Note: Descriptions are shown in the official language in which they were submitted.
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RAPID GENERATION OF T CELL-INDEPENDENT ANTIBODY
RESPONSES TO T CELL-DEPENDENT ANTIGENS
CROSS REFERENCE TO RELATED CASES
This application claims the benefit of U.S. Provisional Application Serial No.
60/948,296, filed July 6, 2007, which is incorporated by reference herein in
its
entirety.
BACKGROUND OF THE INVENTION
Antigens may be characterized as T cell-dependent (TD) or T cell-independent
(TI), depending on whether T cell help is needed to induce an antibody
response.
T-dependent antigens are typically proteins or peptides that are presented by
antigen-
presenting cells to T cells in the context of MHC molecules, leading to T cell
activation. Activated T cells deliver contact- and cytokine-mediated signals
that
promote antibody production, including high affinity antibodies of multiple
isotypes
(Mond et al. (1995) Annu. Rev. Immunol. 13, 655-692; Lesinski & Westerink
(2001)
J. Microbiol. Methods 47, 135-149).
TI antigens are classified into TI types 1 and 2. The TI-1 antigens, such as
LPS, are potent B cell mitogens, which function by non-specifically or
polyclonally
activating most B cells (Lesinski & Westerink (2001) J. Microbiol. Methods 47,
135-
149). The TI-2 antigens, such as polysaccharides, are often large molecules
with
repeated antigenic epitopes, capable of activating the complement cascade, but
lack
the ability to stimulate MHC-dependent T cell help (Mond et al. (1995) Annu.
Rev.
Immunol. 13, 655-692). In an ideal format, TI-2 antigens are typically
flexible, non-
degradable, and hydrophilic, so that they interact simultaneously with
multiple B cell
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receptors (BCRs) (Dintzis et al. (1976) Proc. Natl. Acad. Sci. USA 73, 3671-
3675).
The molecular structure of a classical TI-2 antigen consists of a non-
immunogenic
backbone exhibiting recurring immunogenic epitopes -95-675 A apart. This
periodicity appears to be optimal for simultaneously engaging and cross-
linking
multiple BCRs and rapidly (within -48 h) stimulating IgM responses (Dintzis et
al.
(1983) J. Immunol. 131, 2196-2203; Dintzis et al. (1976) Proc. Natl. Acad.
Sci. USA
73, 3671-3675).
Germinal centers (GC) are microscopically distinguishable structures in
secondary lymphoid tissue where antigen (Ag)-stimulated B cells are induced to
rapidly proliferate, isotype switch, somatically hypermutate, and generate
high-
affinity antibody (Ab)-forming cells and memory B cells. Follicular dendritic
cells
(FDCs) reside in the light zones of germinal centers (GC) and retain Ags in
the form
of immune complexes (ICs). FDCs are prominent in GCs because their numerous
long slender dendrites intertwine and create extensive FDC networks or
reticula.
These FDC networks are fixed in the follicles while T cells and B cells are
free to
circulate. Nevertheless, FDCs release chemokines that attract recirculating
lymphocytes that help organize the follicle and participate in the GC reaction
by
presenting iccosomal antigen that stimulates B cells and provides antigen for
GC B
cells to process and present to GC CD4+ T cells for help. FDCs, residing in
the light
zones of GCs, retain antigens in the form of ICs on numerous long slender
intertwining dendrites. This creates extensive antigen retaining reticula
(ARR),
intimately in contact with numerous mobile B cells (Szakal et al. (1989) Annu.
Rev.
Immunol. 7, 91-109; Szakal et al. (1983) J. Immunol. 131, 1714-1727; Qin et
al.
(2000) J. Immunol. 164, 6268-6275). In GCs, which are typically T-dependent
hot
spots involved in refining humoral immunity, FDC functions include promotion
of B
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cell survival, Ig class switching, production of B memory cells, promoting
somatic
hypermutation, selection of somatically mutated B cells with high affinity
receptors,
affinity maturation, induction of secondary Ab responses and regulation of
high
affinity serum IgG and IgE (Lindhout et al. (1993) Clin. Exp. Immunol. 91, 330-
336;
Lindhout & de Groot (1995) Histochem. J. 27, 167-183; Liu et al. (1991) Eur.
J.
Immunol. 21, 1905-1910; Schwarz et al. (1999) J. Immunol. 163, 6442-6447; Tew
et
al. (1990) Immunol. Rev. 117, 185-211; Qin et al. (1998) J. Immunol. 161, 4549-
4554; Berek & Ziegner (1993) Immunol. Today 14, 400-404; MacLennan & Gray
(1986) Immunol. Rev. 91, 61-85; Kraal et al. (1992) Nature 298, 377-379; Liu
et al.
(1996) Immunity 4, 241-250; Tsiagbe et al. (1992) Immunol. Rev. 126, 113-141;
Tew
et al. (1997) Immunol. Rev. 156, 39-52; Helm et al. (1995) Eur. J. Immunol.
25, 2362-
2369; Kosco et al. (1992) J. Immunol. 148, 2331-2339; Wu et al. (2008) J.
Immunol.
180, 281-290).
TD antigens trapped as ICs on the surface of FDCs are displayed in a periodic
manner, with a characteristic -200-500A spacing (Sukumar et al. (2008) Cell
Tissue
Res. 332, 89-99; Szakal et al. (1985) J. Immunol. 134, 1349-1359). This IC
periodicity on FDCs has been reported in vivo (Szakal et al. (1985) J.
Immunol. 134,
1349-1359) and in vitro (Sukumar et al. (2008) Cell Tissue Res. 332, 89-99).
TD
antigens trapped periodically as ICs on the surfaces of flexible FDC dendrites
with
-200-500A spacing corresponds with T-I-2 antigens with recurring immunogenic
epitopes -95-675 A apart on a flexible backbone (Dintzis et al. (1983) J.
Immunol.
131, 2196-2203; Dintzis et al. (1976) Proc. Natl. Acad. Sci. USA 73, 3671-
3675).
These epitope clusters on FDC dendrites may simultaneously cross-link multiple
BCRs; thus, FDCs may convert TD antigens into TI antigens, capable of inducing
B
cell activation and rapid IgM production in the absence of T cells or T cell
factors.
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Heinemann & Peters (2005) described follicular dendritic-like cells derived
from human monocytes (BMC Immunol. 6, 23; see also WO 2005 / 118779 and EP
04012622.9). These FDC-like cells were derived from their presumed precursors,
monocytes, in vitro. Heinemann & Peters reported a protocol for generating FDC-
like
cells. Using purified human monocytes as a starter population, low
concentrations of
IL-4 (25 U/mL) and GM-CSF (3 U/mL), in combination with dexamethasone (Dex)
(0.5 M) in serum-free medium, triggered the differentiation of monocytes into
FDC-
like cells. After transient de novo membrane expression of alkaline
phosphatase (AP),
such cells highly up-regulated surface expression of complement receptor I
(CD35).
Co-expression of CD68 confirmed the monocytic origin of both the AP+ and CD35+
cells. The common leukocyte antigen CD45 was strongly down-regulated.
Successive
stimulation with TNF-a up-regulated adhesion molecules ICAM-1 (CD54) and
VCAM (CD 106). Both, AP+ and AP- FDC-like cells heterotypically clustered with
and emperipolesed B cells and exhibited the FDC-characteristic ability to
entrap
functionally preserved antigen for prolonged times.
There is long history of immunizing with immune complexes (for review, see,
e.g., Brady (2005) Infection & Immunity 73, 671-678). Further, when immunizing
with ICs, the response is often rapid. "The response of rhesus monkeys to
Venezuela
equine encephalitis vaccine was enhanced by ICs. Remarkably, sustained
protection
was observed in mice just 24 h after ICs that compared with responses 8 days
after
antigen alone." (J. Infect. Dis. 135, 600-610, 1977). Why then are ICs not the
standard for immunization? In short, ICs are not the standard for immunization
because profound suppression is also often observed. The product Rhogam is a
good
example of such suppression (see, e.g., Clynes (2005) J. Clin. Invest. 115, 25-
7).
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SUMMARY OF THE INVENTION
Follicular dendritic cells (FDCs) periodically arrange membrane-bound
immune complexes (ICs) of T-dependent antigens -200-500A apart, leading to the
suggestion that antigen in FDC-ICs can cross-link multiple B cell receptors
(BCRs)
and induce T cell-independent B cell activation. As an example, ovalbumin ICs
on
FDCs were shown to induce purified B cells in vitro and in anti-Thy-1
pretreated nude
mice to produce ovalbumin-specific IgM within -48 h. Moreover, these nude mice
had GL7+ germinal centers (GCs) with IC-retaining FDC-reticula and Blimp-l+
plasmablasts. Rat-anti-mouse IgD (clone 11-26), which did not activate B cells
per se,
was converted to a potent polyclonal B cell activator when loaded as ICs on
FDCs.
FDC-anti-IgD induced high phosphotyrosine levels in caps and patches on
virtually
all purified B cells and strong dose-dependent polyclonal IgM responses within
-48 h.
The present invention comprises the use of FDCs or FDC-like cells to generate
FDC-dependent, but T cell-independent, responses to T cell-dependent antigens,
with
antigen-specific and polyclonal antibody production in -48 h. In embodiments
of the
present invention, ICs were used to load FDCs or FDC-like cells and B cells
were
stimulated in vitro and in vivo in the absence of T cells or T cell factors.
An embodiment of the present invention comprises an in vitro germinal center
(GC) lymphoid tissue equivalent (LTE) where B cells can be induced to produce
specific antibodies, class switch, mutate and produce high-affinity
antibodies. ICs
were used to load FDCs and B cells were stimulated in vivo and in vitro in the
absence of T cells or T cell factors. Our data indicated that IC-challenged
nude mice
produced antigen-specific IgM within -48 h after IC challenge and the response
was
maintained for many weeks. In marked contrast, antigen in adjuvant induced no
antigen-specific IgM at any time. The draining lymph nodes of the IC-
challenged
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mice exhibited well-developed PNA+ and GL7+ GCs associated with Ag-retaining
reticula (ARR) and Blimp-l+ plasmablasts. Moreover, purified FDCs loaded with
ICs
induced purified human and murine B cells to produce antigen-specific IgM in
vitro
in -48 h. Additionally, FDCs loaded with ICs containing anti-delta Abs induced
high
levels of polyclonal IgM within -48 h when cultured with purified B cells.
These anti-
delta-IC stimulated B cells showed capping and patching of intracellular
phosphotyrosine, indicative of B cell signaling. The intensity of
phosphotyrosine
labeling increased, as indicated by increased mean fluorescence intensity, as
the entire
B cell population shifted to the right in flow cytometry. An embodiment of the
present
invention comprises a method of using FDCs, or FDC-like cells, to convert TD
Ags
into TI Ags, capable of inducing B cell activation and Ig production in the
absence of
T cells or T cell factors.
In another embodiment of the present invention, CD4+ T cells were primed
using monocyte-derived dendritic cells (DCs) to present antigen for 10 days in
vitro.
The GC LTE was used to generate specific IgM in the first week, followed by
switching to IgG in response to antigens in the second week. The GC LTE may be
used in predicting problems in immunizing humans when animal experiments fail
to
detect such problems. The GC LTE model of the present invention is a useful
tool for
rapid vaccine assessment.
In another embodiment of the present invention, dual forms of immunogen
were used in the GC LTE, with free antigen being used with the DCs and ICs to
load
FDCs.
In another embodiment of the present invention, this dual immunization
strategy was used in vivo; ICs were targeted to FDCs to initiate an early IgM
response
and expand the specific B cells while free antigen was injected into a
different site to
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target DCs for T cell priming. This dual immunogen strategy resulted in rapid,
specific IgM responses and enhanced IgG responses. Further, ICs promoted
somatic
hypermutation several days earlier in the immune response and this should lead
to
rapid production of high-affinity antibody. These in vivo results were
consistent with
the use of dual forms of immunogen in the GC LTE.
The dual immunization approach of the present invention has wide application
in vaccine design and assessment. For example, people moving to areas with
endemic disease could immunized to provide rapid IgM protection (-24-48 h) and
high-affinity IgG could also be obtained more quickly. Moreover, this
immunization
strategy may be useful for shortening the time need to prepare for booster
immunizations and people with T cell insufficiencies may be immunized, to
rapidly
generate protective IgM.
In another embodiment of the present invention, poor vaccines that are not
currently used may prove to be useful if given as ICs, to induce specific IgM,
or in
dual form, because the resulting Ab response is so much more potent. The
ability of
various poor vaccines to induce specific IgM as ICs can be assessed in the GC
LTE of
the present invention.
In another embodiment of the present invention, we established a germinal
center (GC) lymphoid tissue equivalent (LTE) where B cells could be induced to
produce specific antibodies (Abs), class switch, mutate, and produce high
affinity
antibodies. CD4+ T cells were primed using monocyte derived DCs to present
antigen
(Ag) for -10 days in vitro. Primed CD4+ T cells were mixed with naive B cells
and
FDCs in vitro and media was harvested on days -7 and -14.
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With the GC LTE, specific IgM was obtained in response to ovalbumin
(OVA) in the first week, followed by switching to IgG in the second week. In
addition
to OVA, primary Ab responses with class switching were obtained using
influenza
and anthrax recombinant protective antigen (rPA) as specific antigens.
Moreover,
evidence of affinity maturation was obtained with OVA. In contrast, with HIV
gp 120
a strong IgM response was observed, but we did not see class switching in the
second
week, possibly as a result of gp 120 binding to CD4 and interfering with T
cell
priming. The gp 120-specific IgM response did not class switch and the IgM
response
persisted for 14 days in the GC LTE, suggesting that primed T cells capable of
promoting class switching were lacking. In mice where gp120 does not bind to
CD4
T cells, the murine B cells class switched and normal IgG responses were
obtained.
These gp120 data illustrate how the GC LTE of the present invention may be
useful in predicting problems in immunizing humans when animal experiments
failed
to detect such problems. Thus, the GC LTE model of the present invention is a
useful
tool for the rapid assessment of vaccines and vaccine candidates.
In another embodiment of the present invention, we demonstrated that
T-dependent antigens, such as gp120, can be converted into T-independent
antigens
by presenting them as immune complexes (ICs) for FDCs to trap and arrange in a
periodic fashion on their dendrites. This periodic arrangement allows for
multiple
BCRs to be engaged and IgM responses to T-dependent antigens to be induced in
just
-24-48 h, similar to a TI-2 antigen. These rapid T-independent responses were
demonstrated both in vitro in GC LTEs lacking CD4+ T cells and in T cell-
deficient
animals. Moreover, in normal animals these ICs could induce IgG responses that
were
more than 10 times higher than the responses obtained using free antigen.
