 18, 33, 41, 45, 46, 61, 63, 66, 83, 101,
102, 128, 148, 189, 195, 239, 243, 255, and 257 and expressing the
mutated nucleic acid molecules in host cells; (b) assaying the
modified polypeptide expressed by the host cells for hemolytic
activity; and (c) identifying the modified pneumolysin polypeptides
having substantially similar molecular weight as native wild-type
pneumolysin and which are refoldable.
31. The recombinant nucleic acid molecule of claim 16, wherein the
vector is selected from the group consisting of: a plasmid, cosmid,
bacteriophage and yeast artificial chromosome.
Description
FIELD OF THE INVENTION
[0001] This invention relates to the field of vaccines, and in
particular, methods for the production of modified forms of
pneumolysin and their use in producing compositions for the
immunization of mammals against infections caused by bacteria
including Streptococcus pneumoniae.
BACKGROUND OF THE INVENTION
[0002] Streptococcus pneumoniae is the major cause of bacterial
pneumonia, bacteremia, meningitis, and otitis media (Baltimore et
al. in Bacterial infections of humans: Epidemiology and control
Evans and Brachman eds, Plenum Press, New York, 1989 pp.525-546;
Schuchat et al. N. Engl. J. Med. 1997, 337, 970-976). Even with
appropriate antibiotic therapy, pneumococcal infections have been
estimated to result in as many as 40,000 deaths a year in the
United States (Fedson et al. Archives of Internal Medicine 1994,
154, 2531-2535; Fiebach et al. Archives of Internal Medicine 1994,
154, 2545-2557). In addition, pneumococci have gained increased
resistance to penicillin and other antibiotics making the
development of an effective vaccine to prevent pneumococcal
infections a public health priority (Farr et al. Archives of
Internal Medicine 1995, 155, 2336-2340). Since the current
23-valent pneumococcal capsular polysaccharide vaccine is
ineffective in children less than two years old (Douglas et al. J
Infect Dis 1983, 148, 131-137; Leinonen et al. Pediatric Infectious
Disease Journal 1986, 5, 39-44), numerous groups are developing
multivalent conjugate vaccines to prevent otitis media, the major
indication in this age group.
[0003] Pneumolysin (PLY), a sulfydryl-activated cytolytic toxin, is
produced by all types of Streptococcus pneumoniae (Kanclerski et
al. J Clin Microbiol 1987, 25, 222-225) and is considered a major
virulence factor in pneumococcal infection (Boulnois Journal of
General Microbiology 1992, 138, 249-259). Genetically engineered
PLY-negative mutant strains of S. pneumoniae have been shown to be
significantly less virulent in mice (Berry et al. Microb Pathog
1992, 12, 87-93; Berry et al. Infection and Immunity 1989, 57,
2037-2042). Cytotoxicity of PLY to pulmonary endothelial and
epithelial cells is well demonstrated in vitro (Rubins et al.
Infection and Immunity 1992, 60, 1740-1746). In addition, PLY may
be the principal cause of hearing loss and cochlear damage in a
guinea pig model of pneumococcal meningitis (Winter et al.
Infection and Immunity 1997, 65, 4411-4418).
[0004] As of 1985, an estimated five million children under the age
of 5 died from pneumonia caused by S. pneumoniae in developing
countries each year. Lancet (1985) September 28 2(8457):699-701. S.
pneumoniae employs a number of virulence factors to establish an
initial infection and then produce invasive disease(s). To prevent
systemic infections caused by the various serotypes of S.
pneumoniae, immunization of infants and adults with suitable,
cross-reactive vaccines, capable of eliciting safe, effective, and
long-lasting immunity, is needed.
[0005] In a prospective study of pneumococcal colonization and
infection in children, it was reported that pneumococcal serotypes
6, 14, 19, and 23 are the most commonly carried as well as the most
frequent cause of infection in infants, mainly otitis media (Gray
et al. J. Infect. Dis., 1988, 158, 948-955). In addition, it was
recently found that these same strains are more frequent among the
penicillin resistant clinical isolates (Nesin et al. J. Infect.
Dis., 1998, 177, 707-713). Clinical studies carried out in young
infants with a tetravalent pneumococcal conjugate vaccine including
the above types, report a reduction in the carriage of
vaccine-related strains (Dagan et al. Infect. Dis. J., 1997, 16,
1060-1064).
[0006] Almost all isolates of S. pneumoniae exhibit an external
capsule made up of repeating oligosaccharides. Antigenic
differences in the capsular polysaccharides due to different
saccharide sequences are the hallmark of the different S.
pneumoniae serotypes. Serotype-specific capsular polysaccharides
are the major contributors to the virulence of the pneumococcus.
Existing anti-pneumococcal vaccines are formulated from 23 capsular
polysaccharides selected from the 84 serologically distinct types
currently recognized. Unfortunately, these vaccines are not
effective in all populations, especially those of Asia. A second
shortcoming of the current vaccines is that polysaccharides by
themselves are poor immunogens, especially for infants and the
elderly.
[0007] Polypeptides expressed by S. pneumoniae also play an
important pathogenic role. Some of the defined polypeptides that
appear to contribute to the virulence of this organism include
pneumolysin, autolysin, neuraminidase, pneumococcal surface
polypeptide A (PspA), the 37 kDa polypeptide, adhesion molecules,
hyaluronidase, and an IgA1 protease.
[0008] Virtually all serotypes of S. pneumoniae produce
pneumolysin, one of the major virulence factors. This expression by
the various S. pneumoniae serotypes makes pneumolysin a prime
candidate for use in a protective vaccine against pneumococcal
infections provided its toxicity can be altered.
[0009] Pneumolysin is an intracellular bacterial polypeptide with a
molecular weight of approximately 53-kD. (Kanclerski et al. (1987)
J. Clin. Microbiol. 25:222-225.) It is a member of a family of
thiol-activated hemolysins and has various effects on eukaryotic
cells. Pneumolysin is known to bind to cholesterol molecules in the
eukaryotic membrane, form oligomers, and generate transmembrane
pores. It has also been demonstrated that the respiratory burst,
chemotactic, and phagocytic functions of polymorphonuclear
leukocytes, all of which are critically important for removing
invading pneumococci, are severely compromised in the presence of
pneumolysin.
[0010] Pneumolysin causes both cytolytic and cytotoxic effects, and
can stimulate an inflammatory response by the complement activation
pathway. Nonspecific activation of complement causes depletion of
complement polypeptides and generates nonspecific inflammation.
Inoculation of pneumolysin into lungs of experimental animals
causes pneumoniae-like symptoms. However, pre-immunization with
pneumolysin is protective for experimental animals upon challenge
with pneumococci. Paton et al. (1983) Infect. Immun.
40:548-552.
[0011] Because of pneumolysin's immunogenic activity and capacity
to elicit a protective response in individuals immunized with it,
it has been suggested to use pneumolysin as a component of a
vaccine. See PCT/AU89/00539. However, before pneumolysin can be
included in vaccines for human use, this toxin must be modified so
as to be substantially non-toxic while retaining the capacity to
elicit protective antibodies.
[0012] Modified pneumolysins devoid of toxic activities are
reported to have been generated based on the identification of
amino acid regions of pneumolysin thought to have similar functions
to related thiol-containing polypeptides. (WO 90/06951). The
reported mutations are exclusively in the C-terminal portion of the
polypeptide and were generated using targeted mutagenesis
techniques. Other mutations, including certain specific amino acids
in the N-terminal region have been reported to reduce hemolytic
activity. The most significant reduction in hemolytic activity is
reported as possibly being a result of histidine modification at
position 156. Hill et al. (1994) Infection and Immunity, 62,
757-758. No data is provided concerning whether any of these
substituted pneumolysins were properly refolded. A single mutation,
Thr-172.fwdarw.Ile was reported to be responsible for a pneumolysin
with reduced hemolytic activity. However, anomalous electrophoretic
mobility indicates that the protein is incorrectly folded. Lock et
al. Microb. Pathog. (1996) 21, 71-83.
SUMMARY OF THE INVENTION
[0013] This invention provides a novel method for generating and
identifying stable, genetically modified, substantially non-toxic,
immunogenic pneumolysin polypeptides using random PCR mutagenesis.
Modified pneumolysin (pneumolysoid) which can be used as immunogens
in a vaccine or can be used as an immunogenic carrier polypeptide
for polysaccharide conjugate vaccines against S. pneumoniae or
other bacterial infections are also provided. The modified
pneumolysin polypeptides of this invention, while exhibiting
substantially reduced or none of the toxin's toxic activity, elicit
antibodies which are cross-reactive with those elicited by the
native toxin.
[0014] This invention also relates to nucleic acid sequences
encoding the modified pneumolysins, vectors containing them as well
as transformed host cells capable of expressing the nucleic acid
molecules of this invention.
[0015] Another embodiment of this invention is
polysaccharide-polypeptide conjugate molecules in which the
modified pneumolysin of this invention is covalently coupled to
bacterial polysaccharide to form the conjugate. Such conjugate
molecules are useful as immunogens for eliciting a T cell dependent
immunogenic response directed against the bacterial polysaccharide
conjugated to the modified pneumolysin.
[0016] The invention is further directed to pharmaceutical
compositions containing the modified pneumolysin polypeptides of
the invention which elicit an immune response.
[0017] This invention further relates to a method of eliciting the
production of antibodies reactive to the modified pneumolysin
polypeptides. Such antibodies may be used to elicit both active and
passive immunity. The modified pneumolysins of this invention may
also be used to identify and isolate reactive antibodies.
[0018] It is therefore an object of this invention to provide
genetically stable, modified S. pneumoniae pneumolysin polypeptides
which have substantially attenuated or absent toxicity while
retaining epitopes which cause production of antibodies which also
bind the native toxin molecule.
[0019] It is a further object of this invention to provide a method
for generating genetically modified pneumolysins
(pneumolysoids).
[0020] It is another object of this invention to provide vaccine
preparations comprising a modified pneumolysin polypeptide that can
elicit antibodies and induce protective immunity against
Streptococcus pneumoniae when delivered to a susceptible mammal.
Such vaccines may be based on the pneumolysoid itself, or
conjugates that comprise one or more bacterial polysaccharides
covalently bound to a modified pneumolysin polypeptide of this
invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0021] FIG. 1: Wild-type nucleic acid sequence of type 14
pneumolysin.
[0022] FIG. 2: Non-limiting nucleic acid variations of type 14
pneumolysin. The residue position followed by examples of nucleic
acid substitutions that attenuate hemolytic activity are: 181, C;
443, A; 583, A or G. The residue position followed by examples of
nucleic acid substitutions not observed to attenuate hemolytic
activity are: 50, G; 54, T; 98, C; 122, G; 134, C; 137, C; 187, T;
196, T; 248, C; 276, C; 302, C; 305, G; 351, T; 380, A; 382, C;
459, C; 514, G; 558, C; 566, G; 717, A; 764, G; 770, G; 1038, T;
1138, A; 1212, A; 1296, T; 1386, G; 1395, A.
[0023] FIG. 3: Amino acid sequence of type 14 pneumolysin.
[0024] FIG. 4: Non-limiting amino acid variations of type 14
pneumolysin. The residue position followed by examples of amino
acid substitutions that attenuate hemolytic activity are: 61, Pro;
148, Lys; 195, Ile or Val; 243, Arg, Val, Glu, or Ser; 286, Asp;
446, Ser. The residue position followed by examples of amino acid
substitutions not observed to attenuate hemolytic activity are: 17,
Arg; 18, Asn; 33, Thr; 41, Gly; 45, Ala; 46, Thr; 63, Ser; 66, Tyr;
83, Ser; 101, Thr; 102, Gly; 127, Glu; 128, His; 153, Met; 172,
Ala; 189, Arg; 239, Arg; 255, Gly; 257, Gly.
[0025] FIG. 5: Map of plasmid pNV-19 containing wild-type
pneumolysin nucleic acid sequence. The pNV series of plasmids were
derived from pET-24a by cloning in modified pneumolysin nucleic
acid sequences.
[0026] FIG. 6: Diagram showing the positions of the nucleic acid
and amino acid substitutions in specific modified pneumolysin
polypeptides pNVJ1, pNVJ45, pNVJ20, pNVJ22, pNVJ56, pNV103, pNV207,
pNV111, pNV211.
[0027] FIG. 7: SDS-PAGE showing expression of recombinant
pneumolysin following IPTG induction.
[0028] FIG. 8: Comparison of polysaccharide dose response of
polysaccharide specific IgG following two injections of monovalent
or tetravalent pneumococcal pneumolysoid vaccines in mice.
[0029] FIG. 9: Comparison of polysaccharide-specific IgG following
two injections in mice of tetravalent pneumococcal vaccines
conjugated to pneumolysoid or tetanus toxoid carriers.
[0030] FIG. 10: Pneumolysoid-specific IgG elicited by monovalent
and tetravalent pneumococcal polysaccharide-pneumolysin vaccines in
mice after two injections.
[0031] FIG. 11: Polysaccharide-specific opsonophagocytic activity
elicited by tetravalent pneumococcal PS-pneumolysoid and PS-tetanus
toxoid conjugate vaccines in mice after two injections.
[0032] FIG. 12: Anti-hemolytic pneumolysoid-specific activity
elicited by monovalent and tetravalent pneumococcal conjugates in
mice after three injections.
[0033] FIG. 13: Hemolysis Inhibition Assay. Hemolysis titer of wild
type pneumolysin upon pre-incubation with the indicated mutants.
The bars represent the final hemolytic titer of the wild type
tested against erythrocytes pre-treated with the indicated
mutants.
