Encapsulated high-concentration lipid a compositions as immunogenic agents to produce human antibodies to prevent or treat gram-negative bacterial infections

This invention is directed to the production of antibodies against lipid A by using encapsulating slow-releasing delivery materials or devices containing concentrations of lipid A that are greater than could be given safely to humans in the absence of said materials or devices. The antibodies to lipid A can be used for binding the antibodies to the lipid A that are present in the lipopolysaccharide that coats the surface of the Gram-negative bacteria.

TECHNICAL FIELD OF INVENTION 
This invention relates to encapsulated high-concentration lipid A 
compositions and human monoclonal antibodies as immunotherapeutic agents 
to prevent or treat Gram-negative bacterial infections. 
BACKGROUND OF THE INVENTION 
Gram-negative Bacterial Infection 
Throughout the world, sepsis caused by infection with Gram-negative 
bacteria is one of the major causes of death. Gram-negative bacteria are 
ubiquitous in the environment, and are present in the air, water, food, 
and are present in particularly high concentrations in the intestines. The 
surface of the skin is coated with Gram-negative bacteria and many skin 
infections are caused by these organisms. Because of their widespread 
distribution these bacteria cause opportunistic infections in association 
with other afflictions. For example, it is widely believed that sepsis is 
the most common cause of death in hospitals, and is particularly 
associated with conditions such as cancer, traumatic injury, and burns. A 
major cause of death in association with burns on the skin is infection 
and sepsis caused by gram-negative Pseudomonas organisms. Most urinary 
tract infections are caused by Gram-negative organisms. Individuals who 
are weakened by chronic diseases, or who have massive traumatic or burn 
injuries, or who have penetrating injuries of the intestines, are 
particularly at risk for developing life-threatening Gram-negative sepsis. 
Gram-negative infections that cause diarrhea are one of the major causes 
of death in infants. 
The normal treatment of Gram-negative sepsis is by the administration of 
antibiotics that are active against sensitive organisms. The widespread 
use of such antibiotics has led to the continuous emergence of resistant 
strains of organisms and this has limited the effectiveness of antibiotic 
treatment. In addition, treatment often depends upon prompt administration 
of the appropriate antibiotic, but definitive laboratory tests to prove 
that the organism is sensitive to the antibiotic and that the optimally 
potent antibiotic is being used normally involves isolation and growth of 
the Gram-negative organism and inhibition by candidate antibiotics. 
Sensitivity tests normally require at least 1-2 days to perform, and 
because of urgency of many infections inadequate or suboptimal antibiotic 
therapy is often employed. 
Immunotherapy with Lipid A 
Suboptimal antibiotic use is one of the major factors responsible for 
emergence of antibiotic-resistant organisms. A new strategy of 
immunotherapy has been developed for the treatment of Gram-negative 
sepsis. This technique involves the administration of monoclonal (or 
polyclonal) human antibodies having specificity against lipid A. The 
monoclonal or polyclonal antibodies to lipid A that are used for 
immunotherapy are given by intravenous administration in individuals who 
have sepsis, and are usually used only when a relatively large infection 
has already become established. These individuals are frequently already 
in a toxic condition that renders the treatment by immunotherapy more 
difficult. 
Structure of Lipid A 
The rational for the above immunotherapeutic approach is based on the fact 
that nearly all Gram-negative bacteria contain lipid A as a major element 
in the lipopolysaccharide molecules that coat the surface of all 
Gram-negative bacteria. The general structure of lipopolysaccharide is as 
follows: 
(oligosaccharide repeating unit ).sub.n -(core 
oligosaccharide)-(ketodeoxyoctonate).sub.3 -lipid A 
The structure of lipid A has been completely defined for most Gram-negative 
bacteria, and it has been totally synthesized. It consists of a backbone 
of (B-1,6)-linked D-glucosamine disaccharide which carries phosphate 
residues in the positions 1 and 4'. Amidated or esterified long-chain 
fatty acids (generally D-3-hydroxy and/or acyloxy fatty acids) are present 
in each of the possible sites in the glucosamine moieties. Minor 
differences in structures of lipid A occur in molecules derived from 
various bacteria. In addition, so called "native" lipid A (i.e., 
unmodified lipid A derived by isolation from Gram-negative bacteria) 
usually consists of a mixture of molecules having the same basic structure 
as the "complete" lipid A described above but with differing degrees of 
phosphorylation or different numbers or structures of fatty acids. The 
monophosphoryl lipid A, that has reduced toxicity, lacks the phosphate 
residue at position 1. 