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In another embodiment of the invention, we use dual forms of immunogen in
the GC LTE, with antigen being used with the DCs and ICs to load FDCs. We then
tested such a dual immunization strategy in vivo; ICs were targeted to FDCs to
initiate
an early IgM response and expand the specific B cells, while free antigen was
injected
at a different site to target DCs for T cell priming. In combination, rapid,
specific IgM
responses and enhanced IgG responses were induced. Further, the ICs promoted
somatic hypermutation several days earlier in the immune response, leading to
rapid
production of high-affinity Abs. These in vivo results were consistent with
the use of
dual forms of immunogen in the GC LTE.
The data presented here support the novel concept that FDCs can convert TD
Ags into TI Ags, capable of inducing specific IgM responses in -48 h or less
(see Fig.
1). We reasoned that FDCs may have this ability as a consequence of observing
TD-
Ags in ICs on FDCs periodically spaced -200-500 A apart, consistent with the
recurring epitopes -95-675 A apart on the flexible backbone of an ideal TI-2
Ag.
Thus, we suggest that the repeating epitopes of TD-Ags clustered in ICs on the
surface of flexible dendrites of FDCs, or FDC-like cells, should
simultaneously cross-
link multiple BCRs and rapidly induce specific IgM. In contrast, free Ag, that
will not
decorate FDCs, or FDC-like cells, does not induce Ab responses in the absence
of T
cell or T cell factors, even though it would have unfettered access to BCRs.
We demonstrate here that nude mice, pre-treated with anti-Thy-1 to minimize
any residual T cell activity, responded to ICs by producing specific IgM in -
48 h
while free Ag in adjuvant induced no IgM in nude mice even after many weeks.
In
contrast, normal mice challenged with Ag in adjuvant induced detectible IgM in
4 days, followed by IgG (data not shown) and phenotypically normal nu/+ mice
injected with ICs exhibited a rapid IgM response, followed by a switch to IgG,
which
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we attribute to T cell help that is lacking in the nu/nu mice. Moreover, the
draining
lymph nodes of IC-challenged nude mice exhibited well-developed PNA+ and GL7+
GCs, associated with ARR and Blimp-l+ plasmablasts, further supporting the
concept
that B cells in the follicles were stimulated by the ICs on FDCs. In contrast,
GCs and
plasmablasts were lacking in Ag-immunized nude controls, where the B cells
remained in a resting state, consistent with the lack of T cell help.
These in vivo studies were consistent with in vitro experiments, where highly
purified IC-bearing FDCs and naive B cells from humans or mice were co-
cultured in
the absence of T cells or T cell factors. B cells stimulated with IC bearing
FDCs in
these cultures produced specific IgM in -48 h, while no response was observed
when
ICs were replaced with free Ag. Both the kinetics of the response and the IgM
production are consistent with TI responses. Further, rat anti-mouse IgD mAb
clone
11-26, which by itself does not activate B cells, became a potent B cell
activator when
made into an IC and loaded on FDCs. Activation was indicated by increased
tyrosine
phosphorylation in virtually all B cells, along with patching and capping.
Moreover,
this signaling appeared to be productive, in that FDCs bearing this IC induced
polyclonal IgM in -48 h, consistent with a TI response. Thus, we conclude that
TD
Ags can induce specific IgM responses in an FDC-dependent, but T cell-
independent
fashion.
This concept that TD antigens can trigger B cells when appropriately arranged
is supported by several literature reports. Studies on the requirements for
generation
of Ab responses to repetitive determinants on polymers, polysaccharides and
higher
order structures, such as viral capsid proteins, have indicated that high
molecular
weight arrays of Ag can be efficient in eliciting an Ab response independent
of T-cell
help, while their less ordered counterparts are less immunogenic and require T-
cell
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help (Rosenberg (2006) AAPSJ. 8, E501-E507; Vos et al. (2000) Immunol. Rev.
176,
154-170; Bachmann & Zinkemagel (1997) Annu. Rev. Immunol. 15, 235-270).
Certain bacteria, viruses, mammalian cells, some polymeric proteins, such as
collagen, and hapten-protein complexes have antigenic determinants in multiple
repeats. The multivalent presentation of antigenic determinants extensively
cross-
links BCRs and leads to B cell activation, proliferation, and Ig secretion
that is
characteristic of TI-2 responses. For example, multimerization of monomeric
proteins
by aggregation facilitates presentation of their Ag determinants in a highly
arrayed
structure fit for cross-linking BCRs and inducing Ab responses in the absence
of T
cell help (Rosenberg (2006) AAPSJ. 8, E501-E507).
It is important to appreciate that FDC accessory activity extends beyond
delivering the primary BCR-mediated signal via Ag in the ICs. FDCs also
deliver
secondary or co-stimulatory signals to B cells that are important for optimal
B cells
activation. For example, CD21L on FDCs engages CD21 in the B cell co-receptor
complex and CD21L-CD21 interactions not only promote Ag specific responses but
also polyclonal responses induced by LPS (Carter et al. (1997) J. Immunol.
158,
3062-3069; Qin et al. (1998) J. Immunol. 161, 4549-4554). In addition, FDC-
BAFF
and -8D6 inhibit B cell apoptosis (Li et al. (2004) Blood 104, 815-821; Ng et
al.
(2005) Mol. Immunol. 42, 763-772; Hase et al. (2004) Blood 103, 2257-2265; Qin
et
al. (1999) J. Immunol. Methods 226, 19-27; Schwarz et al. (1999) J. Immunol.
163,
6442-6447); FDCs block IC mediated ITIM signaling in B cells via FcyRIIB and
minimize this inhibitory pathway (Qin et al. (2000) J. Immunol. 164, 6268-
6275;
Aydar et al. (2003) J. Immunol. 171, 5975-5987; Aydar et al. (2004) Eur. J.
Immunol.
34, 98-107); FDCs provide IL-6 for terminal B cell differentiation (Kopf et
al. (1998)
J. Exp. Med. 188, 1895-1906), and FDC-C4BP engages B cell CD40 (Gaspal et al.
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(2006) Eur. J. Immunol. 36, 1665-1673) for a classical activation signal.
Without
wanting to be bound by any mechanism, we believe that the multiple accessory
signals provided by FDCs make it possible to get robust IgM production in the
absence of T cell help.
The short time required to get FDC-dependent TI responses may have
practical application. For example, it may be important in rapidly countering
infectious agents. We note a study showing protection against Venezuelan
equine
encephalitis just 24 h after injecting ICs; in contrast, 8 days were required
for
comparable protection when immunizing with free Ag (Houston et al. (1977) J.
Infect. Dis. 135, 600-6 10). The mechanism for this rapid protection was not
explained, but rapid induction of specific Ab by FDC-ICs could be the
explanation for
a rapid protective response after injecting ICs but not Ag.
Other applications include countering the negative effect of regulatory T
cells
and the non-responder state as a consequence of a limited MHC-II repertoire
that may
be unable to load certain peptides. Individuals who fail to respond to a
vaccine, as a
consequence of problems with Ag presenting cells or the effect of T regulatory
cells,
should mount rapid specific IgM responses when immunized with appropriate ICs.
The ICs should load on FDCs and bypass limitations imposed by MHC and T cells.
Similarly, in other embodiments of the present invention, IgM responses can
be induced in animals or people with congenital and/or acquired T cell
insufficiencies
(Grunebaum et al. (2006) Immunol. Res. 35, 117-126), including HIV-infected
(Cowley (2001) Lepr. Rev. 72, 212-220), aged (Fulop et al. (2005) Drugs Aging
22,
589-603), diabetic ( Spatz et al. (2003) Celllmmunol. 221, 15-26), uremic
(Moser et
al. (2003) Biochem. Biophys. Res. Commun. 308, 581-585), and neonatal (Garcia
et
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al. (2000) Immunol. Res. 22, 177-190; Velilla et al. (2006) Clin. Immunol.
121, 251-
259) animals or people.
The present invention comprises using follicular dendritic cells (FDCs), or
FDC-like cells, to convert T cell-dependent antigens (TD Ags) into T-
independent
antigens (TI Ags), capable of inducing B cell activation and immunoglobulin
production in the absence of T cells and T cell factors, within -48 hours.
Monomeric proteins generally have only a single copy of each antigenic
determinant making them unable to cross-link multiple BCRs and activate B
cells in
the absence of MHC-restricted T cell help. The ability of FDCs to retain ICs
in a
periodic manner allows multimerization of these monomers and facilitates the
multivalent presentation of their antigenic determinants in an array suitable
for cross-
linking multiple BCRs and inducing Ab responses in the absence of T cell help.
Studies on the requirements for generation of Ab responses to repetitive
determinants
on polymers, polysaccharides and higher order structures, such as viral capsid
proteins, have indicated that high molecular weight arrays of Ag are efficient
in
eliciting an Ab response independent of T-cell help, whereas their less
ordered
counterparts are less immunogenic and require T-cell help. Our results are
consistent
with these observations.
Moreover, these results have potential applications in preparing vaccines. For
example, the negative effect of regulatory T cells and the non-responder state
as a
consequence of a limited MHC-II repertoire that may be unable to load certain
peptides may be circumvented. Individuals who fail to respond to a vaccine, as
a
consequence of problems with antigen-presenting cells or the effect of T
regulatory
cells, should still mount rapid specific IgM responses when immunized with
appropriate ICs. The ICs should load on FDCs and bypass limitations imposed by
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MHC and T cells. Similarly, specific IgM responses should be inducible in
animals or
people with congenital and/or acquired T cell insufficiencies, including HIV-
infected,
aged, diabetic, uremic and neonatal animals or people.
The rapidity with which FDC-dependent T cell-independent responses can be
induced also has practical relevance. For example, it may be important in
rapidly
countering infectious or toxic agents, where a response in -24-48 h may be
efficacious. We are impressed by studies showing protection against Venezuelan
equine encephalitis -24 h after injecting ICs; in contrast, 8 days were
required for
comparable protection when immunizing with free Ag (Houston et al. (1977) J.
Infect. Dis. 135, 600-6 10). The mechanism for this rapid protection was not
explained, but rapid induction of specific Ab by FDC-ICs could explain a rapid
protective response after injecting ICs, but not Ag.
The present invention is thus directed to methods for determining whether a
test agent is antigenic, comprising (a) contacting an in vitro germinal center
(GC)
lymphoid tissue equivalent (LTE) with a test agent under conditions promoting
production of IgM, wherein the in vitro GC LTE comprises B cells and
follicular
dendritic cells (FDCs) or FDC-like cells, wherein the follicular dendritic
cells (FDCs)
or FDC-like cells are loaded with immune complexes (ICs) comprising at least a
portion of the test agent, and (b) assaying the in vitro GC LTE of (a) for IgM
production, wherein when production of agent-specific IgM is found in (b), the
test
agent is determined to be antigenic. Preferably the B cells of the in vitro GC
LTE are
exposed to the test agent prior to contacting of the in vitro GC LTE with the
test
agent. Also preferably the test agent is a peptide, a polypeptide, a protein
or a
polysaccharide.
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The present invention is also directed to methods for determining whether a
vaccine formulation is antigenic, comprising (a) contacting an in vitro
germinal center
(GC) lymphoid tissue equivalent (LTE) with a vaccine formulation under
conditions
promoting production of IgM, wherein the vaccine formulation comprises at
least one
antigen and wherein the in vitro GC LTE comprises B cells and follicular
dendritic
cells (FDCs) or FDC-like cells, wherein the follicular dendritic cells (FDCs)
or FDC-
like cells are loaded with immune complexes (ICs) comprising at least a
portion of the
antigen comprising the vaccine formulation; and (b) assaying the in vitro GC
LTE of
(a) for IgM production, wherein when production of antigen-specific IgM is
found in
(b), the vaccine formulation is determined to be antigenic. Preferably, the B
cells of
the in vitro GC LTE are exposed to the antigen prior to contacting of the in
vitro GC
LTE with the vaccine. Also preferably the antigen is a peptide, a polypeptide,
a
protein or a polysaccharide.
The present invention is further directed to methods for determining the
antigenicity of a vaccine formulation, comprising (a) contacting an in vitro
germinal
center (GC) lymphoid tissue equivalent (LTE) with a vaccine formulation under
conditions promoting production of IgM, wherein the vaccine formulation
comprises
at least one antigen and wherein the in vitro GC LTE comprises B cells and
follicular
dendritic cells (FDCs) or FDC-like cells, wherein the follicular dendritic
cells (FDCs)
or FDC-like cells are loaded with immune complexes (ICs) comprising at least a
portion of the antigen comprising the vaccine formulation, and (b) determining
the
amount of IgM produced by the in vitro GC LTE of (a), wherein the amount of
antigen-specific IgM determined in (b) corresponds to the antigenicity of the
vaccine
formulation, thereby determining the antigenicity of a vaccine formulation.
Preferably, the B cells of the in vitro GC LTE are exposed to the antigen
prior to
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contacting of the in vitro GC LTE with the vaccine. Also preferably the
antigen is a
peptide, a polypeptide, a protein or a polysaccharide.
The present invention is additionally directed to methods for determining the
antigenicity of a vaccine formulation, comprising (a) contacting an in vitro
germinal
center (GC) lymphoid tissue equivalent (LTE) with a vaccine formulation under
conditions promoting production of IgM, wherein the vaccine formulation
comprises
at least one antigen and wherein the in vitro GC LTE comprises B cells and
follicular
dendritic cells (FDCs) or FDC-like cells, wherein the follicular dendritic
cells (FDCs)
or FDC-like cells are loaded with immune complexes (ICs) comprising at least a
portion of the antigen comprising the vaccine formulation; (b) collecting IgM
produced by the in vitro GC LTE of (a), and (c) determining the affinity of
the
antigen-specific IgM collected in (b) for the antigen, wherein the affinity of
the
antigen-specific IgM determined in (c) for the antigen corresponds to the
antigenicity
of the vaccine formulation, thereby determining the antigenicity of a vaccine
formulation. Preferably the B cells of the in vitro GC LTE are exposed to the
antigen
prior to contacting of the in vitro GC LTE with the vaccine. Also preferably
the
antigen is a peptide, a polypeptide, a protein or a polysaccharide.
The present invention is also directed to methods for determining whether a
two-component vaccine system is antigenic, comprising (a) contacting an in
vitro
germinal center (GC) lymphoid tissue equivalent (LTE) with a first component
of a
two-component vaccine system under conditions promoting production of IgM,
wherein the first component of the two-component vaccine system comprises an
antigen and wherein the in vitro GC LTE comprises B cells and follicular
dendritic
cells (FDCs) or FDC-like cells, wherein the follicular dendritic cells (FDCs)
or FDC-
like cells are loaded with immune complexes (ICs) comprising at least a
portion of the
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antigen comprising the first component of the two-component vaccine system,
(b)
contacting the in vitro GC LTE of (a) with a second component of the two-
component
vaccine system under conditions promoting production of IgM, wherein the
second
component of the two-component vaccine system comprises the antibody and the
portion of the antigen of the ICs of (a); and (c) assaying the in vitro GC LTE
of (b) for
IgM production, wherein when production of antigen-specific IgM is found in
(c), the
vaccine is determined to be antigenic.