[0034] FIG. 14: Competitive inhibition ELISA studies between a
rabbit polyclonal antibody to wild type PLY and wild type PLY
protein using soluble wild type PLY, PLYD mutant pNV207 (A) and
PLYD mutant pNV103 (B).
[0035] FIG. 15: Fluorescence Spectra of Wild Type Pneumolysin and
Mutants. Fluorescence emission spectra of wild type pneumolysin and
selected mutants recorded in 10 mM sodium phosphate (pH 7.5)
employing an excitation wavelength of 290 nm and monochromator
slits of 2 nm. O represents pNV207, .cndot. represents pNV111,
represents pNV211, +represents pNV103, and .quadrature. represents
wild-type.
[0036] FIG. 16: (A) Far UV CD spectra of mutant pneumolysin
pNV207(upper chart) and type 14 CPS conjugated mutant pneumolysin
pNV207(lower chart); (B) near UV CD spectra of mutant pneumolysin
pNV207 (upper chart) and type 14 CPS conjugated mutant pneumolysin
pNV207 (lower chart).
[0037] FIG. 17: (A) Tetravalent pneumococcal pneumolysoid pNV207
conjugate vaccine in mice: polysaccharide-specific IgG response
over time; (B) tetravalent pneumococcal TT conjugate vaccine in
mice: polysaccharide-specific IgG response over time; (C)
monovalent pneumococcal pneumolysoid pNV207 conjugate vaccines in
mice: polysaccharide-specific IgG response over time.
DETAILED DESCRIPTION OF THE INVENTION
[0038] Pneumolysin is found in virtually all known strains of S.
pneumoniae. Its broad distribution provides the ability to obtain
substantial cross-protection among different S. pneumoniae
serotypes. This invention provides genetically modified pneumolysin
polypeptides which act as toxoids (pneumolysoids) and are therefore
useful for eliciting antibodies and for use in vaccines against S.
pneumoniae. Nucleic acid sequences encoding the modified
pneumolysins, vectors and host cells transformed with vectors
comprising the nucleic acids encoding the modified pneumolysins are
also embodiments of this invention.
[0039] The modified pneumolysin polypeptides of this invention in
which at least one amino acid is substituted, retain sufficient
epitopes to be immunogenic and elicit antibodies which are
cross-reactive with wild-type pneumolysin. In addition, the
toxicity of such modified polypeptides is sufficiently reduced to
allow for their administration to mammals without substantial risk
of dangerous side effect.
[0040] In an embodiment of this invention, specific modified
pneumolysin polypeptides are provided which are covalently bound to
polysaccharides to produce conjugates. By conjugating the modified
pneumolysin polypeptides of this invention to different
polysaccharides, this invention provides compositions capable of
eliciting antibodies to a wide range of serologically distinct
pathogens. By selecting the capsular polysaccharide from specific
bacteria, this invention can be used to provide immunization
against meningococcus, pneumococcus, haemophilus influenzae type b
and Group B streptococcus as well as other bacteria.
[0041] In another embodiment of the invention, genetic
modifications in the pneumolysin genome are generated using random
mutagenesis techniques.
[0042] A. Method For Producing and Identifying Modified
Pneumolysin
[0043] Genetically modified pneumolysin polypeptides of this
invention are produced using conventional recombinant methodology
(Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual 2nd
ed., Cold Spring Harbor Laboratory Press and Ausubel et al. Eds.
(1997) Current Protocols in Molecular Biology, John Wiley &
Sons, Inc.). Minor variant forms of pneumolysin polypeptides have
been reported which show high degrees of conservation of amino acid
and nucleic acid sequences. See, for example, Mitchell et al.
(1990) Nucleic Acid Res. 18:4010 which is incorporated herein by
reference and which reports that isoleucine at position 153 of
pneumolysin of type 1 S. pneumoniae is substituted with methionine
in type 2. Type 14 which also has isoleucine at position 153 has an
asparagine at position 380 rather than an aspartic acid. These
variations may also be included among other substitutions in the
nucleic acid and amino acid compositions of this invention which
provides modified pneumolysin in which at least one epitope is
preserved.
[0044] Modified pneumolysin polypeptides are provided by this
invention which have reduced or no hemolytic activity compared to
the wild-type and retain a sufficient number of epitopes to produce
antibodies cross reactive with native or wild-type pneumolysin.
Identification of such polypeptides is accomplished by first
inserting random mutations into the gene encoding pneumolysin and
then screening the expressed polypeptide products for loss or
reduction of activity associated with toxicity.
[0045] 1. Methods Of Modifying Pneumolysin
[0046] A novel screening system useful for making and identifying
substantially immunogenic, but non-toxic or minimally toxic
pneumolysins useful in immunizing against S. pneumoniae infections
is provided by this invention.
[0047] This method comprises two basic steps: (1) random
mutagenesis and (2) selection.
[0048] Random mutagenesis is one of the suitable techniques for
introducing mutations into pneumolysin. Standard mutagenesis
methods are suitable for use with this invention. In an embodiment,
random PCR is performed in order to randomly incorporate nucleotide
changes into the type 14 pneumolysin genome. The subsequent
selection will identify desirable changes. This method is
applicable with any isolated pneumolysin gene. Preferably, enough
of the nucleic acid sequences is identified to enable production of
oligonucleotide probes. Non-limiting examples of such pneumolysin
genes are those encoding for type 2 and 14 pneumolysin. The
nucleotide sequence encoding type 14 is shown in FIG. 1.
[0049] PCR, or nucleic acid amplification, is described in U.S.
Pat. Nos. 4,183,195, 4,965,188 and 5,176,995, which are
incorporated herein by reference. Generally, PCR is a method for
amplifying one or more specific nucleic acid sequences wherein each
sequence consists of two separate complementary strands. PCR
requires hybridizing each strand with a complementary
oligonucleotide primer. These nucleic acids are templates for
synthesis of complementary strands using primers as described
below. An extension product of each primer is then synthesized
which is complementary to each nucleic acid strand. Next, the
extension products are separated from the templates on which they
were synthesized to produce single stranded molecules. Finally, the
single stranded molecules are again treated with the primers of the
first step under conditions such that an extension product is
synthesized for each of the single stranded molecules produced in
the second step. These steps may be repeated for optimal
amplification of the original nucleic acid and product
synthesis.
[0050] PCR mutagenesis involves incorporation of a "mismatch"
nucleotide into the growing strand and may be facilitated by
reliance on the high error rate of commonly used PCR polymerases.
Other methods, known in the art for creating random mutations may
also be used such as, for example chemical mutagenesis (Eichenleub,
R. (1979) J. Bacteriol. 138:559-566.) Alternatively, the
mutagenesis step may be accomplished by PCR using a "semi-random"
process in which either one or both primers include a random series
of nucleotides but a portion of one or both primers is
complementary and thus "anchored" to at least one known pneumolysin
sequences.
[0051] "Primers," as that term is used herein, refers to an
oligonucleotide, whether occurring naturally as in a purified
restriction digest or produced synthetically, which is capable of
acting as a point of initiation of nucleic acid synthesis when
placed under conditions in which synthesis of a primer extension
product which is complementary to a nucleic acid strand is induced,
i.e. in the presence of nucleotides and an inducing agent such as
DNA polymerase and at a suitable temperature and pH. The primers
are preferably single stranded for maximum efficiency in
amplification, but may alternatively be double stranded. If double
stranded, the primer is first treated to separate its strands
before being used to prepare amplification products. Preferably,
the primers are oligodeoxyribonucleotides but must be sufficiently
long to prime the synthesis of extension products in the presence
of the inducing agent. The exact lengths of the primers will depend
on many factors, including temperature, source of primer and use of
the method. The primers typically contain 10 or more
nucleotides.
[0052] Synthetic oligonucleotide primers may be prepared using any
suitable method, such as, for example, the phosphotriester and
phosphodiester methods (Narang, S. A. et al. (1979) Meth. Enzymol.
68:90; Brown E. L., et al. (1979) Meth. Enzymol. 68:109) or
automated embodiments thereof. In one such automated embodiment,
diethylphosphoramidites are used as starting materials and may be
synthesized as described by Beaucauge et al. (1981) Tetrahedron
Let. 22:1859-1962. One method for synthesizing oligonucleotides on
a modified solid support is described in U.S. Pat. No. 4,458,066
which is incorporated herein by reference.
[0053] It is also possible to use a primer which has been isolated
from a biological source. One such example may be a restriction
endonuclease digest of a large nucleic acid molecule encoding
pneumolysin which is sufficiently complementary to hybridize to the
pneumolysin sequences. Nucleotide substitutions may also be
inserted into primers during chemical synthesis.
[0054] It is to be understood that the nucleotide sequences of this
invention need not be limited to a single mutation within any given
molecule encoding the modified pneumolysin polypeptides. Multiple
mutations are also possible when they preserve the immunogenic
character of native pneumolysin polypeptide (see FIG. 2), while
attenuating or eliminating one or more of its toxic
characteristics. Multiple modifications may therefore be included
in a single polypeptide molecule (see FIG. 4). Multiple
modifications may be useful because they may reduce the likelihood
of reversion to the toxic native sequence. However, a preferred
embodiment of this invention is single mutations in the nucleic
acid sequence which result in single amino acid substitutions.
[0055] The random or semi-random PCR products encoding modified
pneumolysin, may be cloned into an appropriate expression vector
using standard cloning techniques known in the art.
[0056] In an embodiment, the vector includes at least one possible
cloning site, at least one antibiotic selection marker gene,
transcription promoter and an origin of replication. The vector may
be grown in a variety of compatible host cells, allowing a high
degree of expression. Preferred hosts include bacteria such as E.
coli, B. subtilis or yeast such as S. cerevisiae. Other eukaryotic
cells besides yeast such as mammalian cells may also be used, for
example. The cloning plasmid vector/host cell combination may be
any compatible vector and host cell. Any suitable expression vector
and host cell are acceptable provided they are able to support the
expression of the modified pneumolysin. Standard protocols for
cloning and expression may be used as described in Ausubel, F. M.
et al., eds. (1997) Current Protocols in Molecular Biology, John
Wiley & Sons, Inc. which is incorporated herein by
reference.
[0057] 2. Screening Of Modified Pneumolysin
[0058] Following ligation of the modified pneumolysin nucleotide
sequence to the vector in proper reading frame and transformation
into the host cell, screening is performed in order to identify
cell clones expressing modified pneumolysin polypeptides which have
reduced or absent toxicity.
[0059] A method for identifying suitably transformed hosts
expressing the randomly mutated pneumolysin polypeptide is provided
by this invention. Preferred modified pneumolysin polypeptides will
have similar structural features such as size when compared to
native pneumolysin. Therefore selection methods which analyze
polypeptide size such as SDS-PAGE and gel permeation chromatography
maybe used. Transformed hosts expressing modified pneumolysin maybe
identified by analyzing the proteins expressed by the host using
SDS-PAGE and comparing the gel to an SDS-PAGE gel obtained from the
host which was transformed with the same vector but not containing
a nucleic acid sequence coding for pneumolysin or modified
pneumolysin (the "standard host"). Transformed hosts expressing
pneumolysoid will produce a new band when examined by SDS-PAGE and
transformed hosts producing a large band corresponding to
pneumolysoid can be selected as candidates. The modified
pneumolysin polypeptides expressed by these clones may then be
screened for hemolytic activity in the cell extracts to identify
the modified pneumolysin polypeptides that have attenuated
hemolytic activity. Transformed hosts producing non-modified or
modified yet active pneumolysin which are toxic can be eliminated
by this simple screening step.
[0060] Alternatively, modified pneumolysin can be identified by
other methods known to those of ordinary skill in the art such as,
but not limited to, SDS-PAGE, followed by electroblotting or
western blotting analysis, or dot blotting of total cell extracts,
or limited proteolysis of the soluble fraction and further analysis
of the digests by SDS-PAGE or western blotting.
[0061] Factors to be considered in choosing the method of
pneumolysin purification and isolation include whether the modified
pneumolysin is present as a soluble protein or whether it becomes
insolubilized in inclusion bodies. Although not a general rule,
mutations which affect the folding properties of pneumolysin appear
to favor its accumulation in inclusion bodies.
[0062] Modified pneumolysin which has been identified in the
soluble fraction of the cell extracts may be isolated and purified
by conventional methods of purification, such as, but not limited
to: precipitation of nucleic acids, salt fractionation or capture
procedures such as ion exchange chromatography or hydrophobic
interaction chromatography. Gel permeation chromatography may be
used, particularly as a polishing step, following one of the
aforementioned chromatographic procedures. Alternatively, the
recombinant modified pneumolysin may be isolated by affinity
chromatography, or by procedures used for isolation of
thiol-containing proteins, as well as other methods known to those
of ordinary skill in the art (Current Protocols in Protein Science,
1995 John Wiley & Sons).
[0063] Alternatively, modified pneumolysin derived from the
inclusion bodies may be isolated following several inclusion body
washes to remove nucleic acids and other bacterial cell wall
contaminants. This procedure may include, but is not limited to,
washing the pellet with regular buffers, or regular buffers and
detergent additives. The protein may be further purified under
denaturing conditions by dissolving the washed inclusion bodies in
urea or guanidine HCl followed by gel filtration chromatography.
This procedure can be done prior to protein refolding. However,
refolding followed by ion-exchange chromatography represents a
preferred method to achieve maximal yields of refolded and purified
protein.
[0064] Native pneumolysin can be obtained by the procedure
described and used as reference. The hemolytic activity and the
migratory or elution profile of the native counterpart can thus be
used as reference for the isolation of modified pneumolysins from
either the soluble or inclusion body fractions.