Toxicity of Lipid A 
A further useful approach to immunotherapy of Gram-negative bacteria would 
be in the development of a vaccine (i.e., immunoprophylaxis) against 
lipopolysaccharide via its lipid A component. A major theoretical and 
practical impediment to this approach is posed by the extremely high level 
of toxicity of lipopolysaccharide. A synonym for lipopolysaccharide is the 
term "endotoxin", and all of the endotoxic activity of lipopolysaccharide 
is caused by the lipid A component. 
Lipopolysaccharide has literally dozens of biological activities when it is 
studied with in vitro biological systems or when it is injected in vivo. 
Many of these activities are associated with toxicity and are responsible 
for the adverse reactions that commonly observed in the course of 
Gram-negative bacterial infections and sepsis. Among these activities are 
included: pyrogenicity, neutropenia, thrombocytopenia, hypotension and 
shock, shock lung, renal failure, cachexia, and death. All of these toxic 
effects are caused by the lipid A component of lipopolysaccharide. 
Accordingly, there is a need for compositions which contain high 
concentrations of shielded or "hidden" lipid A which permits the slow 
release of lipid A therefrom thereby avoiding the deleterious and lethal 
results caused by the presence of high concentration of neat lipid A in 
the body. 
SUMMARY OF THE INVENTION 
It is therefore an object of the present invention to provide a novel 
immunoreactive composition comprising as an active ingredient a lipid 
A-containing component wherein the lipid A is sequestered, embedded or 
"hidden" in pharmaceutically-acceptable encapsulating-delivery material, 
said material being capable of (a) physically sequestering, "hiding" or 
shielding hydrophobic fatty acid portion of the lipid A from its aqueous 
environment, thereby preventing said portion from expressing its toxic 
properties in the host-mammal and (b) slowly releasing the lipid A from 
said material at a dose level within the range of 185 to 2,200 micrograms. 
The lipid A can be native lipid A, monophosphoryl lipid A or diphosphoryl 
lipid A. It has been found that high concentrations within the ranges of 
185 to 219, 220 to 549, 550 to 1099, and 1100 to 2200 micrograms are 
especially useful in the practice of this invention. The encapsulating 
slow-releasing delivery materials or devices can be a liposome, 
biocompatible-biodegradable polymer, microcapsules, or microspheres, 
mechanical slow drug-releasing devices and combination thereof. 
It is also an object of the present invention to provide a vaccine against 
Gram-negative bacterial infections and immunotherapeutic methods of 
treating a mammal prior to or following infection by Gram-negative 
bacteria including Escherichia, Salmonella, Pseudomonas, Proteus, 
Shigella, Vibrio, meningococcus and gonococcus. 
Because of the toxicity of lipid A and the consequent difficulties in 
producing a satisfactory and effective human vaccine against this 
substance, it has been often proposed to utilize human polyclonal or 
monoclonal antibodies to lipid A as an immunotherapeutic product to treat 
individuals that are afflicted with Gram-negative bacterial infections 
Ziegler, E. J. et al., Treatment of Gram-negative bacteremia and shock 
with human antiserum to a mutant Escherichia coli. New Eng. J. Med. 
307:1225-1230 (1982); Teng, N. N. H. et al., Protection against 
Gram-negative bacteremia and endotoxemia with human monoclonal IgM 
antibodies. Proc. Natl. Acad. Sci. 82:1790-1794 (1984); Baumgartner, J. D. 
et al., Prevention of Gram-negative shock and death in surgical patients 
by antibody to endotoxin core glycolipid. Lancet ii:59-63 (13 Jul. 1985); 
Kirkland, T. N. et al, Analysis of the fine specificity and 
cross-reactivity of monoclonal anti-lipid A antibodies. J. Immunol. 
