Note: Descriptions are shown in the official language in which they were submitted.
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3 1 ~
LIPQSOMAL POL~S~CCH~R7DE VA~C:IN~
This applica~ion is a continuatTon-in-part of U.S. Serial No. 07/699,144 filed
May 13, 1991.
1. Flel~l of Th~ Inver~!~
The presont invention relates to methods and compositions tor inducing a
muccsal immun~ response to polysaccharide antigen encapsulated In a
liposome.
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2~ E3~1~
Despite the availability of intensive care, increasirlgly powerful antimicrobiala~ents, and rigorous prophylactic therapy, infections leading to multiple
organ system failure (MOSF) remain the rnajor cause of late morbidity and
mortality after such immunosuppressive ev~nts as trauma, hemorrhage, and
burns. It has been ~stimated that from 60-88% of deaths occurring more
than 7 days after hemorrhage, bh~rlt trauma, or thermal injury are caused by
sepsis. Nosocomial pneumonla is frcquent after injury, and often contributes
to multiple organ system failure, morbtdlty, and mortality in this setting. As
many as 3~70% of all severely inJured patients develop respiratory tract
infections in the intcnsive care unit.
Secretory immunoglobulin A is the predominant immunoglobulin at mucosal
sur~aces, such as the lung or intestine, and rnicrobial antigen-specific slgA
generated in gastro-intestinal or respiratory mucosa ccnfers protection
against pathogens infectirlg the host at or via these surfacès. Protection
against mucosally associated infection correlat0s best with the number of
antigen-speoific plasma ce!ls and secretory antibody levels in mucosal sites,
and not with serum IgG antibody levels or splenic plasma cell responses.
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21~9~ 2.
In contrast, slgA plays a minor role in prot~cting the host in systemically
invasive infections, such as bacteremia.
T and 8 cells found in mucosal sites such as the lung, Peyer's patches, and
intestinal lamina propria constitute a common mucosal immune system, and
a critical first line of defense against bacterîal infection. The mucosal B cells,
unlike systemic B cells, primarily produce slgA, and antigen-specific antibody
responses in mucosal sites ~ften do not parallel those occurring in systemic
Iymphoid organs, such as t~,9 spleen and Iyrnph nodcs. slgA responses
that originate in one mucosal site, such as the yut assoclated Iyrnphoid
tissue (GALT), dissen~.n~.. e to other rnucosally associated Iymphoid sites,
such as the lung, tonsils, and salivary ~lands. This mi~ration of slgA
secreting plasma cetls betw~en mucosal sites forms the basls for oral and
intranasal immunization strategies aimed at improving pulmonary immunity
to respiratory pathogens. In particular, this strategy has been used
tMestecky, J.Clin.lmrnunol., 7:265, 1~87; Liang, et al., .~,lmrnunol" ~43:484,
1989; and Tamara, et 81., Vaccine, ~:314, 1989) in developin~ vaccines
against protein antigens associated with v~ral infections, such as influenza,
where increased resistance to infections originating at pulmonary surfaces
I~.as been shown to accompany enhanced viral antigen specific pulmonary
secretary antibody production and B cell response.
:
Enhancement of the pulmonary mucosal immune response to protein
antigens, to more than 100 times baseline, has been achieved through oral
or intranasaî administration of the antigen of interest and an adjuvant, such
as cholera toxin (Elson, et al" J,lmmunol,, ~:2736, 1984), glutaraldehyde
deactivated cholera toxin (Liang, et al., J.lmmunol., 141:1495, 198B),
B-subunit of cholera toxin (McKenzie, etal., J.lmmunol., 133:1818, 1984) and
antigen encapsulated in liposomal formulations However, neither oral nor
intranasal immunization with combinations of bacterial polysaccharide
antigens and aquvan~s has been successfully reported to date
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WO g2~20370 PCI~/US92/04055
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The mechanism of action of cholera toxin derived products as adjuvants
appears to center about the affinity of the cholera toxin B subunit for GMI
gangîiosides on cell surfaces. This proper~y of the cholera toxin B subunTt
enhanees deîivery of associated antigens to the surfaces of mucosally
S associated cells initially involved in a localized and then, subsequently, in a
generalized mucosal IgA response to the presented antigen. t)eactivated
cholera toxin and the B subunit of cholera toxin have been proposed to be
appropriate for human oral vaccines since they lack significant toxicity. In
particular, the cholera toxin B subunit has been used successfully as a
component of human and animal~vaccines against protein antigens of
influenza and cholera. An advantage of liposomes is their nontoxic nature.
Further, liposomes have been shown in several systems to have potent
immunoenhancing properties on the mucosal immune response when
administered orally or intranasally (Gregoriadis, Immuno.~ Today, 1~ :89, 1990;
van Rooijen, et al., Immunoiogic~lAdjuvants ~nd Vaccines, Gregoriadis, G.,
Allison, A.C., Poste~G. (eds), Plenum Press, pp 95-106, 1989; Pierce, et ~/.,
Infect.lmmunol. ~:469, 1984). The potent ad)uvant effect of liposomal
delivery systems~ for antigen presentation is thought to be due, at least in
part, to their abil ty to fuse with cell membranes and deîiver their contents
directly to intracellular antigen processing systems.
The immunosuppressed host is ;especially susceptible to infection at
mucosal surfaces. Immunosuppression can be caused, for exampîe, by
chemotherapy,~trauma, bums, and hemorrhage, especially following injufy.
Blood loss is a central factor in the pathophysiologic instability that follows
25 ~ injury, and has~ been shown to increase susceptibility and mortality to
infection. Although blood loss does not result in changes in the absolute
or relative numbers of T!Or B cell subsets (i.e., CD3+, CD4+, CD8+, Ig+
Of B220+) in spleen, Iymph nodes, bone marrow, thymus, or lungs,
widespread changes in T and B~ cell function have been described after
3 0 ~ hemorrhage. Hemorrhage produces multiple abnormalities in immunologic
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function, including alterations in cytokine (IL-1, IL-2, IL-3, IL-5, ~-IFN) release
(Abraham, et ~1., CircShock, 25:33, 1988; Abraham, et al., J.lmmunol.,
~:899, 1989), decreases in mitogen induced T eell proliferation (Abraham,
et ~/., J./mmunol., 142:899, 1989), reduction in IL~2 receptor expression
(Abraham, et a/., J.lmmunol" ~:899, 198~; Stephan, et al" Arch.Surg"
1~2:62, 1987) and changes in splenic (Abraham, et ~I~, Cell.lmmunol.,
~:208, 1989), intestinal (Abraham, et ~I., Cell Immunol., 128:165, 1990),
and pulmonary (Robinson, et Bl., J.lmmunol., ~:3734, 1990), B cell
repertoires.
Studies (Esrig, et al., Rev,Surg, ~:431, 1976; Stephan, e~ al., Arch Surg.,
122:62, 1987) have shown increased susceptibility to infection following
hemorrhage. Unfortunately, the results from those studies are difficult to
interpret both because the models far hemorrhage were severely
immunosuppressive, even if no blood were withdrawn, and the infect~ns
produced were not closely related to those seen clinically. In particular, the
previously described models for hemorrhage involve the placement of
arterial catheters into animals anesthetized for prolonged periods. Such
animal preparations, even without any blood withdrawal, result in greater
than 70% decreases in mitogen induced Iymphoc~te proliteration (Stephan,
et al., Arch.Surg., 1æ:62, 1987), inflammatory response (Abraham, et al.,
Arch.Surg., 9:1154, 1984), ~s well as IL-1 (Ayala, et al., Jmmunology,
70:33, 1990) and~l~2produdion (Stephan, etal., Surg.Forum, 37:i3, 1986).
Many of the pathogenic organisms associated with infection of mucosal
suAaces, such as the respiratory tract and gut, are bacteria where often the
most desirable antigen ~ for purposes of protective immunity is
polysaccharide. Unfortunately! polysaccharide antigens are generally poor
immunogens even when administered systemically ~Schreiber, et al,
J.lmmunol., 146:188, 1991) ~ This is especially true where the host is
immunocompromised.; Enhanoement of the systemic immune response to
.