[0065] Preferred criteria for selecting clones expressing suitable
modified pneumolysin polypeptides include one or more of: (1)
modified pneumolysin expression; (2) at or near full length
expression (based on a molecular weight of about 53,000 for native
pneumolysin); (3) presence of pneumolysoid in the soluble fraction;
(4) low hemolytic activity; and (5) high yield of expressed
polypeptide.
[0066] Although the inclusion of all the above criteria in a
screening protocol would identify the most efficient and likely
useful clones expressing a useful modified pneumolysin polypeptide,
less efficient clones may also produce modified pneumolysins which
are suitable for use in this invention including some that may not
be full length, but are sufficiently long to elicit production of
antibodies cross-reactive with native pneumolysin and/or function
as carrier polypeptides in a polysaccharide-polypeptide conjugate
molecule.
[0067] Although the preferred method for identifying desirable
clones described above directly assays characteristics of expressed
protein including size and hemolytic activity, other methods such
as detecting cross-reactivity with antibodies directed against
native pneumolysin or hybridization to nucleic acid probes may also
be used. In one embodiment, initial identification of host cell
clones transformed with plasmids containing the modified
pneumolysin nucleic acid sequences may be performed using standard
hybridization analysis as known to those skilled in the art. Probes
for modified pneumolysin genes include native pneumolysin nucleic
acid sequences or the amplification primers or other primers
indicating the presence of the amplified sequences. Preferably such
hybridizing probes are 30 to 40 nucleotides in length; more
preferable 10 to 20 nucleotides in length. Stringency should be
relatively low since probes may be hybridizing to sequences
containing altered bases.
[0068] A preferred method of hybridization is blot hybridization.
See Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual
2nd Ed., Cold Spring Harbor Laboratory Press which is incorporated
herein by reference, for additional details regarding blot
hybridization. A probe can be DNA or RNA and can be made detectable
by any of the many labeling techniques readily available and known
to the skilled artisan. Such methods include, but are not limited
to, radio-labeling, digoxygenin-labeling, and biotin-labeling. A
well-known method of labeling DNA is .sup.32P using DNA polymerase,
Klenow enzyme or polynucleotide kinase. In addition, there are
known non-radioactive techniques for signal amplification including
methods for attaching chemical moieties to pyrimidine and purine
rings (Dale, R. N. K. et al. (1973) Proc. Natl. Acad. Sci. USA
70:2238-42), methods which allow detection by chemiluminescence
(Barton, S. K. et al. (1992) J. Am. Chem. Soc. 114:8736-40) and
methods utilizing biotinylated nucleic acid probes (Johnson, T. K.
et al. (1983) Anal. Biochem. 133:125-131; Erickson, P. F. et al.
(1982) J. Immunol. Methods 51:241-49; Matthaei, F. S. et al. (1986)
Anal. Biochem. 157:123-28) and methods which allow detection by
fluorescence using commercially available products. Non-radioactive
labeling kits are also commercially available.
[0069] The screening process includes testing of the
pneumolysoid-expressing cells for low hemolytic activity by methods
which are known in the art. (Bernheimer, A. (1988) Meth. Enzymol.
165:213-217.) A micro-assay may be performed in a 96-well,
U-bottom, micro-titer plate, using an aliquot of culture grown from
colonies positive for pneumolysin (native or modified) expression
determined as described above. The aliquots may be extracted and
normalized for polypeptide content. The extracts may further be
centrifuged and the resulting pellet cell debris and the
supernatant analyzed separately. Further identification of
pneumolysoid expression in the supernatant indicates availability
in the solubilized fraction.
[0070] Aliquots of the cell lysates may be obtained, pelleted by
centrifugation and the supernatant or pellet analyzed for activity.
Screening the pellets for activity involves solubilization with a
denaturant, such as urea, followed by serial dilutions which are
conducted as described for the soluble species. Using this
procedure the protein undergoes refolding and activity, if present,
can be detected.
[0071] Negative activity results imply either an inactive refolded
polypeptide or an improperly refolded polypeptide. To distinguish
between these two conditions, a second screening process can be
used. Activity-negative clones are denatured and refolded before
loading onto an ion-exchange chromatography column. The mutants
which have an elution pattern similar to wild-type pneumolysin can
be further analyzed by gel-filtration chromatography and monomeric
species with a Stokes radius similar to wild-type pneumolysin are
selected.
[0072] The inserted nucleic acid sequence encoding the modified
pneumolysin of selected clone(s) may be sequenced by any of the
methods commonly used in the art and the corresponding amino acid
sequences deduced.
[0073] B. Modified Pneumolysin Polypeptides
[0074] 1. Reduction of Hemolytic Activity
[0075] The modified pneumolysin polypeptides of this invention are
polypeptides that are non-hemolytic or substantially non-hemolytic
and still maintain at least one epitope that binds to antibody
directed against the native polypeptide. Because such hemolytic
activity is associated with the toxicity of pneumolysin, the
modified pneumolysins would therefore also be expected to be less
toxic than native pneumolysin. The modified pneumolysin
polypeptides of this invention contain at least one mutation
relative to S. pneumoniae type 14 wild-type pneumolysin (FIG. 3),
preferably among the first 257 amino acids beginning from the
N-terminus. Modification of as few as one amino acid is required to
result in modified pneumolysin polypeptides which have little or
insignificant toxicity as determined by hemolytic assay. Thus,
substitutions at any one, or more, of positions 61, 148 and 195 may
result in polypeptides having reduced hemolytic activity. Preferred
substitutions for amino acids 61, 148 and 195 are shown below in
Table 1.
1 TABLE 1 Amino Acid Position 61 148 195 Wild-type Ser Met Phe
Substitutions Pro Lys Ile/Val
[0076] Substitutions at these preferred positions with amino acids
other than the preferred ones, for example, those having similar
charge at neutral pH, are also within the scope of this invention.
Accordingly, substitution of the serine at 61 with hydroxyproline;
methionine at 148 with arginine or histidine; phenylalanine at 195
with leucine, glycine or alanine are other non-limiting examples of
possible substitutions.
[0077] Although single substitutions may be sufficient to attenuate
hemolytic activity, such reduction may also be accomplished by
substituting in a single polypeptide specific groups of amino
acids. For example, the collective substitution in a single
polypeptide of the amino acids at positions 33, 46, 83, 239 and 257
produces polypeptides having characteristics of pneumolysin but
with reduced hemolytic activity. Preferred substitutions are shown
in Table 2.
2 TABLE 2 Amino Acid Position 33 46 83 239 257 Wild-type Ile Ile
Leu Ser Asp Substitution Thr Thr Ser Arg Gly
[0078] As with the single substitution, other amino acids in
addition to those which are preferred may also be substituted based
on the same considerations of charge discussed above with the
further non-limiting example that serine and threonine may be
substituted for each other, and that other neutral amino acids such
as those recited above may be substituted for Asp at 257.
[0079] It should be understood that besides the substitutions
disclosed above, which are effective for reducing or eliminating
the hemolytic activity, other substitutions may also be made
provided that at least one epitope capable of binding an antibody
which binds native pneumolysin is retained. Non-limiting examples
of amino acid residues which may be substituted but which alone do
not reduce hemolytic activity include those at positions 17, 18,
33, 41, 45, 46, 63, 66, 83, 101, 102, 127, 128, 172, 189, 239, 255
and 257. Examples of substitutions at these positions include, but
are not limited to those shown in Table 3. Because these sites are
not associated with decreases in hemolytic activity it is expected
that these positions may be more freely substituted with less
regard to size and charge.
3 TABLE 3 Amino Acid Position 17 18 33 41 45 46 63 66 83 Wild- Lys
Lys Ile Asp Val Ile Thr Asn Leu type Substi- Arg Asn Thr Gly Ala
Thr Ser Tyr Ser tution Amino Acid Position 101 102 127 128 172 189
239 255 257 Wild- Ile Asp Val Asn Thr Gln Ser Lys Asp type Substi-
Thr Gly Glu His Ala Arg Arg Gly Gly tution
[0080] It is to be understood that the amino acid substitutions
described above are not exhaustive and that other modified
pneumolysin polypeptides identified according to the methods of
this invention are also within its scope.
[0081] Single point mutations of the native pneumolysin sequence
are preferred because the antigenic nature of the native
pneumolysin polypeptide is more likely to be preserved by the
single point modified form. Alternatively, a combination of
multiple mutations, may be used.
[0082] However, multiple mutations are sometimes unpredictable. The
mutations, in some cases, may act synergistically to abolish
activity or they may be involved in compensation mechanisms during
folding. For these reasons, single point mutations are considered
to be advantageous.
[0083] Although the screening process is based on identifying
modified pneumolysin polypeptides which are substantially
full-length, this invention also encompasses fragments and
truncated forms of the modified pneumolysin polypeptides provided
they retain at least one epitope recognized by an antibody which
binds to the mature pneumolysin. In addition, it is preferred that
such fragments or truncated forms be of sufficient size to produce
polysaccharide-polypeptide conjugates which produce a T cell
dependent immune response.
[0084] The hemolytic activity of the pneumolysoid proteins of this
invention may vary over a wide range depending on how the
pneumolysoid is actually used. For example, conjugation of a
pneumolysoid with reduced hemolytic activity may reduce such
activity further to acceptable levels. Conversely, where a
pneumolysoid is to be introduced into an individual, unconjugated
to another component or where it may be cleaved, it will be
desirable to have the hemolytic activity reduced as close to the
minimum detectable level as possible. For such purposes, levels of
hemolytic activity between about 0.2% and about 0.5%, or more
preferably about 0.2% are suitable. Where some hemolytic activity
may be tolerated, or where such activity may be further attenuated
by, for example, conjugation to polysaccharide, higher levels of
hemolytic activity may be acceptable, i.e. from about 0.5% to about
25%, or more preferably between about 1% and about 10%.
[0085] 2. Protein Structure
[0086] Previous studies report that the C-terminus of PLY contains
the cell-binding site (Owen et al., 1994 FEMS Microbiol. Let. 121,
217-221). The mutagenesis studies of this invention were focused on
the N-terminus which reportedly contains the oligomerization
domain. The finding that pre-incubation of erythrocytes with
certain mutants abrogated the wild type hemolytic activity in a
concentration dependent manner indicates that these mutants are
indeed capable of competing with the wild type counterpart for the
cell binding site. Since the mutants inhibit wild type activity,
these mutants likely retain the structural features of wild-type
pneumolysin. The preservation of the cell binding domain in the
mutant forms, specifically in the case of pNV103 and pNV207 is
significant as these mutants also exhibit the immunological
properties of the wild type molecule, as evidenced in ELISA
inhibition assays. Moreover, antibodies generated against these
mutants possess the ability to neutralize the hemolytic activity of
the wild type counterpart, additional evidence of their native-like
structure.
[0087] The structural features and integrity of wild type
pneumolysin and selected mutants have also been assessed by
circular dichroism and fluorescence spectroscopy. These techniques
offer the unique advantage of providing both qualitative and
quantitative information on the secondary and tertiary structure of
these proteins. Wild type pneumolysin is characterized by a high
content of .beta.-sheet structure, a prominent feature in the far
UV CD spectra of all the mutants selected in the present study. The
shape of the spectra and deconvolution analysis are consistent with
previous studies on recombinant pneumolysin purified from soluble
fractions of E. coli which was structurally and functionally
equivalent to the native pneumococcal pneumolysin (Mitchell et al.,
1989 Biochem. Biophys. Acta 1007, 67-72). Likewise, both the near
UV CD and fluorescence spectra are consistent with the native
structure containing Trp residues (Morgan et al., 1993 Biochem. J.
296, 671-674) whose side chains are partially exposed to solvent,
as evidenced by the emission maximum at .about.345 nm upon
excitation at 290 nm. The unique near UV CD spectra characterized
by a minimum ellipticity at .about.280 and a maximum ellipticity at
.about.290 nm, represents a fingerprint of this (Morgan et al.,
1993) and other cytolysins, such as perfringolysin (Nakamura et
al., 1995 Biochemistry 34, 6513-6520). As such, this characteristic
spectroscopic fingerprint may represent a useful baseline
measurement for subsequent evaluation of batch-to-batch
consistency, particularly for those mutants selected as components
of vaccine candidates.
[0088] C. Nucleic Acid Molecules Encoding Modified Pneumolysin
[0089] The modified pneumolysin polypeptides of this invention are
preferably synthesized by expressing a nucleic acid molecule
encoding the modified polypeptide in a host microorganism
transformed with the nucleic acid molecule. Accordingly, this
invention also encompasses the nucleic acid molecules, including
DNA and RNA encoding the modified pneumolysins discussed above.
[0090] The DNA encoding the polypeptides of the invention may be
used to express recombinant polypeptide in a wide variety of host
cells using a wide variety of vectors. The host cell may be
prokaryotic or eukaryotic. DNA for native wild-type pneumolysin may
be obtained from natural sources, such as Streptococcus pneumoniae,
or alternatively synthesized. The wild-type DNA may then be used as
the starting material for modification, as described above, to
obtain the DNA encoding the modified pneumolysin polypeptides of
this invention. Once identified as encoding desirable modified
pneumolysin polypeptides, the DNA encoding such polypeptides may
then be cloned into various vectors for expression. Alternatively,
the genes encoding such polypeptides may also be synthesized in
whole or in part.
[0091] In one embodiment, the invention relates to a method of
expressing the modified pneumolysin polypeptide in a microorganism
wherein the microorganism is transformed by a vector comprising a
gene encoding the modified pneumolysin polypeptide wherein the
polypeptide so produced comprises more than about 2% of the total
protein expressed in the transformed microorganism. In yet another
embodiment, the modified pneumolysin polypeptide expressed
comprises more than about 40% of the total proteins expressed in E.
coli.