137:3614-3619 (1986); Bogard, W. D. et al., Isolation and characterization 
of murine monoclonal antibodies specific for Gram-negative bacterial 
lipopolysaccharide: Association of cross-genus reactivity with lipid A 
specificity. Infect. Immun. 55:899-908 (1987); Ziegler, E. J. Protective 
antibody to endotoxin core: The emperor's new clothes? J. Infect. Dis. 
158:286-290 (1988); Ward, D.C. et al., Monoclonal antibodies to salmonella 
lipopolysaccharide: functional analysis of anti-lipid A antibodies. Clin. 
Exp. Immunol. 72:157-163 (1988)!. Accordingly, it is a further object of 
this invention is to provide human antibodies in the form of "hyperimmune" 
polyclonal antiserum or a human monoclonal antibody reactive with 
Gram-negative bacteria and providing effective passive prophylaxis against 
or therapeutic treatment of sepsis caused by Gram-negative bacteria, said 
monoclonal antibody produced by a self-reproducing carrier cell containing 
genes that produce a protective human antibody. 
Other objects, features and advantages of the present invention will become 
apparent from the following detailed description. It should be understood, 
however, that the detailed description and specific examples, while 
indicating preferred embodiments of the invention, are given by way of 
illustration only, since various changes and modifications within the 
spirit and scope of the invention will become apparent to those skilled in 
the art from this detailed description. 
It has been discovered unexpectedly that an immunogenic composition 
containing a very high concentration of lipid A encapsulated within slow 
drug release materials such as liposomes, biocompatible-biodegradable 
polymers, microcapsules, microspheres, slow-release devices, or 
combinations thereof, can be injected systemically, including 
intramuscularly or subcutaneously, into a mammalian host. It has been 
established in the prior literature that the dose of lipid A, and 
specifically the dose of monophosphoryl lipid A (MPL), that can be 
administered safely to humans is limited by toxicity (Vosika, G. J. et 
al., Phase-I Study of intravenous modified Lipid-A. Cancer Immunol. 
Immunol. 18:107-112 (1984). The maximum safe intravenous dose of MPL was 
established as 100 ug of MPL per m.sup.2. Assuming a normal surface area 
of 1.7 to 1.8 m.sup.2, the maximum safe dose of MPL is 170 to 180 ug. 
Applicants have discovered that dose levels of the lipid A in the range of 
185 to 2,200 micrograms can be administered to mammalian-hosts without 
toxic effect and result in the production of extremely high titers of 
antibodies, such that the antibody titers are higher than can be obtained 
with lower doses of lipid A. 
The level of antibody activity to lipid A obtained was proportional to the 
dose of liposomal lipid A administered during the original single initial 
immunization. Even when the same total amount of liposomal lipid A was 
given in divided doses in multiple injections at different times the 
resultant antibody activity did not reach the levels achieved when the 
entire liposomal lipid A dose was given at a single time. 
Suppression of Toxicity by using Encapsulating Materials 
Experimental studies in animals have demonstrated that much of the toxicity 
of lipid A is accounted for by one of the phosphate groups (at position 1) 
and by the fatty acid moieties. Monophosphoryl lipid A has greatly reduced 
toxicity as determined by lessened pyrogenicity, but it has not lost all 
of the endotoxic character observed in native (diphosphoryl) lipid A. It 
is generally understood that lipid A that lacks fatty acid groups is 
nonpyrogenic, and the degree of pyrogenicity is related to the number of 
fatty acid groups that are present. Applicants, and others, have 
demonstrated in the literature that much of the toxicity of native lipid 
A, and much of the residual toxicity of monophosphoryl lipid A, can be 
suppressed by inclusion of lipid A into encapsulated materials, such 
liposomes Ramsey, R. B. et al., Effects of lipid A and liposomes 
containing lipid A on platelet and fibrinogen production in rabbits. Blood 
A and liposome-associated lipid A with Limulus Polyphemus amoebocytes. 