WOg2/20370 PCI/US92/04055
21~31.~
5.
polysaccharide antigens has been achieved through coupling to protein
carriers and adjuvants, or through anti-idiotypic manipulation (Schreiber, et
~1., Jlmmunol., ~44:1023, 1990. In contrast, little informatiorl is available
concerning the effects of immunkation with bacterial polysaccharides on the
antigen-specific mucosal immune response. In preyious studies (Abraham,
et ~/., Vaccine, In press), oral immunization with the bacterial polysaccharide
antigens levan or Pseudomonas ~eruginosa polysaccharide type 1
coadministered with cholera toxin, as an adjuvant, was found to prodlJce
increased ~ntigen specific pulmonary slgA titers. However, large amounts
of antigen ~i.e.j 1 mg) as well as the use of a toxic adjuvant were required
in order to enhance the pulmonary piasma cell response.
As a conseq~lence, there is considerable need for an effective non-toxic
method of immunizing mucosal surfaces against various bacterial infections.
The present inwntion provides such method by combining a bacterial
~5 polysaccharide antigen with a liposomal encapsulation carrier.
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SUMMARY OF THE INVENTION
Recognizing the role that pathogenic bacteria play in mucosal infections and
the sevcre th~rapeutic limitations of existTng immunization techniques, th~
inventors produced and evaluated novel Yaccines in an effort to develop a
more ~ffective method of immunkation. These efforts have culminated in the
development of a new method of immunization which is particularly effective
against bacteria which infect or invade the host through mucosal surfaces.
This method was deveioped utilizing compositions which comprise a
bacterial polysaccharide antigen encapsulated in a liposorne. Surprisingly,
these compositions and this method enables the host to mount a significant
antigen-specific secretory immune response to the encapsulated bacterial
polysaccharide antigen while minimizing advsrse reactions to the vaccine.
...
The method of the invention is enhanced by use of novel liposomal
compositions disclosed herein. These compositions comprise a bacterial
polysaccharide antigen encapsulated in a liposome and ~urther including an
adjuvant. The adjuvant can be compartmentalized with the antigen, inserted
into the liposomal membrane, or both
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WO 92/20370 PCI/US92/04055
~ ~J~c~l~
DESCRIPTION OF THE DRAWINGS
FIGURE 1. Anti-l~van slgA titers in lun0 lava~es of mice immunized
intranasally with liposomes containing 38 rng levan. Mice were immunized
at the indicated times following 30% blood volume hemorrhage, with
resuscNation 1 hotJr later. Control (C) mice were immunized 3 days
following ether anesthesia and cardiac puncture, but without blood
withdrawal. Results are shown as OD450 ~ SEM for undiluted lavage
specimens, since the maximal OD was consistently found when the samples
were tested without dilution (~pc0,05 versus Control).
FIGURE 2. Effects of hemorrhage with resuscitation on the numbers of
levan-specific pulmonary plasma cells. Mice were immunized at the
indicated times following 30% blood volume hemorrhage, with resuscitation
1 hour later. Control (C) mice wère immunized 3 days aft~r ether anesthesia
and cardiac puncture, but without blood withdrawal. Results are shown as
the nurnber of total and IgA producîng levan-specific plasma cells per set
of lungs ~ SEM. ~*pcO.05and ~p~0.01 versus Control).
FIGURE 3. Sur~ival of hemorrhaged mice intranasally immunized with 50 ,ul
of empty liposomes (Control), liposomes containing 25 ,ug Pseudomonas
aerug)nosa polysaccharide type 1 (PsA), or liposomes containing ~5 ,ug
Pseudomonas aeruginosa polysaccharide type 1 and 0.2yg (10~g/kg) IL-2
(PsA + IL-2). The mice were immunized 2 hr following 30% blood volume
hemorrhage, and then were inoculated intratracheally with 5 x 107 cfu
Pseudomonas aeruginos~ Fisher-Devlin immunotype 1 (strain 15921 ) 4 days
following blood loss. Each group consisted of 1t animals. At 10 days
following the induction of pneumonia, the rnortality of mice intranasally
immunized with liposomes containing Pseudomonas aeruginosa
polysaccharide and IL-2 was significantly reduced (p<0.005) compared to
the groups of animals immunîzed wtth empty liposornes or immunized with
liposomes containing Pseudomonas aeruginosa polysaccharide, but no IL-~.
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WO 92/20370 PCI'/US92/04055
2 1 ~ 6
DFTAILED DESCRIPTION OF THE PREFERf~ED EMBODIMENT
The inventors have devised eompositions and methods for immunizing an
animal to induee a mueosal tmmune response to a baeterial polysaccharide
antigen whieh represents a signifieant improvement over the prior art
methods intended to aeeomplish this effeet. The present invention
eomprises the administration, to an animal whieh has, or is at risk of having,
an infeetious disease whieh oeeurs via eontaet between the infeetious
organism and a mueosal surfaee.
In the present inventian, liposomes are used to eneapsulate the baeterial
polysaeeharide antigen. When phospholipids are gently dispersed in
aqueous media, they swell, hydrate, and spontaneously form multilamellar
eoneentrie bilayer v~sicles with layers of aqueous media separating the lipid
bilayer. Sueh systems are usually referred to as multilamellar liposomes or
multilamellar Yesieles (MLV's) and have diameters ranging from about 1 00nm
to abo~t 4,um. When MLV's are sonieated, small unilamellar vesieles (SUV's)
with diameters in the range of from about 20 to about 50 nm are formed,
whieh eontain an aqueous solution in the eore of the SUV:
The eomposition of the liposomé i8 usually a eombination of phospholipids,
part eularly high-phase-transition-temperature phospholipids, usually in
eombination with steroids, espeeially eholesterol. Other phospholipids or
other lipids may also be used.
Examples of lipids useful in~ liposome production include phosphatidyl
eompounds, sueh as phosphatidylglyeerol, phosphatidyleholine, phosphati-
dylserine, phosphatidyl~thanolamine, sphingolipids, eerebrosides, and
gangliosides. Particu!arly usefu! are diacylphosphatidylglycerols, where the
lipid moiety eontains from;14-18 earbon atoms, partieularly from 1~18
earbon atoms, and are saturated. I!lustrative phospholipids include egg
WO 92/20370 PCI/US92/04055
2;1~'~3L(;
phosphatidylcholine, dipalmitoylphosphatidylcholine, and distearoylphos-
phatidylcholine .
In preparing liposomes containing the bacterial polysaccharide antigen, such
variables as the efficiency of antigen encapsulation, lability of the antlgen,
homogeneity, and size of the resulting population of liposomes, antigen-to~
lipid ratio, psrrneability and instability of the preparation, and pharmaceutical
acceptability of the formulation should be considered. (Szoka, et al" Anmlal
Reviews of Biophysics ~nd Bio~engineering, ~:467, 1980; C)eamer, et a/" in
Liposomes, Marcel Dekker, New York, 1983, 27; Hope, et al.,
Chem.Phys.Lipids, 40:89, 1986).
The invention is particularly useful in inducing an immune response in an
animal, such as a human, which has been immunocompromised. The term
"immunocompromised" denotes an animal having an immlme system v~hich
is not functioning normally. Examples of conditions which can cause an
animal to become immunocompromised inciude chemotherapy, irradiation,
age, and physical trauma, such as resulting from shock, severe hemorrhage
biood loss, or burns.
Many organisms, especially bacteria, utilize mucosal surfaces as sites of~
infection or initial imasion. In the present invention, the liposomal vacdne
is able to induce a secretory immune response at the mucosal surface in
order to amellorate the pathogenic effect of these various organisms.
