Note: Descriptions are shown in the official language in which they were submitted.
2185568
BACKGROUND OF THE INVENTION
This invention relates to vaccines including oral vaccines
against chlamydial antigens, and a process for making the
vaccine.
Chlamydial infection is a diverse group of conjunctival,
genital, respiratory, and neonatal infections occurring primarily
on mucosal surfaces. The etiologic agent of the infection is an
obligate intracellular bacterial parasite of eukaryotic cells,
chlamydia. There are three genetically different species in this
genus, with certain similarities in morphology, intracellular
developmental cycle and antigenic responses: Chlamydia
trachomatis, Chlamydia psittaci and Chlamydia pneumoniae.
The infection by C.trachomatis is limited to humans.
Fifteen serovars are differentiated based on the antigenic
variations of the major outer membrane protein (MOMP) Grayston
and Wang, J. Infect. Dis., 132:87, 1975), Serotypes D-K, are the
most common cause of sexually transmitted venereal diseases.
Conservatively, more than 4 million cases of chlamydial sexual
infections occur each year in the United States making it more
prevalent than all other sexually transmitted diseases combined.
The diseases include nongonococcal urethritis, mucopurulent
cervicitis, acute epididymitis, ectopic pregnancy and pelvic
inflammatory disease (PID, endometritis, salpingitis,
parametritis and/or peritonitis). The infection in women can be
quite damaging: Among 250,000 cases of pelvic inflammation
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diseases caused by this organism in the U.S. each year, 10°s lead
to infertility. When infants are born to chlamydia-infected
mothers, they are at risk of developing inclusion conjunctivitis
and pneumonia. C. trachomatis serovars A, B, Ba, and C cause
trachoma, an infection of conjunctival epithelial cells. The
chronic and secondary infections induce the infiltration of
subepithelial lymphocytes, forming follicles and the invasion of
fibroblasts and blood vessels to
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the cornea, leading to blindness. On the other hand, the formation of the scar
and malformation of the eyelid, causing trichiasis' constant scraping of the
cornea
by the eyelash can also lead to corneal opacification and blindness. There are
approximately 500 million trachoma cases in the world, and between 7 and 9
million are now blind because of its complications making it the world's
leading
cause of preventable blindness. The prevalence of active trachoma is high in
early age. There are 80 million children in need of treatment. It has been an
enormously important health problem in the Middle East, North Africa, South
Asia
and North India.
C. psittaci mainly affects animals and birds. It had, and still has a great
economic impact in dairy, wool and meat industries. There are 9 serovars from
mammalian species, 7 serovars from avian species and 2 biovars from koala
bears. Mammalian serovar 1, 2, 3, and 9 infect cattle and sheep, causing a
wide
range of disorders from placenta and fetus infection and other reproductive
problems, including polyarthritis-polysisitis, encephalomyelitis,
conjunctivitis as
well as intestinal infections. Although numerous attempts have been made to
produce vaccines, only modest success has been achieved (Schnorr, J. Am. Vet.
Med. Assoc. 195:1548, 1989). Serovars 4, 5, and 6 are the causes of abortions,
pneumonia and polyarthritis in porcine species. Serovar 7 represents
chlamydial
strains of feline conjunctivitis, rhinitis and pneumonitis and serovar 8
includes
guinea pig inclusion conjunctivitis. The avian strains often cause human
infection
in bird handlers and poultry processing workers.
C. aneumoniae is a newly identified species. To date, one serovar has
been identified, TWAR (Grayston, Proceedings of the Seventh International
Symposium on Human Chlamydial Infections, Pg. 89, 1990). Current evidence
suggests that C. oneumoniae is a primary human pathogen that is transmitted
from human to human and causes about 10-20% of community acquired pneu-
monia in adults. It has become the main causative agent of human respiratory
diseases such as pneumonia, bronchitis, pharyngitis, and sinusitis and a
possible
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agent in reactive arthritis. Epidemics have occurred in hospitals, in the
military
and families. The serological finding from many countries have shown that 50-
55% of adults with antibody against TWAR antigen are specific for C. anueun-
oniae. It is the major bacterial cause of illness in newborn. The infection to
elderly persons and those with chronic diseases may cause serious illness or
even death.
The pathogenicity of chlamydial infection is not well understood. It is long
known that different individuals infected by these serovars exhibit different
clinical
manifestations. It has been proposed that it was likely due to the variation
of the
host immune response. It has been shown that immunologic response to the
synthetic Th/B cell epitopes in the various inbred strains of mice is
different,
indicating that the T helper epitope is recognized in the context of the
multiple
major histocompatibility complex.
The target of chlamydial invasion are typically epithelial cells of a host. It
is
still not certain how the chlamydial elementary bodies (EB), (a sporelike,
spheri-
cal particle, about 300 nm in diameter), enter the host cell: receptor-
mediated
endocytosis, and/or non-specific high affinity absorption. It has been
reported
that two proteins, 18 and 32 kD of C. trachomatis bind to Hela 299 cell mem-
brane preparations. Recently, another heat-liable protein membrane protein,
38kD, was proposed a binding to Hela cell line, suggesting a ligand like
mechan-
ism. It has also been proposed that since chlamydia have the ability to infect
a
wide variety of mammalian cells in vitro, there must be some adherence mechan-
ism for the establishment of the infection. The major outer membrane protein
was
proposed as such an adhesion. Recently, it has been demonstrated that a
heparin sulfate-like glycosaminoglycans present on the surface of chlamydia
organisms is required for attachment to host cells. The receptors on the host
cells have also been studied. It was suggested that proteins, 18,000 and
31,000
kD from Hela cells are the receptors due to trypsin sensitivity for the EB
specific
binding. It also has been shown that C. trachomatis and C. pneumoniae bind
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specifically to a lipid on Hela cells. Nuclear magnetic resonance spectroscopy
analysis and atom bombardment mass spectrometry show that it was
phosphatidylethanolamine (PE). At the same time ganglia-series glycolipids
were
found specifically bound to EBs. All those findings suggest that the mechanism
of
endocytosis by epithelial host cells is still a matter of uncertainty. Once
the EBs
enter the host cells by endocytosis, depending upon conditions, they are trans-
formed into a metabolically active, non-infectious reticulate body (RB). The
prime
purpose of RBs is intracellular replication by binary fission using host
metabolites. This occurs in a membrane-bounded vesicle, termed an inclusion.
This inclusion (endosome) can resist the fusion with the lysosomes of host
cells.
Each RB eventually gives rise to one or more EBs which can initiate another
infectious cycle. Host cells may be lysed by release of inclusion bodies or
undamaged by exclusion body exocytons. Surface antigens are thought to direct
both phagocytosis and evasion of phagolysosomal fusion.
The treatment of chlamydial infections has relied on the administration of
antibiotics. This has been proved effective in the early stages of the
infection
depending upon proper timing for diagnosis and screening. The problem is that
the infection can be asymptomatic. Most patients don't realize its presence
until it
has occurred for a period of time. In the chronic stage as in the case of
genital
infection, it has been demonstrated that little can be done to prevent the
damage
of the reproductive tracts in a monkey infection model.
Vaccines employing the whole organism or sub-units of the organism have
been used in an attempt to prevent chlamydia infections caused by members of
the trachoma biovars. These attempts, however, have been disappointing,
partially due to host hypersensitivity in reaction to the vaccines (Grayston
et al,
Clini. Med. J. (Republic of China) 8: 312, 1961, Wang et al, 1967, Am. J.
Opthal-
mol. 63: 1615, Schachter, Pathol. Immunopathol. Res. 8: 206, 1989). The
pathogenesis associated with infections believed to be a process of delayed
hypersensitivity. It is thought that chronic inflammation resulting from
repeated
WO 95124922 218 5 5 ~ ~ pCT~s95/02459
reinfection of humans have an important role in the conjunctiva) infiltration,
blinding sequelae of trachoma and scarring of the fallopian tubes which result
in
infertility and ectopic pregnancy. The surface antigens of elementary bodies
have
been the focus of research attempting to identify a protective antigen.
Surface components of chlamydia actively interact with host cells and with
the host's immune system. They are believed to account for the attachment,
endocytosis and the immune response, but the exact nature and regulation of
these interactions has not yet been fully identified. Several distinct
antigenic
components of C. trachomatis. C. psittaci and C. pneumonia have been in-
vestigated including the identification, characterization and function in
chlamydial
infection. Moreover, it is of importance to determine the mechanism of
infection
and determine the protective antigens. Surface exposed antigens are the main
targets of much research since they are accessible to the immune or other
defense systems. The antigens most actively investigated include major outer
membrane proteins (MOMP) chlamydial lipopolysaccharide, 60-kD heat shock
protein (HSPO 60) adhesions and a glycolipid exoantigen termed the exoantigen
(GLXA).
In the outer membrane of chlamydia there are three cysteine-rich proteins
57, 40, and 12.5 kD which resembles the matrix proteins of gram-negative
bacteria. The 57 and 12.5 kD proteins can not be found in the replicating form
of
the bacteria RBs. As the major outer membrane protein (MOMP), 40 kD, is
abundant in both infectious EBs and RBs. In RBs, the protein could function as
pore-forming proteins that permit exchange of nutrients for the reticulate
bodies.
Genetic and molecular characterization have shown that this protein is
composed
of four variable segments (VS) interspersed among five constant segments.
Those variable segments are surface exposed and have the determinants of
serovar, subspecies and species specificity.
The studies on immune responses to this protein are mostly carried out by
immunization of animals with purified protein. In vitro neutralization
experiments
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have been conducted using the mixture of poly or monoclonal antibodies
specific
to MOMP and EBs to infect cell culture. These experiments indicate that the
antibodies specific to MOMP protein or one single epitope prevent the
inclusion
bodies formation in cell culture. The mechanism of the neutralization does not
involve inhibition of the attachment or penetration, but rather interfere with
the
process after internalization. Using monoclonal antibodies generated by the
whole elementary bodies of serovar B, the monoclonal antibodies which recog-
nized the immuno-accessible MOMP epitope in dot blot assays, neutralized the
infectivity of organisms of monkey eyes. The protection was serovar-specific.
In
a later experiment by using Fab fragments of the monoclonal antibody, it has
been further demonstrated that monovalent Fab neutralized the infection by
preventing the attachment to Syrian hamster kidney (HaK) cells. Confirming
that
the protection is not due to the aggregation of bivalent IgGs. T cell epitopes
of
MOMP have also been investigated. T cell proliferation responses were found in
splenic T cells obtained from A/J mice immunized with MOMP in the presence of
overlapping synthetic peptides which represent primary sequence of serovar A
MOMP. The synthetic peptides which produced T cell response correspond to
surface-accessible serovar-specific epitopes located in variable domains (VD)
VD
I and VD IV. By using a similar approach, it was found in BALB/cByJ mice that
VDIII fragment is T cell dependent. It also has been shown that by using
chimeric
T/B cell peptides derived from two epitopes of MOMP, one is a conserved T
helper cell epitope and the other is a serovar A specific neutralizing
epitope.
Some mice immunized with this peptide produced high-titered serum-neutralizing
antibodies, while others did not. Although MOMP has been a most intensively
studied surface antigen and the neutralization antisera has been produced in
experimental animals, there are still many unsolved questions regarding the
immune response. For example, the neutralization of infection is serovar
specific,
thus, it is limited as a vaccine candidate. The neutralization is still
limited to in
vitro studies, and there has been no convincing in vivo protection from
challenge
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by immunization with an MOMP or chimeric epitopes known.
It has long been known that a chlamydia genus specific antigen was a
glycolipid (Dhir et al, J. Immunol. 109: 116-122, 1972). Much later, it was
found that
lipopolysaccharide (LPS) was in the outer membrane of both EBs and RBs of
chlamydia. It has a chemical structure similar to enterobacterial LPS of the
Re
chemotype (Nurminen et al, Science, 220: 1279-1281, 1983). The monoclonal
antibodies prepared by immunization with EBs of serovar L2 were specifically
against LPS of chlamydia, but not LPS from N. Gonorrhoeae.~yrphimu~~nrn or ~
Sue. However, antibodies produced by Sue. yphimurium Re LPS or lipid A
recognized
chlamydial LPS (Caldwell and Hitchcock, Infect. Immun., 44: 306, 1984)). this
demonstrated that chlamydial LPS has an unique antigenic domain compared to
other gram-negative bacteria. Further characterization has shown that a
chlamydia-
specific domain contained in its saccharide portion, 3-deoxy-D-manno-2-
octulosonic
acid (DKO) with a sequence of a-kDo (2-8)-a kDo(2-4-a-kDo(kDo3). The 2.8
linked
moiety is the structural characteristic of chlamydial LPS. Studies have also
been
carried out in the distribution and the relocation of LPS on the outer
membrane
during the developmental cycle. By immunostaining with a monoclonal antibody
it
was shown that LPS is loosely bound in the membrane during the developmental
cycle, and not shed into media.
Chlamydial LPS was thought to be an ideal antigen for the vaccine candidate
because of its abundance on the surface and its being antigenic. LPS was
suspected as an important virulent determinant in the early steps of the
infection and
the antibodies specific to it serve some function in resolving the chlamydial
infection.
However, little is known concerning the biological function of LPS or the
immunological response to it. It appears that the antibodies which are
specific to
LPS only have been useful in the diagnosis of chlamydial infection and
location of
LPS, but not effective in resolving an infection.
Other Genus-specific chlamydial antigens are 57 to 60 and 75 kD proteins
which have been identified as related to the heat shock protein (HSP) family.
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This was done by comparing the sequence of the operons encoding these
proteins to the groE stress response operon of E. coli or B. megaterium. The
antigenic identity of 57 kD protein was confirmed by the reaction with anti-
HSP-
60 antiserum. The 60 kD protein elicited an ocular hypersensitivity response
in
immune guinea pigs, which was characterized by a predominantly mononuclear
macrophage and lymphocyte cellular infiltration (Watkins, et al, Proc. Natl.
Acad.
Sci. USA 83: 7580, 1986, Morrison et al, S. Exp. Med. 169: 663, 1989). This
was the first indication that an antigen is responsible for delayed
hypersensitivity
in chlamydial ocular infection. The precise involvement of this protein in
stimulat-
ing immunopathogenic responses in human chlamydial diseases has not been
determined. There is evidence that shows a certain percentage of sera taken
from women with PID, ectopic pregnancy and tubal infertility have high anti-
chtamydial antibodies reacting to chlamydial HSPO-60 heat-shock protein.
However, not every patient serum which has high titer to chtamydia reacts with
it,
indicating that either HSP-60 is not surface exposed or antigenicity is MHC
restricted.
The 75-kD protein was found preferentially transcribed during heat stress
of chlamydial organisms. The monospecific antibodies from rabbits raised
against
75-kD protein were found to bind to the organism and neutralized the infection
in
vitro. It is an exposed antigen in the outer membrane.
Genus-specific glycolipid exoantigen (GLXA) was originally isolated from
the supernatants of chlamydia infected cell cultures (Stuart and MacDonatd,
Current Micobiology, 11: 123, 1982). it has been characterized chemically,
biologically and serologically in recent years.(Stuart and MacDonald,
Proceedings
of the Sixth International Symposium on Human Chlamydia Infections, p167,
1986, Stuart et al, Immunology, 67: 527, 1987). Mass spectrographic analysis
indicated that GLXA contains polysaccharides: gulose, (not glucose), mannose
and possibly galactose, white the lipid component has fatty acids of chain
length
C17 and C18:1. There is no KDO or lipid A found in its structure. It is
produced
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218 5 J ~ ~ pCT/US95102459
and released from the infected cells during the growth cycle in vitro.