[0092] Cloning vectors may comprise segments of chromosomal,
non-chromosomal and synthetic DNA sequences. Non-limiting examples
of some suitable prokaryotic vectors include plasmids from E. coli,
such as colE1, pCR1, pBR322, pMB9, and RP4. Prokaryotic vectors
also include derivatives of phage DNA such as M13, fd, and other
filamentous single-stranded DNA phages.
[0093] The modified pneumolysin polypeptides can be expressed
either direct or as fusion constructs. Two non-limiting examples of
fusion constructs are Thiofusion and His-Tag which can be isolated
and purified by conventional methods. Vectors for expressing
proteins in bacteria, especially E. coli, are also known. Such
vectors include, but are not limited to, pK233 (or any of the tac
family of plasmids), pT7, and lambda pSKF. Examples of vectors that
express fusion proteins include the PATH vectors described by
Dieckmann and Tzagoloff (1985) in J. Biol. Chem. 260:1513-1520.
These vectors contain DNA sequences that encode anthranilate
synthesis (TrpE) followed by a polylinker at the carboxy terminus.
Two non-limiting examples of fusion constructs are Thiofusion and
His-Tag which can be isolated and purified by conventional methods.
Other expression vector systems are based on beta-galactosidase
(pEX); maltose binding protein (pMAL); and glutathione
S-transferase (pGST)-(see (1988) Gene 67:31 and (1990) Peptide
Research 3:167). See Ausubel et al., supra.
[0094] Vectors useful in yeast are also available. Suitable
examples are YIp, YRp, YCP, YEp and YLp plasmids. See Ausubel,
Id.
[0095] Suitable vectors for use in mammalian cells are also known.
Such vectors include well-known derivatives of SV-40, adenovirus,
retrovirus-derived DNA sequences and vectors derived from
combination of plasmids and phage DNA. Additional vectors for
eukaryotic expression vectors are reported in (e.g., P. J. Southern
and P. Berg (1982) J. Mol. Appln. Genet. 1:327-341; S. Subramani et
al. (1981) Mol. Cell. Biol. 1:854-864; R. J. Kaufmann and P. A.
Sharp (1982) J. Mol. Biol. 159:601-621; R. J. Kaufmann and P. A.
Sharp (1982) Mol. Cell. Biol. 159:601-664; S. I. Scahill et al.
(1983) Proc. Natl. Acad. Sci. USA 80:4654-4659; G. Urlaub and L. A.
Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220.
[0096] Examples of preferred vectors are plasmids, and some
non-limiting examples of plasmids containing the T7 inducible
promotor, include the expression plasmids pET-17b, pET-11a,
pET-24a-d(+) and pET-9a, all of which are commercially available
from Novagen (565 Science Drive, Madison, Wis. 53711). These
plasmids comprise, operatively linked, in sequence, a T7 promoter,
optionally a lac operator, a ribosome binding site, restriction
sites to allow insertion of the structural gene and a T7 terminator
sequence. See, Novagen catalogue (1993) at 36-43.
[0097] Useful expression hosts include well-known prokaryotic and
eukaryotic cells. Some suitable prokaryotic hosts include, for
example, E. coli, such as E. coli BL21 (DE 3), E. coli SG-936, E.
coli HB 101, E. coli W3110, E. coli X1776, E. coli X2282, E. coli
DHI, and E. coli MRC1, Pseudomonas, and Bacillus, such as Bacillus
subtilis, and Streptomyces. Suitable eukaryotic cells include
yeasts such as Saccharomyces and other fungi, insect, animal cells,
such as COS and CHO cells, human cells and plant cells in tissue
culture.
[0098] In a preferred embodiment, E. coli strain BL21 (DE3) is
employed. The above mentioned plasmids may be transformed into this
strain.
[0099] Selection of E. coli transformed with the desired vectors
may be accomplished using standard selection protocols involving
growth in a selection medium which is toxic to non-transformed
cells. For example, E. coli is grown in a medium containing a
selection agent, e.g. any .beta.-lactam to which E. coli is
sensitive such as ampicillin. The pET expression vectors provide
selectable markers which confer antibiotic resistance to the
transformed organism.
[0100] High level expression of the modified pneumolysin
polypeptide can be toxic in E. coli. Surprisingly, this invention
allows for selection of modified pneumolysin polypeptides which may
be expressed in E. coli to a level of at least about 40% of total
cellular proteins.
[0101] Additional nucleotide mutations may be made that were not
identified in the selection process particularly where the
translated amino acid is the same as the identified amino acid
predicted based on the sequence of the selected clone. In addition,
nucleotide changes may be made which encode conservative amino acid
substitutions, especially where the identified polypeptides exhibit
other amino acid substitutions. Conservative amino acid
substitutions are known in the art and represent substitutions of
"similar" amino acids. Considerations include, but are not limited
to polarity, hydrophobicity, size, and side chain structure.
[0102] The modified pneumolysin polypeptides of this invention are
polypeptides that are non-toxic or substantially non-toxic and
still retain at least one epitope that binds antibody directed at
native pneumolysin. The modified pneumolysin of this invention
contain at least one mutation relative to wild-type pneumolysin,
preferably among the first 257 amino acids of the N-terminus. The
modified pneumolysin may be altered in that the amino acid present
at one, or more than one, of residue sites 17, 18, 33, 41, 45, 46,
61, 63, 66, 83, 101, 102, 127, 128, 148, 172, 189, 195, 239, 243,
255, 257, 286 or 446 of wild-type pneumolysin are replaced, removed
or blocked. As discussed above, additional modifications can be
incorporated from other known, modified pneumolysin polypeptides
such as those at residue sites 367, 379, 384, 385, 397, 428, 433,
434 or 435 which are disclosed in PCT WO 90/06951 which is
incorporated herein by reference. In addition to the amino acid
substitutions of this invention disclosed herein, other amino acid
substitutions that have been reported (Hill et al. (1994) Infection
and Immunity 62, 757-758) for pneumolysin may also be used with
this invention provided they allow for refolding of the pneumolysin
as determined by the methods described herein. Hill et al. reports
four N-terminal region mutations, Arg-31.fwdarw.Cys,
Leu-75.fwdarw.Phe, Val-127.fwdarw.Gly and His-156.fwdarw.Tyr that
result in 75%, 100%, 75% and 2% hemolytic activity, respectively.
They also report four C-terminal region mutations,
Ala-432.fwdarw.Val, Trp-433.fwdarw.Arg, Trp-436.fwdarw.Arg and
Val-468.fwdarw.Leu that result in 100%, <1%, 50% and 100%
hemolytic activity, respectively. However, if any of these
mutations result in improperly refolded pneumolysoids, then it is
preferred they not be used. The preferred modifications of
pneumolysin are those at residue sites 61, 148 or 195 and the most
preferred is at residue 195. Additionally, the combination of
modifications at sites 33, 46, 83, 239 and 257 is also
preferred.
[0103] Specific changes may be introduced into the native
pneumolysin sequence by any of the methods for site-directed
mutagenesis known in the art. In a preferred embodiment, PCR may be
performed using oligonucleotide amplification primers encoding the
desired nucleotide substitution(s) within their sequence.
[0104] Alternatively, the modified pneumolysoid polypeptide may be
constructed by chemical synthesis. (Kent et al. Adv. Exp. Med.
Biol., 1995, 362, 425-438). Such synthesis can be used to make all
or part of a pneumolysoid. In the case of partial synthesis, the
synthetic peptide can be covalently bound to an appropriate portion
of the pneumolysoid peptide, prepared by methods known in the art
or taught herein, to generate a semi-synthetic pneumolysoid.
[0105] D. Vaccine and Antibody Preparations
[0106] This invention is also directed to vaccine and antibody
preparations. According to this invention, the expressed, modified
pneumolysin described above or its derivatives or fragments thereof
may be used as an immunogen to generate antibodies that are
reactive against pneumolysin.
[0107] 1. Antibodies
[0108] The recombinant techniques for polypeptide expression
described above, provide for the production of abundant amounts of
the modified pneumolysin polypeptides of this invention, based on
the nucleic acid sequences of this invention. This facilitates the
generation of antibodies reactive against the modified pneumolysin
polypeptide. However, it should be understood that the polypeptide
may also be synthesized by chemical methods or combinations
thereof.
[0109] In another embodiment, antibodies directed against the
modified pneumolysin polypeptides may be generated by any of the
techniques that are well known in the art. According to one
approach, the antibodies may be generated by injecting an isolated
modified pneumolysin polypeptide preparation or derivatives or
fragments thereof into a host animal. The host animal may be, but
is not limited to, rat, mouse, rabbit, non-human primate, or a
human. Immunological responses may be increased by the use of
adjuvants which are known in the art.
[0110] Monoclonal antibodies directed against the modified
pneumolysin polypeptide may also be prepared by any of the
techniques that are well known in the art. According to one method,
cultures of continuous hybridoma cell lines are used (Kohler and
Milstein (1975) Nature 256:495-497). Monoclonal antibodies directed
against the modified pneumolysin polypeptide may be human
monoclonal antibodies or chimeric monoclonal antibodies made by any
of the techniques that are well known in the art. According to one
approach, chimeric monoclonal antibodies may be generated that have
a non-human (e.g. mouse) antigen-binding domain combined with a
human constant region. (Takeda et al. (1985) Nature 314:452).
[0111] Antibodies directed against the modified pneumolysin
polypeptide may be purified by any of the techniques that are well
known in the art including, but not limited to immunoabsorption or
immunoaffinity chromatography, or other chromatographic methods
(e.g. HPLC, gel filtration or ion exchange). Antibodies may also be
purified as immunoglobulin fractions from serum, plasma or cell
culture medium.
[0112] Antibody molecules of this invention may be intact
immunoglobulin molecules, substantially intact immunoglobulin
molecules, or those portions of an immunoglobulin molecule, for
example Fab fragments, that contain the antigen binding site.
[0113] Fragments of antibodies directed against the modified
pneumolysin polypeptide may be generated by any of the techniques
that are well known in the art. (Campbell (1985) Laboratory
Techniques in Biochemistry and Molecular Biology, Vol. 13, Burdon,
et al. (eds.), Elsevier Science Publishers, Amsterdam).
[0114] 2. Conjugate Molecules
[0115] The modified pneumolysin polypeptides of this invention may
be used to elicit an antibody response to S. pneumoniae in an
individual either alone or when conjugated to another immunogenic
molecule such as a polysaccharide. The other immunogenic molecule,
may be derived from either S. pneumoniae, or from a different
infectious agent against which it is desirable to generate an
immune response. Preferably the other immunogenic molecule to which
the modified pneumolysin is conjugated is a capsular polysaccharide
from a pathogenic bacteria. Such bacteria including for example:
Haemophilus influenzae type b; meningococcus group A, B, or C;
group B or A streptococcus of various serotypes including group B
types Ia, Ib, II, III, V, and VIII; as well as the various
serotypes of S. pneumoniae preferably types 1-23. S. pneumoniae
serotypes 3, 4, 6b, 9v, 14, 18c, 19f and 23 are most preferred.
Such polysaccharides for use to conjugate pneumolysoid may also be
modified themselves in order to be more effective or reduce
cross-reactivity to endogenous epitopes. See, for example, Jennings
et al. U.S. Pat. Nos. 4,727,136, 5,576,002 and U.S. application
Ser. No. 08/484,569 which is published as international application
WO 96/40239 which are incorporated herein by reference for
modification to group B meningococcal polysaccharides.
[0116] Any mode of conjugation may be employed to conjugate the
polysaccharide components with the modified pneumolysin
polypeptide. A preferred method is that described in U.S. Pat. No.
4,356,170, i.e. by introducing terminal aldehyde groups (via
oxidation of cis-vicinal hydroxyl groups) into the polysaccharide
and coupling the aldehyde groups to the polypeptide amino groups by
reductive amination. The polysaccharide and the modified
pneumolysin polypeptides are thereby linked through a
--CH.sub.2--NH-polypeptide linkage.
[0117] It is to be understood, however, that the conjugate vaccines
of the invention are not limited to those produced via reductive
amination. Thus, the vaccines may also be produced by conjugating
the polysaccharide with the modified pneumolysin polypeptide using
an adipic dihydrazide spacer, as described by Schneerson, R., et
al. (1980) J. Exp. Med. 1952:361-476, and in U.S. Pat. No.
4,644,059. Alternatively, the binary spacer technology developed by
Merck may be used, as described by Marburg, S. et al. (1986) J. Am.
Chem. Soc. 108:5282-5287 or, possibly, the reducing ends
methodology.
[0118] Conjugate molecules prepared according to this invention
typically comprise at least one modified pneumolysin polypeptide of
the present invention to which is bound at least one polysaccharide
component. Thus, this invention provides the ability to produce
conjugate molecules wherein the polypeptide is linked to the
polysaccharide through at least two sites to create cross-linked
conjugates.
[0119] The vaccines of this invention may provide active or passive
immunity. Vaccines for providing active immunity comprise a
purified modified pneumolysin polypeptide of this invention.
Preferably the polypeptide of this vaccine comprises at least one
of the following amino acid substitutions in the wild-type
pneumolysin amino acid sequence as shown in Table 1.