In this method, preferably the B cells of the in vitro GC LTE are exposed to
the first component of the two-component vaccine system prior to contacting of
the in
vitro GC LTE with first component of the two-component vaccine system. In a
further preferred embodiment, the B cells of the in vitro GC LTE are exposed
to the
second component of the two-component vaccine system prior to contacting of
the in
vitro GC LTE with first component of the two-component vaccine system.
Also preferably, the antibody of the second component binds the portion of the
antigen of the ICs of (a). Preferably the antigen is a peptide, a polypeptide,
a protein
or a polysaccharide.
The present invention is moreover directed to methods for generating IgM
antibodies, comprising (a) contacting an in vitro germinal center (GC)
lymphoid
tissue equivalent (LTE) with an antigen, wherein the in vitro GC LTE comprises
B
cells and follicular dendritic cells (FDCs) or FDC-like cells, wherein the
follicular
dendritic cells (FDCs) or FDC-like cells are loaded with immune complexes
(ICs)
comprising at least a portion of the antigen; and (b) culturing the in vitro
GC LTE of
(a) under conditions promoting generating of IgM antibodies, thereby
generating IgM
antibodies.
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In preferred embodiment the culturing (b) is for about 48 hours or about 72
hours. The method may also comprise collecting IgM antibodies generated in
(b).
The culturing (b) may continue until antibody class switching is achieved;
preferably
the class switching is switching from IgM production to IgG production. Also
preferably the antigen is a peptide, a polypeptide, a protein or a
polysaccharide.
The present invention is also directed to two-component vaccine systems
comprising a first component and a second component, wherein the first
component
comprises an antigen and wherein the second component comprises an immune
complex of the antigen of the first component. In a preferred embodiment the
first
component further comprises a pharmaceutically acceptable carrier or diluent
and the
second component further comprises a pharmaceutically acceptable carrier or
diluent.
In a related embodiment the present invention is directed to methods of
inducing an immune response in a subject comprising (a) administering a first
component of a two-component vaccine system to a subject, wherein the first
component comprises an antigen and pharmaceutically acceptable carrier or
diluent;
and (b) administering a second component of the two-component vaccine system
to
the subject, wherein the second component comprises an immune complex of the
antigen of the first component, and pharmaceutically acceptable carrier or
diluent.
In preferred embodiments the second component of the two-component
vaccine system is administered to a different location of the subject than the
first
component of the two-component vaccine system. The first and second components
of the two-component vaccine system may be administered concurrently or
sequentially to the subject. The immune response is a rapid production of high-
affinity antibodies, preferably high-affinity IgM antibodies or high-affinity
IgG
antibodies. In one embodiment the high-affinity antibodies are produced within
about
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24 hours after administration of the two-component vaccine system. In a
further
embodiment the immune response is a protective immune response. Preferably the
antigen is a peptide, a polypeptide, a protein or a polysaccharide.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1. Model of FDC-dependent T-independent B cell activation and Ig
production.
A: Monomeric proteins generally express only a single copy of each antigenic
determinant making them unable to cross-link multiple BCRs and activate B
cells in
the absence of T cell help.
B: TI-2 Ags contain numerous periodically arranged epitopes (green
protrusions) attached to a flexible backbone (red curve). This arrangement
allows
extensive simultaneous cross-linking of BCRs (Y-shaped green). The multiple
BCR
cross-linking delivers a signal leading to B cell activation and Ig
production.
C: FDCs express high levels of FcyRIIB (red) and CRs (blue), which trap ICs
containing TD Ags (multi-color clusters) in a periodic arrangement -200-500A
apart.
We reasoned that this spatial arrangement would allow cross-linking of
multiple
BCRs specific for a single epitope, leading to B cell activation and Ig
production as in
panel B.
D: Transmission electron micrograph showing HRP (horseradish peroxidase, a
TD Ag) retained on the FDC surface in IC clusters -200-500A apart. This
facilitates
BCR cross-linking and B cell activation as explained in panels B and C.
E: FDCs accessory activity includes secondary signals or co-signals that
promote B cell activation and Ig production. Specifically, FDCs are decorated
with
the complement-derived CD21 L which will engage B cell CD21. Binding CD21 in
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the CD21-CD19-CD81 complex delivers a positive co-signal for B-cell activation
and differentiation, FDC-derived BAFF ligates BAFF receptors on B cells, and
FDC-
derived C4b-binding protein (C4BP) ligates B cell-CD40, a classical co-signal
in B
cell activation.
Figure 2. Anti-delta IC-retaining FDCs and FDCs-like cells induce rapid (in
-48 h) T-cell independent IgM production in vitro.
Figure 3. OVA-specific IgM after -48 h in a GC LTE without CD4+ T cells.
Figure 4. Rapid T-independent IgM response induced by ICs on FDCs. T
cell-depleted in vitro cultures showing anti-ovalbumin (OVA) IgM production
within
-48 h of culturing naive B lymphocytes with IC-loaded FDCs, indicating the
T-independent nature of the response. However, as indicated in the final
column, the
presence of T cells, likely producing some cytokines, did promote the IgM
response,
without resulting in any IgG.
This figure illustrates a T-independent response in the absence of any T
cells.
Anti-thyl was used to remove T cells; such removal is virtually complete. An
IgM
response was apparent in the absence of T cells. Clearly, the IgM anti-OVA
response
was stronger in the presence of T cells, but it did occur in the absence of T
cells.
Figure 5. Comparison of conventional versus dual immunogen immunization.
Antigen in adjuvant would be expected to target DCs, which would lead to
effective T
cell priming and the ICs would be expected to target FDCs, that would select
and
expand specific B cell populations, such that very rapid and sustained
specific Ab
responses could be developed. By using such dual forms of immunogen, the
promise
of vaccine enhancement via ICs may finally be realized. As an example, we
injected
antigen in adjuvant on one side of an animal, to prime T cells, and ICs on the
other
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side, to target FDCs, and to generate an early IgM response and get specific B
cells
selected and expanded. The ICs were made using OVA haptenated with NIP and
anti-NIP to make the ICs such that the antibody will not interact with OVA on
the T
cell side of the animal and cause any feedback inhibition. The results are
shown in
Figure 5. Note that ICs did give a potent early IgM response at day 2, while
Ag in
adjuvant did not. At day 7, IgM was present for both forms of immunogen, but
by day
14 the IgM response for both forms of immunogen was low, consistent with
helper T
cell activity and class switching. This was not seen at 14 days, or even 28
days, when
only the IC was used as an immunogen. The IgG response is shown in the second
panel. Note that both conventional and dual forms of immunogen gave IgG at
days 7
and 14, but that the dual form of immunogen was stronger. In short, the two
forms of
immunogen resulted in an early IgM response and an enhanced IgG response.
Figure 6. Immune complexes promote Ab production and somatic
hypermutation (SHM). Mice were irradiated with 600 rads and reconstituted with
negatively selected naive k+ B cells and memory T (CGG) cells. These mice were
divided into two groups with one receiving 5 g of NP-CGG in preformed ICs in
the
hind foot pads and front legs. The control group received 5 g of NP-CGG in
each
hind foot or front leg. After 7 days, k+ B cells were isolated from lymph
nodes and
analyzed for VH186.2 mutations.
Panel a illustrates NIP-specific IgG measured by ELISA in 3 mice per group
and the error bar represents SD and the differences are statistically
significant
(p<0.01) and the data are representative of two experiments.
Panel b illustrates the number of mutations per 1000 bases of VH186.2 clones
sequenced. The k+ B cells from the draining lymph nodes of the three mice were
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pooled and RNA extracted. We analyzed 10 clones in each condition and the
difference in mutation frequency was statistically significant (p < 0.01).
Panels c & d show representative illustrations of 10 m thin sections of
draining lymph nodes from the two groups of mice labeled with anti-GL7 to
identify
GC B cells.
Panels e & f shows cumulative data representing total number of GCs and
area of GCs per mid-sagittal section of all six mice challenged with Ag or IC.
Figure 7. Correlation of specific antibodies with somatic hypermutation
(SHM). FDCs enhanced NIP-specific IgG production by B cells isolated 6 days
after
primary immunization, but SHM required FDCs plus an additional encounter with
immunogen. GC reactions were initiated by culturing 1 X 106 unmutated but 6
day
primed B cells, 0.5x 106 T cells, and 0.5 X 106 FDCs in the presence of 100 ng
of
NP(36)-CGG as free Ag or in ICs. The contents in each culture are indicated
across
the bottom and after 7 days of culture, supematant fluids were collected for
NIP-
specific IgG assays and cell pellets were collected for RNA extraction. Panel
a shows
NIP-specific IgG production and Panel b illustrates mutations per 1000 bases
of the
VH186.2 clones recovered from the same cultures as in panel A. The rate of
mutations per 1000 bases in each of the six conditions was calculated after
analyzing:
10, 13, 20, 10, 14, & 14 VH186.2 clones, respectively.
Figure 8. T cells were primed with monocyte-derived DCs. Monocytes were
cultured with IL-4 (1000 U/mL) and GM-CSF (800 U/mL) to generate immature
DCs. After 5 days, OVA (1 g/mL) was added to provide Ag for processing + LPS
(1
g/mL) for DC maturation. This was done in autologous serum to avoid priming
for
antigens in fetal calf serum. After 8 h, CD4+ T cells were added for OVA
priming.
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The priming and maturation for helper T cells was allowed to go for 10 days.
After
6 6
priming the 4X 10 T cells and DCs were mixed with naive B cells (4X 10 cells)
and
6
1 x 10 FDCs. Nine million cells total in a 24 well plate with 3 ml media in
10%
autologous serum. OVA (5 g) + murine anti-OVA (30 g) were complexed (OVA
ICs) and the ICs were placed in vitro to load on FDCs. Supematant fluids were
collected on days 7 and 14. Three mL of media were collected each time. 8A.
Human
in vitro primary Ab response: production of OVA-specific IgM. 8B. shows the
IgG
data. In contrast to IgM, the IgG response was low in the first week and then
the
response switched to all IgG in the second week. Only the combination of FDCs
with
ICs gave a detectible response (by ELISA).
Figure 9. Human IC-driven anti-gp 120 response after blocking CD4 during T
cell priming. A strong IgM response was seen, but class switching did not
occur, as
illustrated in Figure 9. We think the lack of an IgG response was likely
attributable to
gp 120 binding CD4 and interfering with T cell priming. We attribute the
strong
gp120-specific IgM response to the ability of FDCs to arrange ICs on their
surfaces
with periodicity. This periodicity is consistent with the periodicity of
independent
antigens which would give the good IgM responses in the absence of primed T
cells.
Figure 10. Mouse gp120-specific in vivo immune response. A common way
to assess potential immunogens is to start by injecting them into animals.
Those
immunogens that respond well in animals are candidates for further study.
Consider
what happens to Ig class switching when gp120 was injected into mice, as
illustrated
in Figure 10. The murine response to gp129 was good, with IgM responses that
class-
switched to IgG by day 14 as expected. There was no indication that gp120
would
not be a good vaccine candidate from these murine data. The GC LTE predicted
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problems with free, soluble gp 120 that can bind human CD4 when priming human
T
cells and T cell help is necessary for IgG class switching. In contrast,
soluble free
gp120 looks like a good vaccine candidate in an animal model, where IgG class
switching occurred perfectly normally. It should be appreciated that gp 120
will not
bind murine CD4 and would not interfere with T cell priming. Nevertheless, gp
120 on
the virus in vivo does induce a good gp120 response. Perhaps use of gp120 in a
particle, mimicking the virus, might not block T cell priming as well as the
free
molecule that would behave more like a cytokine. Thus, designing the vaccine
differently might give a different result. However, it seems unlikely that
free gp 120 is
going to be a good immunogen in humans and only the in vitro GC LTE provided
that
information.
Figure 11. Effect of alum-pertussis adjuvant on immunogenicy of ICs. FDCs
bear TLR4 and other TLRs on their surfaces. Moreover, LPS activates FDCs and
enhances their ability to stimulate antibody responses in vitro and promote
somatic
hypermutation. We examined whether adjuvant would improve the ability of ICs
to
promote Ab responses. The results illustrated in Figure 11 showed that ICs in
adjuvant and ICs alone appeared to have comparable ability to induce OVA-
specific
IgG. This is an examples where the ICs were able to induce IgG without adding
memory T cells or Ag to prime T cells. Nevertheless, adding adjuvant to the
ICs
resulted in a dramatic enhancement of the IgG responses, consistent with our
data
indicating that FDCs have TLR receptors and are activated by engagement of
these
receptors. In a preferred embodiment of the present invention, both the Ag and
the
ICs should be in adjuvant when immunizing with the dual immunization approach,
based on the results illustrated here.
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Figure 12. T-dependent Ag induced IgM in nude mice and the IgM response
was enhanced by use of adjuvant. IgM responses were rapid and sustained in
nude
mice with ICs as the immunogen (residual T cell activity was blocked with 50
g
anti-Thy-l, i.p., at the time of immunization). OVA in adjuvant failed to
induce a
detectible IgM response in nude mice, as was expected. In marked contrast, OVA
ICs induced a significant IgM response and that response was dramatically
enhanced
by the use of ICs with adjuvant. These data support the concept that use of
ICs may be
able to provide protection in people with T cell insufficiencies where Ag
fails to give
a response, as illustrated here with nude mice, or a very poor response. Human
immunoinsufficiencies are seen, in e.g., AIDS patients, the aged, uremics,
diabetics,
and alcoholics.
Figure 13. Nude mice challenged with OVA ICs, but not with OVA, mounted
OVA-specific immune responses in -48 h and developed GCs.
A: Groups of nu/nu mice, pre-treated with 50 g anti-Thy-1 to block residual
T cell activity, were challenged with alum precipitated OVA with Bordetella
pertussis, OVA-ICs or OVA-ICs with Bordetella pertussis. Serum anti-OVA IgM
levels were determined 48 hours, 1 week and 2 weeks later and results were
recorded
after subtracting background levels using pre-immunization sera. As expected,
anti-
OVA was not detectible in animals immunized with OVA in adjuvant (baseline
tracking). In marked contrast, OVA-specific IgM was present in the sera of all
ICs
injected animals with or without adjuvant in just 48 hrs and was maintained
over a 7-
week assessment period.
B: Mid-saggittal sections from the draining popliteal lymph nodes of IC-
challenged nu/nu mice were labeled for GC B cells with peroxidase-conjugated
peanut agglutinin (PNA) 7 weeks after challenge with ICs. Well-developed PNA+
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GCs were observed in these draining lymph nodes further supporting FDC-IC
mediated B cell activation.