Transmis-
sion electron microscopy utilizing colloidal gold-conjugated goat anti-mouse
second antibody to detect the specifically bound monoclonal antibody revealed
that GLXA is mostly extracellular 48 hours after the infection (Stuart et al,
Immunolgy, 74: 747, 1991) which is different from that found for chlamydial
LPS.
Human sera from patients with clinically defined lymphogranuloma venereum
(LG~ contain IgG antibodies which recognize GLXA (Stuart and MacDonald,
Immunology, 68: 469, 1989), demonstrating the immunoaccessibility in the
natural infection. But, there was little information on its function in the
chlamydial
infection and the immune response to it. The overall immunological reaction to
chlamydial antigens is not well understood. It is still not known how the
chlamydia evade the host immune surveillance. Antibodies found specific to
chlamydial antigens in infected human patients have shown little protection
for
later infection. Although chlamydia mainly affects mucosal surfaces, the
clinical
relevance of the IgA immunity to it has not been completely described. The
feasibility of chlamydia vaccine depends on producing a protective host
defense
which may include S-IgGA response, a cell mediated response and possibly a
humoral antibody. In addition, the ability to produce large quantities of this
antigen indicates a synthetic and/or chimeric antigen may be the method of
choice.
Idiotypes have been intensively studied following Jerne's network theory in
1974. One of his major proposals is the self regulation of the immune system
through a network of idiotype-anti-idiotype interactions (Jerne, 1974). It is
sug-
gested that the idiotopes on a single antibody molecule can mimic ( that is,
be
the "internal image") of any foreign or self epitope at the molecular level.
All idiotypes of a single immunoglobulin molecule have been found to be
located on Fv (fragment variable) region by studies showing that the
inhibition of
binding of anti-idotypic antibodies to the idiotype is the same between Fv and
Fab( Givol, 1991 ). In general, anti-idotypic antibodies are divided into
three types
WO 95/24922 21 ~ 5 ~ 6 ~ pCT~S95102459
Ab2 a , Ab2 ~3 and Ab2s . Only Ab2 Q binds to the complementary determining
region, thus can be the internal image of the antigen. The occurrence of Ab2
displaying internal image of properties must adhere to the following criteria;
(1)
binding onto Ab, and to any other anti-nominal antigen antibodies from another
species and lack of reactivity with Abz to other antibodies; (2) inhibition of
the
binding of Ab, to the specific antigen, the nominal antigen, and (3) the
ability to
elicit the synthesis of Ab3 with anti-antigen specificity in animals without
previous
exposure to the antigen (Ertl and Bona, 1988).
The important role of anti-idiotypic antibodies in vivo has been shown in
numerous experiments. The administration of anti-idiotypic antibodies was
found
to elicit different effects: either suppression or enhancement of the
responses to
the specific idiotype( Hart, 1972, Kennedy, 1983). In autoimmunity, it
certainly
plays an important role. The pathology associated with many autoimmune
diseases is most likely due to (at least in part), a direct idiotype-anti-
idiotype
interaction of the autoimmune antibodies with anti-idiotypic antibodies.
Idiotypic
specificity in a specific antibody were first characterized, by demonstrating
that
specific hapten binding could inhibit idiotype recognition. The first
experimental
support for the validity _of the internal image was presented by Sege and
Peterson in 1978 by using anti-idiotype as a probe to identify cell surface
receptors.
The best information for the exact molecular basis for the mimicking
presently is obtained from the X-ray crystallography of the idiotype-anti-
idiotype
complex. The basis of molecular mimicry of the antibodies can be either local
sequence homology to the original protein as in a reovirus system or, in most
cases, identical conformations from entirely different amino acid sequences as
in
the hemoglobin-myoglobin family of proteins. X-ray crystallography and
sequence
data in the later studies showed that identical, functional conformations can
be
assumed by proteins that differ by as many as 137 of 141 amino acids. The
studies of the crystal structure of idiotope-anti-idiotope complex in the anti-
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2185 5 ~8
lysozyme antibody and the anti-idiotope have demonstrated that a private
idiotope consists of 13 amino-acid residues, most from the complementarily-
determining regions, but including three residues from the third framework
region
of its VL domain. Seven of these residues are common with the paratope of anti-
lysozyme antibody, indicating a significant overlap between idiotope and
antigen-
combining site. Idiotype has been a unique tool in characterization and manipu-
lation of the immune response since it was found and realized: as a clonal
marker to follow B cell development, somatic mutation and fate of clones of B
cells. They have been used as a phenotypic marker for germ-line V genes.
Anti-idiotypic antibodies which bear the internal image of external pathogens
such as virus, bacteria or parasites have been used as surrogate antigens for
vaccine and are being used in treating B cell lymphoma and autoimmune disease
such as encephalomyelitis. In addition, it has been shown that anti-idiotypic
antibodies can induce T-cell responses in which either bytoxic T-cells or T-
helper
cells are produced which recognizes the original antigen.
The provision of a protective vaccine against chlamydial infections of the
eye, genital tract, lung or heart would have worldwide public health benefit.
The
requirements for a successful vaccine candidate against C. trachomatis must
include: (a) immunogenicity after presentation in an clinically safe carrier,
(b)
induction of neutralizing anti-chlamydial antibody, (c) reduction of clinical
and his-
topathologic disease, and (d) absence of chlamydia-specific delayed hyper-
sensitivity. Several chlamydial antigens have been shown to be immunogenic in
patients and animal models. The major outer membrane protein (MOMP) has
been the favored candidate antigen because of its immunogenicity, and the
demonstration of neutralizing anti-MOMP monoclonal antibodies. However,
various formulations of MOMP ranging from membrane extracts to fusion proteins
containing MOMP subunits have been variably immunogenic, but none have
protected against disease in the monkey model of trachoma.
Prior to the present invention, production of neutralizing antibodies by
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using anti-idiotypic idiotype antibody to mimic carbohydrate antigen have been
produced in a bacteria system, Schriver et al, J. of Immunol; 144: 1023, 1990.
Accordingly, it would be desirable to provide a means for preparing a
genus specific oral vaccine capable of providing immunization from chlamydial
infection. It would also be desirable to provide a means for producing such an
oral vaccine in quantity and to provide a process for increasing the
effectiveness
of the vaccine.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is a binding curve of guinea pig antisera to monoclonal GLXA-
Ab, .
Figure 2 is a binding curve of guinea pig anti-monoclonal GLXA-Ab, IgG
antisera to normal mouse IgG before and after immunosorption.
Figure 3 is a curve showing the inhibition of the binding of monoclonal
GLXA-Ab, to GLXA by absorbed guinea pig anti-idiotypic antisera.
Figure 4 is a curve showing fractionation of guinea pig anti-idiotypic IgG.
Figure 5 is a curve showing the inhibition of the binding of monoclonal
GtXA-AB, to GLXA by guinea pig anti-idiotypic isotypes.
Figure 6 is a curve showing the binding of rabbit anti-anti-idiotypic antibody
to guinea pig anti-idiotypic IgG.
Figure 7 is a curve showing the binding of monoclonal GLXA-Ab, to GLXA
by rabbit GLXA-Ab3.
Figure 8 is a curve showing the detection of chlamydial specific ribosomal
RNA from primates.
Figure 9 is a curve showing the effect of GLXA-Ab3 IgG on ocular infection
by clinical disease score.
Figure 10 is a curve showing the inhibition of bending of monoclonal GLXA
Ab,, to GLXA by a hybridoma clone.
Figure 11 is a curve showing the direct binding of chlamydia patient
antiserum to monoclonal GLXA-M Ab2.
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21855~~
Figure 12 is a curve showing that rabbit anti-chlamydia antiserum recog-
nizes monoclonal GLXA-M Ab2.
Figure 13 is a curve showing the protection of mice from chlamydial
infection by immunization with monoclonal GLXA-M Ab2.
Figure 14 is a protection curve by monoclonal GLXA-M Ab2 IgG after a
high dose of ocular infections.
Figure 15 is a curve showing the effect of alum on the protection of mouse
chlamydial infection.
Figures 16A and B show curves of the time course of ocular infectivity
after immunization with monoclonal GLXA-Ab2.
Figure 17 shows a typical timecourse of ocular infection in mice.
Figure 18 illustrates protection wtih anti-idiotypic antibodies against
infection in mice in accordance with this invention.
SUMMARY OF THE INVENTION
The present invention is based upon the discovery that a vaccine, in-
cluding an oral vaccine can be produced with anti-idiotypic antibody
(hereinafter
GLXA-Ab2) which is capable of producing in an animal an anti-anti-idiotypic
(hereinafter GLXA-Ab3) antibody which recognizes GIxA, which is capable of
immunizing an animal against chlamydia and is capable of neutralizing
chlamydia
infection in an animal. In addition, the useful anti-idiotypic antibody for
producing
the vaccine, including an oral vaccine can be either a polyclonal antibody or
a
monoclonal antibody. In addition, it has been discovered that these activities
of
the GLXA- Ab2 surprisingly are obtained despite the fact that its predecessor,
the
idiotypic antibody (hereinafter GLXA-Ab,) from which Ab2 is obtained does not
have any significant activity in either immunizing or neutralizing against
chlamydia infection.
By using the GLXA-Ab2 to mimic carbohydrate of the glycolipid antigen,
the present invention offers an alternative strategy for the conversion of a
thymus-independent antigen into a thymus-dependent immunogen because
14
2185568
idiotype vaccines are proteins. The availability of GLXA-Ab2
which mimics the chlamydial antigen, GLXA, in accordance with
this invention, is of importance: (1) in the elucidation of the
immunological mechanisms associated with the carbohydrate epitope
present on GLXA (2) its function in chlamydial infection such as
the attachment and phagocytosis of the host cells and (3) it
provides an essential tool to identify a GLXA specific receptor
of the host cells. It provides a means for producing a
chlamydial vaccine.
Monoclonal GLXA-Ab2 is an immunogen (i.e., capable of
eliciting an immune response) and binding specifically with the
products of that response whether they be antibodies and/or cell
surface receptors of B cells or T cells. The rapid response to
monoclonal GLXA-Ab2 and the memory demonstrated in the re-
challenge experiment discussed below in Example I and Figs 16A
and 16B indicates that a T cell response has been stimulated.
This means that the T helper cell is responding to an antigenic
determinant (epitope) which differs from the GLXA epitope and is
involved in the stimulation of B cells which produce an antibody
receptor for GLXA-Ab3 that binds GLXA. In addition, the T helper
cell may be of the types which produces cytokines that have been
shown to be involved in protective chlamydia infections. Two of
these are gamma-IFN (gamma interferon) and TNF (tumor necrosis
factor) .
In accordance with this invention, there is provided: (1)
production of polyclonal and monoclonal anti-idiotypic antibodies
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2185568
selected for the internal image of the antigenic epitope on GLXA
(2) characterization of the anti-idiotypic antibodies and (3) a
vaccine including an oral vaccine which provides immunization,
neutralization and protection of chlamydial infection both in
vitro and in vivo and (4) a method for detecting the presence of
chlamydia in a biological sample.
Particular mention is made of an oral vaccine for
administration to an animal in an amount sufficient to effect
immunization against chlamydia which comprises microspheres
having a solid matrix formed of a pharmacologically acceptable
polymer containing a biologically active composition, wherein
said composition is selected from the group consisting of a
monoclonal anti-idiotypic antibody, a Fab fragment of said
monoclonal anti-idiotypic antibody, a (Fab)2 fragment of said
monoclonal anti-idiotypic antibody, and mixtures thereof, wherein
said monoclonal anti-idiotypic antibody, said Fab fragment and
said (Fab)2 fragment induces in an animal anti-anti-idiotypic
antibody which binds an epitope on genus specific chlamydia
glycolipid exoantigen (GLXA).
Mention is also made of use of a biologically active
composition which comprises microspheres having a solid matrix
formed of a pharmacologically acceptable polymer, wherein said
composition is selected from the group consisting of a monoclonal
anti-idiotypic antibody, a Fab fragment of said monoclonal anti-
idiotypic antibody, a (Fab)2 fragment of said monoclonal anti-
idiotypic antibody, and mixtures thereof, and wherein said
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a
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. 2185568
monoclonal anti-idiotypic antibody, said Fab fragment and said
(Fab)2 fragment induces in an animal anti-anti-idiotypic antibody
which binds an epitope on genus specific chlamydia glycolipid
exoantigen (GLXA) in an amount sufficient to effect immunization
against chlamydia infection, for oral immunization of an animal
against chlamydia infection.
DESCRIPTION OF SPECIFIC EMBODIMENTS
In accordance with this invention to produce a vaccine
including an oral vaccine active against chlamydia infection, an
antibody to GLXA is produced in a
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,,
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WO 95/24922 PCT/US95/02459
tPEA/US ~ 9 ocr ~~
first step by any conventional method.
The GLXA is obtained and purified by any presently available method.
Thus GLXA can be isolated on a octylsepharose column and eluted with alcohol
or
isolate on DEAE Sepharose and eluted in low pH aqueous solution or isolated on
a
polyacrylamide bead column and eluted with low pH aqueous solution of KSCN.
(5M). In a preferred method GLXA is obtained and purified from supernatant of
infected cell cultures by presently available methods such as by exclusion
chromatography over a Sepharose 66B-CI column in 0.075 M phosphate, 0.154 M
NaCI, pH 7.2. The GLXA appears in the front fraction of the column and is
detected
by a chemiluminescence assay using an acridinium ester conjugated monoclonal
GLXA-Ab,. The fractions containing the GLXA are usually contaminated by
nucleic
acids, but not protein. The GLXA fractions are pooled, concentrated and
treated
with Rnase and DNAse at pH 8.0 for about 2 hours at 37°C. The mixture
is
rechromatographed over the same column and is now pure. It can be stored at
4°C
(with or without preservative).
The antibody can be polyclonal or monoclonal. In the production of monoclonal
antibody, an idotypic antibody GLXA-Ab, is provided by immunizing an animal,
usually a mouse, with GLXA or the chlamydia bacteria as the antigen. Immune
spleen cells of the animal then are identified, isolated and fused with
lymphoma or
myeloma cells by being contacted with a fusing agent such as polyethylene
glycol
such as by the procedure of Kohler & Milstein, Nature 256: 459, 1975. The
fused
cells then are incubated in a selective medium such as HAT medium which
precludes the growth of unfused malignant cells. The hybridoma cells are
cloned by limiting dilution and supernatants are assayed for secreted
monoclonal antibody of desired specificity. A suitable hybridoma for producing
GLXA-Ab, is deposited in the American Type Culture Collection on March 12,
1993 and identified as ATCC H.B. 11300. Monoclonal antibodies also can
be obtained by ascitic growth of hybridomas in vivo. Alternatively, the
AM~ND~D ~HE~t
2185~~8
WO 95124922 PCTIUS95102459
II'~/~S 1 ~ OCT a99~
lymphocyte cells can be immortalized by exposure to Epstein-Barr virus. The
idiotype antibody, GLXA-Ab, is useful in producing GLXA-Ab2 which,
surprisingly is
active in immunizing against or neutralizing a chlamydia infection. In
additional,
GLXA-Abz is not species specific but is genus specific in that its
immunization and
neutralization activity is useful in many animal species. The monoclonal GLXA-
Ab2
also can be produced by the process utilizing hybridomas set forth above for
GLXA-
Ab, but by utilizing GLXA-Ab, as the antigen. A suitable hybridoma for
producing
GLXA-Abz is deposited in the American Type Culture Collection on March 12,
1993
and identified as ATCC H.B. 11301. GLXA-Ab2 also can be produced as a
polyclonal antibody and, as a polyclonal antibody, it is active for
immunization
against or neutralization of infection by chlamydia. Polyclonal antibodies are
made
in any conventional manner wherein a first animal is immunized with GLXA as
the
antigen and polyclonal GLXA-Ab,, is isolated from the animal's serum. The
polyclonal GLXA-Ab, or monoclonal GLXA-Ab, then is injected into a second
animal
of a different species from the first animal and polyclonal GLXA-Abz then is
recovered from the second animal's serum.