[0120] In another embodiment of this invention, antibodies directed
against the modified pneumolysin polypeptide of this invention may
be used as a pharmaceutical preparation in a therapeutic or
prophylactic application in order to confer immunity from a host
individual to another individual (i.e. to augment an individual's
immune response against S. pneumoniae or to provide a response in
immuno-compromised or immuno-depleted individuals including AIDS
patients). Passive transfer of antibodies is known in the art and
may be accomplished by any of the known methods. According to one
method, antibodies directed against the modified pneumolysin
polypeptides or conjugates thereof of this invention are generated
in an immunocompetent host ("donor") animal, harvested from the
host animal and transfused into a recipient individual. For
example, a human donor may be used to generate antibodies reactive
against the modified pneumolysin polypeptide or conjugate of this
invention, the antibodies transfused in therapeutically or
prophylactically effective amounts into a human recipient in need
of treatment, thereby conferring resistance in the recipient
against not only the pneumolysin toxin, but against S. pneumoniae
and a bacteria which bind antibodies elicited by the polysaccharide
component if the donor was immunized with a conjugate.
[0121] E. Pharmaceutical Compositions
[0122] The pharmaceutical compositions of this invention may
comprise the modified pneumolysin polypeptides, conjugate molecules
comprising the modified polypeptides or compositions comprising
antibodies elicited by one of the modified pneumolysin polypeptide
compositions of this invention. These pharmaceutical compositions
are particularly useful as vaccines.
[0123] For eliciting passive immunity, the pharmaceutical
composition may be comprised of, polyclonal antibodies or
monoclonal antibodies or their derivatives or fragments thereof as
described above. The amount of antibody, fragment, or derivative
will be a therapeutically or prophylactically effective amount as
determined by standard clinical techniques.
[0124] The pharmaceutical preparations of this invention may be
introduced to an individual by methods known to be effective in the
art. Intradermal, intraperitoneal, intravenous, subcutaneous,
intramuscular, oral, and intranasal are among, but not the only
routes of introduction.
[0125] The compositions of the invention may comprise standard
carriers, buffers or preservatives known to those in the art which
are suitable for vaccines including, but not limited to any
suitable pharmaceutically acceptable carrier, such as physiological
saline or other injectable liquids. Additives customary in vaccines
may also be present, for example stabilizers such as lactose or
sorbitol, and adjuvants to enhance the immunogenic response such as
aluminum phosphate, hydroxide, or sulphate and stearyl tyrosine.
The vaccines produced according to this invention may also be used
as components of multivalent vaccines which elicit an immune
response against a plurality of infectious agents.
[0126] Vaccines of the present invention are administered in
amounts sufficient to elicit production of antibodies as part of an
immunogenic response. Dosages may be adjusted based on the size,
weight or age of the individual receiving the vaccine. The antibody
response in an individual can be monitored by assaying for antibody
titer or bactericidal activity and boosted if necessary to enhance
the response. Typically, a single dose is about 0.1 to 10
.mu.g/kg.
[0127] F. Diagnostic Kits
[0128] In another preferred embodiment, the modified pneumolysin
polypeptides of this invention or derivatives or fragments thereof
may be used to produce safer diagnostic kits that do not
incorporate pneumolysin toxin but can still indicate the presence
of antibodies directed against S. pneumoniae. The presence of such
antibodies can indicate prior exposure to the pathogen, and predict
individuals who may be resistant to infection. An antibody reaction
may be identified by any of the methods described in the art,
including but not limited to an ELISA assay. Such knowledge is
important, and can avoid unnecessary vaccination. The diagnostic
kit may comprise at least one of the modified pneumolysin
polypeptides of this invention or derivatives or fragments thereof
and suitable reagents for the detection of an antibody reaction
when the modified polypeptides or derivatives or fragments are
mixed with a sample that contains antibody directed against
pneumolysin.
[0129] Alternatively, the diagnostic kit may further comprise a
solid support or magnetic bead or plastic matrix and at least one
of the modified pneumolysin polypeptides of this invention or
derivatives or fragments thereof.
[0130] In some cases, it may be preferred that the polypeptides or
derivatives or fragments are labeled. Labeling agents are
well-known in the art. For example, labeling agents include but are
not limited to radioactivity, chemiluminescence, bioluminescence,
luminescence, or other identifying "tags" for convenient analysis.
Body fluids or tissues samples (e.g. blood, serum, saliva) may be
collected and purified and applied to the diagnostic kit. The
pneumolysin polypeptides, derivatives (pneumolysoid) or fragments
may be purified or non-purified and may be composed of a cocktail
of molecules. Antibodies within the sample may or may not react
with the pneumolysin.
[0131] Solid matrices are known in the art and are available, and
include, but are not limited to polystyrene, polyethylene,
polypropylene, polycarbonate, or any solid plastic material in the
shape of test tubes, beads, microparticles, dip-sticks, plates or
the like. Additionally matrices include, but are not limited to
membranes, 96-well micro titer plates, test tubes and Eppendorf
tubes. In general such matrices comprise any surface wherein a
ligand-binding agent can be attached or a surface which itself
provides a ligand attachment site.
[0132] All publications, patents and articles referred to within
the specification are herewith incorporated in toto, by reference
into the application. The following examples are presented to
illustrate the present invention but are in no way to be construed
as limitations on the scope of the invention. One skilled in the
art will readily recognize other permutations within the purview of
the invention.
EXAMPLES
Materials and Methods
[0133] Bacterial Strains and Plasmids. Streptococcus pneumoniae
serotype 14 (ATCC, Rockville, Md.) was used in this study for
isolation of genomic DNA. E. coli strain DH5.alpha. (Life
Technologies, Gaithersburg, Md.) was used for initial cloning and
production of plasmid DNA. E. coli strain BL21 (DE3).DELTA.ompA,
used for protein expression, was derived from BL21 (BE3) (Novagen)
(see U.S. Pat. No. 5,439,808 for details). S. pneumoniae was grown
overnight in Todd-Hewitt (TH) broth at 37.degree. C. without
shaking under 7.5% CO.sub.2-. E. coli strains were grown in
Luria-Bertani (LB) broth, supplemented with carbenicillin (50-100
.mu.g/ml) or kanamycin (50 .mu.g/ml) as needed. The plasmid vectors
pUC-19 and/or pBluescript II SK+ (Stratagene) were used for cloning
fragments to be sequenced and the plasmids pET-17b and pET-24a
(Novagen) were used for cloning fragments to be expressed.
[0134] SDS-PAGE. Protein samples were prepared as follows: 1.5 ml
fractions were collected from cultures and the cells harvested by
centrifugation. The cells were resuspended in 150 .mu.l of protein
loading buffer and boiled for 5 min to lyse the cells. Cell debris
were removed by centrifugation and 10 .mu.l of each supernatant
were electrophoresed through an 8-16% gradient Tris-glycine
"Laemmli" polyacrylamide gel (Novex) along with low molecular
weight standards (Bio-Rad). Alternatively, crude extracts prepared
for analysis of hemolytic activity were diluted 1:1 with protein
loading buffer and 10-15 .mu.l loaded onto the gel. The protein
bands were visualized with Coomassie blue staining.
EXAMPLE 1
Expression of Pneumolysin.
[0135] E. coli strain BL21 (DE3) .DELTA.ompa transformed with
pET-17b or pET-24a containing the desired gene was grown with
moderate aeration at 30.degree. C. in LB supplemented with 0.4%
glucose and 100 .mu.g/ml of carbenicillin (for pET-17b constructs)
or 50 .mu.g/ml of kanamycin (for pET-24a constructs). When the
OD.sub.600 reached 0.6, IPTG was added to a final concentration of
0.4 mM (for pET-17b constructs) of 1 mM (for pET-24a constructs)
and the cells were allowed to incubate for another 2 h for
screening, or 5 h for larger scale production. To assay for
pneumolysin levels, 1.5 ml aliquots were removed prior to induction
and at various time points after induction and examined by
SDS-PAGE.
EXAMPLE 2
Cloning of the Pneumolysin Gene for Streptococcus pneumoniae
serotype 14.
[0136] Genomic DNA was isolated from approximately 0.5 g
Streptococcus pneumoniae serotype 14 using the method described
above. This DNA served as the template for two pneumolysin-specific
oligonucleotides in a standard PCR reaction. These oligonucleotides
were designed to be complementary to the 5' and 3' flanking regions
of the pneumolysin gene from S. pneumoniae serotype 2 and to
contain XbaI restriction sites to facilitate the cloning of the
fragment if desired. The sequence of the forward oligonucleotide
was 5' AAC CTT GAT TGA TCT AGA TAA GGT ATT TAT GTT GG 3' and the
reverse oligonucleotide had the sequence 5' TCT TTT TGT CTC TAG AAT
TCT CCT CTC CTA GTC 3'. The PCR reaction conditions were as
follows: 200 ng S. pneumoniae type 14 genomic DNA, the two
oligonucleotide primers described above at 1 .mu.M of each, 200
.mu.M of each dNTP, PCR reaction buffer (10 mM Tris HCl, 50 mM KCl,
pH 8.3), 1.5 mM MgCl.sub.3, and 2.5 units of Taq polymerase, and
QS. to 100 .mu.l with dH.sub.2O. This reaction mixture was then
subjected to 25 cycles of 95.degree. C. for 1 min, 50.degree. C.
for 2 min and 72.degree. C. for 1.5 min. At the end of the cycling
period, the reaction mixture was loaded on a 1.0% agarose gel and
the material was electrophoresed for 2 h after which the band at
1.7 kb was removed and the DNA recovered using GeneClean.RTM. (Bio
101). This DNA was then digested with XbaI, repurified and ligated
to XbaI-digested pUC-19 using T4 DNA ligase. The ligation mixture
was used to transform competent E. coli DH5.alpha.. Recombinant
plasmids were identified and sequenced; many were found to have a
DNA sequence consistent with that of the gene encoding
pneumolysin.
EXAMPLE 3
Expression of the pneumolysin gene in E. coli.
[0137] Plasmids capable of expressing the mature pneumolysin
protein were constructed by amplifying DNA containing the
full-length pneumolysin gene (pST20, pST85, or type 14 genomic DNA)
with nested oligonucleotides designed to isolate the pneumolysin
coding region. The forward oligonucleotide was designed to contain
a NdeI site and would install a start codon at the 5' end of the
coding region. This primer had the sequence 5' TAT TAG GAG GAG CAT
ATG GCA AAT AAA GCA GTA AAT G 3'. The reverse oligonucleotide was
designed to contain an XhoI site and had the sequence 5' GGC CTC
TTT TTG TCT CGA GCA TTC TCC TCT CCT AGT C 3'. This strategy allowed
the cloning of the fragment encoding mature pneumolysin into the
NdeI and XhoI sites of either the pET-17b or pET-24a. Standard PCR
was conducted using a template containing the entire pneumolysin
gene (type 1, 2 & 14) and the two oligonucleotides described
above. This PCR reaction yielded a 1.6 kb product when analyzed on
a 1.0% agarose gel. The DNA obtained from the PCR reaction was gel
purified and digested with the restriction enzyme NdeI and XhoI.
The 1.6 kb product was again gel purified and ligated to NdeI-- and
XhoI-- digested pET-17b or pET-24a using T4 DNA ligase. This
ligation mixture was then used to transform competent E. coli
DH5.alpha.. Colonies that contained the 1.6 kb insert were chosen
for further analysis. The DNA from the DH5.alpha. clones was
analyzed by restriction mapping and the cloning junctions of the
chosen plasmids were sequences. After this analysis, the DNA
obtained from the DH5.alpha. clones was used to transform E. coli
BL21 (DE3).DELTA.ompA. The transformed bacteria were selected on
LB-agar containing 100 .mu.g/ml of carbenicillin, or 50 .mu.g/ml of
kanamycin when using the pET-24a plasmid. Typically, several clones
were screened for their ability to produce the mature pneumolysin
protein.
EXAMPLE 4
Random Mutagenesis To Generate Modified Pneumolysin.
[0138] A portion of the gene encoding pneumolysin comprising amino
acid residues 1-257 was subjected to random mutagenesis using a
modification of the technique as described. (Cadwell, R. C. and
Joyce, G. F. (1994) PCR Methods Appl. 3: pS136-40; Cadwell, R. C.
and Joyce, G. F. (1992) PCR Methods Appl. 2:28-33). An
oligonucleotide complementary to the T7 promoter region of the
pET-24a plasmid (See, FIG. 1a) with the sequence 5'ATT ACG CGA CTC
ACT ATA GGG 3' and an oligonucleotide complementary to a region of
the pneumolysin gene around 1250 bp (See FIG. 1) with the sequence
5'ATT ACG AAC ATT CCC TTT AGG3' were used to define the region of
the gene to be mutated. The random mutagenesis PCR reaction
conditions were as follows: purified plasmid pNV-19.2 (10 ng), the
two oligonucleotide primers described above at 1 .mu.M of each
imbalance dNTP concentrations of 0.2 mM dGTP, 0.2 mM dATP, 1 mM
dCTP, and 1 mM dTTP, PCR reaction buffer (19 mM Tris-HCl, 50 mM
KCl, pH 8.3), 8.0 mM MgCl.sub.2, 0.5 mM MnCl.sub.2, 6 units Taq
polymerase, and QS to 100 .mu.l with dH.sub.2O. This reaction
mixture was then subjected to 40 cycles of 95.degree. C. for 1
minute, 40.degree. C. for 2 minutes, and 72.degree. C. for 3
minutes. After the PCR reaction, fragments were extracted with
phenol/chloroform and ethanol precipitated. The fragment was then
digested with NdeI and HindIII, gel purified and ligated to
pNV-19.2, digested with the same enzymes. The fragments were
ligated and subsequently transformed into competent BL21 (DE3) E.
coli.
EXAMPLE 5
Selection of Modified Pneumolysin Expressing Modified Pneumolysin
Devoid of Toxic Effects.