Infect. Immun. 39:1385-1391 (1983); Dijkstra, J. et al., Modulation of the 
biological activity of bacterial endotoxin by incorporation into 
liposomes. J. Immunol. 138:2663--2670 (1987); Dijkstra, J. et al, 
Incorporation of LPS in liposomes diminishes its ability to induce 
tumoricidal activity and tumor necrosis factor secretion in murine 
macrophages. J. Leukocyte Biol. 43:436-444 (1988); Richards R. L., et al., 
Immunogenicity of liposomal malaria sporozoite antigen in monkeys; 
Adjuvant effects of aluminum hydroxide and non-pyrogenic liposomal lipid 
A. Vaccine 7:506-512 (1989); Alving, C. R. and Richards, R. L., Liposomes 
containing lipid A: A potent nontoxic adjuvant for a human malaria 
sporozoite vaccine. Immunol. Lett. 25:275-280 (1990)!. 
Liposomes consist of bilayers of lipids (such as phospholipids and other 
lipids) in which the hydrophobic regions of the lipids are oriented 
towards each other and the hydrophilic regions of the lipids are oriented 
toward the aqueous phase in which the lipids are suspended. The adjacent 
lipid molecules are held together by van der Waals forces and this results 
in the spontaneous formation of lipid sherules consisting of membranes 
containing lipid bilayers that surround internal aqueous spaces. 
When lipid A in included in the lipid bilayer it can be demonstrated that 
anti-lipid A antibodies that are known to have specificities against the 
lipid headgroup (diglucosamine diphosphate) are readily bound to the 
liposomal lipid A. This can be shown to cause agglutination or complement 
fixation, and complement activation can cause lysis of the lipid bilayer 
resulting in increased permeability of the liposome to marker compounds 
present in the internal aqueous spaces. Other methods for performing the 
immunological studies could include using the liposomes containing lipid A 
as an antigen in enzyme-linked immunosorbent assays, or by using 
fluorescent antibodies or antibodies labeled with dyes, to "light up" and 
visualize the occurrence of the immunological reaction at the surface of 
the liposomes, or by using liposomes as absorbent particles to absorb 
antibodies. It is concluded from this that the lipid A molecule is 
oriented in the expected manner with the hydrophilic portion oriented 
toward the aqueous medium and the hydrophobic (fatty acid) portion buried 
in the lipid bilayer. Similar types of studies can be used to demonstrate 
that anti-lipid A antibodies can bind to lipid A that has been absorbed to 
erythrocytes. 
An expected result of the orientation of lipid A that is described above 
might be that toxic effects caused by the fatty acid groups would be 
reduced or eliminated because of inaccessibility of the fatty acids to the 
aqueous environment. This result has indeed been observed. Our laboratory, 
and other laboratories, have demonstrated that neutropenia, interleukin-1 
secretion, and pyrogenicity induced by lipid A in rabbits are all 
remarkably reduced when lipid A is present in liposomes Ramsey, R. B. et 
al, Effects of lipid A and liposomes containing lipid A on platelet and 
fibrinogen production in rabbits. Blood 56:207-310 (1980); Dijkstra, J. et 
al., Modulation of the biological activity of bacterial endotoxin by 
incorporation into liposomes. J. Immunol. 138:2663-2670 (1987); Dijkstra, 
J. et al., Incorporation of LPS in liposomes diminishes ints ability to 
induce tumoricidal activity and tumor necrosis factor secretion in murine 
macrophages. J. Leukocyte Biol. 43:436-444 (1988); Richards R. L. et al., 
Immunogenicity of liposomal malaria sporozoite antigen in monkeys: 
Adjuvant effects of aluminum hydroxide and non-pyrogenic liposomal lipid 
A. Vaccine 7:506-512 (1989); Alving, C. R. and Richard, R. L., Liposomes 
containing lipid A: A potent nontoxic adjuvant for a human malaria 
sporozoite vaccine. Immunol. Lett. 25:275-280 (1990)!. One widely used 
measure of endotoxic activity is the use of the Limulus amebocyte lysis 
(LAL) test. In this test, the lysate from the amebocyte of Limulus 
polyphemus, is coagulated in the presence of endotoxin. This test is one 
of the most sensitive tests for the presence of endotoxin. We were able to 
reduce the LAL activity more than 10,000-fold by incorporating native 
lipid A in liposomes. However, we found that the LAL activity of liposomal 
lipid A was dependent on the epitope density of lipid A in the liposomes, 
and that by greatly increasing or decreasing liposomal lipid A 
concentration we could produce so-called "limulus-positive" or 
"Limulus-negative" liposomes, respectively Ramsey, R. B. et al., Effects 
of lipid A and liposomes containing lipid A on platelet and fibrinogen 
production in rabbits. Blood 56:207-310 (1980); Richardson, E. C. et al., 
Interactions of lipid A and liposome-associated lipid A with Limulus 
polyphemus amoebocytes. Infect. Immun. 39:1385-1391 (1983); Dijkstra, J. 