Monoclonal GLXA-Ab2 or polyclonal GLXA-Ab2 are both anti-idiotypic antibodies
which mimic an antigen comprising a genus specific chlamydial glycolipid
exoantigen (GLXA). Both forms of GLXA-Abz are useful as a vaccine including an
oral vaccine for immunizing an animal against genus specific chlamydial
infection.
In additional, both forms of GLXA-AbZ are useful for administration to an
animal
infected with chlamydia in order to neutralize genus specific chlamydia
infection.
Also, in accordance with this invention, Fab or (Fab)2 fragments vaccines
suitable for immunizing against or neutralizing chlamlydial infection can be
prepared
from the Fab or (Fab)2 fragments. The Fab fragments can be prepared from GLXA-
Abz by any conventional means such as by exposing the GLXA-Ab2 to papain or by
exposure to trypsin or chymotrypsin followed by reduction and alkylation.
The vaccine of this invention including the oral vaccine, is prepared in a
17
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form which permits it to pass through the stomach of a patient without
degradation of
the vaccine while permitting the vaccine to be taken up by the M cells in the
Peyer's
patches of the patient: The vaccine comprises microspheres having a diameter
between about 1 and about 100 microns, preferably between about 5 and 20
microns. The microspheres are formed from a physiologically acceptable polymer
degradable in a patient including synthetic polymers such as poly(lactic
acid),
polylactide, poly(glycolic acid), poly(glycolide), poly(lactide-co-glycolide),
poly(caprolactone), polyanhydride, poly(ortho esters), polyphosphates,
polyphosphonates, pholyphosphazenes or the like or naturally occurring
polymers
such as gelatin, collagen, albumin, or other proteins, polysaccharides or the
like.
In accordance with this invention an organic solution of the physiologically
acceptable polymer is formed at a polymer concentration of between about 1 and
about 50 w/v %, preferably between about 20 and 40 w/v % in a physiologically
acceptable volatile solvent such as methylene chloride, acetone, acrylonitrile
or the
like.
A second solution is formed from water either in the presence or absence of a
buffer for the polyclonal or monoclonal GLXA Ab2 or the Fab or (Fab)Zfragment
of the
polyclonal or monoclonal antibody GLXA-Abz. Any suitably buffer can be
utilized so
long as it does not degrade either the GLXA-Ab2 or (Fab)2 fragment thereof and
does
not degrade the polymer forming the microsphere shell. Representative suitable
buffers include, phosphate, borate, bicarbonate or the like. The ionic
strength of the
buffer solution should be less than about 10 mM so that excessive salt is not
present
during solvent evaporation and lyophilization. if excess buffer is present,
both the
GLXA-Ab2 and the microencapsulation process can be diversely affected. thus,
if
present, excess buffer is removed from the antibody solution~by any
conventional
means such as by dialysis.
The organic solution of polymer and the aqueous solution of antibody then is
mixed such as by stirring to form a water in oil emulsion wherein the antibody
is
solubilized in the aqueous phase and the polymer is solubilized in the organic
1$
WO 95124922 PCT/U895/02459
phase.
In order to stabilize the emulsion and to form microspheres of a matrix of
the polymer which contains the antibody and/or the FAB or (Fab)2 fragment
thereof, the emulsion is admixed with water containing a surfactant which
stabilizes the emulsion.
A suitable surfactant is polyvinyl alcohol (PVA) such as those having a
molecular weight between about 10,000 and about 35,000, e.g., about 25,000.
The resultant emulsion then is admixed such as by pouring into a second
aqueous solution of the surfactant such as PVA at a suitable surfactant con-
centration such as between about 0.1 and 0.8 weight %, preferably between
about 0.3 and 0.5 weight %. The resultant mixture is stirred for a suitable
time
such as 2 to 10 hours in order to permit the microspheres to form. The micro-
spheres then are recovered such as by centrifugation and lyophilization. The
recovered microspheres containing the GLXA-Abz antibody or the Fab or (Fab)2
fragment thereof in the polymer matrix then is in a form suitable as a vaccine
including an oral vaccine such as by being mixed with water.
The following examples illustrate the present invention and are not
intended to limit the same.
Example 1
Throughout out this study, two serovars of C. trachomatis were used.
Serovar B (Strain Har 36) organisms originally isolated from conjunctival
scraping
of a child, were grown at 37' C on McCoy cell monolayer with Eagle's Minimum
Essential Medium with Hank's balanced salt solution (HMEM, Whittaker, MD) and
stored at -70° C in the same medium with 10% of dimethyl sulfoxide
(Sigma
Chemical Co., MO). Serovar C (TW-3) was grown in mass tissue culture in
McCoy cells. The elementary bodies were purified by centrifugation through
renograffin and resuspended in phosphate-buffered saline and stored at -
70° C.
Chlamydia free Hartley strain inbred guinea pigs were originally obtained
from Jackson Laboratory (Bar Harbor, ME), bred and maintained in an animal
19
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facility. One year old females were used in this study.
Female New Zealand rabbits free of Pasteurella were obtained
from Mill Brock Farm (Hadly, MA). The rabbits were about 6-8
pounds when received and used within one week. BALB/c ByJ (H-2d)
mice were obtained from Jackson Laboratory (Bar Harbor, ME).
Young adult cynomolgus monkeys (Macacca fasicuiareis) were
obtained from Charles River Primates, Inc. (Port Washington, NY)
which were free of clinical ocular diseases. All the primates
had been used for a previous ocular infection with C. trachomatis
serovar C elementary bodies, some primates had been previously
immunized with a chlamydial protein. The primates challenged at
this study, were free of the disease and were not immune. They
were randominized prior to the initiation of the experiment. All
procedures were performed under anesthesia.
For the production of polyclonal anti-idiotypic antibodies,
each of six guinea pigs was immunized subcutaneously with 150 ug
of monoclonal GLXA-Abl (89MS30) IgG in 1 ml of H20 plus 1 ml
Maalox Plus* suspension (William H. Roger, Inc., PA). Three
weeks later the guinea pigs were boosted with 100 ug of
monoclonal GLXA-Abl in 1 ml of Maalox* (aluminum hydroxide, alum)
and H20, and were boosted on a monthly basis.
For the production of anti-anti-idiotypic antibodies, three
rabbits were immunized with absorbed anti-idiotypic guinea pig
isotype IgGI, 300 ug in 1 ml of alum and 1 ml of H20
intradermally on multiple sites and boosted three weeks later
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using the identical protocol. Subsequently, rabbits were boosted
at monthly intervals. For the production of monoclonal anti-
idiotypic antibody, GLXA-Ab2 (91 MS441), fifty ug of KLH
conjugated monoclonal GLXA-Abl IgG was injected intraperitoneally
into five 9-week old female BALB/cByJ mice in an equal amount of
complete Freund's adjuvant (Sigma, MO). The same rabbit was used
in the subsequent booster at day 14 and later on a weekly basis
for five weeks in the presence of incomplete Freund adjuvant. A
final booster injection was given 3 days before the removal of
the spleen. For the protection of chlamydial infection
experiment, two groups of BALB/c ByJ mice (6 to 20 in each group)
were immunized with 50 ug of monoclonal GLXA-Ab2 IgG or 50 ug of
normal mouse IgG per mouse subcutaneously with or without Maalox
and boosted on weekly basis for three weeks.
Monoclonal GLXA-Abl IgG was isolated by protein A affinity
chromatography. The ascites which contains monoclonal GLXA-Abl
IgG was first passed through glass wool to remove lipid and
centrifuged 2000 rpm for 10 minutes to remove precipitates. The
affinity column (1.5 x 9 cm) was packed with 2 ml Protein A
Sepharose CL4B* (Zymed, CA) was first equilibrated in a binding
buffer (1.5M glycine, 3M NaCl, pH 8.9) Jackson Immuno Research
Laboratories, PA). Two ml ascites diluted with equal amount of
binding buffer was loaded onto the column at 0.6-0.8 ml/minute.
The column was stopped for about 30 minutes to allow binding and
then rinsed with about three bed volume of binding buffer. The
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bound IgG was eluted with 0.1 M citric acid, pH 3, into glass
tubes which contained 50 ul 1 M THS, pH 8.0 to equilibrate the
pH. IgG was detected by an absorption at 280 nm. The absorption
larger than 0.1 was pooled and dialyzed against 0.075 M PHA, pH
7.2 overnight. The purity of the IgG isolated was confirmed by
SDS PAGE on a Phastsystem* (Phamacia, NJ).
Direct enzyme-linked immunosorbant assay
The specific response of guinea pig immunized with
monoclonal Abl IgG per well in coating buffer (0.015 M NaHC03,
0.3M glycine, 0.02 NaN03 with 0.06M polyethylene glycol, PEG),
pH 9.6. The plate was stored at 4°C overnight. Each plate was
rinsed three times with 0.05 Tween 20* in 0.075M PHS and blocked
with 1$ HSA/PBS for 2 hours at room temperature. Again, the
plate was rinsed three times. The pre-immune sera or antisera
from guinea pigs were serially diluted with 0.075M PHS and 100 ul
of each was added to each well in duplicate. The mixture was
incubated at room temperature for 1 hour and rinsed. Goat anti-
guinea pig IgG (H & L) horseradish peroxidase conjugate (Jackson
Immuno Research Labs, Inc. PA) 1:1000 was added, incubated for
one hour and rinsed. IMH substrate (Kirkegaard and Perry
Laboratories, MD) was added after rinsing. The absorbance at 405
nm was determined using a Vmax microwell reader (Molecular
Devices Corp., CA). The antisera from rabbits immunized with
guinea pig IgGI was assayed in the similar manner. Each plate
was coated with guinea pig IgGI and the second antibody was goat
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anti-rabbits (H & L) horseradish peroxidase conjugate (Jackson
Immuno Research Labs. Inc., PA).
Preparation of Affinity Chromatography Column
Normal mouse IgG (Jackson Immuno Research Labs Inc., PA) was
conjugated to Affi-Gel IO* (Bio-Rad Laboratories, CA) as
indicated by the manufacturer. Briefly, 5 ml Affi-Gel IO slurry
was transferred to a glass fitted funnel connected to an
aspirator and washed with three bed volumes of isopropyl alcohol
followed by three beds of ice cold deionized water. Normal mouse
IgG (5 mg/ml), 10 ml was mixed with Affi-Gel IO in a vial. The
coupling was done at 4°C overnight with gentle end-to-end
agitation. A column (0.9 x 5m1) was packed with the coupled gel
and rinsed with 0.075M PBS, pH 7.2. The W absorbance at 280 nm
of the effluent was monitored. The highest absorbance of this
portion was used to test for protein content by the Bradford
Assay (Bio-Rad Laboratories, CA) to evaluate the conjugation.
Absorption of guinea pig antisera with normal mouse IgG
A pool of one guinea pig antisera 3, 4 and 5 weeks after the
immunization was absorbed by affinity chromatography on a normal
mouse IgG-agarose column. The column was prepared as above. The
antisera, about one void volume, was loaded onto the column and
incubated for 30 minutes at 4°C and eluted with 0.075M PBS, pH
7.2. The antiserum was absorbed a second time
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WO 95/24922 218 5 ~ 6 ~ p~T~S95/02459
with a freshly prepared column.
Separation of isotypes of guinea pig IgG
Five ml of guinea pig antisera which had been absorbed by normal mouse
IgG, was loaded onto a protein A conjugated sepharose column, and rinsed with
0.02M phosphate-citrate buffer, pH 7.3. Guinea pig immunoglobulin subclasses
IgGI and IgG2 were eluted separately using a step pH gradient of 4.9 until no
protein was detected in the effluent. The column was then eluted with a low pH
gradient, 4.3. After dialysis against 0.02 M phosphate-citrate buffer, pH 7.3,
each
isotype was subjected to a second round of subclass separation.
C. trachomatis serovar B (Har. 36) elementary bodies were propagated in
a McCoy cell monolayer in a 2 liter roller bottle (Corning, PA) in HMEM with
added L-glutamine (final concentration 0.5 mM), NaHC03 (final concentration
0.4
mM) and 10 % of fetal bovine sera (FBS). Briefly, the medium was removed from
the bottle after confluence of the monolayer. Then, 0.5-3.0 ml of EBs
suspension
(depending on the density of the organisms) in 40 ml of cycloheximide overlay
medium (COM) (Whittacker, MD) with added L-glutamine and FBS was inocu-
lated and the bottle was rolled at 3 rpm at 35° C. Two hours later, 200
ml of
COM was added and rolling continued for 48 to 72 hours. The supernatant was
obtained after centrifugation to remove organisms.
Purification of glycolipid exoantigen, GLXA
GLXA was purified by two steps from the supernatant. First, the super-
natant was passed though an octyl-sepharose CL4B column and eluted with 95%
ethanol (Stuart and MacDonald, Current Microbiology, 11:123, 1984). The
antigen in ethanol (antigen content equivalent to 50 ml heavily infected
tissue
culture supernatant) was concentrated to approximately 50 ul and resuspended
in 0.1 M phosphate buffer, pH 7.5. to I ml. Second, antigen thus prepared was
23
. 2185568
further purified by affinity chromatography. The affinity column
was prepared by conjugating monoclonal GLXA-Abl IgG to Affi-Gel
IO (Bio-Rad Laboratories, CA) as instructed by the manufacturer.
The sample was centrifuged and loaded on to the affinity column
and incubated for 30 minutes at 4°C. The column was rinsed with
0.1 M phosphate buffer until the eluant became clear. GLXA was
eluted in approximate 5M of potassium thiocyanate (KSCN,
Mallinckrodt Inc., KY) for about 15-20 ml and dialysed against
0.075M PBS overnight.
GLXA used in the immuno-dot blot assay of binding of GLXA
and monoclonal GLXA-Ab2 by monoclonal GLXA-Ab3 described below
was isolated by a size exclusion method. Sepharose 6BLC column
(2x100 cm) was equilibrated with 0.075M PBS. The supernatant
from C. trachomatis serovar F cell culture was first centrifuged
5000 rpm to remove cell debris and approximately 20 ml was loaded
onto the column. The flow rate was 0.5 ml/minute. GLXA in the
eluant was detected with Magic Lite Analyzer* (Ciba Corning
Diagnostic Corp., MA). The protocol is as described below.