[0139] The transformation described by Example 4 resulted in
numerous colonies (approximately 10.sup.4) of which 400 were
selected randomly for evaluation. The novel screening method
described in this example was used to identify colonies that
expressed modified pneumolysin polypeptides with the following
characteristics: 1) no hemolytic activity, 2) substantially
full-length, 3) partially soluble, and 4) monomeric and refoldable
when isolated from inclusion bodies. This screening method involved
the following steps:
[0140] (a) testing for presence of low hemolytic activity:
[0141] A micro-hemolytic assay was used to evaluate the clones.
Hemolytic activity-assays were conducted in U-bottom micro titer
plates using TBS (Tris-buffered saline, pH 7.4) as an incubation
buffer. Following a pre-incubation period of 5 min with 1 mM DTT,
twofold serial dilutions were performed and the samples incubated
with an identical volume of a 1% suspension of washed sheep
erythrocytes (Cappel) resuspended in the same buffer. The reactions
were conducted at room temperature as a function of time (kinetic
study), and the extent of erythrocyte lysis was monitored by visual
inspection. Each clone undergoing evaluation was scored from 0-5. A
rank of zero indicated no hemolytic activity while a rank of 4-5
indicated hemolytic activity at wild-type levels or above. Two
hundred clones with a score of 0,1,2, were selected and screened
again for other desired properties.
[0142] (b) testing for expression of full-length pneumolysin
polypeptide:
[0143] The polypeptide expression assay was carried out in a
96-well format. Colonies with low hemolytic activity were evaluated
by SDS-PAGE for the presence of a strong band having a molecular
weight of about 53,000 Daltons. Full-length pneumolysin has a
molecular weight of about 53 kD. Fifty-eight out of 200 were found
positive in this assay. These clones were collected for further
selection.
[0144] (c) testing for expression of modified pneumolysin
polypeptides in the soluble fractions:
[0145] Modified pneumolysin polypeptides expressed in both the
soluble fraction and inclusion bodies are more likely to be
refoldable. Ten ml cultures from 2h IPTG-induced E. coli cells
harboring plasmids containing mutant pneumolysin sequences lacking
or exhibiting reduced hemolytic activity were harvested and
resuspended in 1.5 ml of TEN buffer; the cells are lysed by a
sequential freezing/thawing/sonication procedure until the
supernatant exhibits significant levels of protein, as indicated by
the Bradford protein assay, which is indicative of successful
lysis. The lysed cell suspension is centrifuged (14,000 rpm/10 min)
and aliquots of both, the pellet and supernatant are analyzed by
SDS-PAGE. An aliquot of the soluble fraction is tested for
hemolytic activity and the hemolytic titer is determined to confirm
the reduced activity observed in the kinetic qualitative study
conducted in the initial phase of screening. Clones were found that
contained soluble, modified pneumolysin polypeptides that had
little hemolytic activity.
[0146] (d) High yields of refoldable and monomeric, modified
pneumolysin polypeptides:
[0147] Clones containing soluble pneumolysin are selected for the
next step in the screening procedure, which consists of discarding
the supernatant by aspiration, washing the pellet with TEN buffer
twice, and solubilizing the pellet in 5 ml of 8 M urea prepared in
TEN buffer. After sonicating for 2 min, the urea solution is
quickly centrifuged to remove aggregates and added dropwise to 45
ml of refolding solution, under constant stirring at 4.degree. C.
The refolding solution is then loaded onto a 2 ml DEAE-Sepharose-FF
column, pre-equilibrated in Buffer A (25 mM Tris.HCl, pH 8.0). The
column is washed with Buffer A and the bound protein is eluted with
a gradient of 0 to 1 M NaCl. The properly refolded pneumolysin
mutant should elute as a single peak between 13 and 20% Buffer B
(25 mM Tris.HCl, 1 M NaCl, pH 8.0) similarly to what is observed
for the wild-type. The protein peak is further analyzed by HPLC on
a Superose 12 column and both elution time, aggregate/monomer
ratio, and hemolytic activity are evaluated (see Table 4). The
selected mutant(s) should present a single monomeric species with a
Stokes radius comparable to the wild-type. Five clones (pNVJ1,
pNVJ20, pNVJ22, pNVJ45, pNVJ56) with high yields of monomeric
modified polypeptides were selected for further analysis including
nucleic acid sequencing. The amino and nucleic acid substitutions
of these clones are shown in Tables 5A and 6. Throughout the
specification and claims, proteins are given the name of the vector
that encodes them.
4TABLE 4 Comparison of Wild-Type (pNV19) And Mutant Pneumolysin
Polypeptides Pure HPLC Hemolytic Activity Monomer (Elution activity
(% wild Protein (mg/L) time) (U/mg) type) pNV19 63 20.1 10.sup.6
100 pNV111 92 19.3 2,555 (9) .sup.1 0.25 pNVJ22 86 20.7 2,440 (9)
0.24 pNVJ20 90 19.8 1,961 (6) 0.20 pNVJ1 66 20.2 1,536 (2) 0.15
pNVJ45 86 18.7 1,360 (5) 0.14 pNVJ56 104 19.8 2,000 (2) 0.20 pNV211
n.d. 20 1800 (2) 0.18 pNV207 100 20.5 800 (2) 0.08 pNV103 104.7 20
950 (2) 0.10 .sup.1 Numbers in parenthesis indicate number of
experiments.
[0148]
5TABLE 5A Amino Acid Sequence of Wild-Type (pNV19) Pneumolysin and
Modified Forms X.sub.aa17 X.sub.aa18 X.sub.aa33 X.sub.aa41
X.sub.aa45 X.sub.aa46 X.sub.aa61 X.sub.aa63 X.sub.aa66 X.sub.aa83
X.sub.aa101 X.sub.aa102 pNV19 Lys Lys Ile Asp Val Ile Ser Thr Asn
Leu Ile Asp pNVJ1 Arg Asn -- -- -- -- Pro -- Tyr -- Thr -- pNVJ45
-- -- -- Gly -- -- -- -- -- -- -- -- pNVJ20 -- -- -- -- -- -- --
Ser -- -- -- -- pNVJ22 -- -- Thr -- -- Thr -- -- -- Ser -- --
pNVJ56 -- -- -- -- Ala -- -- -- -- -- -- Gly pNV103 -- -- -- -- --
-- -- -- -- -- -- -- pNV207 -- -- -- -- -- -- -- -- -- -- -- --
pNV111 -- -- -- -- -- -- -- -- -- -- -- -- pNV211 -- -- -- -- -- --
Pro -- -- -- -- -- X.sub.aa127 X.sub.aa128 X.sub.aa148 X.sub.aa172
X.sub.aa189 X.sub.aa195 X.sub.aa239 X.sub.aa255 X.sub.aa257 pNV19
Val Asn Met Thr Gln Phe Ser Lys Asp pNVJ1 -- -- -- -- -- -- -- --
-- pNVJ45 -- -- -- Ala -- Ile -- Gly -- pNVJ20 Glu His Lys -- -- --
-- -- -- pNVJ22 -- -- -- -- -- -- Arg -- Gly pNVJ56 -- -- -- -- Arg
Val -- -- -- pNV103 -- -- -- -- -- Val -- -- -- pNV207 -- -- -- --
-- Ile -- -- -- pNV111 -- -- Lys -- -- -- -- -- -- pNV211 -- -- --
-- -- -- -- -- --
[0149]
6TABLE 5B Amino Acid Sequence of Modified Pneumolysin Polypeptides
Protein Mutation Hemolytic activity pNV19 wild-type 100% pNV21 446
P to S 25% pNV46 286 E to D 12% pNV22 243 G to R, 446 P to S <1%
pNV38 243 G to V <1% pNV39 243 G to E <1% pNV40 243 G to S
<1% pNV20 243 G to R <1%
[0150]
7TABLE 6 Nucleic acid substitutions to the wild-type (pNV19)
pneumolysin gene which resulted in dramatically reduced hemolytic
activity. N.sub.50 N.sub.54 N.sub.98 N.sub.122 N.sub.134 N.sub.137
N.sub.181 N.sub.187 N.sub.196 N.sub.248 N.sub.302 pNV19 A G T A T T
T A A T T pNVJ1 G T -- -- -- -- C -- T -- C pNVJ45 -- -- -- G -- --
-- -- -- -- -- pNVJ20 -- -- -- -- -- -- -- T -- -- -- pNVJ22 -- --
C -- -- C -- -- -- C -- pNVJ56 -- -- -- -- C -- -- -- -- -- --
pNV103 -- -- -- -- -- -- -- -- -- -- -- pNV207 -- -- -- -- -- -- --
-- -- -- -- pNV111 -- -- -- -- -- -- -- -- -- -- -- pNV211 -- -- --
-- -- -- C -- -- -- -- N.sub.305 N.sub.380 N.sub.382 N.sub.443
N.sub.514 N.sub.566 N.sub.583 N.sub.717 N.sub.764 N.sub.770 pNV19 A
T A T A A T T A A pNVJ1 -- -- -- -- -- -- -- -- -- -- pNVJ45 -- --
-- -- G -- A -- G -- pNVJ20 -- A C A -- -- -- -- -- -- pNVJ22 -- --
-- -- -- -- -- A -- G pNVJ56 G -- -- -- -- G G -- -- -- pNV103 --
-- -- -- -- -- G -- -- -- pNV207 -- -- -- -- -- -- A -- -- --
pNV111 -- -- -- A -- -- -- -- -- -- pNV211 -- -- -- -- -- -- -- --
-- --
EXAMPLE 6
Site Directed Mutagenesis of Pneumolysin Gene With Single
Mutation.
[0151] To dissect whether a single mutation or multiple mutations
are responsible for the loss of hemolytic activity in specific
peptides (Table 4), each mutation was introduced into the wild-type
allele as a single-site mutation using oligonucleotide directed
mutagenesis. Table 7 presents the oligonucleotides used to
introduce these specific mutations. Polypeptides carrying desired
mutations were identified and their nucleic acid sequences
confirmed. The following polypeptides with single base changes that
resulted in a loss of hemolytic activity from these site-directed
polypeptides were identified (See Table 5A): nucleic acid sequence
103 contains a single base change at 583 from wild-type T to
modified G (195-Phe.fwdarw.Val); nucleic acid sequence 207 contains
a single base change at 583 from wild-type T to modified A
(195-Phe.fwdarw.Ile); nucleic acid sequence 111 contains a single
base change at 443 from wild-type T to modified A
(148-Met.fwdarw.Lys); nucleic acid sequence 211 contains a single
base change at 181 from wild-type T to modified C
(61-Ser.fwdarw.Pro).
[0152] The polypeptides shown in Table 5B exhibited poor refolding
yields, explaining their reduced hemolytic activity. Single
mutations introduced into pneumolysin polypeptide at positions 243,
286 and 446 or a combination of substitutions introduced at
positions 243 and 446 produced species found exclusively in the
insoluble fraction as inclusion bodies. Attempted refolding of
these mutants yielded mostly aggregated species.
8TABLE 7 Modified Pneumolysin Sequences MUTATION POSITION AA #
Primer Sequence 443 148 Forward
5'ggtcaggtcaataatgtcccagctagaaAgcagtatg3' Met-Lys Reverse
5'gctgtgagccgtgattttttcatac- tgcTttctagctg3' 583 195 Forward
5'gcagattcagattgttaatGttaagcagatttattata3' Phe-Ile Reverse
5'atctgcttaaCattaacaatctgaatctgcttttcgcc3' 583 195 Forward
5'cagattgttaatAttaagcagattta- ttatacagtcagc3' Phe-Val Reverse
5'aatctgcttaaTattaacaatctgaatctgcttttcgcc3' 181 61 Forward
5'acaagtgatattCctgtaacagctaccaacgacagtcgc3' Ser-Pro Reverse
5'agctgttacagGaatatcacttgt- atttgtcgacaagct3'
EXAMPLE 7
Expression and Purification of Modified Polypeptides.
[0153] These single mutated genes were cloned into expression
vectors (pET-24a) individually to overexpress the modified
polypeptides in E. coli. The expression level is .about.40%. Novel
purification and refolding processes were developed to purify these
recombinant modified pneumolysins.
[0154] Pneumolysin expressed in E. coli cells harboring the
expression vector pNV19 was isolated from inclusion bodies by
resuspending and lysing the cells in TEN buffer (50 mM Tris-HCl,
100 mM NaCl, 10 mM EDTA pH 8.0), with an air driven cell disrupter
(Stansted Fluid Power Ltd.) under a pressure of 8,000 psi. The cell
lysate was centrifuged at 13,000 rpm at 4.degree. C. for 20
minutes; both pellet and supernatant were saved for isolation of
soluble and aggregated pneumolysin, respectively. The inclusion
bodies were washed three times with TEN buffer and stored at
-70.degree. C. Purification and subsequent refolding were achieved
by solubilizing the inclusion bodies in an 8 M urea solution
(freshly prepared in TEN buffer), followed by PEG-assisted
refolding. Polypeptide solutions in 8 M urea (200 .mu.g/ml) were
diluted 10-fold by drop-wise addition to a refolding solution,
consisting of 20 .mu.M of PEG 8,000 in 25 mM Tris-HCl, pH 8.0,
under constant stirring at 4.degree. C. The sample was clarified
and loaded into a DEAE-Sepharose Fast Flow ion exchange column
(Pharmacia) equilibrated in 25 mM Tris-HCl, pH 8.0. A gradient of
0-1 M NaCl was applied and pneumolysin containing fractions were
identified by detection of hemolytic activity, as described below,
and by SDS-PAGE. The purified fractions were concentrated by using
an Amicon concentrator and PM30 membrane. Aliquots of purified
polypeptide were tested for hemolytic activity, and analyzed by
SDS-PAGE and size exclusion chromatography, using a Superose 12
column.