C: Phenotypically normal heterozygous nu/+ mice with competent T cell
compartment also responded to ICs by producing OVA-specific IgM within 48
hours,
although, these IgM levels declined over time
D: The phenotypically normal nu/+ also produced IgG and the increase in this
isotype correlated with a decrease in IgM. Note that neither class switching
nor OVA-
specific IgG was detectable in nu/nu mice lacking T cell help (baseline
tracking).
Figure 14. Purified OVA-IC-bearing FDCs induced OVA-specific IgM
production by purified B cells within -48 h in the absence of T cells.
Purified murine
or human B cells were incubated with purified OVA-IC-loaded FDCs at a ratio of
1FDC:2B cells and OVA-specific Abs were assessed after 48 hours. A: murine and
B:
human B cells. B cells stimulated with FDCs bearing OVA ICs produced OVA-
specific IgM in -48 h. Control conditions, that failed to produce a detectable
response, included FDCs with B cells stimulated with free OVA that would have
had
free access to BCR. The data are representative of two experiments of this
type.
Figure 15. Purified FDCs bearing anti-IgD ICs on their surfaces induced
polyclonal IgM production by purified B cells within -48 h. Given that B cells
are
signaled by anti-delta ICs on FDCs, we reasoned that the simultaneous
engagement of
multiple B cell receptors should signal, at least some of these B cells
adequately, to
rapidly produce IgM (models on left). FDCs to B cells was held constant at
1FDC :
4 B cells. Rat anti-mouse IgD (mAbl 1-26) was held constant at 0.1 or 1 g/mL
with
FDCs. The goat anti-rat to form ICs with the rat anti-Ig delta was used at a
ratio of 6
goat antibodies to 1 rat anti-mouse IgD mAb. The anti-IgD immune complexes in
the
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second, third and fourth columns showed almost nothing over the level without
any
IgD, indicated in the first column. This was the expected result, given that
there was
no second signal from IL-4 or anti-CD40 for the B cell. However, addition of
FDCs
with the ICs gave a potent response. Here, we show a TI response without any
factors
beyond those provided by ICs and FDCs. Results showed that B cells stimulated
with
as low as 100 ng anti-IgD ICs loaded on FDCs produced IgM within -48 h in a B
cell
number-dependent fashion. In the absence of FDCs, anti-IgD ICs did not induce
production of IgM even at doses of 10 g/mL (data on right).
DETAILED DESCRIPTION OF THE INVENTION
One of the primary embodiments of the present invention is methods for
determining whether a particular agent, an antigen or a vaccine formulation
might
function in the production of protective immunity in a subject upon
administration of
the agent, antigen or vaccine formulation.
Many of the methods of the present invention use in vitro germinal center
(GC) lymphoid tissue equivalents (LTEs). As used herein, GC LTEs are comprised
of a co-culture of B cells and follicular dendritic cells (FDCs) or FDC-like
cells
(Heinemann & Peters (2005) BMC Immunol. 6, 23; WO 2005 / 118779; EP
04012622.9). In addition to B cells and FDCs, the GC LTEs of the present
invention
may comprises T cells. In preferred embodiments, all the cells are human
cells.
GC LTEs are described, for example, in US 2007/0218054 (WO 07/075979),
which discloses the incorporation of GCs into three-dimensional (3D)
engineered
tissue constructs (ETCs). The preparation of GC LTEs is described in the
Examples of
US 2007/0218054. In an embodiment of the invention described therein, the GC
was
incorporated in the design of an artificial immune system (AIS) to examine
immune
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(especially humoral) responses to vaccines and other agents. In a further
embodiment
of that invention, development of an in vitro GC added functionality to an
AIS, in that
it enabled generation of an in vitro human humoral response by human B
lymphocytes that is accurate and reproducible, without using human subjects.
The
invention also enabled the evaluation of, for example, vaccines, allergens,
and
immunogens and activation of human B cells specific for a given antigen, which
can
then be used to generate antibodies. Embodiments of that invention comprised
placing follicular dendritic cells (FDCs) in an ETC, such as a collagen
cushion,
microcarriers, inverted colloid crystal matrices, or other synthetic or
natural
extracellular matrix material, where they could develop in three dimensions.
FDCs in
the in vivo environment were attached to collagen fibers and did not
circulate, as most
immune system cells do. Thus, placing FDCs in, for example, a collagen matrix
ought to be more in vivo-like.
FDCs are localized to the lymph follicles and they assist in B cell maturation
by the presentation of intact antigen to the B cells. Such presentation occurs
in the
germinal centers of peripheral lymphoid organs and also results in class
switching and
B cell proliferation. FDCs present antigens to B cells in the form of an
immune
complex (IC), which is comprised of antigens and antibodies bound thereto. In
vivo,
immunogens are quickly converted into immune complexes (ICs) by antibodies
persisting in immune animals from prior immunization(s) and ICs form in
primary
responses as soon as the first antibody is produced. These ICs are trapped by
FDCs
and this leads to GC formation. Immune complexes are typically poorly
immunogenic in vitro, yet minimal amounts of antigen (converted into ICs in
vivo)
provoke potent recall responses.
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FDCs render ICs highly immunogenic. In fact, in the presence of FDCs, ICs
are more immunogenic than free antigen (Tew et al. (2001) Trends Immunol. 22,
361-
367). A high density of FcyRIIB on FDCs bind Ig-Fc in the IC and consequently
the
ITIM (immunoreceptor tyrosine-based inhibitory motif) signal delivered via B
cell-
FcyRIIB may be blocked. Antigen-antibody complexes cross-linking BCRs initiate
this inhibitory signal and FcyRIIB on B cells. BCR is not cross-linked with B
cell
FcyRIIB in the model and thus a high concentration of FcyRIIB on FDCs
minimizes
the negative signal to the B cell. In addition, FDCs provide IC-coated bodies
(iccosomes), which B cells find highly palatable. The iccosome membrane is
derived
from FDC membranes that have antigen, CD21L, and Ig-Fc attached. Iccosomes
bind
tightly to B cells and are rapidly endocytosed (Szakal et al. (1988) J.
Immunol. 140,
341-353). Binding of BCR and CD21 of the B cell to the iccosomal antigen-CD21L-
Ig-Fc complex is likely important in the endocytosis process. The B cells
process this
FDC-derived antigen, present it, and thus obtain T cell help (Kosco et al.
(1988) J.
Immunol. 140, 354-360). Thus, these ligand-receptor interactions help
stimulate B
cells and provide assistance beyond that provided by T cells.
ICs trapped by FDCs lead to GC formation. GC formation is involved in the
production of memory B cells, somatic hypermutation, selection of somatically
mutated B cells with high affinity receptors, affinity maturation, and
regulation of
serum IgG with high affinity antibodies (Tew et al. (1990) Immunol. Rev. 117,
185-
21 l; Berek & Ziegner (1993) Immunol. Today 14, 400-404; MacLennan & Gray
(1986) Immunol. Rev. 91, 61-85; Kraal et al. (1982) Nature 298, 377-379; Liu
et al.
(1996) Immunity 4, 241-250; Tsiagbe et al. (1992) Immunol. Rev. 126, 113-141).
The GC is generally recognized as a center for production of memory B cells;
we have found that cells of the plasmacytic series are also produced (Kosco et
al.
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(1989) Immunol. 68, 312-318; DiLosa et al. (1991) J. Immunol. 1460, 4071-4077;
Tew et al. (1992) Immunol. Rev. 126, 1-14). The number of antibody-forming
cells
(AFCs) in GCs peaks during an early phase (about 3 to about 5 days after
secondary
antigen challenge) and then declines. By about day 10 when GCs reach maximal
size,
there are very few AFCs present (Kosco et al. (1989) Immunol. 68, 312-318).
During
the early phase, GC B cells receive signals needed to become AFCs. The GC
becomes
edematous and the AFCs leave and we find them in the thoracic duct lymph and
in the
blood. These GC AFCs home to bone marrow where they mature and produce the
vast majority of serum antibody (DiLosa et al. (1991) J. Immunol. 1460, 4071-
4077;
Tew et al. (1992) Immunol. Rev. 126, 1-14; Benner et al. (1981) Clin. Exp.
Immunol.
46, 1-8). In the second phase, which peaks about 10-14 days after challenge,
GCs
enlarge, and the memory B cell pool is restored and expanded. Thus, production
of B
memory and fully functional and mature antibody responses appears to require
GCs
and FDCs.
Potentiating B cell viability can be done with or without FDCs present to
enhance in vitro GC efficacy. A method is to add fibroblasts or other stromal
cells,
such as synovial tissue-derived stromal cell lines, the effects of which are
to prolong
B cell viability in vitro through cell-cell co-stimulation (e.g., Hayashida et
al. (2000)
J. Immunol, 164, 1110-1116). Another soluble agent that has been shown to
increase
naive and memory B cell viability is reduced glutathione (GSH), perhaps
through
anti-oxidant activity (see Jeong et al. (2004) Mol. Cells 17, 430-437).
Although
Jeong et al. did not see enhanced viability of GC B cells, they did
significantly
enhance naive and memory B cells with fibroblasts and GSH, suggesting that
peripheral B lymphocytes can be used to populate the in vitro GC. Other
soluble
factors, such as IL-4, CD40L and anti-CD40 have been shown to potentiate B
cell
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viability (L. Mosquera's work and M. Grdisa (2003) Leuk. Res. 27, 951-956).
Ancillary factors and cells that increase B cell viability with or without
FDCs will
enhance in vitro GC performance.
In vivo FDCs exist in networks linked to collagen and collagen associated
molecules. This linkage allows networks of FDCs to remain stationary while B
cells
and T cells move in and out of contact with the FDCs and associated antigen.
This
arrangement has been reconstructed in the in vitro GCs of the present
invention.
Vaccination Site Model. Dendritic cells (DCs) are among the most potent
antigen-presenting cells (APCs) and are the only known cell type with the
capacity to
stimulate naive T cells in a primary immune response. Peripheral blood
monocytes
are widely accepted as a reliable source of precursor cells for DC generation
in vitro.
Such monocyte-derived DCs (mo-DCs) posses the overall phenotype and antigen-
presenting abilities found in DCs in vivo.
A common generation technique for mo-DCs is based on using the cytokines
GM-CSF and IL-4 for 5 days, leading to cells with an immature phenotype. After
antigen priming for a subsequent 2 days, mo-DCs increase their co-stimulatory
and
antigen-presenting capabilities to a state called maturation.
Interestingly, Randolph et al. (1998) (Science 282, 480-3) found that the
likely
naturally occurring process of monocyte transendothelial migration induces a
process
of differentiation into DCs in just 2 days, without addition of exogenous
cytokines.
This process starts with monocytes traversing a monolayer of endothelial cells
in the
luminal to abluminal direction, followed by a reverse transmigration to the
luminal
surface after a period of 48 h of resting (interaction) within the
extracellular matrix
(susceptible of containing specific antigens).
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In 1968, Szakal and Hanna (J. Immunol. 101, 949-962; Exp. Mol. Pathol. 8,
75-89) and Nossal et al. (J. Exp. Med. 127, 277-290) published the first
descriptions
and electron micrographs of what are now known as follicular dendritic cells
(FDCs).
Both groups used i2sI-labeled antigens and examined autoradiographs of the
follicles
in rodent spleens or lymph nodes using electron microscopy. Both groups found
that
radiolabel persisted on or near the surface of highly convoluted fine cell
processes of
dendritic-type cells with peculiar, irregularly shaped, euchromatic nuclei.
The fine
cell processes formed an elaborate meshwork around passing lymphocytes,
allowing
extensive cell-cell contact. Several names have been used for these cells but
a
nomenclature committee recommended the name "follicular dendritic cell" and
the
abbreviation "FDC" and these have been generally adopted (Tew et al. (1982) J.
Reticuloendothelial Soc. 31, 371-380).
The ability of FDCs to trap and retain antigen-antibody complexes, together
with their follicular location, distinguishes them from other cells, including
other
dendritic cells (DCs). FDCs bearing specific antigens are required for full
development of GCs (Kosco et al. (1992) J. Immunol. 148, 2331-2339; Tew et al.
(1990) Immunol. Rev. 117, 185-211) and are believed to be involved in Ig class
switching, production of B memory cells, selection of somatically mutated B
cells
with high affinity receptors, affinity maturation, induction of secondary
antibody
responses, and regulation of serum IgG with high affinity antibodies (Tew et
al.
(1990) Immunol. Rev. 117, 185-211; Berek & Ziegner (1993) Immunol. Today 14,
400-404; MacLennan & Gray (1986) Immunol. Rev. 91, 61-85; Kraal et al. (1982)
Nature 298, 377-379; Liu et al. (1996) Immunity 4, 241-250; Tsiagbe et al.
(1992)
Immunol. Rev. 126, 113-141). Many researchers have worked with FDCs in culture
in
2D with the general idea of mimicking an in vivo GC. An appreciation of the
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accessory functions of FDCs and regulation of these functions is important to
an
understanding of fully functional and mature antibody responses.
FDC development is B cell-dependent; FDCs are not detectable in, for
example, SCID mice, mice treated with anti-mu (to remove B cells), or mice
lacking
the mu chain (where B cells do not develop) (MacLennan & Gray (1986) Immunol.
Rev. 91, 61-85; Kapasi et al. (1993) J. Immunol. 150, 2648-2658). In T cell-
deficient
mice (e.g., nude mice), FDCs do develop, although the development is retarded
and
the FDCs do not appear to express many FDC markers (Tew et al. (1979) Aust. J.
Exp. Biol. Med. Sci. 57, 401-414).
Reconstitution of FDCs in SCID mice occurs best when both B cells and
T cells are adoptively transplanted, suggesting that T cells are also involved
in FDC
development (Kapasi et al. (1993) J. Immunol. 150, 2648-2658). Disruption of
LT/TNF or the cognate receptors disrupts lymph node organogenesis and
interferes
with the development of FDC networks (De Togni et al. (1994) Science 264, 703-
707;
Rennert et al. (1996) J. Exp. Med. 184, 1999-2006; Chaplin & Fu (1998) Curr.
Opin.
Immunol. 10, 289-297; Endres et al. (1999) J. Exp. Med. 189, 159-168; Ansel et
al.
(2000) Nature 406, 309-314). As summarized by Debard et al., it is known that
a lack
of LTa, LT(3, TNFaRl, and LT(3R interferes with the development of FDC
networks
(19). B cells are an important source of LTa/(3 heterotrimers, consistent with
data
indicating that FDC development is B cell-dependent (Endres et al. (1999) J.
Exp.
Med. 189, 159-168; Ansel et al. (2000) Nature 406, 309-314; Fu et al. (1998)
J. Exp.
Med. 187, 1009-1018).
The functional element of a mammalian lymph node is the follicle, which
develops a GC when stimulated by an antigen. The GC is an active area in a
lymph
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node, where important interactions occur in the development of an effective
humoral
immune response. Upon antigen stimulation, follicles are replicated and an
active
human lymph node may have dozens of active follicles, with functioning GCs.