Chemluminometric immunoassay (Inhibition Assay)
Inhibition of the binding of monoclonal GLXA-Abl IgG to GLXA
by guinea pig GLXA-Ab2 or rabbit GLXA-Ab3 (antisera, IgG, IgG
isotypes) was detected by chemiluminometric immunoassay with a
Magic Lite Analyzer (Ciba Corning Diagnostic Corp., MA). The
protocol for the inhibition was as follows: Affinity purified
GLXA was diluted 1:5 with reagent A (Ciba Corning), mixed and
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aliquoted into plastic tubes (200 ul/tube, Sarstedt, NJ). A
neutralizing solution Reagent B (Ciba Corning), 100 ul was added
to each tube and mixed well. Serial dilutions of guinea pig
GLXA-Ab2 or rabbit GLXA-Ab3 IgG, 50 ul were added to each of the
above tubes and incubated at room temperature. One hour after
the incubation, 100 ul acridium ester-labeled (AE-labeled)
monoclonal GLXA-Abl IgG in 1% BSA/PBS (between 220,000 to 240,000
relative light units, RLU) was added into the mixture and
incubated for one hour. Then, polyclonal anti-chlamydial
antibodies which were covalently bound to paramagnetic particles
(500 ul) was added to each tube and incubated for another hour.
The bound immune complex (paramagnetic particles-GLXA-AE labeled
monoclonal GLXA-Abl IgG) is separated from the unbound by
subjecting the mixture to a magnetic field. The particles were
rinsed 3 times. Bound AE-labeled antibody was measured by the
Magic Lite Analyzer and RLU were recorded. The percentage of
inhibition of binding was calculated as follows:
Inhibition =RLU control-RLU test
RLU control X 100
where control represents PBS and test represents either pre-
immune or antisera (or IgG) added respectively.
Immuno-dot blot Assay
A Bio-rad dot blot apparatus was used for this assay.
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Polyvinylidene fluoride (PVDF) membrane, 0.45 um, Immobilon-NC*
(Millipore, MA) was coated with the affinity-purified GLXA or
chlamydial rLPS for 18 hours at 4°C. The membrane was then
washed with 0.05 Tween 20 in 0.075M PBS buffer, pH 7.2 and
blocked for 2 hours at room temperature with 3~ BSA/PBS. After
rinsing, serial dilutions of rabbit GLXA-Ab3 IgG (90MS699)
(isolated by protein A affinity chromatography, method see above)
or monoclonal Abl IgG (89MS30) were added (100 ul per well),
incubated at room temperature for 2 hours, and washed to remove
unbound IgG. The PVDF sheet was removed from the apparatus and
blocked with 3 ~ BSA/PHS for two hours with gentle agitation,
then probed with 1:1000 dilution of peroxidase conjugated goat
anti-rabbit IgG (H and L) or rabbit anti-mouse IgG (H and L)
(Jackson Immuno Research Labs., PA) for 1 hour at room
temperature. Unbound conjugated antibody was removed by washing
in a petri dish for 4 times, 5 minutes with agitation. The
binding was detected by 4 CN (4-chloro-I-naphthol) membrane
peroxidase substrate system
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(Kirkegaard & Perry Laboratories Inc., MD). Assays were carried out in
duplicate.
Biotinylation of IgG
Monoclonal GLXA-Ab, or rabbit GLXA-Ab3 IgGs were labeled with biotin.
Briefly, the GLXA-IgG was first dialysed against 0.1 M sodium borate, pH 8.8
for
4 hours at 4° C. Then, 200 ug of biotinamidocaproate N-
hydroxysuccinimide ester
(Sigma Chemical Co., MO) per mg of IgG was added, and incubated at room
temperature for 4 hours. Finally, 20 ul of 1 M NH4C1 per 250 ul of ester was
used
and incubated for 10 minutes at room temperature to stop the reaction. Labeled
IgGs were dialysed against 0.075M PBS for 3 days at 4% C.
Immunofluorescence staining of infected McCoy cells
Confluent McCoy cell monolayers were grown on the cover slips in a 24
well plate (Falcon, NJ) for 24 hours. The medium was taken out from each well
before 200 ul of C. trachomatis serovar B elementary bodies (Har 36) or 200 ul
of HMEM alone were applied to each well. The inclusions forming units (IFU)
was
approximately 1000/200 ul. Immediately after the application, 200 ul of
cycloheximide overlay medium (COM) with L-glutamine and FBS was added to
each well. The mixture was incubated at 37° C for two hours and the
medium
was replaced by 1 ml COM per well. The plates were then cultured for 48 hours
at 37° C in a humidified incubator with 5% C02.
After the incubation, cell cover slips were washed with 0.075M PBS twice,
minutes each. The coverslips were then blotted with tissue carefully to remove
buffer on the cover slips. Then 400 ul of 50 ug/ml biotin-labeled monoclonal
GLXA-Ab, or 400 ul of 1.20 mglml of biotin-labeled GLXA-Ab 3 was added to the
cover slips and incubated at 37° C for 1 hour. The cover slips were
rinsed twice
with PBS (five minutes each rinse). Four hundred ul of fluorescein-
streptavidin
26
WO 95!24922 PCT/US95/02459
1:50 in 1 % of BSA/PBS was added and incubated for 1 hour. The unbounded
fluorescein-streptavidin was rinsed out as above. Fluorescence was detected by
photograph with an Olympus 25, 35mm camera (ASA 400 TMAX black and white
KodaK film) mounted on a Zeiss A.7082 Oberkichen microscope, illuminated with
a 12v/IOOz halogen lamp.
Neutralization of Chlamydia by GLXA-Ab3 (90MS699 ) in vivo
Inoculums and specimens
The rabbit pre-immune and GLXA-Ab 3 IgG, normal mouse and monoclonal
GtJCA-Ab, IgG were filter sterilized. The IgGs (0.2 mg/ml) were mixed (1:1)
respectively with C. trachomatis serovar C (TW 3) elementary bodies (2000
inclusion-forming units120u1). The mixtures and non-treated EBs (as a control)
were incubated at 37' C for 45 minutes, with gentle shaking every 15 minutes.
After the incubation, approximately 2 ug IgG plus 1000 IFU in 20 ul was
inoculated onto inferior fornix of each eye of eight monkeys, four with GLXA-
Ab3
IgG plus EBs, two with pre-immune IgG plus EBs and two with EBs only. At the
same time, 100 ul of each type of mixture was used to infect 10 wells of 2-day-
old McCoy cell monolayer coverslip in a 48 well tissue culture plate for
determination of the infectivity of the inoculum. Culture method are described
below.
Conjunctiva! swabs were taken by sweeping the interior tarsus and fornix,
the lateral fornix, the superior tarsus and fornix, and the medial fornix on
the day
prior to inoculation, and on days 2, 6, 9, 13, and 20 after the inoculation.
The
swabs were immediately immersed in 2 ml collection medium and disrupted by
vortexing for 2 minutes in the collection medium.
Determination of chlamydial infectivity by cell culture
27
2185568
Conjunctival swabs were first disrupted by vortexing for two
minutes in the collection medium to collect EBs from the swabs.
Then 200 ul of this collection medium was inoculated onto McCoy
cell monolayer cover slips. The cells in a 48 well tissue
culture plate were grown for two days, 5 wells for each sample.
For an in vitro neutralization experiment, 100 ul of the mixture
was inoculated onto the monolayer cover slips of 10 wells for
each sample. The inoculated plates were then centrifuged at
1,000 x g for 1 hour at room temperature. The plates were
incubated at 37°C for 2 hours and the medium was replaced by 1 ml
of 10 % FBS in COM (Whittaker, MD). The plates were cultured at
37°C in a humidified incubator with 5% C02 for 48 hours. The
culture medium was aspirated from each well after the incubation.
The monolayer coverslips were washed once with 0.075M PBS, fixed
with ethanol for 5 minutes and then rinsed with H20 twice. The
fluorescein-labeled rabbit anti-chlamydial monoclonal antibody
with Evans blue counterstain (Syvo, Co., CA.) 25 ul per well was
added and incubated for 30 minutes at 37°C. After the
incubation, the wells were rinsed with H20 twice and a cover slip
was mounted to each well with mounting fluid. The inclusion
bodies were counted by inverting the plate on a fluorescence
microscope.
Direct Florescent antibody-stained cytology (DFA)
The conjunctival swab (scraping) from each eye of infected
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primate was pressed on alcohol-precleaned glass slide. The
slides were air dried and fixed with cold acetone for 5 minutes.
Slides were then dried and 30 ul of fluorescein-labeled
monoclonal antibody reagent with Evans blue counterstain (Micro
Trak* Chlamydia Direct Reagent, Syvo Co. CA) were overlaid. The
incubation was carried out in a covered, moist chamber. After 15
minutes, slides were turned on edge to remove excess stain and
rinsed with deionized water for 10 seconds. Slides were allowed
to completely air dry. A cover slip was placed on each slide
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with mounting medium (Syva Co., CA) and sealed with fingernail polish. Slides
were examined under a fluorescence microscope at 500x and 1250x. The
infectious titer was scored on a semi-quantitative scale 0-4+: 0 is negative
0.5 is
negative on first passage and positive to any degree of second passage, 1 is
for
1 to 9 inclusions per well on first passage and so on.
Clinical examination and scoring
The clinical response of each eye was graded as ten clinical signs (Taylor
et al, Invest. Opthalmol. mss. Sci. 29:847, 1988): the follicular response in
the
lumbar, timbal, superior tarsal, superior fornix, and inferior fornix portion
of the
conjunctiva hyperemia or injection of the bulbar, superior tarsal, superior
fornix,
and inferior fornix conjunctiva and ocular discharge. The clinical response
was
graded on a scale of 0 to 3 for each of 10 signs of conjunctiva) inflammation
to
obtain a total inflammatory score for each monkey. The examiner was unaware
about the allocation of the monkeys. The means of the scores were used to
describe the response of a group of monkeys.
Analysis of Conjunctiva) Swabs via RNA-directed Hybridization
Total RNA was prepared from conjunctiva) swab samples taken from
GLXA-Ab3 or normal rabbit IgG treated monkeys on the day before the challenge
and on day 2, 6, 9, 12, and 20 after the challenge. The swab samples were
first
homogenized in phenol at 65% in the buffer containing 50 mM Tris (pH 8.0), 150
mM NaCI, 10 mM ethylenediaminetetraacetic acid (EDTA), 1 % SDS. RNA was
precipitated and redissolved in the same buffer. This preparation of RNA was
extracted several times with phenol:chloroform (3:1 ), resuspended and treated
extensively with DNase I. The quality of RNA preparation was monitored by
ethidium bromide-UV visualization after separation on formaldehyde-agarose
gels.
29
WO 95124922
218 5 5 ~ 8 p~~s95/02459 °-°
The probe used was a DNA fragment containing the 16S and 23S
ribosomal RNA and flanking sequences which was excised from the chlamydial
genomic plasmid clone pL2, 434Sc1-IA (Cheema et al, The Amer. J. Med. Sci.
302:261-268, 1991). The DNA fragment was labelled by nick translation using
32PdCTP, 800 Ci/m mole (Amersham Corp., IL). The specific activity for all
restriction fragment probes was about 106 cpm/ug DNA. Slots on each blot
included 1 ug of monkey or human-derived total RNA, 10 pg pure C. trachomatis
or C serovar RNA (positive control), 3 ug yeast or rat RNA, buffer alone and 1
ug
RNA from swabs of each monkey taken prior to infection (negative control). RNA
was fixed to 0.22 um filter (Schleicher and Schuell Corp., NH). The
hybridization
results were visualized via autoradiography at -70' C using X-GMAT AR film
(Kodak, New York).
Production and Characterization of Monoclonal Anti-idiotypic Antibodies
(monoclonal GLXA-Abz)
Conjugation of keyhole limpet hemocyanin with monoclonal GLXA
Monoclonal Ab, (89MS30) IgG was isolated from a protein A chromatog-
raphy column as set forth above. The keyhole limpet hemocyanin conjugation
was carried out by using glutaraldehyde. Briefly, 1.5 mg of IgG was mixed with
0.05 mg KLH, (Sigma Chemical Co., MO) (approximately 1 molar of IgG per 50
amino acid of KLH) in equal volume of 0.2 % of glutaraldehyde in PBS, in-
cubated at room temperature with gentle stirring. One hour later, 1 M of
glycine
was added to make a final concentration of 0.02M and incubated for another
hour at room temperature. The conjugate was then dialysed against PBS
overnight.
Anti-monoclonal Ab, hybridoma production
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WO 95/24922 PGT/US95/02459
Four days after the last boost (see immunization), spleen cells were isolated
from
two mice which had the highest titer. Fusion was made Hfith mouse myeloma cell
line Sp2/0-Agl4 according to the techniques initially developed by Kohler and
Milstein (Nature, 1975) and modified (Goldsby, A Practical Guide to Making
Hybridomas In Nucleic Acid & Monoclonal Antibody Probes, Swaminathn &
Prokask, Eds. Deller. N.Y. pg. 367, 1989). Feeder cell layer were from spleens
of
mice 8 weeks old.
Screening of Monoclonal anti-idiotypic antibody, 91 MS441 (monoclonal GLXA
Ab2)
Anti-idiotypic antisera from immunized mice and supernatants from cloned
wells were detected by a sandwich ELISA (Uytdehaag et al, J. Immunol, 134:
1225, 1985, Hiroshima et aI,J. Immunol. 144: 224, 1990). Briefly, polystyrene
Immulon II microtiter plates (Dynatech Laboratories Inc., VA) were coated with
100 ul of 1 ug monoclonal Ab, IgG in 0.1 M carbonate buffer, pH 8.9 overnight
at
4° C. The unbound IgG was removed and the wells were blocked by 3%
BSA/PBS for 1 hour at 37° C. After washing, serial diluted antisera or
100 ul of
culture supernatant from wells with hybridoma cells was added. After 1 hour
incubation at 37° C and washing, 1 ug of biotin conjugated monoclonal
Ab, IgG
was added to each well and the reactivity was detected by the addition of
streptavidin-horseradish peroxidase (Jackson immuno Research Labs.,PA). One
hour later, TMB peroxidase substrate (Kirkergaard & Perry Laboratories inc.,
MD)
was added. The absorbance was read at 405 nm in a microplate reader. As
positive and negative controls, immune sera taken before the fusion (1:10
dilution) and medium alone were used.
Inhibition of binding Assay
The inhibition of the binding of monoclonal GLXA-Ab, to GLXA by mouse
antiserum and the supernatant of the clones was determined by immuno-chemi-
31
2185568
luminometric assay as described above.
Generation of ascites
Ten BALB 3/cByJ mice, age not strictly required, were
injected with Pristane* (2,6,10,14-tetramethylpentadecane, Sigma
Chemical Co., MO), 1 ml per mouse intraperitoneally. Ten days
later, the mice were injected with approximately 2 x 108
monoclonal GLXA-Ab2(91 MS441) producing hybridoma cells. The
ascites was harvested by using a Vacutainer* 20 g blood
collection needle (Becton Dickinson, NJ.) about 10 minutes and
stored at -20°C until use.
Isotyping of anti-idiotypic IgG
The isotyping was carried out by ELISA. The supernatant
from clone 91 MS441, (100 ul) was coated in each well and
incubated at 4°C overnight. After rinsing and blocking with 3°s
BSA/PBS for 2 hours, 100 ul of rabbit antiserum specific to mouse
subclass; IgGI, IgG2a, IgG2b, IgG3, IgM, IgA, K chain or ~ chain
(Bio-Rad Laboratories, CA) was added to each well in duplicate.