et al., Modulation of the biological activity of bacterial endotoxin by 
incorporation into liposomes. J. Immunol. 138:2663-2670 (1987)!. Based on 
this it is clear that liposomes containing very high concentrations of 
lipid A can exhibit biological activities that are qualitatively different 
than liposomes containing lesser amounts of lipid A. Furthermore, 
liposomal lipid A per se can have properties that are qualitatively 
different than those of nonliposomal lipid A. 
Lack of Toxicity of Liposomal Lipid A in Humans 
Based on the perception that liposomes containing lipid A would be expected 
to have reduced toxicity in vivo, monophosphoryl lipid A was included in 
liposomes that were employed as a candidate vaccine for human malaria 
(Plasmodium falciparum). These liposomes also contained a recombinant 
antigen that was used for immunizing against the malaria. The liposomal 
lipid A served as an adjuvant for enhancing the immunogenicity of the 
recombinant antigen and the liposomes themselves were also absorbed with 
another adjuvant, aluminum hydroxide (alum). The candidate vaccine was 
injected three times, for example at 0, 8-12, and 16-20 weeks, into five 
groups of human volunteers (5 volunteers were in group 1, 6 in groups 2-4, 
and 7 in group 5). Increasing doses were sequentially injected in order to 
test the safety of the vaccine but each group received exactly the same 
dose of alum. Vaccine doses were as follows, based on dilution of the 
liposomes: group 1, 1:100; group 2, 1:10; group 3, 1:4;, group 4, 1:2; 
group 5, 1:1 (i.e., undiluted). Group 5 received 2.2 mg of monophosphoryl 
lipid A during each intramuscular injection, and to our knowledge this is 
the highest dose of lipid A that has ever been purposely given to a human 
subject. A previously published study with free monophosphoryl lipid A 
given intravenously to human volunteers demonstrated that the highest safe 
dose that could be used in humans was approximately one-twelfth of the 
maximum dose that we administered intramuscularly in liposomes. At the 
one-twelfth dose of intravenous free monophosphoryl lipid A a considerable 
level of reactogenicity and toxicity was observed (Vosika et al., Phase-I 
study of intravenous modified Lipid-A. Cancer Immunol. Immunol. 18:107-112 
(1984). In contradistinction, applicants found that even at the highest 
does of monophosphoryl lipid A no significant acute toxic effects were 
observed. 
Induction of Antibodies to Lipid A in Humans 
Upon examining the sera of individual volunteers for the presence of IgG 
antibodies to lipid A we observed that most of the sera contained 
antibodies against lipid A. We conducted our immunological test by 
solid-phase enzyme-linked immunosorbent assay (ELISA) with purified lipid 
A as an antigen in microtiter plate wells. Other methods for studying the 
immune reaction could have included numerous other standard procedures for 
measuring reactivity of antibodies with purified antigen, including 
radioimmunoassay, flocculation of antigen or of liposomes or cells 
containing antigen, complement fixation, immunofluorescence assays or 
other assays utilizing dyes as markers, and hemagglutination of 
erythrocytes coated with lipid A, and hemagglutinin-inhibition assays with 
the same type of erythrocytes. All of the above assays, and others similar 
to them, are variations on the same theme of antigen-antibody reactions 
and are standard in the art. We made the quite unexpected discovery that 
although each group of volunteers contained individuals whose serum 
contained both IgG and IgM antibodies to lipid A, and that elevations were 
invariably based on comparison with preimmunization control samples drawn 
prior to injection of the vaccine, the IgG antibody titers increased 
substantially among the individuals injected with increasing doses of 
lipid A, and the highest levels of antibodies were observed in group 5. We 
concluded therefore that the optimal immune results of antibody levels 
against lipid A were achieved only with doses of lipid A that could not be 
safely given in the absence of liposomes. We concluded that this procedure 
is the only apparent safe method yet described for achieving maximum 
levels of IgG antibodies, and such levels of antibodies were achieved even 
at the first bleeding time (two weeks). 