Because of the interreiatedness of the various limbs of the mucosal immune
system, it is possible to ;vaccinate the animal at one mucosal site and
produce a secretory imm~me`response at a different site. Examples of
tissues which can! be irnmunized andlor can produce a secretory immune
response include, the respiratory tract, the gastro-intestinal tract, and the
urinary tract, as~well as glands such as the mammary glands, salivary
glands ~and tear ducts. ~ Ihus,~it ~is possible to induce a secretory immune
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10.
response against a gastro-intestinal pathogen by immunizing intranasally.
Alternatively, it is possible to induce a secretory immune response in the
respiratory tract by immunization of th~ gut, such as the Peyer's patches or
lamina propria.
The terrn "immunogenically effective amount" as used in the invention
denotes that amount of polysaccharide antigen which is necessary to induce
the animal to produce antibodies which will bind to epitopes present on the
polysaccharide antigen.
The method and compositions of the invention are especially useful in
allowing immunization of bacterial polysaccharide antigens. Among the
various bacterial polysaccharide antig~ns which are preferred are those
derived from species or strains of Aero~cter, Klebsiella, Proteus,
Salmonella, Shigella, ~ Campylobacter, Pseudomonas, and Streptoco~cus.
Especially preferred are bacterial potysaccharides derived from strains of
Ps~udomonas aeruginosa and Strepfococcus pneumoni~e. These bacterial
polysaccharide antigens can be derived from a single strain or serstype or
they may be polyvalent, i.e., a pool of antigens from various serotypes. The
use sf a polyvalent bacterial polysaccharide antigenic mixture is especially
~ useful for organisms such as Pseudomonas aeruginosa and Streptococcus
pneumoniae where clifferent serotypes are implicated in the etiology of
disease. Techniques for the preparation of polysaccharide antigens from
various bacteria~ are well known to those of skill in the art or can be readily
ascertained without undue experimentation.
It is also possible for the liposomal preparations containing the bacterial
polysacc,haride antigen to include an adluvant Adjuvants are substances
that can be used to nonspeciflcally augment a specific immune response.
Normally, the adjuvant and the antigen are mixed prior to presentation to the
immurle system, or presented separately, but into the same site of the
WO 92/20370 PCI/USg2/04055
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11.
animal being immunized. Adjuvants can be loosely divided into several
groups based on their composition. These groups include oil adjuvants (for
example, Freund's ~omplete and Incamplete), mineral salts (for example,
AIK~5O~2, AlNa(SO4)2, AINH4~SO~), silica, alum, Al(OH)3, Ca3(PO4)2, kaolin,
and carbon) polynucleotides (for example, poly IC and poly AU acids), and
certain natural substances (for example, wax D from MycobacteriL~m
t~Jberculosis, B subunit of cholera toxin, glutaraldehyde treated cholera toxin,as well as substan~es found in Corynebacterium pan/Llm, Bordetella
pertussis, and members of the genus Brucella).
Other adjuvants which cah also be incorporated in the liposomal
preparations include immunomodulators and other biological response
modifiers. The term "biological response modifiers" is meant to encompass
substances which are involved in modifying the immune response in a
manner which enhances the immune response to the polysaccharide
antigen. Examples of biological response modifiers include Iymphokines
such as the interleukins, macrophage activating factors, migration inhibition
factor, colony stimulating factors and the interferons. Preferred interleukins
are those which display a direct affect on in vitro B-cell function, such as
proliferation, antibody production, or activation. Especially preferred are IL-2and IL-6. Other biological response modifiers are known, or readily~
ascertainable, by those of skill in the art.
The liposoma! preparations of the invention can also be modified to contain
an adjuvant in the membrane of the liposome. Preferred compounds are
;~ those characterized as lipoidal amines which are disclosed in Chang, et ~I.,
Anhrjtis`~nd Rheumatism, ~:169, 1978 and Chang, et al., Arthritis and
Rheumatj~sm, ~:62, 1980, which are hereih incorporated by refererlce.
Especially preferred among the lipoidal amines is avridine (MN-dioctadecyl-
N'j N~-2-hydroxymethyl-propane-diamine). Liposomes containing avridine
can encapsulate multiple antigens, and the presence of avridine in the
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12.
liposornal membrane potentiates the adjuvant effect of the liposome, so that
a greater mucosal immune response can be achieved after the
administration of antigen in avridine containing liposomes. Avridine and
bacterial polysaecharide containing liposomes can be prepared by the
detergent dialysis techrlique ~as modified by Philippot, et al., Biochem
Biophys Acta, ~:137, 1983) to produc~ large unilamellar liposomes,
capable of encapsulating large amounts of antigen. (:)ther techniques for
liposome preparation such as the dehyclration-rehydration modification
(Kirby and Gregoriadis, Biotechnology, 2:979, 1984) also yield high
eneapsulation percentages. Regardless of the adjuvant which is utilized
accordin~ to the inv0ntion, it is possible for the adjuvant to be present withinthe liposome itself, i.e., compartmentalized wlth the polysaccharide antigen,
in the membrane of the liposome vesicle, or both. Those of skill in the art
are familiar with techniques for the inclusion of adjuvant within the liposome
(for example, complexed to the bacterial polysaccharide antigen) or xvithin
the liposomal mernbrane.
Many different techniques exist ~or the timing of the immunizations when a
muitiple immunization regimen is utilized. It is possible to use the liposomal
preparation of the invention more than once to increase the levels of the
ameliorating secretory immune response.
Generally, the~ dosage of polysaccharide anti~en administered to an animal
will vary depending upon su~h factors as age, condition, sex, and extent of
the disease, if any, and other variables which can be adjusted by one of
ordinary skill in the art. Howevet, it is preferred, at least in instances wherethe animal has been subject to an immunosuppressive event due to trauma
such as severe blood loss, to administer the imrnunization within 24-~8
hours, preferably within 24 hours.
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Th~ antigenic preparations of the invention can be administered as either
single or multiple dosages and can vary from 10 yy/rnl to 10,000 ~g/ml,
more prefcrably from 50 ,ug/ml to 5,000 ~g/ml antigen per dose, most
preferably 100 ~g/ml to 600 ,ug/ml antigen per dose. Generally, those
dosa~es which elicit the highest levels of slgA (secretory immune response)
to the bacterial polysaccharide antigen are preferred.
When a biological response modifier, such as an interleukin is included with
the antig~n in the liposome, dosages of modifier oan vary from about
0.001 ,ug/ml ts about 1000,u~/ml, preferably from about 0.01 ,uglml to about
200 ,ug/ml, most pr~ferab!y from aboLt 0.05 ~g/ml to about 50 ,ug/ml.
Having now generally described th~ invention, a more complete
understanding can be obtained by reference to the following specific
examples. These examples are provided for the purpose of illustratior~orlly
and are not intend~d to be limRin~ unless otherwise specified
EXAMPLE 1
IMMUNOGENIC STUDIES OF LIPOSONIE ENCAPSUI ATED
BACTERIAL PO~YSACCHARIDE ANTIGENS
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Studies were done in 8-12 week ol~ rnale BALB/C mice ~ackson Labs, Bar
Harbor, ME) to determine the secretory immune response to bacterial -
antigen encapsulated in liposomes. ~ ~
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Uposomes were prepared by the detergent dialysis technique, as modified
by Philippot, et al. (Biochem Biophys Acta, ~:137, 1983) to produce large
unilarnellar liposomes, capable of encapsulating high amounts of antigen.
Cholesterol, phophatidyls~nne, and phosphatidylcholine (8 ~JM each) were
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combined, dried to a film under N2, and then placed in a Iyophilizer for
another 60 minutes. The bacterial polysaccharide to be encapsulated was
suspended in 0.5 ml of buffer (150 mM NaCI, 10 mM HEPES (GIBCO), 1
mM EDTA, pH 7.4) and added to the dry lipids. C~4-labelled inulin t20 ,u1),
to monitor encapsutation efficiency, was added to the suspension. After 30
minutes, 0.24 ml of 1 M octylgiwoside (Calbiochem) was added to the lipid
and antigen mixture, and shaken vigorously. The sample was transferred
into dialysis tubing (molecular weight cutoff 3S00), and dialyzed against 100
ml of buffer containing 2.4 g SM-2 Biobeads (Biorad). After 24 hours, the
liposoms preparation was placed on the A5M ~Biorad) column, and the
liposome fraction (in the void volume) collected. Encapsulation efficiency
was determined by comparing the C14 counts in the initial lipid suspension
to that in the final liposome fractions. In generaî, encapsulation efficiencies
of approximately 40% (levan and Pseudomonas ~er~ginosa typ~ 1) and 10%
Streptococcus pneumoniae type 3 polysaccharide ~PPS 3) were obtained.