The plate was incubated at room temperature for one hour. The
rabbit antiserum was detected by horseradish peroxidase
conjugated goat anti-rabbit (H and L) and TMB substrate
(Kirkegaard & Perry Laboratories Inc. MD.)
Purification of monoclonal GLXA-Ab2 IgG was purified by affinity
chromatography on a protein-G-sepharose column. Protein G-
* Trade-mark
- 32 -
71915-4
2185568
Sepharose 4B (Zymed, CA.) 5 ml was packed in a 0.5 x 9 cm column.
The ascites (2 ml) was diluted 1:1 with 0.02 M phosphate buffer
pH 7.3 and loaded on the column and washed with the same buffer
until no protein was detected. The bound IgG was eluted with 0.1
M citric-glycine buffer, pH 2.6. The eluent was collected in 1
ml fraction which contained 50 ul of 1 M tris-saline buffer, pH
8.0 to balance the eluent. Fractions having UV
- 32a -
71915-4
WO 95/24922 218 5 5 J ~ PCT/US95/02459
absorption above 0.1 were pooled and dialysed in PBS overnight at 4° C.
The
purified IgG was identified by SDS-PAGE for confirmation.
Immunoprecipitation of monoclonal GLXA-Ab2 by anti-chlamydial antisera from
other species
Polyclonal antibodies from a human patient diagnosed with a chlamydial
infection and chlamydial EBs injected rabbits were tested for the recognition
of
monoclonal GLXA-Ab2 by ELISA. The procedure was essentially the same as
described above with the following exceptions. Briefly, the ELISA plate was
coated with 1 ug of monoclonal GLXA-Ab2 per well in 0.075M PBS without
coating buffer overnight at 4° C. The wells were rinsed with 0.05%
Tween 20 in
0.075M PBS and blocked with 3% BSA/PBS for 2 hours at room temperature.
Serial dilution of the patient's serum (92MS273), control human serum
(88MS356), rabbit antisera (88MS188) or control rabbit serum (92MS450) 100 ul
was added to each well in duplicate. After 1 hour incubation at room tempera-
ture, the walls were washed 3 times and 100 ul of peroxidase conjugated goat
anti-human or goat anti-rabbit IgG (Jackson ImmunoResearch Labs, MD) was
added. TMB substrate was added after one hour incubation. The plate was read
on a Vmax microplate reader (Molecular Devices Corp., CA).
Protection from Chlamydia Infection by Immunization of Monoclonal GLXA-Abz in
a Mouse Infection Model
Immuno-dot blot assay of binding of GLXA and GLXA Ab3 raised by monoclonal
GLXA-Ab2
Immuno-dot blot assay was done by the same method as described
above. The exception is that a PVDF sheet was coated with purified GLXA (100
ul) per lane in 0.075M PBS. Antisera taken from the mice which were immunized
with monoclonal GLXA-Abz IgG or normal mouse IgG were serially diluted with
33
WO 95124922 218 5 J J ~ PCT/US95/02459
3% BSA/PBS. The second antibody was horseradish peroxidase conjugated
rabbit anti-mouse IgG (H and L). The photograph was taken by Kodak TMAX
100. The staining intensity of dot blot was scanned by a densitometer.
Inoculation and specimens
C. trachomatis serovar C (TW-3) elementary bodies 5000/20u1 were
inoculated onto each eye of the mice which were immunized with monoclonal Ab2
or normal mouse IgG. On the day before the inoculation and on day 7, 10, 14,
21, 28 and 35 after the inoculation, conjunctiva were swabbed from each eye.
The area included the inferior tarsus and fornix, the lateral fornix, the
superior
tarsus and fornix, and the medial fornix. The conjunctiva) swabs were
immediate-
ly immersed in the collection medium and disrupted for two minutes by vortex
and kept on ice until culturing.
Identification of Receptor on Host Cells by Monoclonal GLXA-Ab 2 IgG
FACS analysis of the specific binding of monoclonal GLXA-Ab2 to HECEC cells
Human endometrial gland epithelial cells (HECEC) were grown in a 75
mm2 flask at 37° C with 5% C02. When confluent, cells were scraped off
the
flask using a cell scraper (Baxter, IL) and centrifuged 200 x g for 5 minutes.
The
cells were rinsed once with 20 % FBS in Hanks buffer (Whittker, MD) and
passed through a 19 G syringe needle four times to obtain single cells. Serial
dilutions of biotin labeled monoclonal GLXA-Ab2 or biotin labeled normal mouse
IgG in Hanks buffer, (100 ul) were added to each vial which contained
approximately 1.5 x 106 HECEC cells and incubated on ice for 30 minutes. Each
vial was rinsed twice with 0.02 % azide in Hanks buffer. Later, 100 ul of FITC
conjugated streptavidin was added and incubated for 30 minutes on ice. After
washing twice, the cells were kept in 400 ul of sheath buffer on ice. Cells
plus
FITC conjugated streptavidin and cells alone were used as background control.
34
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WO 95124922 PCT/US95/02459
Single color flow cytometry was performed immediately by FACS scan (Becton
Dickinson).
Detection and Characterization of Polyclonal Anti-idiotypic Antibodies
Generation of anti-idiotypic antibodies against monoclonal GLXA-Ab, in guinea
pigs
The immunogen which was used to produce the anti-idiotypic antibodies in
guinea pigs was a monoclonal antibody identified as 89MS30 (monoclonal GLXA-
Ab,). It was originally produced by immunization of BALB/cByJ mice with
chlamydial
elementary bodies propagated in embryotic egg. Mice spleenocytes were fused
with
Sp2/0-A14 myeloma cells and the clone was screened. The clone (89MS30)
reacted to all 15 serovars of C. trachomatis. C. pneumoniae. and C. ~~ sittaci
6BC and
mouse meningopneumonitis by EIA, demonstrating recognition of a genus specific
antigen. The IgG was isotyped as IgG2Bb by ELISA using rabbit anti-mouse
antiserum (Bio-Rad Laboratories, CA). The monoclonal GLXA-Ab IgG was isolated
from the ascites with rec-protein A sepharose 48 conjugate column (ZYMED, CA).
Inbred guinea pigs (Hartley 13) were immunized and boosted with monoclonal
GLXA-Ab, IgG, 150 ug each in the presence of Maalox, as an adjuvant. Pre-
immune sera and antisera were obtained by heart puncture and centrifugation.
Five
immunized guinea pigs demonstrated strong immune responses against monoclonal
Ab, IgG by ELISA. It was demonstrated that the anti-monoclonal GLXA-Ab IgG
titer
was more than 1 to 20,000 one week after the first boost by ELISA for anti-
idiotype.
Pre-immune sera (~); antisera 3 weeks (~) after the immunization; 1 week (~)
and 2
weeks after the boost (~). Each value represents the mean of the duplicated
determinations. These guinea pig antisera kept increasing two weeks after the
boost as shown in Figure 1.
Complete absorption of guinea ig antisera with normal mouse IgG.
In order to remove guinea pig anti-mouse antibodies that are directed against
epitopes other than the idiotopes present on the hypervariable regions of
RECTIFIED SHEET (RULE 91)
WO 95/24922 j PCT/US95/02459
the monoclonal Ab, IgG molecules, the guinea pig antisera was absorbed with
normal mouse IgG. The absorption was carried out by affinity chromatography.
The
column was made by conjugating normal mouse IgG to Affi-Gel 10. The
conjugation
was evaluated by detecting the amount of unbound protein at absorbance at 280
nm. The highest optical density from a fraction was 0.23, containing 0.18 mg
of
protein by the Bradford Assay (Bio-Rad Laboratories, CA). The total mouse IgG
used in the conjugation was 50 mg" demonstrating that the conjugation was
successful. The guinea pig antisera were loaded onto the column and eluted
with
0.075M PBS, pH 7.2. this procedure was repeated using a newly prepared column.
The antisera before and after the absorption was tested and compared with pre-
immune sera for the reactivity to normal mouse IgG by ELISA. The concentration
of
each antiserum for the assay was equilibrated. Each well was coated with 1 ug
of
normal mouse IgG. Serial dilutions of pre-absorbed (~); pre-immuned (~) and
absorbed guinea pig natisera (~) were added. PBS was background control (O).
Horse radish peroxidase conjugated goat anti-guinea pig IgG (H&L) was used as
the
second antibody. Each value represents the mean of duplicate determinations.
The
result showed that sera after the absorption had the same reactivity against
normal
mouse IgG as pre-immune sera (Fig. 2). The unabsorbed antisera had 5 times
more
reactivity, indicating that all antibodies specific to epitopes other than
idiotypes have
been completely removed.
Inhibition of the binding of monoclonal GLXA-Ab, to GLXA by guinea pig
antisera
If guinea pig antisera contain the specific anti-idiotypic antibody against
monoclonal GLXA-Ab, IgG molecules, the direct effect is that it would inhibit
the
binding of antigen, GLXA, to the monoclonal antibody, monoclonal GLXA-Ab,. In
other words, the antisera bind to the complementary-determining region of
monoclonal GLXA-Ab, IgG, thus preventing GLXA from binding to the active site.
To
test this, competition of
RECTIFIED SHEEP (RULE 9'll
WO 95!24922 218 5 ~ 6 ~ pCT~S95/02459
binding was performed by chemiluminometric immunoassay. Serial dilutions of
guinea
pig pre-immune sera, unabsorbed antisera, or absorbed antisera were incubated
with GLXA which was isolated by immunoaffinity chromatography. After one hour
at
room temperature, the mixtures were incubated with monoclonal GL.XA-Ab, IgG
conjugated with acridium ester for
RECTIFIED SHEET (RIFLE 91)
2185568
an additional hour. Solid phase paramagnetic particles were added and a RLU
was
determined. The inhibiting percentage is calculated as mentioned in Material
and
Method. Guinea pig anti-idiotypic antisera exhaustively absorbed with mouse
IgG
(~) inhibits the binding of mAb, to GLXA at approximately the same dilutions
as
unabsorbed guinea pig antisera (O) by chemiluminometric immunoassay. Pre-
immune (O); mAb, (~); and ZX PBS (~) were included as controls. Percent
inhibition
in the binding was calculated as described in the text. As shown in Figure 3,
when
the guinea pig antisera was diluted 1:40, 95% of the binding of monoclonal Ab,
to
GLXA was inhibited by both unabsorbed and absorbed antisera, compared with 15
for pre-immune serum. In addition, the unabsorbed antisera had nearly
identical
inhibition as absorbed antisera at equivalent protein concentration,
indicating that no
anti-idiotypic antibodies were removed by absorption. Therefore, a competitive
anti-
idiotypic antibody had been generated in guinea pigs.
Profile of subclasses of guinea pig anti-monoclonal GLXA-Ab, IgGs
Previous experiments had shown that different isotypes of guinea pig
antiidiotype exert different effects on the idiotype production. IgGI isotype
enhances
the idiotype production, whereas IgG2 isotype inhibits the idiotype clone. To
test the
inhibition of different isotypes of guinea pig anti-idiotypic IgG, subclasses
of IgG
were isolated from absorbed guinea pig anti-idiotypic antisera. A step pH
gradient of
phosphate-citrate buffer was used. IgG, and IgG2 were separated and isolated
on a
protein A affinity column. The elution profile of the isotypes, IgG, and IgG2
is shown
in Figure 4. The purification of IgG was accomplished by protein A affinity
chromatography. Separation of the guinea pig IgG, and IgG2 was carried out by
a
step pH gradient of phosphate-citrate buffer. Each peak was identified by
immunoelectrophoresis (IEP) with goat anti-guinea pig IgG to immuno-
precipitate
bands (Bethyl, TX). The first peak which is composed of IgG, was precipted by
both
goat anti-guinea pig IgGI and lgG2, but formed a single band by goat anti-
guinea pig
antisera. The first peak was then repurified by the same method.
2185568
Inhibition of the binding by subclasses of guinea pig IgG
The competition by different subclasses of IgG was carried out again by
chemiluminometric immunoassay using guinea pig anti-idiotypic antibodies of
either
IgG, or IgG2 isotypes. Figure 5 shows that 0.4 ug IgG, was able to inhibit the
binding
of monoclonal GLXA-Ab, to GLXA by 50 %. As shown in Figure 5 Inhibition of the
binding of mAb, to GLXA by guinea pig anti-idiotypic isotypes. The binding of
mAb,
to GLXA was inhibited by guinea pig anti-idiotypic IgG, (~) and partially by
IgG2 (~) in
chemiluminometric immunoassay. Homologous unconjugated mAb, (o) or PBS (O)
were used as controls. Percent inhibition in the binding was calculated as
described
above. The total inhibition
37A
WO 95124922 218 5 5 ~ ~ p~/Ug95/02459
occurred when 100 ug was used. This was essentially the same concentration
needed when unlabeled monoclonal GLXA-Ab, IgG was used. IgG2 showed little,
if any, inhibition, approximately 25 % of the binding of monoclonal GLXA-Ab,
to
GLXA with L0 ug. These data demonstrate that the IgG, subclass of anti-
idiotypic
IgG was at least 5 times more inhibitory than the IgG2 subclass.
Generation of Anti-anti-idiotypic Antibodies (GLXA-Ab3) in Rabbits by Guinea
Pig
IgG1
The evidence obtained showed that guinea pig anti-idiotypic IgGI was
against the hypervariable region of monoclonal GLXA-Ab, IgG and also inhibited
its binding to GLXA. The final criteria of an internal image of the anti-
idiotypic
antibodies is to confirm structurally that the Ab2 is Ab2 , not Ab2 or Ab2 .
Since
Ab2 binds to the framework portion of immunoglobulins, it can also inhibit the
binding of Ab, to the cognate antigen. To confirm that guinea pig anti-
idiotypic
IgGI is Ab2, the isotype IgG1 was used to produce an anti-anti-idiotypic
antibody
(GLXA-Ab3) which can recognize the GLXA epitope in an animal which has never
been exposed to GLXA antigen, (Ertl et al,Proc. Natl. Acad. Sci. USA 81:2850,
1988). Three New Zealand white rabbits were immunized with guinea pig anti-
idiotypic IgGI in the presence of adjuvant, Maalox (alum). The antisera from
one
rabbit (S2) were tested against IgGI by ELISA (Fig. 6). The titer increased
with
time after the immunization. The titer was much higher than 20,000 compared
with pre-immune sera three weeks after the boost.
Anti-anti-idiotypic antibodies from rabbits recognize GLXA
The dot blot apparatus (Bio-Rad Laboratories, CA) was used to detect the
reactivity of rabbit antisera to GLXA. Antisera from two rabbits were tested
by
immuno-dot blot assay. Since monoclonal GLXA-Ab, is cross-reactive to
chlamydial LPS, polyvinylidene fluoride (PVDF) membrane was coated with both
38
WO 95124922
21 ~ 5 ~ ~ ~ p~~s95/02459
GLXA and chlamydial rLPS. Both rabbits antisera recognized GLXA and LPS
with high reactivity. When IgG from rabbit (S2) (90MS699) was used, the dots
were positive at a concentration of 0.006 ug per lane. Pre-immune IgG did not
react. The IgG isolated from this antisera was tested for its capacity of
inhibiting
the binding of monoclonal GLXA-Ab, to GLXA, since GLXA-Ab3 produced by the
internal image, GLXA-Ab2 should exhibit a similar binding. This is confirmed
as
shown in Figure 7. The inhibition by GLXA-Ab3 increases with concentration,
but
about five times less inhibitory compared to unlabeled monoclonal Abp. This
demonstrates that the guinea pig anti-idiotypic antibody IgGI is the internal
image
of antigen, GLXA. GLXA-Ab3 IgG was further tested for the recognition of
elementary bodies of C. trachomatis in vitro.