[0155] Hemolytic activity assays were conducted in U-bottom
micro-titer plates using TBS (Tris buffered saline, pH 7.4) as an
incubation buffer. Following a pre-incubation period of 5 minutes
with 1 mM DTT, twofold serial dilutions of normalized proteins were
performed and the samples incubated with an identical volume of a
1% suspension of washed sheep erythrocytes (total volume 200 .mu.l)
(Cappel) resuspended in the same buffer. The reactions were
conducted at 37.degree. C. for 30 minutes and the extent of
erythrocyte lysis was monitored spectrophotometrically by spinning
down the U-plates transferring the supernatant to flat-bottomed
plates and measuring the extent of hemoglobin release at 450 nm.
The end point was set to be the concentration at which 50% lysis
occurred and was based on comparison with a 0.5% cell suspension
that was lysed hypotonically (see Tables 4 and 5B).
[0156] Another method of assaying the modified pneumolysin
polypeptides is to conduct a hemolysis inhibition assay of the
modified polypeptides. This assay consists of determining the
ability of the mutant proteins to reduce or eliminate the hemolytic
activity of the wild-type protein by pre-incubating erythrocytes
with the modified pneumolysin polypeptides and assessing the
hemolytic titer of the wild-type pneumolysin toward the pre-treated
erythrocytes. The results from using this assay with four modified
polypeptides are given in Table 8, and a detailed description of
the procedure appears in Example 11.
9TABLE 8 Hemolysis inhibition assay of pneumolysin by the
pneumolysin mutants Designation Mutation End point (*) pNV19
wild-type 512 pNV103 Phe.sup.195Val 64 pNV111 Met.sup.148Lys 128
pNV207 Phe.sup.195Ile 32 pNV211 Ser.sup.61 Pro 512 (*) Reciprocal
of the hemolytic titer of a wild-type pneumolysin preparation in
the presence of the indicated mutant
[0157] The antigenic cross-reactivity of the selected single site
pneumolysin mutants was determined by immunizing rabbits (n=2) with
each of the mutant proteins shown in Table 9 by conventional
immunization procedures. Immunization of rabbits: New Zealand White
rabbits (Covance, Denvers, Pa.) weighing 2-3 kg were immunized
subcutaneously with 100 .mu.g of wild-type or mutant pneumolysin
emulsified with complete Freund's adjuvant, (Vol/Vol). Booster
doses of vaccine mixed with incomplete Freund's adjuvant were
administered by the same route 21 and 42 days after the primary
dose. Sera were collected on day 0, 21, 42, and 52. The sera were
tested for the presence of antibodies against wild-type
pneumolysin. The antigenic titer of pooled sera (n=2) towards type
14 pneumolysin was determined by ELISA. In brief, plates were
coated with wild-type pneumolysin and incubated with serial
dilutions of each of the anti-mutant pneumolysin sera. Significant
binding of wild-type pneumolysin to antibodies elicited by the
modified pneumolysin polypeptides was observed as shown in Table
9.
10TABLE 9 Reactivity and hemolysis neutralizing titer of mutant
pneumolysin rabbit antisera towards type 14 pneumolysin by ELISA
Antibody Neutralizing Designation Mutation Titer Titer pNV19
wild-type 892,647 256 pNV211 Ser.sup.61Pro 432,100 128 pNV111
Met.sup.148Lys 296,113 128 pNV103 Phe.sup.195Val 2,505,208 512
pNV207 Phe.sup.195Ile 402,426 128 PBS -- -- 8
[0158] As can be seen in Table 9, antisera to each of the above
polypeptides, in addition to their strong cross-reaction with the
wild-type pneumolysin as measured by ELISA, have significant
neutralizing, anti-hemolytic titers as measured in a hemolysis
inhibition assay.
EXAMPLE 8
Preparation of Pneumolysoid Conjugates Preparation of
Polysaccharide for Conjugation.
[0159] PnC type 14 polysaccharide (ATCC Lot #2016107) (390 mg) was
dissolved in 16 ml of 0.5 N NaOH, and the solution was heated at
70.degree. C. for 3 hours. Following cooling of the solution, 1.93
ml of glacial acetic acid was added to bring the pH to 4. After
addition of 3 ml of 5% (w/v) NaNO.sub.2, the reaction mixture was
kept stirring at 4.degree. C. for 2 hours. The sample was then
diluted to 50 ml with deionized water and the pH was adjusted to 7
with 0.5 N NaOH. Excess reagents were dialyzed out by diafiltration
with DI water through a Spectra/Por molecularporous membrane tubing
(MWCOL:3,500), and the retentates freeze-dried. The deaminated type
14 polysaccharide was then molecular sieved on a Superdex G-200
(Pharmacia) column using PBS as eluent. Fractions eluting from the
column with molecular weight between 5000 and 15,000 as determined
by Chromatography/Multiangle Laser Light Scattering using a
Superose 12 column (Pharmacia) were pooled and dialyzed against DI
water through a Spectra/Por molecularporous membrane tubing (MWCOL
3,500) and freeze-dried. Preparation of conjugates.
[0160] Each of the PnCPS were first depolymerized and functional
aldehydes were introduced into the fragmented CPS by oxidation with
sodium metaperiodate. Following the oxidation process, the excess
periodate was destroyed with ethylene glycol, the oxidized
polysaccharides were dialysed against DI water and lyophilized.
[0161] Modified pneumolysin polypeptides in 0.2 M phosphate buffer
(pH 8) at a concentration of 5 mg/ml were mixed with 2.5
equivalents (by weight) of PnC 14 polysaccharide-fragment together
with 2 equivalents (by weight) of recrystallized sodium
cyanoborohydride. Reaction mixtures were incubated at 37.degree. C.
for 24 hours. Conjugates were then purified from the free
components by passage through a Superdex G200 (Pharmacia) column
using PBS containing 0.01% thimerosal as an eluent. Fractions
eluting from the column were monitored on a Waters R403
differential refractometer and by UV spectroscopy at 280 nm. The
fractions containing the conjugates were pooled, sterile-filtered
through a 0.22 .mu.m Millipore membrane and then stored at
4.degree. C. Polypeptide and carbohydrate content were measured by
the methods of Bradford and Dubois respectively. Polysaccharide
content in the resulting conjugates were approximately 30%.
[0162] Tetanus toxoid conjugates for use as control, were also
produced as described above and as follows: Tetanus toxoid (Serum
Statens Institute) was first passed through a molecular sieve
column (Superdex G-200 Pharmacia) in order to obtain the monomer
form of the toxoid. For conjugation, 12 mg of the monomer and 36 mg
of the PnC 14 polysaccharide-fragments were dissolved in 600 .mu.l
of 0.2 M phosphate buffer pH 7.2. Recrystallized sodium
cyanoborohydride (24 mg) was then added to the solution which was
then incubated at 37.degree. C. for 3-days. The conjugate was
purified as above. The conjugates had polysaccharide contents in
the 25-30% range (see Table 10).
11TABLE 10 Composition of Tetanus-Toxoid and Modified Pneumolysin
type 14 Conjugates Carrier Approx. Polypeptide PS % PS in
Polypeptide MW of PS (mg/ml) (mg/ml) Conjugate pNV103 9,000 0.170
0.079 32% #195 Phe-Val pNV207 9,000 0.117 0.048 29% #195 Phe-Ile
pNV111 9,000 0.145 0.062 30% #148 Met-Lys pNV19 9,000 0.115 0.049
30% Wild-type Tetanus Tm 9,000 0.245 0.098 28%
EXAMPLE 9
Immunization With Modified Pneumolysin Conjugates.
[0163] Groups of 20 CD1 female mice (age 6-8 weeks), from Charles
River Laboratories, were immunized subcutaneously (S. C.) with 2
.mu.g of the conjugated polysaccharides of Example 8 adsorbed on
aluminum (Aluminum hydroxide, Superfos, Denmark) at a concentration
of 1 mg of elemental aluminum per ml of PBS containing 0.01%
thimerosal. Mice received the vaccine at day 0, 28, and 49. Sera
were collected at day 0, 42, and 59, and stored at -70.degree.
C.
[0164] ELISA.
[0165] Micro titer plates (Nunc Polysorb ELISA plates) were
sensitized by adding 100 .mu.l of type 14 polysaccharide-fragment
(MW ca: 10,000)/HSA conjugate (2.5 .mu.g/ml) in PBS. The plates
were sealed and incubated at 37.degree. C. for 1 hour. The plates
were washed with PBS containing 0.05% Tween 20 (PBS-T) and blocked
with 0.5% (w/v) BSA in PBS for 1 hour at room temperature. The
wells were then filled with 100 .mu.l of serial two-fold dilutions
in PBS-T plates, 100 .mu.l of peroxidase labeled goat anti-mouse
IgG (H+L) (Kirkegaard and Perry Laboratories), and then washed five
times with PBS-T. Finally, 50 .mu.l of TMB peroxidase substrate
(Kirkegaard and Perry Laboratories) were added to each well, and
following incubation of the plates for 10 minutes at room
temperature, the reaction was stopped by the addition of 50 ul of 1
M H.sub.3PO.sub.4. The plates were read at 450 nm with a Molecular
Device Amex microplate reader using 650 nm as a reference
wavelength.
[0166] Inhibition ELISA Assay.
[0167] Microtiter plates (NUNC Polysorp) were coated with PLY (20
ng in 100 .mu.L to each well) in PBS (50 mM sodium phosphate, 150
mM NaCl, pH 7.4) for one hour at 37.degree. C. After washing the
plates with PBS+0.05% Tween 20 (PBST), the plates were post-coated
with 150 .mu.L of PBS+0.1% BSA, rewashed, and stored at 4.degree.
C. until used.
[0168] Hyperimmune rabbit anti-PLY was diluted in PBST, added to
the PLY coated plates, and incubated at room temperature for 1 h.
After washing, 100 .mu.L of goat anti-rabbit Ig-HRP conjugate (KPL)
diluted in PBSTween according to the manufacturer's instructions
were added to each well. The plate was incubated at room
temperature for one hour and then washed again. 100 .mu.L of TMB
microwell substrate (KPL) were added to each well. The reaction was
stopped after 10 minutes by the addition of TMB one-component stop
solution (KPL) and the OD 450 nm was immediately read. The dilution
corresponding to 1/2 the maximum signal was chosen for the
inhibition study. PLYD mutants as well as PLY as a control were
diluted serially in three-fold ingrements in PBST containing the
rabbit antiserum diluted such that the final mixture contained the
dilution which gave half-maximal activity and applied immediately
to the coated microtiter plates in duplicate. The plates were
incubated at room temperature for one hour and processed.
Inhibition was determined as percent of maximum signal achieved
with dilute antiserum in the absence of any inhibitor.
[0169] Opsonic Activity of Conjugate Antisera.
[0170] The opsonic ability of mice antisera to the PnC type 14
conjugates was tested in an in vitro opsonophagocytic killing assay
using the human promyelocytic leukemia HL-60 cell line (ATCC
#CCL240). (See Table 11). Briefly, 200 cfu of PnC type 14 (12-8-95
CB) cells were mixed in equal volume with serially diluted
antibodies and incubated 15 minutes under shaking at 37.degree. C.
in a 5% CO.sub.2 incubator. Baby rabbit complement and HL-60 cells
(5.times.10.sup.5) cultured 5-days in the presence of 90 mM
dimethylformamide were added to the mixture which was then
incubated at 37.degree. C. for 1 hour under shaking. Aliquots were
removed for quantitative culture and plated on chocolate agar.
Titers were determined by extrapolating the antibody dilution
corresponding to 50% live bacteria.
12TABLE 11 Immunogenicity of PnC 14 Polysaccharide Conjugates ELISA
titer Op+ to wild-type Titer pneumolysin Carrier ELISA Titer at Day
at Day at Day Polypeptide 0 42 59 59 59 Tetanus <50 287,000
170,000 28,000 <50 Toxoid pNV103 <50 209,000 178,000 18,000
124,000 #195 Phe-Val pNV207 <50 175,000 149,000 31,000 111,000
#195 Phe-Ile pNV111 <50 137,000 127,000 10,500 84,000 #148
Met-Lys pNV19 <50 275,000 241,000 29,000 124,000 Wild-type PBS
<50 <50 <50 <100 * PnC 14 polysaccharide-specific
antibody titer + Opsonophagocytic Titer
[0171] As can be seen from the data in Table 11, all of the
modified pneumolysin conjugates elicited antibodies which had
opsonophagocytic activity in the presence of complement. Mice
immunized with all the above conjugates, in addition to a strong
IgG anti-PS response, mount a very strong IgG response against the
pneumolysoid carriers and to the same extent as that raised against
the conjugated wild-type pneumolysin.
Example 10
Preparation of Tetravalent 6B/14/19F/23F Pneumolysoid Vaccines.
[0172] Preparation of Conjugates.
[0173] The hydrolysis of polysaccharides was carried out as
follows: type 6B PS was depolymerized with 0.1 N HCl at 60.degree.
C. for 3 hrs and 45 min; type 14 was depolymerized with 0.1 N HCl
at 60.degree. C. for 7 hrs; type 19F was depolymerized with a 10 mM
NaOAc buffer of pH 4.1 at 70.degree. C. for 2 hrs and 20 min; and
type 23F was depolymerized with 0.2M acetic acid solution at
100.degree. C. for 30 minutes.