It should be pointed out that one of the striking observations that we made 
is that we produced antibodies against native lipid A by immunizing with 
liposomes containing monophosphoryl lipid A. 
In the prior art it has been demonstrated that the phosphate residue in the 
C1 position of lipid A can contribute to the specificity to antibodies 
against lipid A. Indeed, polyclonal antisera to native lipid A have been 
described in which antibodies in the antisera did not react with analogues 
of lipid A that contained the position C4' phosphate but lacked the 
position C1 phosphate (Brade et al., Infection and Immunity, vol 55, pp 
2636-2644, Nov. 1987; see the summary of FIG. 3 and Table 11 therein that 
summarizes the "type e" antibody specificity). Likewise, in the same 
publication it is taught conversely that antisera can be demonstrated that 
cannot react with lipid A analogues that include the C4' phosphate 
(antibody type d in the above Table 1). Based on this it is evident from 
the prior art that it is not established that antibodies to monophosphoryl 
lipid A (i.e., lipid A that lacks the position 1 phosphate) will 
necessarily react with native lipid A. In at least some instances position 
1 phosphate is a critical immunodominant factor that mainly determines the 
specificity of the antibodies against native lipid A. In of the complex 
specificities that are generated by immunizing with lipid A it is not 
obvious that immunizing with a derivative of lipid A lacking a phosphate 
group (e.g., monophosphoryl lipid A) would induce antibodies against the 
native lipid A containing the phosphate group. Brade et al. have 
demonstrated that antibodies to lipid A can be produced (type e 
antibodies) that require the presence of the C1 phosphate group that is 
lacking in monophosphoryl lipid A. It is not obvious that such 
anti-C1-phosphate antibodies would not be uniquely induced by liposomal 
native lipid A. Accordingly, if anti-C1-phosphate antibodies were 
exclusively induced by Liposomal Lipid A they could not have been induced 
by immunizing with liposomal monophosphoryl lipid A that lacks the 
C1-phosphate group. 
Proposed Strategies for use of Liposomal Anti-Sepsis Vaccine 
We anticipate that a liposomal anti-sepsis vaccine could be used either for 
immunoprophylaxis (as with other types of vaccines in their conventional 
usage forms) or as an immunotherapeutic agent in individuals who have 
already developed sepsis or individuals at risk for developing sepsis 
(e.g., patients subjected to traumatic injuries or to burns, or patients 
who are debilitated because of old age or chronic diseases such as cancer 
or diseases that might cause a prolonged bedridden state). The results 
indicate that the vaccine would be effective within two weeks after 
administration, but the extremely high levels achieved in group 5 suggest 
that the vaccine could be effective even at earlier times. The vaccine 
could be used in conjunction with other therapeutic measures, such as 
correction of the underlying inciting condition (e.g., removal of abscess, 
removal of cancer, closing of perforated intestines, removal of infected 
intravenous catheters, replacement of infected heart valves, etc.), or 
with concurrent administration of antibiotics, steroids, anticancer drugs, 
or immunotherapeutic or any other therapeutic measures that would normally 
be expected to have therapeutic benefit in the absence of the liposomal 
anti-sepsis vaccine. 
Another unexpected benefit from our anti-sepsis vaccine appeared to be that 
the high titers of antibody activity that were achieved at the highest 
initial dose of vaccine (group 5) could not be duplicated simply by giving 
multiple immunizations with a smaller dose of vaccine. The IgG anti-lipid 
A levels of group 5 observed after two weeks were higher than the highest 
levels ever observed in any of the other groups, even after subsequent 
immunizations that resulted in doses of lipid A that were equivalent to 
those that were given initially in group 5. In the FIG. 1, each group 
received a boosting immunization at either 8 weeks (groups 1-3) or 10 
weeks (groups 4 and 5) that was identical to the original immunization 
dose. Therefore, after the second (boosting) immunization group 4 received 
a total dose of liposomal monophosphoryl lipid A in two injections that 
was identical to the dose of lipid A that was present in the single 
initial immunization dose of group 5. Despite this, when measured 2 weeks 
after the boosting immunization, group 4 achieved an IgG antibody level 
that was only slightly more than half as high as the level achieved 2 
weeks after the initial immunization with the twice the antigen dose given 
to the volunteers in group 5. 