Interactions between B cells, T cells, and FDCs take place in GCs. Various
studies of
GCs in vivo indicate that the following events occur there:
= immunoglobulin (Ig) class switching,
= rapid B cell proliferation (GC dark zone),
= production of B memory cells,
= accumulation of select populations of antigen specific T cells and B cells,
= hypermutation,
= selection of somatically mutated B cells with high affinity receptors,
= apoptosis of low affinity B cells,
= affinity maturation,
= induction of secondary antibody responses, and
= regulation of serum immunoglobulin G (IgG) with high affinity antibodies.
Similarly, data from in vitro GC models indicate that FDCs are involved in:
= stimulating B cell proliferation with mitogens and it can also be
demonstrated
with antigen (Ag),
= promoting production of antibodies including recall antibody responses,
= producing chemokines that attract B cells and certain populations of T
cells,
and
= blocking apoptosis of B cells.
While T cells are necessary for B cell responses to T cell-dependent antigens,
they are not sufficient for the development of fully functional and mature
antibody
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responses that are required with most vaccines. FDCs provide important
assistance
needed for the B cells to achieve their full potential (Tew et al. (2001)
Trends
Immunol. 22, 361-367).
Humoral responses in vaccine assessment can be examined using an artificial
immune system (AIS). Accessory functions of follicular dendritic cells and
regulation
of these functions are important to an understanding of fully functional and
mature
antibody responses.
Important molecules have been characterized by blocking ligands and
receptors on FDCs or B cells. FDCs trap antigen-antibody complexes and provide
intact antigen for interaction with B cell receptors (BCRs) on GC B cells;
this antigen-
BCR interaction provides a positive signal for B cell activation and
differentiation.
Engagement of CD21 in the B cell co-receptor complex by complement derived FDC-
CD21L delivers an important co-signal. Coligation of BCR and CD21 facilitates
association of the two receptors and the cytoplasmic tail of CD 19 is
phosphorylated
by a tyrosine kinase associated with the B cell receptor complex (Carter et
al. (1997)
J. Immunol. 158, 3062-3069). This co-signal dramatically augments stimulation
delivered by engagement of BCR by antigen and blockade of FDC-CD21L reduces
the immune responses -10- to -1,000-fold.
Test A~4ent
As used in the methods of the present invention, a test antigen is a molecule
for which information regarding its ability to induce an immune response is
desired.
As further indicated herein, the ability of a test antigen to induce an immune
response
can be determined based on the ability of the test antigen to induce
production of IgM
or IgG using the methods described herein. The test antigens used in the
methods of
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the present invention are limited only in that they can be administered to the
GC LTEs
of the present invention.
In a preferred embodiment the test agent is an antigen against which it is
desired to induce an immune response in a subject (upon administration of the
antigen
in a vaccine formulation to a subject). Such antigens include polypeptides,
peptides,
proteins and polysaccharides. In preferred embodiments the test agents are
proteins or
polysaccharides derived from a bacteria or virus having the ability to infect
and cause
disease in a human. Thus, for example, test agents may be surface or integral
membrane proteins of bacteria or coat proteins of viruses. The test agent may
be an
entire polypeptide or polysaccharide, or a portion of thereof. In one
embodiment, the
test agent may be the entire organism (e.g., bacteria virus) against which it
is desired
to raised an immune response. In this embodiment, preferably the organism is
attenuated such that it can no longer cause disease or an infection in the
subject to
which it is administered.
In other embodiments the test agent may be a non-biological molecule, for
which information regarding the molecules antigenicity is desired.
Immune Complexes
An embodiment of the present invention concerns antigen-antibody complexes
(immune complexes, ICs) that can be used, for example, in in vitro GC LTEs and
which may be used, for example, for pre-clinically evaluating vaccine
candidates and
other immunomodulatory agents.
Immune complexes play an important role in the function of follicular
dendritic cells (FDC), which are principally responsible for regulating the
differentiation of antigen-specific B cells into high-affinity antibody
producers in the
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generation of a humoral immune response. In vitro experiments have shown that
B
cells stimulated to produce antibody in the absence of IC-loaded FDC are not
capable
of fully differentiating into high-affinity antibody producers. Consequently,
specific
IC will be important in eliciting a humoral immune response within the AIS.
As used herein, an immune complex or IC comprises an antibody and an
antigen to which it is bound. The skilled artisan will understand that there
are no
limitations on the identities of the antibodies and antigens that comprise the
immune
complexes of the present invention. For example, the antibodies may be
obtained
from any species of animal, though preferably from a mammal such as a human,
simian, mouse, rat, rabbit, guinea pig, horse, cow, sheep, goat, pig, dog or
cat.
Preferably the antibodies are human antibodies. Nor is there a limitation on
the
particular class of antibody that may comprise the immune complex, including
IgGl,
IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD and IgE antibodies. Antibody fragments
of
less than the entire antibody may also be used, including single chain
antibodies,
F(ab')2 fragments, Fab fragments, and fragments produced by an Fab expression
library, with the only limitation being that the antibody fragments retain the
ability to
bind the antigen.
The antibodies may also be polyclonal, monoclonal, or chimeric antibodies,
such as where an antigen binding region (e.g., F(ab')2 or hypervariable
region) of a
non-human antibody is transferred into the framework of a human antibody by
recombinant DNA techniques to produce a substantially human molecule.
For the production of antibodies, various hosts including, but not limited to,
goats, rabbits, rats, mice, humans, etc., can be immunized by injection with a
particular protein or any portion, fragment, or oligopeptide that retains
immunogenic
properties of the protein. Depending on the host species, various adjuvants
can be
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used to increase the immunological response. Such adjuvants include, but are
not
limited to, detoxified heat labile toxin from E. coli, Freund's, mineral gels
such as
aluminum hydroxide, and surface active substances such as lysolecithin,
pluronic
polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and
dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum are
also potentially useful adjuvants.
Antibodies and fragments thereof can be prepared using any technique that
provides for the production of antibody molecules, such as by continuous cell
lines in
culture for monoclonal antibody production. Such techniques include, but are
not
limited to, the hybridoma technique originally described by Koehler and
Milstein
(Nature 256:495-497 (1975)), the human B-cell hybridoma technique (Kosbor et
al.,
Immunol Today 4:72 (1983); Cote et al., Proc Natl. Acad. Sci 80:2026-2030
(1983)),
and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer
Therapy, Alan R. Liss Inc, New York N.Y., pp 77-96 (1985)).
Techniques developed for the production of "chimeric antibodies," i.e., the
splicing of mouse antibody genes to human antibody genes to obtain a molecule
with
appropriate antigen specificity and biological activity, can also be used
(Morrison et
al., Proc Natl. Acad. Sci 81:6851-6855 (1984); Neuberger et al., Nature
312:604-
608(1984); Takeda et al., Nature 314:452-454(1985)). Alternatively, techniques
described for the production of single chain antibodies, such as disclosed in
U.S.
Patent No. 4,946,778, incorporated herein by reference in its entirety, can be
adapted
to produce Aap-specific single chain antibodies. Additionally, antibodies can
be
produced by inducing in vivo production in the lymphocyte population or by
screening recombinant immunoglobulin libraries or panels of highly specific
binding
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reagents as disclosed in Orlandi et al., Proc Natl. Acad. Sci. USA 86: 3833-
3837
(1989); and Winter G. and Milstein C., Nature 349:293-299 (1991).
Antibody fragments such as F(ab')2 fragments can be produced by pepsin
digestion of the antibody molecule, and Fab fragments can be generated by
reducing
the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression
libraries
can be constructed to allow rapid and easy identification of monoclonal Fab
fragments
with the desired specificity. (Huse W. D. et al., Science 256:1275-1281
(1989)).
The antigens that comprise the immune complexes of the present invention are
limited only in that they are bound by the antibody of the immune complex.
Thus, in
general terms, the antigen is a small molecule, such as a peptide of 10-15
amino acids.
Generally the antigens comprising the immune complexes of the present
invention
will be a portion of a larger antigen that is present in the vaccine
formulations of the
present invention or a portion of a test agent of the present invention. For
example, as
explained further herein the vaccine formulations of the present invention
include an
antigen and a pharmaceutically acceptable carrier or diluent. Such antigens
found in
the vaccine formulations may be any antigen against which it is desired to
induce an
immune response in a subject (upon administration of the vaccine formulation
to a
subject). Such antigens include polypeptides, peptides, proteins and
polysaccharides.
The skilled artisan will thus understand that while the immune complexes of
the
present invention comprise an antibody and at least a portion of an antigen,
to which
the antibody is bound.
As indicated above, the development of ICs first requires the generation of
antibodies reactive against the antigen of interest. Specific antibodies can
be elicited
by immunizing animals with antigen directly, but this can be a costly, slow,
and
inconvenient procedure. While it is also possible to generate reactive
antibody by
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stimulating naive B cells in vitro, this also can be a laborious technique
that typically
yields only small quantities of specific antibody.
An embodiment of the present invention thus comprises approaches to
generating ICs by artificially coupling antibody to antigen in a non-specific
manner.
This offers the following advantages over existing techniques:
= pre-existing antibodies can be used,
= a large amount of IC can be produced without the need of generating a
specific
immune response,
= one antibody can be coupled to many different antigens, and
= IC development will be much quicker than is possible with current methods.
In an embodiment of the present invention, ICs can be generated by coupling a
hapten to the antigen of interest, which can then be bound by a specific
antibody. As
an example, fluorescein isothiocyanate (FITC) of Fluorescein-EX dyes can be
conjugated to primary amino groups on a target protein, using literature
procedures
In this regard, Fluorescein-EX or other derivatives bearing elongated linkers
may be
advantageous over tight linker-antigen conjugates formed by FITC and other
haptens.
Commercially available high-affinity anti-FITC antibodies can then be used to
bind
the antigen-hapten conjugate, forming a complete IC. Tetanus toxoid can be
used as a
model antigen, because most adults are immunized against it and the humoral
and
cell-mediated immune responses generated against this antigen are well known.
In
other embodiments, other linkers (e.g., digoxin) and antigens (e.g.,
ovalbumin) can be
used. In another embodiment, the antibody can be chemically coupled to the
antigen
using, for example, the amine-thiol cross-linking method that is often used to
form
protein heteroconjugates. Using these non-specific chemistries does not
require an
agglutination step, making them useful for polyclonal antibodies.
Additionally, the
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stoichiometry of the IC can be manipulated without affecting the size or
density of
this complex.
Vaccine Formulations
As indicated above, the present invention is also directed to methods for
utilizing and testing vaccine formulations. As used herein, a vaccine
formulation
comprises at least one antigen and a pharmaceutically acceptable carrier or
diluent.
The antigens comprising the vaccine formulations of the present invention
may be any antigen against which it is desired to induce an immune response in
a
subject (upon administration of the vaccine formulation to a subject) or for
which
information regarding its antigenicity is desired to be known. Such antigens
include
polypeptides, peptides, proteins and polysaccharides. In preferred embodiments
the
antigens comprising the vaccine formulations of the present invention are
derived
from a bacteria or virus having the ability to infect and cause disease in a
human.
Thus, for example, antigens comprising a vaccine formulation may include
surface or
integral membrane proteins of bacteria or coat proteins of viruses. The
antigen may
be an entire polypeptide or polysaccharide, or a portion of thereof. In one
embodiment, the antigen may be the entire organism (e.g., bacteria virus)
against
which it is desired to raised an immune response. In this embodiment,
preferably the
organism is attenuated such that it can no longer cause disease or an
infection in the
subject to which it is administered.
The amount of the antigen present in the vaccine formulation will vary based
on the identity of the antigen and will thus be determined by the skilled
artisan.
However, in certain methods of the present invention the amount of antigen in
a
vaccine formulation will typically be an amount sufficient to induce an immune
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response in a subject, preferably a protective immune response to the organism
from
which the antigen was derived.
The vaccine formulations used in the methods of the present invention will
preferably be in a formulation that is similar to or identical to the
formulation that
would be administered to a subject. However, the skilled artisan will also
understand
that the methods of the present invention may utilize a vaccine formulation
comprising at least one antigen and an inert carrier or diluent, such as water
or
buffered solution.
Two-Component Vaccine Systems
The present invention is also directed to a two-component vaccine system and
to methods for utilizing and testing a two-component vaccine system. As used
herein,
a two-component vaccine system comprises two components, wherein the first
component comprises at least one antigen, preferably in a pharmaceutically
acceptable carrier or diluent, and wherein the second component comprises an
immune complex comprising the antigen of the first component and an antibody
bound thereto, preferably in a pharmaceutically acceptable carrier or diluent.
The antigens comprising the two-component vaccine systems of the present
invention may be any antigen against which it is desired to induce an immune
response in a subject (upon administration of the vaccine formulation to a
subject) or
for which information regarding its antigenicity is desired to be known. Such
antigens include polypeptides, peptides, proteins and polysaccharides. In
preferred
embodiments the antigens comprising the two-component vaccine systems of the
present invention are derived from a bacteria or virus having the ability to
infect and
cause disease in a human. Thus, for example, antigens comprising a two-
component
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vaccine system may include surface or integral membrane proteins of bacteria
or coat
proteins of viruses. The antigen may be an entire polypeptide or
polysaccharide, or a
portion of thereof. In one embodiment, the antigen may be the entire organism
(e.g.,
bacteria virus) against which it is desired to raised an immune response. In
this
embodiment, preferably the organism is attenuated such that it can no longer
cause
disease or an infection in the subject to which it is administered.
The amount of the antigen present in the two-component vaccine system will
vary based on the identity of the antigen and will thus be determined by the
skilled
artisan. However, in certain methods of the present invention the amount of
antigen
in a two-component vaccine system will typically be an amount sufficient to
induce
an immune response in a subject, preferably a protective immune response, to
the
organism from which the antigen was derived.
Each of the components of the two-component vaccine systems of the present
invention will preferably be in a formulation that is similar to or identical
to the
formulation that would be administered to a subject. However, the skilled
artisan will
also understand that the methods of the present invention may utilize a two-
component vaccine system wherein each component comprises an inert carrier or
diluent, such as water or buffered solution.
In a preferred embodiment each of the components of the two-component
vaccine systems of the present invention are separately formulated and in
separate
containers. However, it is envisioned that in certain embodiments the two
components could be mixed in the same container.
In a preferred embodiment of the methods directed to inducing an immune
response in a subject using the two-component vaccine system of the present
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invention, the two components are administered to separate sites of a subject.
Thus,
when the components are administered via an injection, the injection sites are
different locations, for example, the left arm and the right arm of an animal,
such as a
human, or the left leg and right of an animal, such as a human. The components
may
be administered at the same time, or sequentially. In a preferred embodiment
the
components are administered within less than 15 minutes, 30 minutes, 45
minutes,
one hour, two hours, three hours, four hours, five hours or more, of each
other.