[0174] Oxidized 6B PS was prepared as follows: the partially
depolymerized PS (35 mg) was dissolved in 1750 ml DI water and
treated with 250 ml of 10 mM NaIO.sub.4 in the dark for 2 hrs at
room temperature. The excess NaIO.sub.4 was destroyed with ethylene
glycol, and after extensive dialysis the oxidized PS was
lyophilized. Oxidized 14 PS was prepared as described above for
type 6B. Oxidized 19F was prepared as follows: 50 mg of
depolymerized PS was dissolved in 0.2 M sodium phosphate buffer pH
7.5 (5 ml) and treated with 41 ml of 100 mM NaIO.sub.4 at 4.degree.
C. overnight in the dark. Excess NaIO.sub.4 destroyed with ethylene
glycol and after extensive dialysis the oxidized 19F PS was
lyophilized. Oxidized 23F was prepared as follows: 68 mg of
partially depolymerized PS was dissolved in 3.4 ml of 3 mM
NaIO.sub.4 solution at room temperature in the dark for 1 hour. The
excess NaIO.sub.4 was destroyed by the addition of ethylene glycol,
and after extensive dialysis, the oxidized PS was lyophilized to
dryness.
[0175] The oxidized PSs were separately coupled to recombinant
pneumolysoid mutant 207 in which amino acid Phe residue 195 was
replaced by Ile. In brief, the oxidized PSs and the protein (5
mg/ml) in 0.2 M sodium phosphate buffer were combined at a
PS/protein ratio of about 2.5:1 by weight at room temperature and
sodium cyanoborohydride (2 equivalents by weight) was then added.
The conjugation mixtures were incubated at 37.degree. C. for 2
days. After reduction of the residual aldehydes of the conjugated
PS, with excess NaBH.sub.4, the conjugates were purified from the
reaction mixtures by passage through a column of Superdex 200 PG
(Pharmacia) eluted with PBS containing 0.01% thimerosal as the
preservative, except for the type 23 conjugate where the conjugate
was loaded onto a Q Sepharose Fast Flow column, and eluted with 10
mM Tris-HCl, pH 7.5 using a gradient of 0.5 M NaCl. Fractions
corresponding to the conjugates were pooled and analyzed for
protein and carbohydrate content as described in example 8 (see
Table 12).
13TABLE 12 Composition of pneumolysoid conjugates Pneumococcal
Approx. Polypeptide PS % PS in Serotype MW of PS (mg/ml) (mg/ml)
conjugate 6B 41,000 0.24 0.14 37 14 41,000 0.13 0.08 38 19F 10,000
0.46 0.14 23 23F 90,000 0.44 0.06 12
Example 11
Immunization with Pneumolysoid Tetravalent Vaccines.
[0176] Immunization of Mice.
[0177] Six to 8 weeks old female outbred CD-1 mice (Charles River,
Raleigh) were immunized with monovalent or tetravalent vaccines.
Streptococcus pneumoniae polysaccharides types 6B, 14, 19, and 23
were conjugated to tetanus toxoid or pneumolysin mutant (0.5 .mu.g
PS/0.2 ml to 2 .mu.g PS/0.2 ml) in 1 mg/ml 1 alum. The vaccines
were administered subcutaneously, on days 0, 28, and 49, and blood
samples were collected on days 0, 14, 28, 38 and 59. ELISA titers
against polysaccharides and the carrier protein were determined
using HSA-PS conjugates and wild-type pneumolysin (FIGS. 8, 9 and
10). The opsonic activity of the sera was determined in a
phagocytic assay using HL-60 cell line as described in Example 9
(FIG. 11).
Hemolysis Assay.
[0178] The pneumolysin activity was assessed according to Paton et
al. (1993) Infect. Immun. 40:548, with some modifications. In
brief, on standard U-bottomed microtiter plates, wild-type and
mutant pneumolysin proteins were twofold serially diluted in TBS
(15 mM Tris, 0.15 M NaCl, pH 7.5) plus 1 mM DTT as cofactor, in a
final volume of 100 .mu.l. One hundred microliters of 1% sheep
erythrocyte suspension in TBS were added and the reaction conducted
at 37.degree. C. for 30 minutes. After spinning down the unlysed
cells, the extent of the erythrocyte lysis was monitored in the
supernatant at 405 nm using a microtiter plate reader. The end
point of the assay was taken as the well in which 50% of
erythrocytes were lysed, based on a 0.5% cell suspension lysed
hypotonically.
[0179] Hemolysis Inhibition Assay of Murine Antisera.
[0180] Inhibition of the hemolytic activity was tested according to
Paton et al. (1993) Infect. Immun. 40:548, with some variations.
Before dilution, the mouse antisera were treated twice with
chloroform, to eliminate cholesterol. A twofold serial dilution of
50 .mu.l of the mice antisera were performed and 50 .mu.l of toxin
stock solution at 4HU (hemolytic units) were added. The hemolytic
activity of the toxins were assessed immediately before the
neutralization assay. After 15 min incubation at 37.degree. C. to
allow serum antibody to bind to the pneumolysin, 100 .mu.l sheep
red blood (1% in TBS) (ICN, Costa Mesa, Calif.) was added in each
well. The plates were incubated 30 min at 37.degree. C. and the
unlysed cells were pelleted by centrifugation. The extent of the
erythrocyte lysis released in the supernatant was monitored at 405
nm using a microtiter plate reader. The antibody titers were taken
as the highest dilutions of sera which gave complete inhibition of
the hemolysis (FIG. 12).
[0181] Hemolysis Inhibition Assay by Modified Pneumolysin.
[0182] Modified pneumolysin polypeptides can be tested for their
ability to inhibit the hemolytic activity of wild-type pneumolysin
when pre-incubated with erythrocytes. A suspension of erythrocytes
(3 ml) was incubated with 1 .mu.l (1 mg/ml) of each of the modified
pneumolysin polypeptides for 10 min and the suspension added to
wells of a microtiter plate containing serial dilutions of
wild-type pneumolysin. The plate was incubated at 37.degree. C. for
30 min and the hemolytic titer compared with a control incubation
performed with normal erythrocytes. The selected mutants exert
variable degrees of inhibition of the wild type hemolytic activity
upon pre-incubation with erythrocytes (FIG. 13), suggesting that
these mutants are capable of competing with the wild type for the
binding site, but are unable to insert into membranes to form lytic
channels. The mutants pNV103 and pNV207 represent the most
effective inhibitors, followed by pNV111. The mutant pNV211
apparently does not exhibit such inhibition properties. Additional
corroboration of the structural integrity and identity of the PLYD
mutants is that most of their antigenicity is retained when
compared to native PLY as shown in FIG. 14.
Circular Dichroism (CD) Spectroscopy.
[0183] The secondary and tertiary structures of the free wild type
and mutant pneumolysin and the respective conjugate were evaluated
by circular dichroism (CD) spectroscopy in the far UV (180 to 250
nm) and near UV (250 to 350 nm) regions, respectively. Concentrated
stock solutions of protein were dialyzed exhaustively against a
buffer system comprised of 10 mM NaPO.sub.4 (pH 8.0). Spectra of
samples containing 1.0 mg/ml protein were recorded at 0.1 nm
wavelength intervals on a JASCO Model 710 circular dichroism
spectropolarimeter (JASCO, Easton, Md.) employing a scan speed of 5
nm/min and average response time of 1 s. A minimum of four
consecutive scans were accumulated and the average spectra stored.
The temperature of the samples was maintained at 25.degree. C.
through the use of water-jacketed 0.01 cm and 1.0 cm pathlength
cells in the far and near UV, respectively.
[0184] Fluorescence Spectroscopy.
[0185] Fluorescence measurements were performed on an SLM
AMINCO-Bowman 8100 Series 2 spectrofluorometer. Fluorescence
spectra of samples containing 100 .mu.g/ml protein in 10 mM
NaPO.sub.4 (pH 7.5) were recorded over the range of 300 to 500 nm
employing an excitation wavelength of 290 nm and slit widths of 2
nm. Temperature stability was maintained through use of a
water-jacketed 1.0 cm quartz cuvette thermostatted at 25.degree.
C.
[0186] A comparison of the fluorescence spectra of wild type
pneumolysin and selected mutants has been performed under
experimental conditions in which these proteins adopt a native
folded conformation. As evidenced in FIG. 15, the fluorescence
spectra of all the proteins are characterized by a maximum emission
intensity at .about.345 nm, with somewhat higher amplitudes
observed for the mutant proteins when compared to the wild type.
Overall, the results indicate that all the proteins are in a native
conformation, which is characterized by a significant number of Trp
residues exposed to solvent. These results have been observed
previously for perfringolysin, and are consistent with the presence
of a Trp-rich cell-binding domain in the C-terminus of these
cytolysins.
[0187] Basic Structural and Immunological Features of Pneumolysin
(PLY), Pneumolysoid (PLYD) and CPS-PLYD Conjugates as Assessed by
Circular Dichroism.
[0188] PLY overexpressed in E. coli and refolded from inclusion
bodies exhibits a typical far UV CD spectrum characteristic of a
high content of .beta.-sheets with a minimum observed at .about.215
nm (Minetti et al. Biophys. J., 1998, 74, A233) which does not
significantly change in the single point mutation PLYD. Likewise,
chemical conjugation of either PLY or PLYD with PnCPS does not
affect the overall secondary structure of the proteins (FIG. 16A).
The near UV CD spectrum (FIG. 16B) which derives from the relative
assymetry of tyrosyl and tryptophanyl residues in the protein has
also been assessed in the free versus conjugate protein and reveals
a highly ordered structure resembling the wild type free protein.
The conjugate, however exhibits minor changes in the near UV CD
profile as a result from the presence of the polysaccharide on the
surface of these complexes, interfering particularly with the
specific Tyr signal (i.e., negative ellipticity with a minimum
centered at .about.280 nm), reduced in the respective conjugate.
Additional corroboration of the structural integrity and identity
of the PLYD mutants is that most of their antigenicity is retained
when compared to native PLY as shown in FIG. 14.
[0189] Spectroscopic methods represent a powerful tool in the
evaluation of the integrity of proteins. In the particular case of
conjugate vaccines which employ proteins as carriers, these
methods, in conjunction with functional and immunological
techniques may facilitate monitoring batch-to-batch variations as
well as the molecular basis for vaccine efficacy (Crane et al. Eur.
J. Biochem. 1997, 246, 320-327; Jones et al. Dev. Biol. Stand.
1996, 87, 143-151). The mutations render the protein atoxic, but it
retains the ability to refold to a native-like structure,
indistinguishable from the parent molecule. The nearly
superimposable far UV CD spectra of the free mutant protein (i.e.,
pNV207) and the corresponding Pn 14 conjugate, as seen by both
amplitude and crossover points, are indications that the secondary
structure of the protein within this macromolecular complex remains
intact. These results contrast with previous studies conducted with
other polysaccharide-protein conjugates in which light variations
in the secondary structure were noticeable, following conjugation
(Crane et al. Eur. J. Biochem. 1997, 246, 320-327).
[0190] The tyrosyl residues in the vicinity of the conjugation
sites may be perturbed by the presence of the polysaccharide as
indicated by the differences observed in the near UV CD spectra in
the region around 280 nm. However, the tryptophanyl peak,
characteristic at 290 nm, remains unaffected by the conjugation,
another indication that the Tyr-containing regions are not affected
by the reductive amination procedure.
[0191] Overall the spectroscopic in conjunction with the
serological results provide excellent evidence that PLYD-CPS
conjugates represent suitable vaccine candidates for the prevention
of pneumococcal diseases.
[0192] Immunogenicity Time Course Studies.
[0193] An immunogenicity time course study for the tetravalent
PLYD-PnCPS was carried out and is shown in FIG. 17A. The animals
received three injections at days 0,28 and 49, and blood samples
were obtained at days 0, 14, 28, 38, and 59. Each dose contained
0.5 .mu.g PnCPS of each type. The PnCPS-specific IgG response to
each type increased over time to peak just after the second
injection (titers ranging between 20,000 and 50,000) and then
reached a plateau. Significant booster effects were observed after
the second injection.
[0194] In FIG. 17B is shown the time course of the PnCPS-specific
IgG response of the tretravalent TT conjugate. Like for the PLYD
combination vaccine, the animals were similarly immunized using the
same schedule and same dose of vaccine. Again, the IgG response to
each type polysaccharide increased after each injection with
similar magnitude (final titers between 50,000 and 200,000) except
for type 23F which gave a significantly lower titer (ca: 5,000).
Booster effects were also observed for each type after the second
injection, except for 23 which gave a much less pronounced
effect.
[0195] For comparison with the above PLYD-PnCPS combination
vaccine, the immunogenicity time course of the monovalent
PLYD-PnCPS conjugates are shown in FIG. 17C. The animals received
the same doses of 0.5 .mu.g of PnCPS and had the same immunization
schedule as mentioned above. The time course of the IgG response to
the PnCPS in those monovalent conjugates was almost identical to
the one observed for the combination except for the type 23 PnCPS
which gave rise to a less steeper time course curve, with antibody
titers an order of magnitude lower than those of the three other
types.
[0196] The preclinical studies demonstrate that conjugates
consisting of polysaccharides derived from four pneumococcal
strains (6, 14, 19, and 23) and PLYD are highly immunogenic in
animals, and they elicited PnCPS-specific antibodies titers which
compared well with those raised with a TT tetravalent conjugate. In
addition, the PLYD tetravalent conjugate was able to generate high
levels of PLY-specific IgG antibodies that neutralized hemolytic
activity of wild type PLY. In the light of a recently published
report on the clear pathogenic role of PLY in the hearing loss and
cochlear damage in a pneumococcal experimental meningitis model, it
is clear that PLYD vaccine-induced antibodies will be a useful
adjunct to the capsular antibodies to ameliorate or even prevent
the feared complications associated with otitis media (Winter et
al. Infection and Immunity 1997, 65, 4411-4418).
Sequence CWU 1
1
* * * * *