Monoclonal GLXA-Ab, and GLXA-Ab3 IgG were conjugated with biotin by
glutaldehyde. Labeled monoclonal GLXA-Ab, or GLXA-Ab3 was incubated with
McCoy cell monolayer on coverslips which were infected with C. trachomatis
serovar B (Har 36) elementary bodies for 48 hours. Non-infected monolayers
were used as control. The florescence staining pattern of the elementary
bodies
by monoclonal GLXA-Ab, and polyclonal GLXA-Ab 3 IgGs demonstrated that
GLXA-Ab3 not only recognized the purified form, but also native form of GLXA.
Neutralization of the Chlamydial Infection in Primates by GLXA-Ab3 IgG
(90MS699)
The next question concerned the ability of monoclonal GLXA-Ab, or
GLXA-Ab3 to neutralize chlamydia elementary bodies and thus protect host cells
from infection. The experimental approaches utilized to answer this question
involved neutralization of infection in cultured cells and neutralization of
the
infection in primate conjunctiva.
39
WO 95124922 J PCT/U895/02459
In vivo GIJCA-Ab3 neutralizes chlamydial infection whereas monoclonal GLXA-Ab,
does not.
Neutralization in primate conjunctivae was determined by preincubating
organisms with antibody IgG and detection of the effect on ocular infection.
The
IgG fraction of monoclonal GLXA-Ab, or Ab3 was incubated with elementary
bodies of C. trachomatis serovar C. Normal mouse or rabbit pre-immune IgG as
well as no immunoglobulin added served as controls. The mixture was inoculated
to each eye of the primate. On the day before the and after the inoculation,
con-
junctiva) swabs were taken by sweeping different areas of the conjunctivae
(see
above). Ocular chlamydial infection in primates was determined by cell culture
assay which included second-passage on days post-infection. As shown in Table
1, three primates were infected with monoclonal GLXA-Ab, treated EBs on the
left eyes, GI~CA-Ab3 treated EBs on the right. At the same time, two primates
were infected with normal mouse tgG treated EBs (shown as NI ) or EBs alone
on the left and right eyes alternatively. The same method applied to pre-
immune
rabbit IgG (shown as N3). All eyes inoculated with monoclonal GLXA-Ab, treated
EBs were positive at least once (primate No. 515, 84, and 26). However, only
one eye of the GLXA- Ab3 treated eye was positive once, at day 10 post-infec-
tion, two of them were never positive (primate No. 515, 84 and 26). Eyes
inoculated with normal mouse or pre-immune rabbit IgG treated EBs were all
positive at feast once (primate No. 563, 20, 17 and 329). The untreated EBs
(shown as C) produced infection in two of the four eyes involved (primate No.
563, 20, 17 and 329). Although the data points are diminutive, they do show
that
monoclonal GLXA-Ab, does not neutralize chlamydia in primate conjunctiva. On
the other hand, it suggests that GLXA-Ab3 is neutralizing. In order to confirm
that
GLXA-Ab3 does neutralize, a number of tests were carried out, including: (A,)
neutralization in cell culture (B), neutralization in primate conjunctiva (C),
detec-
tion by clinical culture assay (D), detection by direct fluorescence antibody
cytology (E), chlamydia specific RNA probe hybridization and (F) determination
of
WO 95124922 2 i 8 5 J ~ ~ pCT/LTS95/02459
the severity of ocular infection by clinical scoring.
Table 1.
Neutralization of chlamydial infection in primate conjunctivae
by Ab3 but not mAb,
Primate Eye Aba Day Following Infection
0 3 7 10 14
515 L mAb, - - + + -
R Ab3 _ _ _ _ _
563 L C - - -
R N, _ - _ + _
84 L mAb, - - - + +
R Ab3 - - - + -
20 L N3 - - - + -
R C - - - - -
26 L mAb, - - + - -
R Ab3 _ _ _ _ _
17 L N, - - - + +
R C - - - + +
329 L C - - + - -
R N3 - - - + +
aEBs treated with normal mouse IgG(N1), pre-immune rabbit IgG (N3) or EBs
alone (C) were controls.
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GLXA-Ab3 neutralizes the chlamydial infection in vitro.
In vitro, cell culture assay was carried out with GLiCA-Ab3 and pre-immune
rabbit antibody. The pre-immune rabbit and GLXA-Ab 3 (90MS699) IgGs were
isolated by protein A affinity chromatography. The mixture of 10 ug of each
IgG
and 100 ul serovar C EBs (1000 IFU/ml) or EBs alone were inoculated onto wells
containing McCoy cell monolayers. Ten wells per sample were used. Inclusion
bodies were detected by FITC-conjugated monoclonal anti-chlamydia antibody 48
hours after the incubation. IFU/ml was based on 15 fields per well. As is
shown
in Table 2, Ab3 IgG reduced the infectivity 3 times higher compared to pre-
immune IgG, 5 times higher compared to EBs alone, indicating neutralization by
G LXA-Ab3.
Table 2.
In vitro neutralization of chlamydial infection by Ab3 IgG
E$s trPatPrt with Mpan I I/ 5 m ric +
Ab3 34.1 + 7.2
Normal IgG 93.8 + 22.4
None 155.3 + 5.5
GLXA-Ab3 neutralizes the chlamydial infection in primates
Eight primates were randomly divided into three groups in this experiment. In
the
eyes, four primates received with purified EBs previously incubated with GLXA-
Ab3
IgG (eight eyes), two received EBs previously incubated with pre-immune IgG
(four
eyes) and two received untreated EBs (four eyes). On the examining day, the
con-
junctiva) swabs were taken and cell cultured. Since there is no significant
differ-
ences between the recipients of pre-immune IgG and EBs alone, the results are
presented as 8 experimental eyes and 8 control eyes. Cell culture results are
expressed as IFU/ml based on counting inclusions in 15 fields for two wells
per
sample. As shown in Table 3, 20 days after the challenge, 1 of eight eyes was
42
PCT/US95102459
WO 95124922 218 5 ~ f~ 8
positive compared to eight out of eight eyes that were positive in the control
group.
When the accumulated results were examined, with GLXA-Ab3, 9 of 40 22.5%)
were positive, in the contrast, 36 of 40 (90%) were positive without GLXA-Ab3.
Table 3.
Neutralization of chlamydial infection in primate conjunctivae by
Ab3 IgG by cell culture assays
Day of No. of Eye Culture Positive
Experiment Ab3 Preimmune Noneb Combined
IgG Control
0 0/8 0/4 0/4 0/8
2 3/8 2/4 3/4 5/8
6 3/8 4/4 4/4 8/8
9 1/8 3/4 4/4 7/8
1 2 1/8 4/4 4/4 8/8
20 1IR did did R/R
9/40 17/20 19/20 36/40
a Only one first passage negative sample was second-passage positive.
bEBs were previously incubated without antibody.
In an parallel assay, direct fluorescence antibody cytology assay (DFA) was
also carried out to evaluate the numbers of EBs from the conjunctiva) swabs.
The
conjunctiva) swab samples were fixed onto slides and stained with FITC-labeled
monoclonal antibody against C. trachomatis. It was considered to be positive
when
or more characteristic elementary bodies were seen on each slide (Micro Trak,
Syva Co. CA). As shown in Table 4, EBs were detected in the GLXA-Ab 3 treated
group on only two occasions, day 6 and 12 post-infection, while the remainder
were positive through day 20. Only one eye was EB negative in the non-treated
43
WO 95!24922 PGT/US95/02459
2185568
group at day 2, that was probably an artifact. EBs were detected in all eyes
in this
group for the remainder of the experiment. On the last examination day (day
20),
none of the eyes treated with GLXA-Ab3 was positive, while 8 of 8 were
positive in
the combined control group. In addition, DFA and culture results are
completely
congruent.
Table 4.
Neutralization of chiamydial infection by Ab3 using direct
fluorescence antibody cytometry assay (DFA)e
Experiment Ab3 Preimmune Noneb Combined
IgG Control
0 n/R nid ma niR
2 0/8 2/4 0/4 5/8
6 1/8 2/4 4/4 6/8
9 0/8 4I4 4I4 8/8
12 1/8 4/4 4/4 8/8
20 flL8 d/d d/4 Rlf~
2/40 16/20 19/20 35/40
D was consi ere positive i s were oun on a s i e.
bEBs were considered previously incubated without antibody
GLXA-Ab3 substantially attenuates the chlamydial reprication in conjunctiva!
infection
To further understand the mechanism of the neutralization, chlamydial
specific ribosomal RNA had been examined from those primate conjunctiva) swabs
by a DNA probe (Cheema et al,The Ameri. J. Med. Sci. 302:261-268, 1991). Total
RNA was extracted from conjunctiva) swabs taken from primate eyes. RNA from
serovar C EBs, human or yeast were used as control. 32P-chlamydial DNA encod-
44
WO 95!24922 2 l ~ 5 5 ~ ~ pCT~S95/02459
ing ribosomal RNA16S and 23S genes was used to detect chlamydial specific
RNA in a Northern slot-blot hybridization assay. As shown in Figure 8, control
eyes
(infected either with pre-immune rabbit IgG plus EBs or EBs alone) uniformly
show
significant levels of chlamydial RNA at all time points examined, similar RNA
samples prepared from the eyes of GLXA-Ab3-treated organism show significantly
attenuated levels of chlamydial RNA. This indicates that the neutralization
happens
at the very early stage of the infection.
The degree of conjunctiva) inflammation after inoculation with EBs pre-
viously incubated with either GLXA-Ab3 or pre-immune IgG or without previous
incubation was evaluated by clinical response. The clinical response was
graded
based on a total clinical disease scores (TCDS) derived from 10 clinical
features of
inflammation (Taylor et al, Invest. Opthalmol. Vis. Sci. 29: 1847, 1988). The
accumulative disease scores were obtained for each group of primates by examin-
ing 10 signs existing in the conjunctiva. As shown in Figure 9, recipients of
GLXA-
Ab3 developed very little clinical disease and this declined after day 8.
Control
animals continued to develop severe disease through day 21 post-challenge.
This
pathological finding is consistent with cell culture, DFA and RNA
hybridization
data.
Generation of And Characterization of Hybridoma Cell Lines Producing Anti-
idiotypic Antibody.
Production of anti-idiotypic hybridoma cells
Five syngeneic mice (BALB/cByJ) were immunized intraperitoneally with
KLH conjugated monoclonal GLXA-Ab, IgG in the presence of Freund's complete
adjuvant. The anti-idiotypic anti-sera against monoclonal GLXA-Ab ~ IgG from
these
mice were detected by sandwich ELISA. The titer was about 1:1000 three weeks
after the immunization (data not shown). Fusions were made between Sp2/0-Agl4
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WO 95/24922 PCT/US95/02459
cells and spleen cells from two mice which had the highest titer against
monoclonal
GLXA-Ab, . The size of the spleens were almost twice the size of normal
spleen.
The clones were screened by the same method for mouse antisera testing. The
pool of mice antisera or pre-immune sera diluted 1:10 was used as controls in
all
screening tests. Among 283 wells screened, 8 were considered positive against
mAb, with a titer two times or higher than negative control. These hybridomas
were
cloned. One clone (91 MS441 ) had the highest titer in ELISA and was
propagated.
The ascites was produced and IgG isolated. The isotype of IgG of this clone
was
identified to be IgGI, having a K light chain (Bio-Rad Laboratories, CA). The
fusion
results was summarized in Table 5.
Table 5.
Summary of fusion for anti-idiotvoic antibodies
Immunized Anti-id TiterTotal Wells Wells PositiveStable Cloned
in
BALB/cByJ Supernatant with Growing of Anti-id Anti-id Lines
(%)
Hybridoma (% of Total)
2 1:1000 283 8 1
(2.8) (0.35)
Light chain:K Heavy
chain:IgG,
Hybridoma (91 MS441 ) secreting IgG inhibits the binding of monoclonal GLXA-
Ab, to
G LXA
The supernatant from positive clones were further tested for the inhibition of
the binding of monoclonal GLXA-Ab, to GLXA by chemiluminometric immunoassay.
Total inhibition of the binding was found from the supernatants of 4 clones
without
dilution. Two weeks later only one clone (91 MS441 ) was found stable. As
shown in
Figure 10, at a dilution of 1:10, the supernatant from this clone inhibits the
binding of
monoclonal GLXA-Ab, to GLXA by 80% compared to 0% of a non-positive clone (91
MS442) from the same fusion. Serial dilutions of culture supernatant from
clone
91 MS441 (~) and negative clone 91 MS442 (O) were tested by chemiluminometric
immunoassay. The percent is obtained form the mean of the duplicate. this
demonstrates that an anti- .
RECTIFIED SHEET (RULE 91)
218 5 5 J ~ PCT/US95/02459
WO 95/24922
idiotypic clone was produced. It is identified as monoclonal GLXA-Ab2.
Monoclonal
GLXA-Ab2 is recognized by patient and rabbit anti-chlamydia antisera.
As an internal image of an antigenic determinant, the anti-idiotypic antibody
should be recognized by any specific antisera which is against this epitope
regardless of species. In order to test this property of the IgG produced by
clone
(91MS441), human serum from a patient clinically diagnosed as chlamydia
positive
was used. Since the isotype of monoclonal GLXA-Ab2 is IgG,, the purification
of the
IgG was carried out by protein G affinity chromatography. The isolated
monoclonal
GLXA-Ab2 IgG was coated on a microtiter plate and tested for reactivity with
the
patient's serum (91 MS253) by ELISA. Human serum without clinically diagnosed
chlamydial infection (88MS356) was used as a negative control. The results
showed
that the patient's serum had a titer which was more than 1:2000 against
monoclonal
GLXA-Ab2 (Fig. 11 ). The test was carried out by ELISA. Serial dilutions of
the
antiserum obtained from a patient diagnosed as chlamydial infection (~) and
non-
diagnosed individual (O) were incubated with mAb2lgG coated on the polystyrene
wells. The second antibody is goat anti-human peroxidase conjugate. Data is
presented as the means for duplicates. This demonstrates that infected human
antisera has the specific antibody against monoclonal GLXA-Ab2, implying that
monoclonal GLXA-Ab2 is the internal image of GLXA.
The specific recognition of monoclonal GLXA-Ab2 by a rabbit anti-chlamydial
antisera (88MS 188) was also tested in the similar way. The rabbit antiserum
was
produced by inoculating EBs. As shown in Figure 12, rabbit antisera has higher
binding to monoclonal GLXA-Abz than pre-immune serum, but lower than expected.
The difference between the binding is seen to the dilution factor of 1:1000.