As indicated above, each of the components in the two-component vaccine
system may be formulated with a pharmaceutically acceptable carrier or
diluent. As
such each of the components in the two-component vaccine system can be
formulated
in a variety of useful formats for administration by a variety of routes.
Administration
of the components of the two-component vaccine system can be by any means
generally used in the art, and includes intravenous, intraperitoneal,
intramuscular,
subcutaneous and intradermal routes, nasal application, by inhalation,
ophthalmically,
orally, rectally, vaginally, or by other means that results in the vaccine
components
contacting mucosal tissues.
Injectable formulations of the components of the two-component vaccine
system for administration via intravenous, intraperitoneal, intramuscular,
subcutaneous and intradermal routes may include various carriers such as
vegetable
oils, dimethylacetamide, dimethylformaamide, ethyl lactate, ethyl carbonate,
isopropyl myristate, ethanol, polyols (glycerol, propylene glycol, and liquid
polyethylene glycol) and the like. Intramuscular preparations can be prepared
and
administered in a pharmaceutical excipient such as Water-for-Injection, 0.9%
saline,
or 5% glucose solution.
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Solid formulations for oral administration may contain suitable carriers or
diluents, such as corn starch, gelatin, lactose, acacia, sucrose,
microcrystalline
cellulose, kaolin, mannitol, dicalcium phosphate, calcium carbonate, sodium
chloride,
or alginic acid. Disintegrators that can be used include, without limitation,
micro-
crystalline cellulose, cornstarch, sodium starch glycolate, and alginic acid.
Tablet
binders that may be used include acacia, methylcellulose, sodium
carboxymethylcellulose, polyvinylpyrrolidone, hydroxypropyl methylcellulose,
sucrose, starch, and ethylcellulose. Lubricants that may be used include
magnesium
stearates, stearic acid, ailicone fluid, talc, waxes, oil, and colloidal
silica.
In one embodiment of the present invention, each of the components in the
two-component vaccine system may exist as atomized dispersions for delivery by
inhalation. The atomized dispersion typically contains carriers common for
atomized
or aerosolized dispersions, such as buffered saline and/or other compounds
well
known to those of skill in the art. The delivery of the components via
inhalation has
the effect of rapidly dispersing the vaccine components to a large area of
mucosal
tissues as well as quick absorption by the blood for circulation. One example
of a
method of preparing an atomized dispersion is described in U.S. Patent No.
6,187,344, entitled, "Powdered Pharmaceutical Formulations Having Improved
Dispersibility," which is hereby incorporated by reference in its entirety.
The components in the two-component vaccine system described herein can
also be formulated in the form of a rectal or vaginal suppository. Typical
carriers
used in the formulation of the inactive portion of the suppository include
polyethylene
glycol, glycerine, cocoa butter, and/or other compounds well known to those of
skill
in the art.
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Additionally, the components in the two-component vaccine system may be
administered in a liquid form. The liquid can be for oral dosage, for
ophthalmic or
nasal dosage as drops, or for use as an enema or douche. When the vaccine
components are formulated as a liquid, the liquid can be either a solution or
a
suspension of the vaccine components. There are a variety of suitable
formulations
for the solution or suspension of the vaccine components that are well know to
those
of skill in the art, depending on the intended use thereof. Liquid
formulations for oral
administration prepared in water or other aqueous vehicles may contain various
suspending agents such as methylcellulose, alginates, tragacanth, pectin,
kelgin,
carrageenan, acacia, polyvinylpyrrolidone, and polyvinyl alcohol. The liquid
formulations may also include solutions, emulsions, syrups and elixirs
containing,
together with the active compound(s), wetting agents, sweeteners, and coloring
and
flavoring agents. Various liquid and powder formulations can be prepared by
conventional methods for inhalation into the lungs of the mammal to be
treated.
Each of the components of the two-component vaccine system of the present
invention may be administered in a single dose or in multiple doses over
prolonged
periods of time, such as up to about one week, and even for extended periods
longer
than one month or one year. In some instances, administration of the
components
may be discontinued and resumed at a later time. For example, a second dose
can be
administered 28 days later, or at some other time interval to be determined.
Serum for
antibody assessment can be collected prior to immunization and fourteen days
following each dose. Sera is then assessed for antibodies against the antigen
in the
vaccine system.
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A kit comprising the necessary components of the two-component vaccine
system for inducing an immune response in a subject and instructions for their
use are
also within the purview of the present invention.
Measuring Antibody/Anti~4en Interactions
Antibody affinity is the strength of the reaction between a single antigenic
determinant and a single combining site on an antibody. It is the sum of the
attractive
and repulsive forces operating between the antigenic determinant and the
combining
site of the antibody. Most antibodies have a high affinity for their antigens.
Avidity
is a measure of the overall strength of binding of an antigen with many
antigenic
determinants and multivalent antibodies. Avidity is influenced by both the
valence of
the antibody and the valence of the antigen. Avidity is more than the sum of
the
individual affinities.
Antibody affinity can be assessed, for example, by determining the
equilibrium KD, which can be estimated for moderate to high affinity
interactions
using a series of antibody/antigen concentrations (see, e.g., Daugherty et al.
(1998)
Protein Engineering 11, 101-108 and Nolan & Sklar (1998) Nature Biotechnol.
16,
633-8).
The ease with which one can detect antigen-antibody reactions will depend on
a number of factors, including affinity (the higher the affinity of the
antibody for the
antigen, the more stable will be the interaction), avidity (reactions between
multivalent antigens and multivalent antibodies are more stable and thus
easier to
detect), the antigen to antibody ratio (the ratio between the antigen and
antibody
influences the detection of antigen-antibody complexes because the size of the
complexes formed is related to the concentration of the antigen and antibody),
and the
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physical form of the antigen (e.g., if the antigen is a particulate,
generally,
agglutination of the antigen by the antibody is used, whereas if the antigen
is soluble,
generally, the precipitation of the antigen after the production of large
insoluble
antigen-antibody complexes is used).
When an antigen is particulate, the reaction of an antibody with the antigen
can be detected, for example, by agglutination (clumping) of the antigen. The
general
term agglutinin is used to describe antibodies that agglutinate particulate
antigens. All
antibodies can theoretically agglutinate particulate antigens but IgM, due to
its high
valence, is a particularly good agglutinin and it can sometimes be inferred
that an
antibody may be of the IgM class if it is a good agglutinating antibody.
Agglutination tests can be used in a qualitative manner to assay for the
presence of an antigen or an antibody. The antibody is mixed with the
particulate
antigen and a positive test is indicated by the agglutination of the
particulate antigen.
Agglutination tests can also be used to measure the level of antibodies to
particulate
antigens. In this test, serial dilutions are made of a sample to be tested for
antibody
and then a fixed number of red blood cells or bacteria or other such
particulate antigen
is added. Then the maximum dilution that gives agglutination is determined.
The
maximum dilution that gives visible agglutination is called the titer. The
results are
reported as the reciprocal of the maximal dilution that gives visible
agglutination.
Passive hemagglutination - The agglutination test only works with particulate
antigens. However, it is possible to coat erythrocytes with a soluble antigen
(e.g. viral
antigen, a polysaccharide or a hapten) and use the coated red blood cells in
an
agglutination test for antibody to the soluble antigen, referred to as passive
hemagglutination. The test is performed just like the agglutination test.
Applications
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include detection of antibodies to soluble antigens and detection of
antibodies to viral
antigens.
If the antigen is soluble, generally, the precipitation of the antigen after
the
production of large insoluble antigen-antibody complexes is used. Such
precipitation
tests include the radial immunodiffusion assay of Mancini kMancini et al.
(1965)
Immunochemistry 2, 235-54; Mancini et al. (1970) Immunochemistry 7, 261-41. In
radial immunodiffusion, antibody is incorporated into an agar gel as it is
poured and
different dilutions of the antigen are placed in holes punched into the agar.
As the
antigen diffuses into the gel, it reacts with the antibody and when the
equivalence
point is reached a ring of precipitation is formed. The diameter of the ring
is
proportional to the log of the concentration of antigen because the amount of
antibody
is constant. Thus, by running different concentrations of a standard antigen a
standard
curve is prepared, from which the amount of an antigen in an unknown sample
can be
quantitated; thus, it is a quantitative test. If more than one ring appears in
the test,
more than one antigen/antibody reaction has occurred. This could be due to a
mixture
of antigens or antibodies. This test is commonly used in the clinical
laboratory for the
determination of immunoglobulin levels in patient samples.
Another technique is that of immunoelectrophoresis. In
immunoelectrophoresis, a complex mixture of antigens is placed in a well
punched
out of an agar gel and the antigens are electrophoresed so that the antigens
are
separated according to charge. After electrophoresis, a trough is cut in the
gel and
antibodies are added. As the antibodies diffuse into the agar, precipitin
lines are
produced in the equivalence zone when an antigen/antibody reaction occurs.
This test
is used for the qualitative analysis of complex mixtures of antigens, although
a crude
measure of quantity (thickness of the line) can be obtained. This test is
commonly
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used for the analysis of components in a patient's serum. Serum is placed in
the well
and antibody to whole serum in the trough. By comparisons to normal serum, one
can
determine whether there are deficiencies on one or more serum components or
whether there is an overabundance of some serum component (thickness of the
line).
Radioimmunoassays (RIA) are assays based on the measurement of
radioactivity associated with immune complexes. In any particular test, the
label may
be on either the antigen or the antibody.
Enzyme-linked immunosorbent assays (ELISAa) are based on the
measurement of an enzymatic reaction associated with immune complexes. In any
particular assay, the enzyme may be linked to either the antigen or the
antibody.
Specific determination of, for example, mouse, rabbit or human IgG or IgM
concentrations can be made with commercially available reagents, such as the
Easy-
Titer Antibody Assay Kits (Pierce), or by ELISA. The Easy-Titer Antibody Assay
Kits include antibody-sensitized microspheres to measure the specific
concentration
of mouse, rabbit and human antibodies by an easy and rapid microagglutination
technique using standard microplates and UV-Vis plate reader
(spectrophotometer).
Each kit is specific for a particular species and class of immunoglobulin and,
unlike
total protein assays, can specifically measure the concentration of target
antibody in
samples (e.g., serum, plasma, culture supematant) that contain other proteins.
The kits
are sensitive, requiring very small sample volumes. Antibody concentration is
determined from the assay response (absorbance) by comparison to a standard
curve
prepared using dilutions of a known antibody sample. Easy-Titer Assay Kits
detect
and measure specific target antibodies using agglutination of microspheres
that are
coated ("sensitized") with the specific anti-IgG or IgM polyclonal antibodies.
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Examples
Example 1. FDC-like cells function like FDCs
FDC-like cells can be derived from human monocytes using published
techniques (Heinemann & Peters (2005) BMC Immunol. 6, 23; see also WO 2005 /
118779 and EP 04012622.9. Use of these FDC-like cells is an advantage over
isolating FDCs from human tonsils, which are not always readily available. An
alternative is isolating FDCs from secondary lymphoid tissues of animals, but
isolating functionally active FDCs from secondary lymphoid tissue requires
considerable skill and there are times when introducing animal cells into a
human
system is not acceptable. Thus, it is desirable to be able to use readily
available
human FDC-like cells that have accessory activity comparable with FDCs.
We examined whether FDC-like cells could trap ICs like FDCs. To test this,
FDCs and FDC-like cells were incubated with labeled ICs, the cells were washed
to
remove unbound ICs, and incubated overnight (- 15 h). Phagocytic cells can
trap ICs,
but such ICs will be endocytosed and destroyed during the overnight
incubation. In
contrast, FDCs trap ICs on their surfaces and the ICs persist on the cell
surface for
many months to years in vivo. Both FDCs and FDC-like cells trapped and
retained
ICs after overnight incubation (data not shown).
We next examined whether IC-bearing FDC-like cells had accessory activity
and could promote the production of antibodies. To test this, FDCs and FDC-
like
cells were loaded with rat anti-mouse IgD, complexed with specific anti-rat
IgG. We
reasoned that the anti-delta in the ICs would bind membrane IgD on the B cells
and
provide a potent signal, causing the B cells to begin making IgM. When FDCs or
FDC-like cells were loaded with ICs and incubated with purified B cells, they
rapidly
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formed similar clusters with B cells that were typical of GC reactions in
vitro. After
-48 h the supernatant fluids were collected and the IgM response was
determined.
The results are illustrated in Figure 2.
FDCs are on the left side and FDC-like cells are on the right, at a ratio of 1
FDC or FDC-like cell to 2 B cells. A high number of FDC-like cells was chosen
to
ensure that we would see accessory activity, even if it was weaker in FDC-like
cells
than in FDCs. IgD ICs were used over a range, from - 100 ng to - 10 g.
However,
-100 ng appeared to be adequate, as there were not a significant increase in
Ab
production with higher levels of IC. The B cells were used in 10-fold
increases, from
_10000, _100,000 to _106 B cells. The Ab response followed with the 10-fold
increases in B cells corresponding increases in IgM production (from -10
ng/mL, to
- 100 ng/mL, to - 1000 ng/mL at the highest dose of B cells). It was observed
that the
FDC-like cells gave similar results, from -5 ng/mL, to 50 ng/mL, to 500 ng/mL,
at the
highest dose of B cells. In short, the patterns were very similar with the two
cell
types, there being about a two-fold increase in Ab production in favor of the
FDCs,
under these experimental conditions.
Example 2. Rapid in vitro antigenicity assessment
Using FDCs or FDC-like cells, the in vitro GC LTE of the present invention
can be used to rapidly assess the antigenicity of antigens. FDCs or FDC-like
cells
loaded with IC can be used to induce a rapid (-48 h) IgM response. The B cell
repertoire can be assessed, as can the antigenicity of the antigens.
Example 3. Rapid in vitro vaccine assessment
Using FDCs or FDC-like cells, the in vitro GC LTE of the present invention
can be used to rapidly assess the antigenicity of vaccine candidates. FDCs or
FDC-
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like cells loaded with IC can be used to induce a rapid (-48 h) IgM response.
The B
cell repertoire can be assessed, as can the antigenicity of the antigens.
Example 4. Using the enhancing effect of ICs
A problem with the use of ICs is that they do not always activate DCs and
prime T cells. In an embodiment of the present invention, a dual immunization
strategy is used, in which ICs are targeted to FDCs to initiate an early IgM
response
and expand the specific B cells. Free antigen is then also injected into a
different site
to target DCs for T cell priming. With this dual immunization strategy, rapid
specific
IgM responses and enhanced IgG responses are induced. As an example, we looked
at what happened when T cell help is provided with the ICs. This should bypass
DCs
and the need for T cell priming.
Example 5. Dual immunization
Activating and inhibitory FcyRs appear to regulate signaling in DCs. For
example, selective blockade of inhibitory FcyRIIB enables human dendritic cell
maturation (Dhodapkar et al. (2005) Proc. Natl. Acad. Sci. USA 102, 2910-
2915).