The average ske of the unilamellar liposomes was 995 :~ 80 nm, as
determined by dynamio laser light scattering using a Coulter NS4D
instrument. .
.,
Animals were immunked intranasally by applying 0.1 ml of liposomal
suspension to the nares and allowing this to be inhaled. Antigens tested~
were levan (38 ~g) from~Aerob~ct~er levanious ~Sigma, St. Louis, MO),
polysaccharide type 1 (33 ~g) from Pseudomonas ~eruginosa, and
pneumococcal polysacd~aride type~ 3 (33 ,ug) from Streptococcus
pneumoniae.
Pulmonary lavages were obtained by pooling bronchoalveolar washings
produced by injecting 0.,5 ml of phosphate buffered saline (PBS) 3 times in~o
the trachea and lungs !n general, a final volume of approximately 0.4 ml
~; was obtained. The washings were centrifuged to remove cellular debris and
stored at -20- C until tested.
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Pulmonary lavages and serum were tested for production of antibody
a~ainst the bacterial polysaccharide antigens using an ELISA technique
(Portnoi, et al., Eur.J.lmmunol., ~:571, 1988). The 96 well ELISA plates
~Costar, 3590, Cambridge, MA) were coated overnight with antigen (10
,~lg/ml) in 0.05 M phosphate buffer, pH 8. After washing the plates with PE~S,
the wells were saturated with PBS containing 1% ovalbumin for 30 minutes
at 37- C. ASter further washing with PBS, 50 ,ul of pulmonary lavage fluid or
serum in serial 2-fold dil~tions was~transferred to the ELISA plates, and the
plates incubated for 2 hours at room temperature. After washing,
peroxidase-coupled goat anti-mouse antibody was a~ded to each well. After
a f~rther 1 hour incubationj the bound antibody was revealed with
chromogen containing H2O2 and orthophenyldiamine. The reaction was
stopped 20 minutes later by addition of 50 yl ot 10% SDS to each well and
the OD measured at 450 nm in a photometer (MR 600, Dynatech
Instruments, Torrance, CA) with a 410 nm correction filter.
: .
: ` TABLE 1
QRGANISM
Aerob~cter
~ nkus ~ levan 0.046 ' 0.017 0.126 + 0.023
Pseudomon~s
~eruginosa ~: type 1 0.255 ~ 0.061 0.660 ~ 0.051
: ~ Streptococcus :~
pneumoniae PPS 3 0.127 + 0.044 0.293 + 0.115
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The data in Table 1 shows that animals vaccinated via the mucosal immune
system produce significant titers of slgA specific for a given bacterial
polyæaccharide antigen when the antigen is presented in the form of a
vaccine which utilizes a liposome carrier system to encapsulate the bacteriai
polysaccharide antigen.
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IMMUNIZATION OF IMMUNOCOMPROMISED ANIMALS
The effect of immunization with liposomally encapsulated bacterial
polysaccharide antigen was studied in immunocornpromised animals.
Immunocompromization was induced in mice using the murine hemorrhagic
model (Abraham, e~ ~I., J.lmmunol., ~g2:899, 1989). Briefly, mice were
anesthetized with inhaled ether after being placed into a covered b~aker.
Cardiac puncture was performed with a 3~gauge needle and 30% of the
~ calculated blood volume (approximately 0.55 ml for a 20 g mouse) was
withdrawn over a 6~second period. ~ blood was collected into a syringe
containing 1Q0 U heparin~in 0.1 ml, and then was kept at 37OC for 1 hour
until reinfusion into the unanesthetized mouse through a retroorbital plexus
injection. Using this method, it was possible to resuscitate all hemorrhaged
mice without complieation.~ The mouse then was allowed to recuperate in
~20 its cage. The total period of ~ether anesthesia was less than 2 minutes in all
eases. The mortality rate~ with this hemorrhage protocol is approximately
- ~ 12h, with all deaths~;oeeuning ;during the first 24 hours post hemorrhage,
and most deaths occurring within the 1 hour post hemorrhage.
With this hemorrhage rnodel, ether anesthesia and cardiac puncture without
blood withdrawal result;in no~changes in mitogen-induced Iymphocyte
proliferation, IL-2R~ expression, ~pheno~rpic characteristics (CD3, CD4, CD8,
B220"u, Ly-1 expression)~of B or T lymphocytes, lymphokine (IL-2, Ik3, IL-5,
-
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, -
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17.
y-lFN) release, or splenic, intestinal, or pulmonary B cell clonal precursor
frequencies (Robinson, et al" J.lmmunol., 145:3734, 19g0). In particular,
previous experiments had found no alterations in the numbers or
frequencies of pulmonary B cell clonal precursors specific for levan or
Pseudomonas aeruginos~ polysaccharide type 1 in mice after ether
anesthesia and cardiac puncture, but no blood withdrawal (Robinson, et ~
J.lmmunol., ~:3734, 1990). Similarly, no evidence of hemothorax, bleeding
into the pericardiai space, lung, or cardiac contusion has been found in
surviYing mice with this method of hemorrhage (Abraham, et al" J./mmunol.,
142:899, 1989). Animal selection, immunization, and ELISA techniques were
performed as described in Example 1.
,
Intraparenchymal lung Iymphocytes were isolated as previously described
(Abraham, et al., J.lmmunol., 144:2117, 1990). In brief, 3 mice were used
for each experiment, and the lung cells pooled for analysis. After the rnouse
was killed by cervical dislocation, the chest was opened and the lung ,~vascular bed was flushed by injecting 3-5 ml of chilleci (4~C) PBS into the
right ventricle. The lungs were then excised, avoiding the peritracheal Iymph
nodes, and washed twice~ in RPMI 1640 (GIBCO). The lungs were minced
finely, and the tissue pieces placed~ in RPMI 1640 with 5% FCS (GIBCO),
penicillin/streptomycin, 10 mM HEPES, 50 ,uM 2-ME (GIBCO), 20 mM L~
glutamine (GlBCOj, containing 20 U/mi collagenase (SIGMA), and 1 ,uglml
DNase (SIGMA). Foliowing incubation for 60 minutes at 37 C any rernaining
intact tissue was disrupted by passage through a 21 gauge needle. rlssue
~ ~ fragments and the ~majority o f dead cells were removed by rapid filtration
25~ th~ough a glass wool column, and cells collected by centrifugation. The cell
pellet was suspended in 4 ml of 40% Percoll (Pharmacia, Uppsala, Sweden)
and layered onto 4 ml of 80h Percoll. After centrifugation at 600 g for 20
minutes at 15-C the cells at the interface were collected, washed in RPMI
1640, arld counted~ ~ V iability was consistently greater than 98% as
determined by trypan blue~ :exclusion.
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Detection of total numbers and levan-specific plasma cells in the lung was
performed using a technique similar to that described by Sedgwick and Holt
(Sedgwick, et ~I., J.Jmmunol. Methods, 57:301, 1983 Briefly, .96 well flat
bottom ELISA plates were coated by overnight incubation at 4- C with levan
(10 ~ug/ml) in 50 ~JI/well 0.05 M potassium phosphate buffer, pH 8 The
plates then were washed with PBS, and the wells sa~urated with PBS
containing 1% gelatin Sor 1 hour at 37- C After further washing with PBS,
the wells were seeded with a titration of intraparenchymal lung Iymphocytes
from immunized or unimmunked mice in 100 ,~l RPMI containing glutamine,
2-ME, 10 mh~l HEPES, penicillin/streptomycin, and 1% fCS, The number of
cells per well was adjusted to be from 102 to 106.