Expectation of Similar Results with Other Strategies 
The net result of the use of liposomes containing lipid A is that the fatty 
acids of lipid A are "hidden" from the aqueous environment. This result 
presumably could also be accomplished by other types of particles or 
strategies. For example, polymers of various types, microcapsules, and 
microspheres all would be expected to accomplish the same result. Although 
the terms "liposomes", "microcapsules", "microspheres", "polymeric drug 
delivery vehicles", "slow release devices", or other similar terms are 
often used informally in the literature to describe various types of 
carrier systems for drugs and proteins, there is no universal agreement, 
nor are there any generally accepted national or international 
conventions, that define the meanings of such nomenclature. Liposomes are 
ordinarily understood to consist of lipid membranes that are capable of 
enclosing an internal aqueous space and the membranes may consist of a 
variety of types of lipids. Methods for manufacturing liposomes are 
described in U.S. patent application Ser. No. 07/202,509 filed 2 Jun. 
1988, of which the present specification is a continuation-in-part. There 
is no necessary requirement for the presence of a closed membrane, only a 
requirement that the lipids self-associate such that they form a 
particulate structure. Among the lipids that have been used either alone 
or in combination with other lipids to construct liposomes are included 
phospholipids, glycolipids, glycophospholipids, diglycerides, 
triglycerides, sterols, steroids, terpenoids, free fatty acids, and 
lipoidal vitamins. Numerous liposomal constructs have been made that 
contain synthetic fatty acyl groups conjugated to proteins or other 
polymers. Liposomes may also be formed that use lipids to reconstitute 
insoluble hydrophobic proteins. The terms "microcapsule" or "microsphere" 
in the present specification refer to particulate constructs that are used 
as carriers for antigenic molecules (such as lipid A), and the molecules 
that form the constructs, whether they are liposomes, lipids, proteins, or 
various other types of polymers, provide a diffusion barrier to prevent 
immediate release of the antigenic material. Slow release of the antigenic 
material to the immune system is promoted by these constructs. 
Release of encapsulated materials from particles such as those comprised of 
polymers most commonly occurs by diffusion. The diffusion mechanisms of 
materials from polymers are multitudinous, including: release from a 
reservoir surrounded by a polymeric film; release from a matrix in which 
the material to be released is distributed uniformly through the polymer; 
cleavage of the material from a polymer backbone; dissolution of the 
polymer by exposure to an environmental solvent; permeation by water 
leading to leakage due to osmotic swelling either by creating pores in the 
polymer or through preformed pores; and release of material by imposition 
of external magnetic fields. The net result of each of these mechanisms is 
initial sequestration and "hiding" of the material to be released, 
followed by a relative degree of slow release that is regulated by the 
release mechanism and by the nature of the polymeric microcapsule or 
microsphere chemical and physical composition. These mechanisms are 
described in further detail by in Langer, R., New methods of drug 
delivery. Science 249:1527-1533 (1990). Langer also points out that the 
diffusion and release mechanisms outlined above can apply to immunological 
applications including antibody production against encapsulated or 
sequestered antigen. We also have evidence that liposomes containing lipid 
A serve as "slow release" devices. We found that a considerable fraction 
(perhaps as much as half) of a formulation of fluorescence-labeled 
liposomes containing lipid A that we injected intramuscularly in mice 
remained at the injection side for at least a week. Slow release of the 
toxic lipid A might cause a nontoxic but highly immunogenic stimulus. The 
slow release of lipid A could be accomplished by any type of particle or 
device that is designed to release drugs or other therapeutic substance in 
vivo.

DETAILED DESCRIPTION OF THE INVENTION EXAMPLES 
The herein offered examples provide methods for illustrating, without any 
implied limitation, the practice of this invention in the production of 
immunoreactive compositions containing extremely high dose levels of lipid 
A that can be used to produce vaccines and human mono- and polyclonal 
antibodies reactive with lipid A. 
The profile of the representative experiments have been chosen to 
illustrate methods of making immunotherapeutic compositions that result in 
the production of antibodies to lipid A that would be expected to be 
useful in the prevention and treatment of infections (sepsis) caused by 
Gram-negative bacteria. 