Accordingly, to avoid FcyRIIB on DCs, free antigen was used rather than ICs
while priming T cells for the in vitro primary in our germinal center (GC)
lymphoid
tissue equivalent (LTE). We used the same approach in vivo by putting antigen
in a
different location, far away from the ICs.
Example 6. Dual immunization in vitro
A balance between activating/inhibitory FcyRs may regulate signaling in DCs.
For
example, selective blockade of inhibitory FcyRIIB enables human dendritic cell
maturation (Dhodapkar et al. (2005) Proc. Natl. Acad. Sci. USA 102, 2910-
2915).
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Accordingly, to prime T cells we used free antigen rather than ICs to avoid
FcyRIIB on DCs. Note the superiority of ICs over free antigen when presented
to B
cells by FDCs. In the experiment shown in Figure 3, there are no T cells, but
there
was a specific IgM response with just B cells and FDCs. In Figure 4, the
presence of
T cells, which likely made some cytokines, did improve the IgM response, but
did not
result in any IgG, as expected (Fig. 3,4).
Example 7. Dual immunization in vivo
We tested a dual immunization strategy in vivo. ICs were targeted to FDCs to
initiate an early IgM response and to expand the specific B cells, while free
Ag was
injected into a different site to target DCs for T cell priming. In
combination, rapid
specific IgM responses and enhanced IgG responses were induced and this
appeared
to be a consistent result (Fig. 5). Furthermore, ICs promoted somatic
hypermutation
several days earlier in the immune response and this should lead to rapid
production
of high affinity Abs (Fig. 6,7).
Example 8. Purified FDCs can re-attach to an ETC matrix
Purified FDCs can re-attach to an ETC matrix and attract B and T cells to
form lymph node-like follicles in vitro. We showed that FDCs adhere to
collagen and
to collagen-associated molecules in vitro. Moreover, on collagen type 1, we
found
that the FDCs would extend dendrites and form FDC-reticula. See El Shikh et
al.
(2007) Cell Tissue Res. 329, 81-89. We observed lymph node-like follicles in
the GC
LTE.
Example 9. Human B cell responses
To determine whether specific primary in vitro human B cell responses could
be generated in 3-D in vitro GCs, we used an ETC matrix using naive human B
cells
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and human memory T cells in combination with antigen-bearing FDCs. We observed
anti-tetanus responses using memory T cells to induce anti-tetanus responses
including responses with IgM-bearing B cells that we believe are naive. Our
success
with this model allowed us to examine whether monocyte-derived dendritic cells
could be used to prime naive human T cells and whether these primed T cells
could be
used to create a complete primary immune response in vitro.
The germinal center LTE has been used to generate specific IgM followed by
switching to IgG in response to OVA (Fig. 8a). Figure 8b shows the IgG data
and is
consistent with class switching. After the first week, the IgM response was
maximal,
with a small IgG response, but by the end of the second week the response had
class
switched, giving minimal IgM production, and the IgG response was maximal.
Additionally, we generated similar data with influenza antigens and anthrax
rPA
(recombinant protective antigen) (data not shown). However, with HIV gp120 we
got
a strong IgM response, but failed to get class switching (Fig. 9).
This lack of an IgG response may be attributable to gp 120 binding to CD4 and
interfering with T cell priming. We attribute the good gp120-specific IgM to
the
ability of FDCs to arrange ICs on their surfaces with periodicity. This
periodicity is
consistent with the periodicity of T-independent antigens that give the good
IgM
responses in the absence of primed T cells.
Simultaneous binding of multiple BCRs gives a signal adequate to give
specific IgM responses in the absence of specific T cells. The ability of FDCs
plus ICs
to induce T-independent responses is illustrated in nude mice, below.
Example 10. Assessment of potential immunogens
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The response in the GC LTE is instructive regarding the use of free gp 120 as
an immunogen in humans. A common way to assess potential immunogens is to
start
by injecting them into animals. Those immunogens that cause responses in
animals
are candidates for further study.
Regarding Ig class switching, when gp l20 was injected into mice (Fig. 10),
the murine response to gp120 was good, with IgM responses that class-switched
to
IgG by day 14, as expected. There was no indication that gp120 would not be a
good
vaccine candidate from these murine data.
In short, the GC LTE predicted problems with free, soluble gp l20 that can
bind human CD4 when priming human T cells, and T cell help is necessary for
IgG
class switching. In contrast, soluble, free gp1201ooks like a good vaccine
candidate
in an animal model, where IgG class switching occurred normally. It should be
appreciated that gp 120 will not bind murine CD4 and would not interfere with
T cell
priming. Nevertheless, gp120 on the virus in vivo does induce a good gp120
response.
It may be that use of gp 120 in a particle, mimicking the virus, may not block
T cell
priming as well as the free molecule.
Thus, designing the vaccine differently may give a different result. However,
it seems unlikely that free gp120 would be a good immunogen in humans, and
only
the in vitro human artificial immune system of the present invention provided
that
information.
Example 11. Assessment of vaccines, in vitro and in vivo
We compared the magnitude and quality of primary and secondary immune
responses in vivo with responses obtained in the in vitro AIS using both
established
and experimental vaccines. We demonstrated that dual forms of immunogen can
lead
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to a rapid IgM response, as well as a T cell response, leading to class
switching and
high-affinity IgG production (data not shown).
ICs do not always activate DCs and prime T cells. The dual immunization
approach of the present invention targets ICs to FDCs to initiate an early IgM
response and expand specific B cells, in combination with antigen targeted to
DCs to
prime the T cells, resulting in rapid, specific IgM as well as more rapid and
enhanced
IgG responses.
Priming of naive T cells requires DCs that are regulated by a balance between
activating/inhibitory FcyRs that control signaling in DCs. For example,
selective
blockade of inhibitory FcyRIIB enables human dendritic cell maturation, as was
first
shown by Dhodapkar et al. (2005) Proc. Natl. Acad. Sci. USA 102, 2910-2915.
In short, ICs may or may not prime T cells and that appears to have frustrated
attempts to use ICs in vaccines. The final assessment from a review discussing
that
fact that ICs often give rapid and enhanced immune responses was that,
"[b]ased on
reports published to date, it is difficult to predict whether a given antibody
will have
an enhancing or suppressive effect on the magnitude or efficacy of the
subsequent
immune response to the antigen." (Brady (2005) Infection & Immunity 73, 671-
678).
Given the known problems with ICs in priming naive T cells, in an
embodiment of the present invention, we used free antigen rather than ICs to
avoid
FcyRIIB on DCs for the in vitro primary. We used the same approach in vivo. We
injected free antigen into a separate site, to target antigen to a separate
lymph node
from the ICs, allowing for T cell priming in the absence of ICs. In contrast,
with T
cells, ICs were much better with FDCs than free antigen in vitro. When
presented to
B cells by FDCs, ICs appear to be much better in vivo.
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Example 12. Use of adjuvant to promote a strong antibody response
We have previously shown that FDCs bear TLR4 and other TLRs on their
surfaces. Moreover, LPS activates FDCs and enhances their ability to stimulate
antibody responses in vitro and promote somatic hypermutation.
We next looked to see whether adjuvant could improve the ability of ICs to
promote Ab responses (Fig. 11). ICs in adjuvant and ICs alone appeared to have
comparable ability to induce OVA-specific IgG. In this example, the ICs were
able to
induce IgG without there being memory T cells or antigen to prime T cells.
Nevertheless, adding adjuvant to the ICs resulted in a dramatic enhancement of
the
IgG responses, consistent with our data indicating that FDCs have TLR
receptors and
are activated by engagement of these receptors. Thus, preferably both the
antigen and
the ICs are in adjuvant when immunizing with the dual immunization approach of
the
present invention.
We have shown that T-dependent antigens can be converted into T-
independent antigens by loading them on FDCs in the form of ICs. These results
and
the adjuvant results above prompted us to examine whether T-dependent antigens
could induce IgM in nude mice and whether the IgM response would be enhanced
by
use of adjuvant. The results of this experiment are illustrated in Figure 12.
OVA in adjuvant failed to induce a detectable IgM response in nude mice, as
was expected. In contrast, OVA ICs induced a significant IgM response and that
response was enhanced by the use of ICs with the adjuvant.
Thus, in an embodiment of the present invention, ICs are used to provide
protection in people with T cell insufficiencies where antigen fails to give a
response
(as shown here with the nude mice) or a very poor response. Such human
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immunoinsufficiencies include AIDS patients, the aged, uremics, diabetics, and
alcoholics. In an embodiment of the present invention, a method for generating
rapid
(-24-48 h) protection is provided by injecting the antigen as an IC.
Example 13. IC-bearing FDCs
Epitope clusters on FDC dendrites may simultaneously cross-link multiple
BCRs; thus, FDCs may convert TD antigens into TI antigens capable of inducing
B
cell activation and rapid IgM production in the absence of T cells or T cell
factors. To
test this, IC-bearing FDCs were used to stimulate B cells in vivo and in vitro
under
conditions lacking T cell help. Nude mice (nu/nu) were challenged with OVA-ICs
and
the OVA-specific Abs were measured after -48 h (Fig. 13). GC development was
also
studied in these mice using light and confocal microscopy. Moreover, purified
FDCs
loaded with OVA-ICs or anti-delta (anti-mouse IgD) ICs were cultured with
purified
murine and human B cells in vitro and the OVA-specific and total IgM responses
were measured respectively. Confocal microscopy and flow cytometry were used
to
visualize and quantify tyrosine phosphorylation indicative of signaling in B
cell by
IC-bearing FDCs in vitro.
Our data indicated that OVA-IC challenged nude mice produced OVA-
specific IgM within -48 h and the response was maintained for -7 weeks (Fig.
13).
The draining lymph nodes of these mice exhibited well developed PNA+ and GL7+
GCs associated with antigen retaining reticula (ARR) and Blimp-l+
plasmablasts.
Moreover, OVA-IC loaded FDCs induced purified human and murine B cells to
produce OVA-specific IgM in vitro in -48 h. FDCs loaded with anti-delta
induced
high levels of total IgM within -48 h when cultured with purified B cells.
Anti-delta
IC-stimulated B cells showed characteristic capping and patching of
intracellular
phosphotyrosine and the intensity of phosphotyrosine labeling was increased in
all
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stimulated B cells as indicated by increased mean fluorescence intensity and
total
population shift in flow cytometry. FDCs trapped and retained ICs on their
surfaces,
as shown by confocal microscopy and were able to induce rapid IgM production
by
purified B cells in vitro within -48 h. In short, we have shown the ability of
FDCs to
convert TD antigens into TI antigens, capable of inducing B cell activation
and Ig
production in the absence of T cells or T cell factors.
Thus, in an embodiment of the present invention, immune responses are
induced to TD antigens in patients with congenital and acquired T cell
insufficiencies,
including infants, the aged, AIDS patients, diabetic, and uremic patients.
Example 14. Immune response to ovalbumin (OVA)
Homozygous athymic NCr-nu/nu and heterozygous NCr-nu/+ mice were
purchased from The National Cancer Institute at Frederick (NCI-Frederick).
Mice
were housed in standard plastic shoebox cages with filter tops and maintained
under
specific pathogen-free conditions, in accordance with guidelines of the
Virginia
Commonwealth University Institutional Animal Care and Use Committee.
Challene of nu/nu mice with OVA immune complexes
Mice were injected with 20 g ovalbumin (OVA), 5 g in each limb, in the
form of (1) alum precipitated OVA (Sigma-Aldrich, St. Louis, MO, A5503) with
Bordetella pertussis, or (2) OVA immune complexes (ICs) made of NIP(4-Hydroxy-
3-iodo-5- nitrophenylacetyl)-OVA (Biosearch Technologies, Novato, CA, N-5041-
10) + goat polyclonal anti-tri-nitro-phenol Abs (Anti-TNP, Biomeda corp.,
Foster
City, CA, J05) or (3) OVA ICs made of alum precipitated NIP-OVA with
Bordetella
pertussis + anti-TNP. Anti-TNP Abs effectively bind the OVA-conjugated NIP
forming ICs. Azide-free Functional-Grade Purified anti-mouse CD90 (50 g, Thy-
l,
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eBioscience, 16-090 1) were given IP per mouse to inhibit residual T cell
activity,
especially y-b T cells, that may be present in these animals. Animals were
bled after
48 h, 1 week, and 2 weeks. Homozygous nu/nu mice were also bled after 7 weeks
and
mid-saggittal sections in the popliteal lymph nodes were labeled for GC B
cells with
peroxidase-conjugated peanut agglutinin (PNA-HRP, Sigma-Aldrich, St. Louis,
MO,
L7759). Ova-specific IgM was assessed in the collected sera and levels were
recorded
after subtracting the pre-immunization background levels.
Enzyme immunohistochemistry and light microscop
To test for GC development in OVA-ICs challenged nu/nu mice, popliteal
lymph nodes (LNs) were collected and frozen in CryoForm embedding medium
(IEC). Frozen sections of 10 m thickness were cut on a Leica cryostat (Jung
Frigocut
2800E) and air dried. Following absolute acetone fixation, the sections were
dehydrated and the endogenous peroxidase activity was quenched with the
Universal
Block (Kirkegaard & Perry Laboratories, 71-00-61). Mid-saggital sections were
washed, saturated with 10% BSA, before incubation with HRP-conjugated PNA. The
sections were washed then developed using diaminobenzidine substrate kit (BD
Pharmingen, San Jose, CA, 550880). The sections were washed, mounted, and
coverslipped; images were captured with an Optronics digital camera on an
Olympus
light microscope.
Confocal microscopy
Although they lack reactivity to OVA (Schuurman et al. (1992) J. Exp. Anim.