Polystyrene plate was coated with mAb2lgG, 1 ug/well. Serial dilutions of
rabbit
antiserum specific to chlamydial EBs (~) or pre-immune rabbit serum (O) were
added to each well in duplicate. The specific binding of the antiserum was
detected
by mouse anti-rabbit IgG (H&L) HRP conjugate and TMB substrate. This shows
that
rabbit antiserum which is
47
RECTIFIED SHEET (RULE 91)
"~' WO 95124922 218 5 5 ~ ~ pCT/US95/02459
against chlamydia has the specific antibody recognizing monoclonal GLXA-Ab2,
an
additional evidence for monoclonal GLXA-Ab2 as an internal image.
Antisera monoclonal GIxA-Ab3 from syngeneic mice recognize GLXA
The anti-idiotypic antibody as an internal image can raise antigen specific
antisera in animals that have never been exposed to that antigen. In this
case, if
monoclonal GLXA-Abz (91 MS441 ) is the internal image of GLXA, (that is
bearing
REC11F1ED SHEET (RULE 91)
_ PCTIUS95/02459
WO 95124922 218 ~ J ~ 8
the same antigenic structure), it should produce an anti-GLXA antibody in the
same strain BALBIc ByJ mice in spite of the fact that they have never been
exposed to GLXA. In an initial experiment, 8 mice were immunized with mono-
clonal Ab2 IgG, four mice with normal mouse IgG. No adjuvant was used in the
subcutaneous injection. Seven days and 14 days after the immunization and one
boost, antisera were obtained from these mice by sacrificing half of the mice
in
each group. Immuno-dot blot assay was carried out with purified GLXA. The dot
blot showed that mice immunized with monoclonal GLXA-Ab2 IgG had a titer as
high as 1:800 compared to 1:100 or less of the mice immunized with normal
mouse IgG. The blots were further quantified by a densitometer. When diluted
to
1:100 after the first boost, the antisera from all four mice bind to GLXA
whereas no
binding was seen in the control group even when diluted 1:50. This
demonstrates
that this antisera is solely elicited by the paratope of the anti-idiotype
because the
recipients are syngeneic.
GLXA-Ab3 from syngeneic mice inhibits the binding of monoclonal Ab, to GLXA.
GLXA-Ab3 antisera which specifically bind to GLXA were also tested for the
inhibition of the binding of monoclonal Ab, to GLXA by chemiluminometric im-
munoassay. At a dilution of 1:25, GLXA-Ab3 inhibits 90% of the binding
compared
to 18 % of inhibition by the control antisera day 8 post immunization. This is
equal
to the same inhibition demonstrated by 10 ug of unlabeled monoclonal GLXA-Ab,.
The same inhibition was found in the antisera on day 14, one week after the
first
boost with the same amount (50 ug/mouse) of IgG. It is noted that inhibition
percentage dropped from 70% on day 7 to about 50 % on day 14 when diluted
1:100.
Immunization with Monoclonal GLXA-Ab2 Protects Mice from Chlamydial Infection
BALBIc ByJ mice were used as an ocular chlamydial infection animal model
48
2185568
in this study. The first experiment was carried out with 39 mice. Twenty mice
were
immunized subcutaneously with 50 ug of monoclonal GLXA-Ab2 and nineteen with
normal mouse IgG without any adjuvant. They were given a total of three
injection,
one week apart. One week after the last injection, the mice were inoculated
with ~,,
trachomatis serovar C elementary bodies (5000 IFU in each eye). The
conjunctiva)
swabs were taken from each eye of the mice on day 0, 7, 14, and 21 post
infection.
Samples from conjunctiva) swabs were collected and cultured in McCoy cell
monolayers for 48 hours. Inclusion bodies were counted, and 5 inclusions were
considered as positive in 15 fields. As shown in Figure 13, mice immunized
with
monoclonal GLXA-Ab2 had half the infectivity compared with mice immunized with
normal mouse IgG. In a second experiment (twenty mice in each group), the
immunization was repeated in the similar manner. However, they received 10g
IFU
per eye, 100 times the number of EBs as used in the first experiment. Twenty
mice
were immunized with mAb2 (~) and nineteen with normal mouse IgG (O)
subcutaneously (50 ug IgG/mouse). they were ocular challenged with ~,
trachomatis serovar C. EBs after three times immunization in a week interval.
Each
eye received 5000 IFU. One day before and after the challenge, conjunctiva)
sample was taken from each eye and cell cultured. percentage of positive eyes
are
based on the number of the positive eye among all the'eyes examined in a
group.
Surprisingly, almost the same protection curve found in the mice (Fig. 14),
suggesting that monoclonal GLXA-Ab2 elicits a vigorous host defense response.
After immunization three times in a week interval, twenty mice immunized with
mAb2
IgG (~) and twenty with normal mouse IgG (O) received approximate 5x10' to
5x106
chlamydia trachomatis serovar C. EBs per eye. Conjunctiva) chlamydial
inclusions
were tested by cell culture obtained from cojunctival swabs. Values are the
means
of first and second passage culture results except day 28.
In a third experiment, aluminum hydroxide (alum) was used in the
immunization: Ten mice were immunized with monoclonal GLXA-Ab2 in the presence
of Maalox as
.~
~,:
4~
2185568
adjuvant, eight mice immunized without Maalox and sixteen mice immunized with
normal mouse IgG in the presence of Maalox. The immunization procedure is the
same as the previous ones. When Maalox was used in the immunization; the titer
of
the anti-anti-idiotypic antisera in mice was doubled as tested against GLXA in
immuno-dot blot. The higher titered antisera in the adjuvant treated mice
demonstrate more effective protectibn in the mice from the ocular infection
compared to the ones immunized without alum (Fig. 1 ~). Ten mice were
immunized
with mAbZIgG without alum (O) and sixteen with normal mouse IgG plus alum (~).
One week after the last boost, mice were ocularly infected and conjunctiva)
infection
was detected by cell culture on day as shown. However, mice immunized with
monoclonal GLXA-AbZ had significantly less infection than those receiving
normal
mouse IgG plus alum. In addition, the infection was cleared by post challenge
day
28. In the fourth experiment, eight mice were immunized with
49A
WO 95/24922 PCT/US95/02459 -~
monoclonal GLXA-Abz and eight with normal mouse IgG in the presence of alum.
The immunization and inoculation protocols are the same as before. However, as
shown in Figure 16 (A), on day 7, both groups had the same degree of
infection,
50 % of the eyes are infected. However, beginning on day 10, a significant
difference of the infection is seen between the two groups. For the group im-
munized with monoclonal GLXA-AbZ IgG, on day 14, two out of eight eyes were
positive. On day 22, one out of eight. No eye was positive on day 28, showing
a
clear regression of the infection. In contrast, mice immunized with normal
mouse
IgG, on day 14, six out of six eyes were positive and continued to be positive
until
day 42. The number of infected eyes began to fall by day 28 seen in this group
is
believed to be a natural recovery process. In a final experiment, the mice
with
completely cleared infection from the above experiment were rechallenged with
EBs in order to evaluate whether the immunity was associated with memory. A
significant factor in terms of protection is the ability of the immunized mice
to clear
the organisms from their eyes much earlier than the non-immunized ones. This
is
clearly shown to be the case with mice immunized with monoclonal GLXA-Ab2
(Figure 16 (B)). This shows that monoclonal GLXA Ab2 not only is able to evoke
a
protective immune response but also a memory immune response.
This example illustrates that an anti-idiotypic antibody which mimics GLXA,
protects mice as an animal model from chlamydial ocular infection. The mono-
clonal antibody mAb, (89MS30) which was used to produce anti-idiotypic an-
tibodies was produced by immunization with whole EBs and screened for its
reaction to a genus specific antigen. It has been used to identify GlxA and
demonstrated specific binding to the polysaccharide portion of GLXA. This
antibody was also found cross-reactive to cLPS.
First GLXA and cLPS are distinctly different genus specific antigens. GLXA
was found on RBs, EBs, inclusion membranes, host cell membranes and shed into
the inclusion space, cytoplasm of the infected cells and into the
surroundings. It
was obtained from the supernatant of infected cell culture. Whereas cLPS was
"' WO 95!24922 21 ~ 5 ~ ~ g p~T~s95/02459
found both on EBs and RBs (mainly RBs). They are not secreted or shed from
infected cells, but loosely bound to the RBs. Structurally, they have
different
polysaccharide moietys. GLXA has a unique sugar residue: gulose or gulose
derivatives, mannose and galactose, probably arranged in repeating units of
guluoronic and mannuronic acids. Only two fatty acids were found associated
with
the antigen compared to at least 12 in cLPS. Whereas cLPS has typical linear 2-
keto-3-dexyoctonoic acid (KDO) trisaccharide (common core) and polysaccharides
like glucosamiane and heptose. Serologically, monoclonal GLXA-Ab, specifically
bind to gulose and mannose repeating block. Whereas, cLPS genus specific
epitope is on KDO, the specific determinant is the 2-8 linkage. Obviously, it
is very
unlikely that GLXA and cLPS share the same epitope. It is known that
antibodies,
whether poly-or monoclonal, antibodies produced either by whole EBs or by
purified cLPS which are specific to cLPS have no neutralizing or protective
functions. From the composition analysis, it is suggested that gulose together
with
mannose and galactose form the specific epitope of GLXA. Whereas, cLPS, in
addition to KDO trisaccharide as an epitope, has other epitopes in KDO region
and
also other saccharide portions. These later structures have shown a broad
cross-
reaction with LPS from other gram negative bacteria. Since the protective
epitope
of GLXA consists of an array of sugar residue, it is more reasonable to
believe that
some of which cLPS is partially shared with GLXA.
There are some other possibilities about this cross-reaction. For example,
(1) GLXA and cLPS do not share any primary similarity, but structurally form
similar binding motif; (2) although monoclonal GLXA-Ab, binds to cLPS, GLXA
and cLPS do not have the similarity in antibody binding site. The monoclonal
antibody can be multispecific, that is, it recognizes a quite different
epitope.
From the discussion above, it is believed that monoclonal GLXA-Ab, is
specific to GLXA epitope, the possibility that GLXA and cLPS share same sugar
residues or merely structure similarity may explain the cross-reactivity.
51
WO 95/24922 ~ PCT/US95102459
Anti-idiotypic antibodies, GLXA-Ab3 and monoclonal GLXA-Ab2
Anti-idiotypic antibody is a potent and long lived immunogen
Monoclonal GLXA-Ab, was injected into guinea pigs subcutaneously in the
absence of conjugate or Freund's adjuvant. All four immunized guinea pigs
developed high titered anti-idiotypic antibodies specific to monoclonal Ab,,
which
were found as early as 3 weeks after the first immunization. The titer was ap-
proximately 1:5000 by ELISA. The production of the anti-idiotypic antisera
contain
a relatively high concentration of GLXA-Ab2 which is specific to the
hypervariable
region of monoclonal GLXA-Ab,. This was shown after two absorptions by normal
mouse IgG. When the IgG1 isotype from guinea pig anti-idiotypic antibodies was
used as an immunogen in three rabbits, the titer of anti-anti-idiotypic
antibody was
more than 1:20,000 two months after immunization. This demonstrates that
immunoglobulin itself is a very potent immunogen. This is true not only for
interspecies immunization, but also syngeneic immunization. With monoclonal
anti-idiotypic antibody monoclonal GLXA-Abz, the immunization was carried out
in
syngeneic mice without KLH-conjugation or Freund's adjuvant. The mice de-
veloped high titered GLXA-Ab3 in a short time after immunization (9 days). The
protocol used in this study is different from most methods which use either a
conjugate or Freund's adjuvant for a higher immunogenicity. This indicates
that
immunoglobulin as an antigen is more immunogenic compared to most isolated or
synthetic peptide antigens.
A successful vaccine not only requires that it be a good immunogen but that
it is long lasting (preferably for the lifetime off the host). The immunity
produced
by idiotype is tong lived. The ability to inhibit the binding of monclonal
GLXA-Ab,
to GLXA by guinea pig GLXA-Ab2 from three immunized guinea pigs have been
monitored for as long as 77 weeks. With only three boosts, the inhibition one
year
post immunization is almost equal to antisera collected in the early stages
after the
immunization. This indicates that the immunity elicited by the idiotypic
antibody
52
"a WO 95/24922 ~ ~ ~ ~ J O ~ PCT/US95/02459
monoclonal GLXA-Ab, has a long term memory. Since the half life of an antibody
molecule or the majority of antibody-producing cells is about a few weeks, the
boosting interval (six months) is far beyond the life span of the B cells and
the
immunoglobulins. It is the constant stimulation within the idiotypic network
that
keeps this anti-idiotypic antibody at a certain level. The change of idiotypic
specificity during this period has not been seen in this case.
An internal image of chlamydial GLXA, isotypic difference
In this study, guinea pig GLXA-Ab2 IgG1 and IgG2 were separated. The
regulatory function of these two isotypes to the idiotype was not evaluated.
However, the difference between IgG1 and IgG2 subclasses have been found in
inhibition of the binding of monoclonal GLXA-Ab, to GLXA. With a novel system,
chemiluminometric immuno-assay, the incubation and the final detection were
all
carried out in solution rather than solid phase as in ELISA, thus greatly
lessening
the possibility of the inhibition by hindrance. The results have shown that
GLXA-
Ab2 IgG1 inhibited 100% of the binding whereas IgG2 50% at the same concentra-
tion. This suggested that GLXA-Abz IgG1 has a high affinity in binding to the
idiotype or being more like the antigen, GLXA. This demonstrates an isotypic
difference in their binding ability to the idiotype which reflects a
difference in their
respective active sites. IgG1 has a different idiotype binding ability from
IgG1.
There are a number of examples of dominant idiotypes, for example, A5A
idiotope
of anti-strep-A carbohydrate antibodies or the T15 idiotope of phosphoryl
choline
antibodies. It is not clear if there is any isotype preference of anti-
idiotypic
antibody in different systems. This finding suggests that a certain isotype of
GLXA-Ab2 is the internal image while others are not.
Monoclonal GLXA-Ab2 as an immunogen of chlamydial GLXA
53
WO 95/24922 PCT/US95/02459
2 ~ s5~~a
The purpose of making monoclonal anti-idiotypic antibodies is to: (1) have a
constant source of anti-idiotypic antibody for vaccine study; (2) identify a
possible
receptor for GLXA on host cells; and (3) further characterize the epitope on
GLXA.
This enables an understanding of biological functions of GLXA in terms of
epitope
density, its role in mechanism of infection and the protective function
against chla-
mydial infection in vivo. The production of monoclonal anti-idiotypic
antibodies
was carried out in the syngeneic BALB/cBYJ mice. In the first fusion, one
stable,
highly inhibitory clone (91MS441) from 283 clones screened was selected. In
the
second fusion, another clone (91 MS442) was selected though it is not as
inhibitory
as 91 MS441 clone in chemiluminometric immunoassay. The monoclonal GLXA-
Ab2 produced by this clone (91 MS441 ) has been shown to be the internal image
of
the chlamydial antigen, GLXA.
It is interesting to note that the inhibitory ability of mouse GLXA-Ab3
slightly
but obviously decreases over time. The dosage of the anti-idiotype has been a
factor in either enhancing the idiotype or suppressing the idiotype. This
inhibition
results by GLXA-Ab3 has shown that after administration of monoclonal GLXA-Abz
IgG twice, the inhibition is higher than the sera obtained after
administration three
times. This indicated 50 ug is either too much for one dose or too much for
repeated administrations. On the other hand, it shows that a low amount is
enough for protective immunity. The reason for choosing normal mouse IgG as a
negative control in the immunization rather than a non-relative clone is that
it
would prevent any possible bias from a specific clone.