Experimental Methods and Result 
Preparation of Liposomes. Liposomes for immunization were prepared from a 
mixture of dimyristoyl phosphatidylcholine (DMPC), dimyristoyl 
phosphatidylglycerol (DMPG), and cholesterol (Chol) in molar ratios of 
1.8/0.2/1.5. DMPC and DMPG were purchased from Avanti Polar Lipids, 
Pelham, Al.. Chol was purchased from Sigma Chemical Co., St. Louis, Mo. 
and was recrystallized three times from ethanol before use. Monophosphoryl 
lipid A (Ribi Immunochem Research Inc.) was incorporated in the liposomes 
at a concentration of 52.6 ug per umole of phospholipid. All lipids were 
prepared as solutions in redistilled chloroform. Following the additions 
of the appropriate volumes of lipid solutions into round bottom flasks, 
they were rotary evaporated until all the solvent was evaporated and a 
thin film of lipid was left on the flask wall. The flask was further dried 
in a desiccator under high vacuum overnight. The lipids were then hydrated 
with sterile pyrogen free water (USP) so that a phospholipid concentration 
of approximately 40 mM was achieved. The hydrated lipids were then 
aliquotted into vaccine bottles and freeze dried. After lyophilization, 
the bottles were stoppered under vacuum and stored at 4 degrees C. in the 
dark. At a later time the drug substance, SK & F 105154 (R32NS1), was 
injected into each bottle. The phospholipid concentration in the bottle 
was 250 mM after the reconstitution with the R32NS1. After an eighteen 
hour incubation, the bottles were hand shaken until all the lipid was 
suspended. The bottles were then washed with sterile PBS, pH 6.4 and 
centrifuged. After two more washes the liposomes were resuspended with 
PBS, pH 6.4. The liposomes were then dispensed, using aseptic technique, 
into vaccine bottles at 0.9 ml per bottle. Alum concentrate (Rehsorptar, 
Lot #C17502, Armour Pharmaceuticals, Kankakee, Ill.), diluted to the 
1.2-1.8 mg Al/ml range with PBS, pH 6.4, was dispensed into each vaccine 
vial at a volume of 0.7 ml. The phospholipid concentration of the final 
product was 55 mM. The lipid A concentration was 2.9 mg/ml. 
ELISAS. Enzyme-linked immunosorbent assays were performed utilizing lipid A 
from S. Minnesota R595 (List Biologics, Campbell, Calif.) as antigen. 
Wells of "U" bottom polystyrene microtitre plates (Immulon II, Dynatech 
Laboratories, Inc., Alexandria, Va.) were coated with 50 ul of an 0.02 
ug/ul ethanolic solution of lipid A. Wells were allowed to air dry in a 
fume hood for approximately 1 hour. Plates not used immediately were 
stored at -20 degrees C. until use. Blocking buffer (PBS with 10% 
heat-inactivated fetal calf serum) was added to all wells at a volume of 
100 ul and held at room temperature for 2 hours. Blocking buffer was 
removed by inversion of the plates followed by a sharp tap of the plate to 
remove all the well contents. Human sera were diluted 1:100 in blocking 
buffer and added to wells in triplicate at a volume of 50 ul per well. 
Following an eighteen hour incubation overnight at 4 degrees C, the 
contents of the wells were aspirated, the plates were washed three times 
with PBS alone, and 50 ul of the appropriate alkaline phosphatase- 
conjugated goat anti-human Ig (Kirkegaard & Perry Laboratories, Inc., 
Gaithersburg, Md.) was added to the wells. The conjugate was diluted in 
blocking buffer and added to the wells to give a final concentration of 5 
ng per well. After an incubation of 2 hours at room temperature, the 
contents of the wells were aspirated, and the plates were washed three 
times with PBS alone. Then 50 ul of the substrate, p-nitro 
phenylphosphate, prepared in diethanolamine buffer was added to the wells 
and incubated 1 h at room temperature in the dark. The absorbance was read 
at 405 nm using a UV max kinetic plate reader (Molecular Devices, Palo 
Alto, Calif.) Values were reported after subtracting out values in wells 
which lacked only antigen.