The plates then were incubated at 37- C in 5% CO2 for 5 hours The cells
were removed by flicking the plate, followed by Iysis with 0.05% Tween 20
in H2O. After washing with PBS~ containing 0 05% Tween, goat anti-mouse
total lg coupled with biotin was added to the wells in PBS 1% gelatin, and
the plates left at 4 C overnight The plates then were washed in PBS,
0.05% Tween, streptavidin coupled with alkaline phosphatase added to the
wells. Aner a 1 hour incubation at 37 C, the plates were washed and the
revealing substrate,~ 2.3 mM Sbromo~4-chloro-3-indolyl phosphate ~Sigma,
St. Louis, MO) diluted in 2-amino-2-methyl-l-propanol (Sigma, St. Louis, MO),
containing 0.75h agarose was added to each well After a 1 to 2 hour
incubation period at~ 37~C, the antibody secreting cel!s were revealed as
blue spots which could~be counted. The dilution of cells producing 20 to
40 spots/well was used to~quan~tate the total number of antibody-specNic
B cellsjsample. The~ sensitivity and specificity of this ass Iy has been
documented in Ag-inhibition tests and in studies involving Ag-specific
hybridoma cell lines (iai~man, et ~l., J.lmmur~ol., 141:801, 1988; Sedgwick,
et ~1., J.lmmunol. Methods, 87:37, 1986.
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Pneumonia was induced using Fisher-Devlin immunotype 1 strain 15921 of
Pseudomonas ~eruginosa obtained from Dr Gerald Pier, Harvard University,
Bostorl, MA After f esh plating on tryptic soy agar plates, the bacteria were
transferred to tryptic soy broth and incubated at 37 C until a concent-ation
of 109 cfu was achieved. The bacteria were centrifuged, washed twice in
chilled PBS, then resuspended to a concentration of 5 x 10B, 108, 2 x 109,
or 3 x 109 cfulml in PBS Mice were anesthetized with pentobarbital (50
mglkg) i.p, vertically suspended, and 40 ,ul of the bacterial suspension
introduced into the trachea using a blunt 22 gauge needle passed through
the mouth. The mice were returned to their cages and permitted free
access to food and water. Mortality was assessed twice each day Mice
were observed for a 7 day pariod, but no mortality was found after day 5
, . .
In experiments examining antigen~specific pulmonary slgA titers and
pulmonary plasma cells, Z groups of mice (each group consisting of 3 mice)
were examined for each experimental condition ELISA was perfo~med on
pulmonary lavages f om each mouse (6 lavages examined fo- each
condition) For~ the ELISA Spot assay, pooled lung Iymphocytes were
isolated from each group of 3 ~ nice The numbe- of antigen-specNic plasma
- cells per set of lungs was calculated by multiplying the numbe- of antigen-
~20 ~ specific spots per 106 iung Iymphocytes, as determined by the ELISA Spot
Assay, by the t:otal~ numbe~ r~ of; cells isolated per set of lungs.
Data are p-esented~ as mean ~ standard error ~SEM) for each experimental
group Comparisons between means of ~roups were performed by the
Student t test for differenc~es be~Heen 2 groups or by using one way
analysis of variance for examining differences for experiments with more than
2 groups! Survival data were analyzed by Chi-square and Fisher's Exact
analysis A p value less than 0 05 was considered to be significant
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A. RESULTS OF INTRANASAL IMMUNIZATION
.
Because of the existence of a common mucosal immune system ~Mestecky,
et ~1, J.Clin.lmmunol" 7:26S, 1987), where secretory antibody responses
generated at one mucosal surface subsequently are found at other nwcosal
sites, mice were intranasally immunized with incr~asing arnounts (1, S, 10,
and 38 /ug) of the bacterial polysaccharide antigen levan encapsulated in
liposomes. One week later the animals wer~ sacrUiced, the lungs lavaged
as described above, and the bacterial anti~en specific slgA titers measured
by ELISA. In addition, lung Iymphoc~rtes were isolated and the number of
bacterial antigen specifio plasma cells per set of lungs was enumerated
using the ELISA Spot assay. Serum also was obtained, and anti-levan titers
measured by ELISA.
In unimmunized mice and in animals intranasally immunized with bet~veen
1 and 10 ,ug of liposome encapsulated levan, no ievan-specific plasma cells
wsre found ~mong lung Iymphocytes. In contrast, 33 ' 5 levan-speclfic
plasma ceîls~per set of lungs were present following intranasal immunization
with 38 ,ug of the levan containing liposomes. Similarly,~increases in the
levan specific slgA titers were found only in rnice immunized with 38 ~g of
the liposome encapsulated ievan (OD450-0.126 * 0.023versus 0.046 ~ 0.017
20 - in unimmunized mice). No changes in serum anti-levan titers were found in
any group of intranasally immunized mice as compared with the
unimmunized group.
B. EFFECTS OF HEMORRHAGE ON PULM()NARY PLASMA
CELL RESPONSE
In order to examine the eMects of hemorrhage induced immunosuppression
on the pulmonary secretory ahtibody response to bacterial antigens, 30%
of mouse blood volume was~ removed, then returned 1 hour later.
,
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Liposomes containing 38 ~g levan were administered intranasally at
predetermined timepoints post hemorrhage. One week following
immunization, lung lavages and pulmonary Iymphocytes were collected for
analysis of levan-specific slgA titers and plasma cell numbers. Serum also
was collected 1 week post immunization, and anti~levan titers measured.
The results of these experlments are shown in FIGURES 1 and 2.
Anti-levan slgA titers in lung lavages from mice immunized 3 days after
blood loss were not different from those in normal or control animals.
However, anti-levan slgA titers were significantly decreased in mice
immunized 7 and 14 days following hemorrhage (FIGURE 1). Reduced
numbers of levan-specific pulmonary plasma cells also were found after
hemorrhage and resuscitation, but with kinetics somewhat different from that
present for anti-levan slgA titers in lung lavages. More than 70% decreases
in the numbers of levan-specific plasma cells, both total and IgA producing,
were present between 1 and 7 days after hemorrhage, with return to
prehemorrhage values present 14 days after blood loss ~FIGURE 2).
Essentially no levan-specific pulmonary plasma cells were found when
immunization was performed 3 days post hemorrhage.
Hemorrhage, rather than other components of the model, was responsible~
for alterations in the ~anti-lévan response among pulmonary plasma cells.
Immunkatbn of mice 3 days; following ether anesthesia and cardiac
puncture, but without blood !Nithdrawal (control), resulted in no significanS
changes in either anti-l~van slgA titers in lung lavages (OD4so 0.115 ~ 0.009
in controls versus Q126 ~ 0.023 in~normals) nor in numbers of levan-specific
pulmonary plasma cells ~35 ~ 7~in controls versus 33 ~ 5 in normals). No
changesjin serum anti-levan titers as compared to normal, unhemorrhaged
mice were found in cor~rol animals or at any time point following blood loss.
.
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C. EFFECT OF HEMORRHAGE ON PNEU~IONIA SURVIVAL
The previous experiments demonstrated that the pulmonary secretory
antibody and plasma cell responses to a bacterial polysaccharide antigen
were decreased between 1 and 14 days following hemorrhage and
resuscitation. Antibodies directed against bacterial polysaccharides,
including PseLIdomQnas aeruginoss polysaccharides, are protective against
infection with the organisms from which these antigens were isolated (Pier,
et al., Infect.lmmunol., ~:919, 1978; Stein, et al., J.Exp.Med., 160:1001,
1984). The present results therehre sùggested that pulmonary resistance
to infection might be reduced at this time point following blood loss In
order to examine this hypothesis, experimental Pseudomonas ae~uginosa
pneumonia was induced in normal, hemorrhaged, and hemorrhaged and
resuscitated mice using intr~tracheal injection of increasing numbers of
Pseudomonas aer~ginosa Fisher-Devlin immunotype 1 organisms. ~
In unmanipulated mice or ~mice subjected to ether anesthesia and cardiac
puncture, but no blood withdrawal, no mortaiity was found when groups of
mice (n = 6) were given 2 x 107 or 4 x 10~ organisms. However, when mice
(n = 6) were given 8 x 107 organisms intratracheally, the mortality rate was
83%, and when 12 x 107 organisms were injected, the mortality rate was
100%. AII mice ~lied~ between~24 and 96 hours after being infected.