Monoclonal GLXA-Ab, and GLXA-Ab3 bear the same antigen binding structure.
GLXA-Ab3 from immunized rabbits and mice recognized affinity purified
GLXA by immuno-dot blot assay, though there is no previous exposure to GLXA or
infection. The binding of monoclonal GLXA-Ab, to GLXA is inhibited as the
concentration of GLXA-Ab3 increases. This indicates that the anti-anti-
idototype
has equivalent antigen reactivity as idiotype, that is they recognize the same
54
°
'° WO 95/24922 218 5 ~ ~ ~ PCT/US95l02459
epitope. The same antigen reactivity of monoclonal GLXA-Ab, and GLXA-Ab 3 from
rabbits to GLXA was further proved in the immuno-fluorescent staining of the
inclusions in infected McCoy cell culture. The experiment suggested the
structural
similarity of antibodies. Using monoclonal GLXA-Ab2 which mimic GLXA to
identify its role would be valuable in understanding the mechanism of anti-
idiotype
protection. The experiment was carried out with human epidermoid carcinoma
cells (A431 ) as well as human endometrial gland epithelial cells (HEGEC).
Those
cell lines were used because of their human origin. A preliminary binding
experi-
ment was performed by direct antibody staining detected with FACScan (Becton
Dicknson, N.J.) flow cytometer. Since the separation of these epithelial cells
into a
single cell suspension is very difficult without using enzyme or harsh
separation,
especially HEGEC cells, the cell population consists of singlets, doublets and
multiplets. Single cell population was gated right above the cell debris.
Since the
control, cells stained with normal mouse IgG was at exactly the same gate, it
is
believed that the two groups are comparable. In addition, the binding is
direct,
monoclonal GLXA-Ab2 or normal mouse IgG was directly biotin labeled. The
intensity of monoclonal GIxA-Ab2 bound increases with the concentration
specifi-
cally to the host cells (HEGEC). While normal mouse IgG has no such binding
even at the highest concentration. If this finding can be repeated and proved
to be
valid, GLXA is an adhesin or a ligand which binds to HEGEC cells. There are
some reasons to believe that GLXA is an adhesin or a ligand. First, as
mentioned
above, GLXA-Ab 3 neutralization occurs at the very early stage. This is proved
by
finding apparently no significant chiamydial specific ribosomal RNA in
conjunctiva
samples taken from primates inoculated with GLXA-Ab 3 treated EBs. This sug-
gested that GLXA-Ab 3 blocked EBs from getting into the host. Second, GLXA was
found to enhance the infection by approximately three fold when McCoy cells
were
pre-incubated with GLXA. It is only if GLXA as an adhesin or ligand which
facilitate the attachment of EBs to host cells that this result can occur.
Actually,
EBs may well use this mechanism to infect host cells because GLXA are secreted
WO 95124922 PCT/US95/02459
218558
from infected cells.
A possible mechanism of the role of GLXA may be the following: EBs attach
to the host cells either by GLXA on EBs or by free GLXA which were secreted by
the infected cells into the surroundings. This makes it much easier and
efficient
for GLXA to absorb to surrounding cells. Ebs can then attach to the host cells
either by GLXA on EBs, thus binding to the host cells. This mechanism seems
much more efficient than one to one attachment.
Neutralization of chlamydial infection by GLXA-Ab3
The preliminary neutralization test in vitro has shown that more chlamydial
inclusions were found with GLXA-Ab, treated than rabbit Ab3 treated C.
trachoma-
tis serovar B EBs. This showed that GLXA-Ab3 neutralized the infection,
whereas
monoclonal GLXA-Ab, did not. G(xA-Ab3 neutralized the infection in cell
culture
whereas monoclonal GLXA-Ab, did not. This in vitro result was repeated in vivo
in
two later experiments with the primate infection model. It was confirmed that
GLXA-Ab3 effectively neutralized chlamydial infection both in vitro and in
vivo.
The understanding of the mechanism of this neutralization comes from the
chlamydia specific RNA hybridization experiment. The primates were ocularly
inoculated with GLXA Ab3 or normal rabbit IgG treated EBs. Chlamydial RNA was
detected from the conjunctival swabs taken from primates on different days
prior
and post inoculation. The RNA hybridization assay used in this study is an
extremely sensitive way to detect chlamydial infection in samples which are
unequivocally negative by either cell culture of DFA or both. The
substantially low
RNA found in Ab3 treated primates provides the evidence that the
neutralization
occur at very early stage of the infection, before the internalization of the
or-
ganisms. This suggests that GLXA may be an adhesin or ligand functioning at
the
stage of attachment.
The role of humoral immunity in protection against chlamydial infection has
long been discrepant. However, it has been shown both in vitro and in vivo
that
56
WO 95124922 PCT/US95102459
21855~~
humoral immunity may play some role in preventing chlamydial infection.
Protection of Mice from chlamydial infection by immunization with monoclonal
G LXA-Ab2
The chlamydial ocular infection model in BALB/cByJ mouse was employed
in a vaccine study with monoclonal anti-idiotypic antibody monoclonal GtXA-
IgG.
It is model for C. trachomatous serovars which only infect humans. In the im-
munization experiments, monoclonal (GIxA-Ab2) IgG (50 ug/mouse) was used to
immunize 6 to 20 mice in each group subcutaneously either in the presence of
adjuvant or with no adjuvant. Mice immunized with monoclonal GLXA-Ab2 mount a
distinctive GIxA-Ab3 titer detectable one week after a single immunization.
The
system used in this study is syngeneic: monoclonal GLXA-Ab,, monoclonal GLXA-
Ab2 or GIxA-Ab3 are all produced in the same strain of mice. They bear the
same
immunoglobulins genetically. The only "foreign structure" is the specific
binding
site of each type antibody (monoclonal GIXA-Ab, or monoclonal GIxA-Ab2).
Normal mouse IgG from the same strain was used as a negative control. Con-
sistently, mice immunized with monoclonal GLXA-Ab2 developed a significantly
high GtxA-Ab3 titer against purified GLXA compared to the control group. The
average titer was as high as 1:3200 comparing with 1:200 in the mice immunized
with normal mouse IgG. This clearly demonstrates that the production of GIXA-
Ab3 antibody in synergenic mice is elicted by the hypervariable region, the
struc-
ture which mimics the GLXA. In addition, the GLXA-Ab3 from these mice
inhibited
the binding of monoclonal GLXA-Ab, to GIxA as efficiently as unlabeled mono-
clonal GIxA-Ab,. These experiments shows that monoclonal GIxA-Ab2 mimics
one single epitope of GIXA.
The protection of mice from ocular infection by C. trachomatous was
consistently demonstrated in four individual experiments in vivo. The mice
immunized with monoclonal GLXA-Ab2 or normal mouse IgG (three times on a
weekly basis) were infected in the eyes by inoculation of EBs (5000 IFU/eye).
The
57
WO 95/24922 PCT/US95/02459
2785568
conjunctiva) swabs were taken on different days before and after the
infection.
Inclusions were detected by cell culture. The eyes of the mice immunized with
monoclonal GLXA-Ab2 IgG have significantly short recovery (9 days after ocular
inoculation comparing to 28 days of the control immunized with normal mouse
IgG). The GLXA-Ab3 titer is well correlated with cell culture results. This
demon-
strates that monoclonal GLXA-Ab 2 which mimics a single GLXA epitope, is an
effective immunogen, and evokes a strong protective immune response against
chlamydial infection. This shows that a single protective epitope resides on
chlamydial GLXA.
Prospective of the mechanism of the neutralization and protection
This example is the first demonstration that a non-neutralizing monoclonal
GLXA-Ab, can produce a neutralizing GLXA-Ab 3 in vivo through an anti-
idiotypic
antibody (GLXA-Abz). The central issue is that monoclonal GLXA-Ab, produced by
immunization with chlamydial EBs does not neutralize or protect primates from
reinfection whereas GLXA Ab3 produced by guinea pig GLXA-Ab2 neutralized the
infection and immunization with monoclonal GLXA-Ab2 protects mice from reinfec-
tion.
ORAL VACCINE
(a) Infection of mouse eyes: Eyes of BALB/c mice were infected by topical
application of 2500-106 IFU/5 ul of Percoll purified elementary bodies (EB)
from
serovar C (TW/3) suspended in SPG or PBS. Concentration was reconfirmed by
titration at each challenge. The course of infection was followed by clinical
exam,
microbiologic assays, histopathologic exams, and immunologic assay. Modified
grading scales for clinical scoring and histopathology were developed. Con-
junctiva) specimens were collected with sterile urethra) swabs and tested in
culture
or direct fluorescent antibody (DFA) assays for the presence of chlamydial or-
ganism by standard methods, Infect, Immun., 1989, Vol 57, Pgs. 2977-2983, with
the exception that blind second passages were performed on all specimens.
58
WO 95124922 PCT/US95/02459
21855~~
(b) Test of the anti-idiotypic antibody (Ab2) as a protective vaccine: BALB/c
mice
were immunized subcutaneously 1-3 times with either 50 or 100 ug Ab2 in
alumina
(Maalox). Ten to 12 days after the last immunization, an Ab3 response was
verified by a chemiluminescent or GLXA dot blot assay, Curr. Microbiol; 1933,
Vo1.28, Pgs. 885-890, after which all eyes were challenged with infectious C-
EB
(5000 IFU). The course of infection was followed as described above. Control
mice received the same regimen of purified normal ascites IgG2 and infectious
challenge.
Ab2 or IgG incorporated into biodegradable microspheres was used to
immunize BALB/c mice, either orally or subcutaneously, with 50 ug Ab2
equivalent
(w/w). Microspheres were prepared by using as polymer, polylactic acid, Bio/-
Technology, 1933, Vol. 10, Pgs. 1446-1449 and Pharmaceut. Res.., 1991, Vol. 8,
Pgs. 713-720. Retention of Ab2 function was confirmed by the ability of micro-
spheres containing the Ab2 to induce an Ab3 response after subcutaneous injec-
tion. Control mice received IgG2 in microspheres, soluble Ab2 in alumina, or
control IgG in alumina. Five-10 micelgroup were challenged on day 0 and
followed for protection against ocular infection.
Results
(a) A reproducible inbred mouse model of ocular infection by a human biovar.
BALB/c mice developed reproducible ocular infection after single or repeated
innoculation with serovar C of C. trachomatis. While clinical disease was most
evident with repeated infection (daily, repeated weekly or once weekly), even
a
single inoculation of infectious chlamydia induced lid thickening and exudate.
Histopathologically, intensity of inflammatory mononuclear infiltrate loss of
goblet
cells, and exudate were dose-dependent. The mean histopathologic disease score
at day 12-14 was 6.8 t 0.8 compared to 0 t 0 for normal tissue. A typical
microbiologic timecourse obtained with conjunctival swabs from 10 BALBIc mice
is
shown in Figure 17.
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WO 95/24922 PCTlUS95/02459
2i85~68
(b) Soluble anti-idiotvpic antibod~protects agiainst ocular chlamvdial
infection
Ab2 in alumina successfully immunized mice as evidenced by prechallenge Ab3
dot blot titers ranging from 1:200-:800 compared to <1:100 for controls. After
infectious ocular challenge, conjunctiva) swabs were collected for culture at
days 7,
10, 14, 21, 28, 35 and 42. Culture results from a typical experiment are shown
in
Figure 18. One hundred percent of control mice (n=10) which received IgG with
alumina became infected by day 12-14. In contrast, 50% of Ab2 recipient eyes
were totally protected and those eyes which were culture positive at day 7
cleared
infection 2-4 weeks earlier than controls. Histopathologic disease scores were
reduced from 9.2 ~ 0.8 (IgG) to 2.9 ~ 0.8 (Abz). A higher infectious challenge
dose (106 IFU/5ul) or a lower dose (50 ug of Ab2 ) protected similar numbers
of
eyes. Similar protection was observed after rechallenge of animals after they
became culture-negative.
(c) Ab~ in microspheres protects after oral or subcutaneous immunization.
In order to optimize presentation of the anti-Id to the mucosal immune system,
experiments were performed with Ab2 incorporated into biodegradable micro-
spheres. Groups of 5-10 mice recieved 50 ug Ab2-microspheres either orally
(p.o.)
in 200 ul bicarbonate buffer, subcutaneously (s.c.) in PBS, soluble Ab2 in
alumina
(Maalox) s.c., or oral IgG-microspheres. After one boosting immunization, all
mice
received an infectious ocular challenge of 5000 IFU C-EB. On day 12, all eyes
were swabbed for culture assay. The results are shown in Table 6.
Microbiologic
protection was highly significant in recipients of Ab2-microspheres based on
reduced mean inclusions per group compared to recipients of IgG-microspheres
(p< .003). Second passage outline confirmed first passage results.
WO 95124922 PCTIUS95102459
2i85~68
Table 6
Test of Ab~ in Microsaheres as an anti-Chlamvdial ~ Vaccine
Passage I (%Positive/Grp) Passage 2% Positive Grp)
Grp-Treatment (#mice) % Mice Mean IFU P %Mice Mean IFU P
2 icrosp eres ~ . t . <. 7 . t . c.
(n=8)
B:S.C. Ab2 Microspheres 50 0.9410.4 (c.01) 70 1.1810.4 (<.001)
(n=10)
C:S.C. Ab2 in Maalox 80 0.6610.3 (<006) 100 1.1510.5 (c.004)
(n=5)
D:ORAL IgG-Microspheres 100 3.0~0.5 100 4.2110.6
(n=6)
All animals received 50 ug Abz or IgG twice; P-values in comparison to Group
D.
This example demonstrates that inbred BALB/c mice can be used as a
model of ocular chlamydial infection. A single infectious inoculation of a
human
biovar of C. trachomatis (ClTIN-3) leads to ocular infection in BALB/c and
C3HIHeN mice which can be detected clinically, histopathological, microbiologi-
cally, and by immunoassay.
The monoclonal anti-idiotypic antibody (mAb2) to anti-GLXA mAb, was
tested as a potential anti-chlamydial vaccine candidate in the mouse model.
mAb2
competes with mAb, for binding to GLXA and immunization with mAb2 induces Ab3
which recognizes the same epitopes on GLXA as recognized by mAb,. The
animals immunized with mAb2 had no previous exposure to GLXA or whole
chlamydia, yet the Ab3 induced by mAb2 reacted with GLXA in dot blot and ELISA
essays.
The results demonstrate unequivocally that Ab2 can successfully immunize
against ocular chlamydial infection, even after systemic immunization with
soluble
anti-Id in alumina. Ab2 incorporated into microspheres protected 50% of eyes
from
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WO 95!24922 PCT/US95/02459
~1855~8
infection after systemic immunization, and 44% of eyes after oral presentation
of
the same dosage. The mean inclusion counts for recipients of Ab2 microspheres
were reduced by 65-72% compared to IgG-microsome controls. The results
strongly support the usefulness of an anti-idiotypic antibody as an anti-
chlamydial
vaccine and demonstrate the feasibility of Ab2 incorporated into microspheres.
In
addition to being highly immunogenic, protection by the criteria of reduced
clinical,
histopathologic, and microbiologic disease were also fulfilled for both
soluble Ab2
and AbZ delivered in microspheres.
62