Sci. 35, 33-48), the fact that environmentally-induced GCs have been reported
in old-
age un-immunized athymic rats (Schuurman et al. (1992) J. Exp. Anim. Sci. 35,
33-
48) prompted us to confirm that GCs developing in athymic nu/nu mice
challenged
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with OVA-ICs co-localize with OVA-ICs retaining reticula and express
phenotypic
markers characteristics of GCs in normal mice. To test this, two groups of
nu/nu mice
were challenged with: a) OVA-specific rabbit serum (Meridian Life Science Inc,
Cincinnati, OH, W59413R) plus alum-precipitated OVA and B. pertussis or b)
normal
(non-specific) rabbit serum (Gibco, Grand Islands, NY plus alum-precipitated
OVA
and B. pertussis. Mid-saggittal sections (10 m-thick) in the axillary lymph
nodes
were triple labeled with GL7-FITC (BD Pharmingen, San Jose, CA, 553666) (for
GC
B cells), B220-Cy5.5 (Pharmingen, San Jose, CA, 552771) (general B cell
marker),
and Rhodamine Red-X-AffiniPure Goat Anti-Rabbit IgG (Jackson ImmunoResearch
Laboratories, West Grove, PA, 111-295-144) multiple adsorbed to minimally
cross
react with mouse, rat and human serum proteins (to label the OVA ICs retained
in the
FDC reticulum). To label for GC-associated plasmablasts, the Rhodamine Red-X-
AffiniPure Goat Anti-Rabbit IgG was replaced in some sections with Blimp-l-PE
(Santa Cruz Biotechnology Inc, Santa Cruz, CA. sc-13203 PE). Sections were
mounted with anti-fade mounting medium, Vectashield (Vectashield, Vector
Laboratories, Burlingame, Calif.), cover-slipped, and examined with a Leica
TCS-SP2
AOBS confocal laser scanning microscope fitted with an oil plan-Apochromat 40X
objective. Three lasers were used, Argon (488 nm) for FITC, HeNe (543 nm) for
Rhodamine-Red X or PE, and HeNe (633 nm) for Cy5.5 (shown as pseudo-color
magenta). Parameters were adjusted to scan at 512X512 pixel density and 8-bit
pixel
depth. Emissions were recorded in three separate channels and digital images
were
captured and processed with Leica Confocal and LCS Lite software.
In vitro stimulation of purified B cells with purified IC-bearing FDCs
Naive untouched human B cells were purified by negative selection on LS
MACS separation columns using The Naive B Cell Isolation Kit II (Miltenyi
Biotec,
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Auburn, CA, 130-091-150). Murine B cells were purified by positive selection
on LS
MACS separation columns using CD45R (B220) MicroBeads (Miltenyi Biotec,
Auburn, CA, 130-049-501).
FDCs were isolated by positive selection from LNs (axillary, lateral axillary,
inguinal, popliteal, and mesenteric) of irradiated adult mice, as previously
described
(Sukumar et al. (2006) J. Immunol. Methods 313, 81-95). One day before
isolation,
mice were irradiated with 1000 rad to eliminate most lymphocytes, and then
sacrificed, and LNs were collected, opened, and treated with 1.5 mL of
collagenase D
(22 mg/ml, C-1088882; Roche), 0.5 mL of DNase I(5000 U/mL, D-4527; Sigma-
Aldrich), and 2 mL of DMEM with 20 mM HEPES. After 45 min at 37 C in a COz
incubator, released cells were washed in 5 mL of DMEM with 10% FCS. Cells were
then sequentially incubated with FDC-specific Ab (FDC-Ml) (BD Pharmingen, San
Jose, CA, 551320) for 45 min, 1 g of biotinylated anti-rat ^L chain (BD
Pharmingen, San Jose, CA, 553871), for 45 min, and 20 L of anti-biotin
microbeads
(Miltenyi Biotec, Auburn, CA, 130-090-485) for 15-20 min on ice. The cells
were
layered on a MACS LS column and washed with 10 mL of ice-cold MACS buffer.
The column was removed from the VarioMACS, and the bound FDCs were released
with 5 mL of MACS buffer.
Purified FDCs were loaded with 100 ng/mL OVA ICs made of OVA/rabbit
anti-OVA at a ratio of 1:6. IC-loaded FDCs were used to stimulate 20x 106
purified B
cells at a ratio of 1FDC:2B cells. Cells were cultured in 10 mL culture medium
and
OVA-specific Abs were assessed after 48 h.
The rat anti-mouse IgD mAb clone 11-26 (SouthernBiotech, Birmingham,
Alabama, 1120-14) was complexed with Fc-specific rabbit anti-rat IgG (Jackson
ImmunoResearch Laboratories, West Grove, PA, 312-005-046) at a ratio of 1:4
and
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ICs were used to load purified FDCs or FDC-like cells. This monoclonal
antibody per
se does not induce proliferation of mature B cells in vitro, nor does in vivo
injection of
the monoclonal antibody have any effect on activation of B lymphocytes. FDCs
and
FDC-like cells were loaded with anti-delta ICs at doses of 0.1, 1.0, and 10
g/mL and
used to stimulate 104, 105, and 106 purified murine B cells in 1 mL cDMEM.
Culture
supernatants were assessed after 48 h for total mouse IgM production using
ELISA.
ELISA
Total and OVA-specific IgM were assessed in sera and culture supernatants
-48 hours after stimulation of B cells with OVA or anti-IgD IC-bearing FDCs in
vivo
and in vitro. Samples were loaded on 96-well plates coated with 100 g/mL OVA
(for
OVA-specific Abs) or goat anti-mouse IgM (for total IgM). Samples were left
overnight, washed and captured mouse IgM was detected with biotinylated goat
anti-
mouse IgM followed by streptavidin-alkaline phosphatase. Alkaline phosphatase
was
developed with pNPP alkaline phosphatase substrate system (KPL, Gaithersburg,
Maryland, 50-80-00) and read on ELISA reader at 405 nm.
Visualization and quantification of intracellular phosphotyrosine in
stimulated
B cells usin confocal microscopy and flow c ometry
Purified B cells were stimulated with anti-IgD IC-bearing FDCs for 45 min.
Cells were washed, fixed, and permeabilized using the Fix & Perm cell
permeabilization kit (Caltag Laboratories). Intracellular phosphotyrosine was
detected
with FITC-conjugated Anti-Phosphotyrosine, clone 4G10, (Upstate Biotechnology,
Lake Placid, NY, 16045). Cells were washed and analyzed with flow cytometry or
deposited onto polylysine-coated glass slides for visualization with confocal
microscopy using argon beam emitting 488 nm laser.
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Nude mice challenged with OVA ICs, but not OVA, developed GCs and
OVA-specific IgM
If periodically arranged FDC-ICs can induce specific IgM in the absence of T
cells, then nude mice should rapidly produce specific IgM when challenged with
a TD
antigen in the form of ICs but not with TD antigen alone. This hypothesis was
tested
in nu/nu mice given 500 g of a-Thy-l, to block any residual T cell activity,
and
challenged with OVA in adjuvant or OVA in ICs with or without adjuvant. As
expected, anti-OVA was not detectible in animals immunized with OVA over a
7-week period, even with adjuvant. (Fig. 13). In marked contrast, OVA-specific
IgM
was present in the sera of all ICs-injected animals with or without adjuvant
in just
-48 h. The highest OVA-IgM levels were induced using adjuvant-supplemented
OVA-ICs and these IgM levels were maintained over a 7 week assessment period.
This is not unexpected, as LPS will activate FDCs and promote their accessory
activities (El Shikh et al. (2007) J. Immunol. 179, 4444-4450). Well-developed
PNA+
GCs were observed in the draining lymph nodes of the IC-challenged animals,
further
supporting FDC-mediated B cell activation (Fig. 13). Phenotypically normal
heterozygous nu/+ mice also responded to ICs by producing OVA-specific IgM
within -48 h (Fig. 13), although, these IgM levels declined as the isotype
switched
from OVA specific IgM to IgG, in the presence of T cell help (Fig. 13).
IC-induced GCs in nude mice are associated with well-developed ARR and
plasmablasts
As expected for a T-dependent protein antigen, GCs were not detected in
athymic nude mice, nu/nu mice challenged with OVA (data not shown). The B cell
follicles labeled with B220, but not with the GC B cell marker GL7. In marked
contrast, the follicles in nu/nu mice challenged with OVA-ICs developed large
GCs.
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In an overlay, GL7 bright GC B cells surrounded by a zone of un-activated B220
bright B cells were seen. There was an area of dim B2201abeling. Activated B
cells
tend to downregulate B220 and this dim B220 area correlated with the
expression of
the activation marker GL7. In some images, a well-developed crescent-shaped
ARR
labeled with anti-rabbit IgG (identifies the rabbit IgG in trapped ICs made of
OVA +
rabbit-anti-OVA IgG) was seen. A funnel-shaped antigen transport site
extending
from the sub-capsular sinus into the lymph node cortex was apparent.
Purified OVA IC-bearing FDCs induced OVA-specific IgM production by
purified B cells within -48 h in the absence of T cells and T cell factors
If periodically arranged FDC-ICs can induce specific IgM in the absence of T
cells, then purified FDCs, bearing a TD antigen in the form of ICs, but not
with TD
antigen alone, should rapidly stimulate specific IgM by naive B cells in
vitro. FDC-B
cell interactions are not MHC or species restricted and murine FDCs can
stimulate
human B cells effectively (Fakher et al. (2001) Eur. J. Immunol. 31, 176-185).
Purified murine (Fig. 14A) or human (Fig. 14B) B cells stimulated with FDCs
bearing
OVA IC in cultures lacking T cells and T cell factors produced OVA-specific
IgM in
-48 hours. Both the kinetics of the response and the IgM production were
consistent
with a T-independent response. Control conditions, that failed to produce a
detectable
response, included FDCs with B cells stimulated with free OVA that would have
had
unfettered access to BCR.
Purified B cells were signaled by FDCs bearing anti-ND ICs as indicated by
increased levels and distribution of intracellular 12hosl2holyrosine
Extensive cross linking of BCRs can lead to B cell signaling, as indicated by
increases in and redistribution of intracellular phosphotyrosine in caps and
patches.
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We sought for evidence that ICs arranged on FDCs can signal B cells. We
reasoned
that anti-IgD loaded on FDCs in the form of ICs should engage multiple BCRs
and
induce B cell phosphotyrosine in caps and patches near the membrane surface.
The
anti-IgD mAb (rat anti-mouse IgD clone 11-26) was selected for this study
because it
does not induce B cell activation. This mAb was complexed with Fc-specific
rabbit
anti-rat IgG (to leave the Fabs free to engage BCRs) and loaded on the surface
of
FDCs. Phosphotyrosine labeling in unstimulated B cells was low and evenly
distributed. In contrast, B cells stimulated with FDCs bearing ICs labeled
more
intensely and the phosphotyrosine was capped (fluorescence localized at one
pole of
the cell surface), or patched on the membrane indicating a marked
redistribution (data
not shown). Most B cells exhibited the patched or capped intracellular
phosphotyrosine pattern consistent with being signaled. Moreover, flow
cytometric
analysis confirmed that the B cells exhibited higher levels of intracellular
phosphotyrosine (increased MFI) and the entire B cell population had clearly
shifted
to the right suggesting that virtually the entire B cell population had been
signaled by
anti-delta bearing FDCs. Higher magnification imaging revealed that the
phosphotyrosine labeling was intense at areas of contact between the B cell
membrane
and the AMCA-labeled IC-bearing FDCs.
Purified B cells stimulated with FDCs bearin anti-ND ICs on their surfaces
produced IgM within 48 h
Given that B cells are signaled by anti-delta ICs on FDCs, we reasoned that
the simultaneous engagement of multiple B cell receptors should signal, at
least some
of these B cells adequately, to rapidly produce IgM. Moreover, this should be
possible
in the absence of T cells, as was seen in Figure 14 where OVA ICs induced OVA-
specific IgM responses in vitro in the absence of T cells or T cell factors.
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Accordingly, we sought to test the hypothesis that anti-BCR bearing FDCs
induce
substantial polyclonal IgM responses in the absence of T cells or T cell
factors. As
shown in Figure 15, _1 X 104-_1 X 106 B cells stimulated with 0.1-10 g/mL
anti-IgD
ICs loaded on FDCs produced IgM within -48 h in a B cell dose-dependent
fashion,
although -100 ng of IC stimulated as well as 10 g. In the absence of FDCs,
anti-IgD
ICs did not induce production of IgM, even at doses of 10 g/mL.
T-I type 2 Ags show periodically arranged epitopes attached to a flexible
backbone. Their structure allows extensive cross-linking of BCRs and
activation of B
cells. Although T-D Ags possess multiple epitopes on their surfaces, each
particular
epitope is not repeatedly presented and accordingly BCRs specific for that
epitope are
not cross-linked and B cells are not activated. We believe that, if T-D Ags
can be
spatially approximated so that similar epitopes are close enough to cross-link
multiple
BCRs specific for the epitope, B cells can be activated without the need for T
cell
help.
FDCs express high levels of FcyRIIB and CRs, which trap ICs containing TD
Ags (multi-color clusters) that are periodically arranged on FDCs with -100-
500A
spacing that is ideal for extensive BCR cross-linking and B cell activation.
Transmission electron micrographs showed HRP (TD Ag) loaded on the surface of
FDCs as ICs with distances between IC clusters ranging between -200-500 A.
this
arrangement is fit to cross-link BCRs and activate B cells.
FDCs can stimulate B cells not only by cross-linking their BCRs, but
secondary accessory signals can also be delivered. As detailed previously FDCs
provide a complement-derived CD21L for B cell CD2 1; its interaction with the
CD21-CD19-CD81 complex delivers a positive co-signal for B-cell activation and
differentiation (Tew et al. (2001) Trends Immunol. 22, 361-367; Fakher et al.
(2001)
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Eur. J. Immunol. 31, 176-185; Qin et al. (1998) J. Immunol. 161, 4549-4554;
Qin et
al. (1997) Adv. Exp. Med. Biol. 417, 493-497; Qin et al. (2000) J. Immunol.
164,
6268-6275; Aydar et al. (2004) Eur. J. Immunol. 34, 98-107; Aydar et al.
(2003) J.
Immunol. 171, 5975-5987). Coligation of BCR and CD21 facilitates association
of the
two receptors, and the cytoplasmic tail of CD 19 is phosphorylated by a
tyrosine
kinase associated with the BCR complex. Additionally, the high density of
FcyRIIB
on FDCs binds Ig Fc in the Ag/Ab complex saving the B cells from the
inhibitory
signal delivered by the immunoreceptor tyrosine-based inhibition motif (ITIM)
if the
ICs were left to cross-link the BCR and the FcyRIIB on B cells. FDC-derived
BAFF
(Hase et al. (2004) Blood 103, 2257-2265; Ng et al. (2005) Mol. Immunol. 42,
763-
772) that ligates BAFF receptors on B cells, and FDC-derived C4b-binding
protein
(C4BP) (Gaspal et al. (2006) Eur. J. Immunol. 36, 1665-1673) that ligates CD40
on B
cells are other molecules that can deliver accessory activation signals to the
B cells.
Without wanting to be bound by any mechanism, from these experiments, we
propose an FDC-dependent T cell-independent multi-signal model for B cell
activation and Ig production. FDCs deliver a first BCR-mediated signal via
extensive
cross-linking of multiple BCR clusters helped by the flexibility of FDC
dendrites that
can geometrically fit the contour of B cells, in addition to FDC-derived
accessory
signals, known for their ability to co-stimulate B cells (see Fig. 1).
All documents, publication, manuals, article, patents, summaries, references
and other materials cited herein are incorporated herein by reference in their
entirety.
Other embodiments of the invention will be apparent to those skilled in the
art from
consideration of the specification and practice of the invention disclosed
herein. It is
intended that the specification and examples be considered as exemplary only,
with
the true scope and spirit of the invention being indicated by the following
claims.
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