Histologic exarnination of the tungs in mice having died after intratracheal
introdùction of Pseu~omonas ~eruginosB organisms showed changes
consistent with an acute and cGnsolidative pneumonia.
Next, mice (n = 19) were bled 30/O of their total blood volume. In one
9 roup of hemorrhaged mice ~n~- 7), the removed blood was reinfused i
hour following hemorrhage. The remaining mice ~n = 12) were left
unresuscitated. A controi group (n = 13) of mice subjected to ether
anesthesia and cardia~ puncture, but no blood withdrawal was included
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23.
After 4 days, 2 x 107 Pseudomorlas aeruginosa organisms were inoculated
into the trachea of each mouse.
TA~L~ 2
NUMBER NUMBER %
~::ONDITION QI~D SURVIVED RT~
Controt 0 13 0
Hemorrhage 8 4 67
Hemorrhage and
Resuscitation 4 3 57
~,.
a p~0.01 vs, Control
No animals in the control group died following infection. In contrast, the
mortality rate was 67% in the mice bled 4 days prior to infection, and was
57% in the hemorrhaged and resuscitated mice (~able 2~. The mortality rate
in hemorrhaged animals which had been resuscitated was not significantly
different from that in the hemorrhaged, but unresuscitated group. ~
Another experirnent was done to study the ~ffect of imrnunization on survival
of animals which were immunosuppress~d by hemorrha~e. In this study,
both control and imrnunized anima!s were h~morrhaged and resuscitated as
described above. Ths 9 animals in th~ immunized group were vaccinated
using Pseudomonas ~eruginosa type nurnber 1 polysaccharide (33 yg)
encapsulated in liposomes 1j hqur after hemcrrhage and resuscitation. Both
groups of animals were exposed to 2 x 107 viable cells of Pseudomonas
aeruginosa 96 hours after hemorrhage and resuscitation. The results of this
study are shown in Tab!~ 3.
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TAB~E 3
NUMBERNVM~ER %
C :)~LDITION N ~IEDS!I~VIVED MOF~T~ITY
Unimmunized 13 13 0 100
Irnmunized 9 6 3 67*
~ p~0.05
As shown in Table 3, imrnunized animals showed a significantly increased ,
level of survival (33%) as compared to anirnals in the unimmunized group.
Thus, intranasal immunization with liposomally encapsulated bacterial ~ .
0 polysaccharide antigen can significantly cnhance the survival of
immunosuppressed animals at risk of pathogenic or nosocomial infection or .
colonization.
.
The experimental results illustrated in Tables 2 and 3 demonstrate that
hemorrhage, even when followed by resuscitation, results in marked and
long lasting depression in pulmonary antibactarial B cell function. Reduced
numbers of pulrnonary plasma cells producing antibody against the
immunizing bacterial polysaccharid~ antigen were found between 1 and 14
days following blood loss, and titers of bacterial antigen specific slgA were
decreased for more than 2 weeks after hemorrhage. The difference in post
hemorrhage kinetics betw~en anti-levan slgA and levan-specific plasma cells
is not surprisin~. Because secreted antibodies can persist at mucosal
surfaces for several days, antigen-specific antibody titer~ would not be
expected to immediately reflect changes in plasma cell numbers, but ra~her
to ~oltow the pattern of plasma cell alteration in a delayed fashion. The
significance ~f these abnormalities in pulmonary B cell function was
WO ~2/20370 PCI/US92/0405S
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demonstrated by an increased susceptibility to Pseudomonas Qeruginosa
pneumonia at a time point 4 days following hemorrhage, when bacterial
antigen-specific pulmonary plasma cell numbers were at their lowest point.
Previous studies by the inventors had shown that hemorrhage without
S resuscitation resulted in marked decreases Tn the nurnbers and percentages
of pulmonary, intestinal, and splenic small resting B cells (clonal precursors)
committed to the production of antibodiès to baeterial antigens. Because
plasma cells develop from B cell clonal precursors, alterations In clonal
precursor repertoires are frequently reflected by parallel chang0s In antigen-
specific plasma cell frequencies. This situation following unresuscitated
hemorrhage had been previously demonstrated, where decreases in the
frequency of bacterial antigen-specific splenic and intestinal clonal
precursors were reflected~ by a sirrilar decrease in frequency of antigen-
specific plasma cells from these anatomic sites ~Abraham, et ~I., Cell
Immunol.,lZ:208,1989;Abraham,et~l.,Cel~ Immunol.~8:165, 1990). In
hemorrhaged mice, decreased numbers of pulmonary B cell clonal
precursors committed to producing antibody to the bacterial antigens levan
and Pseudomonas :~eruginosa polysaccharide type 1 occur between 3 and
10 days after blood loss (Robinson,~et 81.l J.tmmunol., 145:3734, 1990). In
the present experiments, the tim~e course for.post hemorrhage decreases of
bacterial antigen-specific pulmonary plasma cell numbers and slgA
production is quite similar to that found among antigen-specific B cell clonal
precursors, and therefore suggests that these hemorrhage induced
atterations in pulmonary~plaSma cell number and function reflect alterations
in the numbers and~frequencies of bacterial antigen-specific B cell clonal
precursors among the resting, smail B cell population.
Decreased numbers of ~ bacterial antigen-specific clonal precursors, able to
be recruited into the plasma c~li compartment, may be limiting in terms of
host defense following exposure to a bacterial inoculum. The present
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experiments suggest that such a rnechanism may b~ responsible for the
increased susceptibility to infection after blood loss, since increased mortality
from Pseudomon~s ~eruginosa pneumonia following hemorrhage occurred
at a time point associated with decreased numbers of bacterial
polysaccharide antigen-specific B cell clonal precursors in the îung and a
diminished ability to produce bacterial antigen sp~cific secretory antibodies.
Animals resuscitated 1 hour following hemorrhage showed the same
increased susceptibility to Pseudomon~s ~eruginosa pneumonia as did
hemorrhaged, but unresuscitated mice. Thes~ results therefore suggest that
abnormalities in immun~ response are rapidly and irreversibly induced by
hemorrhage, and that resuscitation has limited effects in modifying these
immunologic alterations.
Because of the ability of B and T cells to migrate from one mucosal surface
to another, it was not surprising that the post hemorrhage time cours~e of
t 5 alteration in bacterial antigen-specific pulmonary plasma cells resernbled that
found previously ~Abraham, et al., Cell Immunol., 128:165, 1990) among
plasma cells isolated from intestinal lamina propria. In particular, in both
mucosal sites, decreased numbers of bacterial antigen-specific plasma cells
were found between 3 and 14 days post hemorrhage.
Th~se studies show that increased bacterial antigen-spe iific antibody titers
can be achieved in the lungs when the bacterial polysaccharide antigen
levan was administered intranasally in a liposomal formulation. The amount
of antigen encapsulated~in liposornes and given intranasally, 38 ~g, was
approximately 30-fold less than that required with oral immunization to
produce the same increase in numbers of levan-specific pulmonary plasma
cel!s. As expected withj a mucosal immunization techni~ue, no change in
serum anti-levan titers were found in immunized animals. Because
liposomes are nontoxic and, as shown in this stud~, can act as potent
adjuvants in enhancing the mucosal immune response to polysaccharide
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antigens, they would be particularly useful in intranasal and oral vaccination
formulations aimed at preventing nosoeomial infection in
immunoeompromised, critieally ill patients.
,,,:,
Nosoeomial pneumonias are frequent following injury and other critical
illnesses. The present experiments demonstrate that hemorrhage, even if
resuscitated, results in alterations in pulmonary B cell ~nction which are
long lasting, and associated with increased susceptibility to infection at this
mucosal surfaee. Interestingly, the disappearanee sf bacterial antigen~
specific pulmonary B cell clonal preeursors and plasma eells does not occur
immediately post hemorrhage, so that significant numbers of B eell clonal
precursors, able to be recruited into the plasma eell population and to
produee bacterial antigen-specifie antibody, are still present in the lungs
during the first 24 hours post hemorrhage. These results suggest that
immunization immediately post h~morrhage would be able to enhance
pulmonary slgAtiters direetedtoward important baeterial antigens and might
be able to inerease resistanee to pneumonia foilowing hemorrhage or injury.
The present experiments, showing that intranasal immunization with baeterial
polysaeeharides eneapsulated in liposomes produce inereased pulmonary
baeterial antigen-speeifie slgA titers, suggest that this approach may be
useful in eritieally ill patients thro~lgh enhandng the anti-bacterial mucosal
immune response and thereby decreasing the ineidenee of nosocomial
pneumonia, as weil as morbidity and mortality assoeiated with these
. .
respiratorytraet infedons. ~ ~ ;
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EXAMPLE 3
IMMUNOGENIC: EFFEC:T OF LIPOSOME ENCAPSUI~TED BACTERIAL
POLYSA~CHARIDE ANTIG~AND INTERLEIJK!N
The effect of encapsulation of IL-2 into liposomes containing ba~terial5polysaccharides on the antigen-spechic secretory antibody response in the
lungs was studied by intranasally immunizing mice with liposomes containing
25 ~g of levan and incr0asing amounts of IL~2 (0 to 02 ,~lg, i.e., 0 to 10
~g/kg). One week later, the animals were sacrificed, the lungs lavaged, and
levan-specific Ig titers in lavage fluid measured by ELISA, as described
10above. In addition, lung Iymphocytes were isolated and the number of total
and levan-specific plasma cells per set of lungs enumerated using the ELISA
spot assay (see EXAMPLE 2, Sedgwick, et ~I, J. Immunol. Methods, ~7:301,
1983). Serum was obtained and anti-levan titers measured by ELISA.
.
~L~
15 ~LEVAN SPECIFIC IMMUNE RESPONSE IN PRESENCE OF IL-2
LEVAN~SPECIFIC
TOTAL IgA
SECRETORY: PULMONARY PllLMONARY -
IL-2a laA TlTERSb PLASMA CELLS PLASMA CELLS q
0.00.172 1~ 0.057 : ~4.0 L 0.~ 3.6 + 0.9
0.10.152 + ;0.~029 ~ 2.4 ~ 1.0 2.4 ~ 1.0
: 1.0 0.290 + 0.028 9.3 1 1.0 8.3 ~ 1.0
10.0 ~1.023~+ 0~169 ! ' !'333,0 + !i;Q.0296.0 ~ 430
a ,ug/kg, equivalent to 0, 0.002, 0.02, and 0.20 ~g, respec~ively
b A4so _ SEM
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As shown in TABLE 4, significant increases in anti-levarl slgA titers in lung
lavagPs (p<0.01) wers found in rnice immunized with liposomes containing
10 ~/kg IL-2. There also was small, but si~nificant (p~0.013 increase in
pulmonary anti-levan titer~ in mic~ a~ter imrnunization wîth liposomes
containing 1 ~fg/kg IL-2 (OD450 0.290 + 0.02g versus 0.172 ~ 0.057 in control
mi~e immunized with liposomes containing levan, but no IL-2). No
alternation in pulmonary anti-levan slgA titers were found in mice immuniz~d
with a smaller dose of IL~2 (i.e., 0.1 ,~g/kg). In contrast to the large
increases in antigen-specific slgA titers in lung lavages from immunked
mice, no~changes in antigen-specifiG IgM or lgG titers occurred ~ollowing
intranasal immuniza~ion with IL-2 contaTning liposomes. Similarly, serum anti~
levan titers were not significantly altered in any group of rnice intranasally
immunized with the IL-2 containing liposomes as compared to control,
unimmunized miee.
': ,"
1~ Intranasal immunization with liposomes containin~ levan and 10 ~lg/kg IL-2
- produced more than 8~fold incrsases in the number of 10van-spec~ic
pulmonary plasma cells as compared to numbers pres~nt following
vaccination with liposomes containing levani but no IL-2. More than 90% of
the levan-specific pulmonary plasma cells produced IgA. The number of
levan-specific pulmonary plasma cells was increased by approximately 2-fold
in animals imrnunized with liposomes cor taining 1 ~lg/k~ IL-2. I~o alterations
in the numbers of levan-specific plasma cells in lung digests were found in
animals receiving lower doses ot IL-2.
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TABLE 5
EFFECT OF IL~ ON F~E~UENCY OF PLASMA CE~Ls
PULMONARY
PLASMA ÇELLS
~1~2- T.OTAL laA
0.0 3712 ~ 34~ 3392 ~ 290
- 0.1 2460 ~ 250 2Z80 * 210
1.0 78B5 ~ 660 7470 ~ 717
10.0 740~ ~ 6B8 6698 :~: 858
,.
8 ~lg/kg, equivalsnt to 0, O.OOZ, 0.02, and 0.20 ~g, respsctiv~ly
Irnmunization with liposomes cQntaining IL-2 resulted in increases both in ths
relative frequ~ncy of levan-specific plasma cells in the lungs and in the total
numbers o~ pulmonary plasma ceils, producing antibody of all specificities.
In animals immunized with liposomes containing levan alone, without IL-2,
there were 4 ~ 1 leYan-specific plasma cells per 106 isola~ed lung
Iymphocytes, and 0.14% of all lung plasma cells were 10van specific. In
cQntrast, in mice immunized with liposomes cont~ining 10 ~ug/kg IL-2, there
were 93 ~ i 1 levan-specific plasma celis i~er 1 o6 isolated lun~ Iymphocytes,
and these levan-spec-~ic plasma cells comprised approximately 4.1% of the
lung plasma celi population. In adciition, the total number o~ plasrna cells
isolated per set of lung~ in~reased from 3712 ~i 340 in mice immunized with
liposomes containing levan alone to 7408 1 688 in mice imrnunked with
liposomes containing 10 ~g/kg IL-2. Of note, the enhancing effects of
liposome eneapsuiated IL-2 on pulmonary plasma cell numbers appear to
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be somewhat distinct from the adjuvant effects of IL-2 on pulmonary plasrna
cell numbers appear to be somewhat distinct from the adjuvant effects of
IL-2 on increasing antigen-specific plasrna cell numb0rs. Intranasal
immunization with liposomss containing 1 ,ug/kg IL-2 r0sulted in incr0asss
in total numbers of pulmonary plasma cells comparable to those seen when
10 yg/kg IL-2 was included in the liposomes (7885 ~ 660 plasma cells/set
of lungs for liposomes containing 1 yg/kg IL-2 versus 7408 ' 688 plasma
cells/sct of lungs for liposomes containing 10 ~Jgtkg IL-2). In contrast, the
number of levan-specific plasma cells per set of lungs increased by more
than 3~fold, from 9 ~ 1 to 333 ~ 50 when the amount of IL-2 included in the
liposomes was changed from 1 ,ug/kg to 10 yg/kg.
Because the previous experiments demonstrated potent adjuvant properti0s
of liposome encapsulated IL-2 on bac~erial antigen-specific secretwy
antibody response in the lungs, the possibility that inclusion of JL-2 into
liposomes would permit use of a decreased dose of antigen, whil~ still
achTeving enhancement of antigen-specific slgA titers. To address this issue, ;;
liposomes containing 2.5 ~g of levan and 0.2 yg Ik2 ~i.e., 10 yg/kg) were
prepared. No significant increase in levan-spcGific slgA titers in ,oulmonary ;-~
lavages as compared to unimmunized controls, and no levan-speclfic
pulmonary plasma cells were found foilowing immunization with this - ~ i
liposomal preparation, indicating that inclusion of Ik2 was unable to
sufficiently enhance antibody production to compensate for a 1~fold
reduction in the immunizing antigen.
,:
In order to determine if the enhancement of bacterial polysaccharide
antigen-specific secretory antibody response associated with liposomal
encapsulation; of IL-2 andll0yan could b~ achieved with other bacterial
polysaccharides, mice were intranasally immunized with liposomes
containlng either Pseudomonas aeruginosa polysaccharide type 1 (25 yg)
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alone or with 0.2 ~g IL-2 (i.e., 10 ~g/k0). The resutts of these studies are
presented in TABLE 6.
TAB~E 6
EFFEI::T OF IL-2 O~ÇT3ETORY Içt~ RESPONSE TO
~omonas aerualnosa POLYSA~::CHQ~IDE ~A)
C;ROUP P8A ~ S~CRFrO~q~b
- - 0.46B :t: 0.139
2 ~5~1g -~ 0.584 I~ 0.07!;
3 25~Jg 0.21J9~0.941 ~: 0.167
a 10 ~Iglkg
b A4So ~ SEM
,
As was shown in EXAMPLE 1, intran~sal immunization with liposomes
containing Pseudomonas 2eruginosa polysaccharid0 alone increased
antigen-specific slgA titers in pulrnonary lavages, compared to those in
animals immunized with empty liposomes (OD450 9.4666 + 0.139 for empty
liposomes versus 0.584 ~ 0.075 for liposomes containing Ps~udomonas
~eruginos~ polysaccharide). Addition of IL-2 to liposomes produced
significant increase (pc0.01 ) in anti-Ps~udomon~s aeruginosa polysaccharide
sigA titers when compared eithsr to those found in animals immunized with
empty liposom~s or in mice immunized with liposomes c~ntaining
Pseudomonas oen ginosa polysaccharide, bu~ no IL-2.
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Because Ik4 has ~een shown to affect B cell maturation and progression
to antibody production, the possible adjuvant effects of encapsulation with
IL-4 was investigated. However, no significant increases in anti-levan slgA
titers in lung lavages were found in mice intranasally immunized with
liposomes containing 25 ~g levan and 0.2 llg (.i.e., 10 ,ug/kg) IL-4 (OD,,50
0.183 ~ 0.075 in controls versus 0.144 ~ 0.081 for mice immunized with Ik4
containing liposomes). Further, no levan-specific pulmonary plasma cells
were found on ELISA Spot analysis in animals imm~nked with the IL-4
containing IjPQSOmeS.
While not wanting to be bound to a particular theory, because of the relative
predominance of Th2 cells at mucosaî sites, it is possible that sufficient
levels of IL~, able to support B cell responses, are alread~r present in these
anatomic locations, and h~rther increasing local IL-4 levels produces no
additional effect. In addition, there is evidence that IL-4, while capa61e of
stimulating the early steps in the B~cell response, also has inhibitory
properties on B cell proliferation, particularly that driven by IL-2. If this is the
case ~n vivo, then providing increased local concentration of IL-4 may
actually have an inhibitory effect on antiboc~y prod~ction;~
Further studies were done evaluating the effect of Ik2 containing liposome~
~20 to protect against pneumonia using the hemorrhage model previously
desaibed. In these experiments,~ mice were bled 30% blood volume, then
2 hr later intranasal!y administered liposomes contàining either 25 ,ug
Pseudomonas aeruginosa~ potysaccharide type 1 alone, or 25 ~lg
Pseudomon~s aeruginosa polysaccharlde and 0.2 ~Jg IL-2, or 25 ,~g levan
25 ~ and 0.2 ,ug IL-2. Fo~r days later, the animals were infected intratrachealiy
with P$eudomonas,aeruginosa; i~
In unimmunized micè or mice ~immunked with liposomes containing levan
and ~ IL-2, the mortality rate~ ~ was 100% following the induction of
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Pseudomonas aeruginosa pneumonia (FIGURE 3). No significant
improvement in survival was found in mice immunked with liposomes
containing Pseudomonas ~eruginoss polysaccharide, but no IL-2. In
contrast, the mortality rate was reduced to 45% in mice intranasally
immunked with liposomes containing Pse~Jdomon~s ~eruginosa
polysaccharide and 10 ,ug/kg IL-Z (p~a.005). Histopathologic studies
showed acute consolidative and hemorrhagic pneumonla with the presence
of gram negative rods in the lungs of both immunized and unimmunized
mice which died following intratracheal introduction of bacteria ;;
, . ,
Intranasal immunkation of mice with liposomes containing IL-2 and
Pseudomonas ~eruginosa pobsaccharide provided significant protection in
a pneumonia model which is 100% lethal in unimmunéed mice. Of note,
vaccination with liposomes containing IL-2 and a bacterial polysaccharide
(levan) derived *om~another organism~ ~,4ero~cter fevanicum) was not
effective against Pse~domon~s sentginos~ pneumonia, demonstrating that
the protection achieved~ by immunization with liposomes Is not due to
nonspecific enhancement of pulmonary immune response. The dose of
bacteria used to produce pneumonia in the present experiments, 5 x 107
cfu, was more than twice as high as~we have used previously, when
mortality rates of 100% in unimmunized animals also were found. In~
pr~ous studies, d was found that !iPosomallY encapsulated Pse~domonas
; ~ aeruginQsa polysacGharide, without lL-2, was able to provide partial
protection from mortality due to pneumonia. The lack of protection of this
formulation in the present experiments is probably due to the higher dose
25 ~ ~ of ~bacteria used to produce;pneumonia and to the smaller amount of
polysaccharide incorporated into liposomes (25 yg versus 33 yg used in
previous studies).~
~ '
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YVC) 92/20370 PCI'/US92/M055
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35. .
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In orcier to evaluate the effectiveness o~ other interleukins, studies similar to
those described for IL-2 and IL-4 were performed using recombinant human
IL-6 in combination with polysaccharide antigen. Results are sho~,vn in
TABLE 7.
TA81"E 7
EFFEC:T OF IL-6 ON LE\/QN SPECIFIC lMMUNE RESPONSE
Levan~Sp~clflc Pla~ma
Cells/Set of Lunas
Amount of IL-6a % Levan Speclfic .
10Adminlstered Total ~9Q Pulmonarv Plasma Cells
0.0 9 8 0.13
0.1 29 19 2,04"
1.0 106 96 1.89
5.0 61 41 1,77 . ~;
a ~g/kg, equivalentto 0, 0.002, 0.02, 0.10~g, respectively
..":
s .. .. .
In each case, male BALB/c mice were immunized intranasally with 50 ~1 of
suspension containing 25,ug !evan and the above~n~ted an~aunt of human
recombinant IL-6. ~ Five days !ater the lungs were digested, lung :
Iymphocytes purNied, and the number of levan-specific pulmorlary plasma
cells per sst of lungs determined using the ELISA Spot assay as previously
clescribed. As seen here, the incorporation of IL-6 into liposomes resulted
in rnarked incre~es in the inumber of levan-sp~cUic! lung plasma cells,
showing a potent adjuvant effect of IL-6 incorporated into liposomes. ~
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36.
,
The data shows that the percentage of pulmonary plasma cells which are
antigen specific rises approximately 1~fold when IL-6 is incorporated into
liposomes used for intranasal administration. The percentage of levan-
specUic pulmonary plasma cells were calculated by dividing the number of
levan-specific pulmonary plasma cells per 104 isolated lung Iymphocytes by
the number of pulmonary plasma cells of all specificities per 106 isolated ;
lung Iymphocytes.
The invention now being fully described, it will be apparent to one of
ordinary skill in the art that rnany changes and modifications can be made
without departing from the spirit or scope of th0 invention. ~i
,
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