U.S. patent application number 10/971219 was filed with the patent office on 2005-05-26 for immunogenic composition and method of developing a vaccine based on factor h binding sites.
This patent application is currently assigned to NMK Research, LLC. Invention is credited to Karp, Nelson M..
Application Number | 20050112139 10/971219 |
Document ID | / |
Family ID | 34520141 |
Filed Date | 2005-05-26 |
United States Patent
Application |
20050112139 |
Kind Code |
A1 |
Karp, Nelson M. |
May 26, 2005 |
Immunogenic composition and method of developing a vaccine based on
factor H binding sites
Abstract
An immunogenic composition able to provide an immune response to
Human Complement Factor H binding on one or more HIV glycoproteins
is disclosed, which is characterized by at least one binding site
epitope of the glycoproteins. Such immunogenic composition wherein
the glycoprotein comprises gp120, gp41, or both glycoproteins gp120
and gp41 is hereby disclosed. Sialic acid is removed to enhance
immune recognition of the composition and to impair Factor H
binding. A medication having an inhibitive action on autoimmune
response by specific inhibition of the cleavage of C3b by Factor H
into inactive cell fragments.
Inventors: |
Karp, Nelson M.; (Virginia
Beach, VA) |
Correspondence
Address: |
WILLIAMS MULLEN
222 CENTRAL PARK AVENUE
SUITE 1700
VIRGINIA BEACH
VA
23462-3035
US
|
Assignee: |
NMK Research, LLC
|
Family ID: |
34520141 |
Appl. No.: |
10/971219 |
Filed: |
October 22, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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60513827 |
Oct 23, 2003 |
|
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Current U.S.
Class: |
424/188.1 ;
424/199.1 |
Current CPC
Class: |
A61P 31/18 20180101;
A61K 47/12 20130101; A61P 31/00 20180101; A61K 9/0014 20130101;
A61K 2039/523 20130101; A61K 2039/5256 20130101; A61K 2039/6018
20130101; A61P 37/04 20180101; Y02A 50/466 20180101; A61K 39/12
20130101; A61P 37/00 20180101; A61K 39/00 20130101; C12N 2740/16034
20130101; C12N 2740/16122 20130101; A61K 39/21 20130101; C12N
2740/16222 20130101; Y02A 50/30 20180101; A61K 2039/55583 20130101;
A61K 2039/55511 20130101; C12N 2710/16243 20130101; A61K 2039/5252
20130101; A61P 43/00 20180101; A61K 2039/55594 20130101; C12N 7/00
20130101; C12N 2740/16063 20130101; A61K 2039/53 20130101; C07K
14/005 20130101; A61K 9/0019 20130101; A61K 2039/5258 20130101 |
Class at
Publication: |
424/188.1 ;
424/199.1 |
International
Class: |
A61K 039/21; A61K
039/12 |
Claims
What is claimed is:
1. A composition for eliciting an immune response to HIV
comprising, in a pharmaceutically acceptable support, an effective
quantity of at least one binding site of Factor H on at least one
HIV glycoprotein.
2. A composition according to claim 1, wherein said at least one
HIV glycoprotein is selected from the group gp120 and gp41.
3. A composition according to claim 2, in which said binding site
is expressed by a recombinant carrier.
4. A composition according to claim 3, wherein said recombinant
carrier is a virus.
5. A composition according to claim 4, wherein said virus is a
herpes virus.
6. A composition according to claim 5, wherein said herpes virus is
Epstein Barr virus.
7. A composition according to claim 4, wherein said virus is a
poliovirus.
8. A composition according to claim 4, wherein said composition has
been treated with neuraminidase, trypsin, or other appropriate
enzyme to remove sialic acid.
9. A composition according to claim 3, wherein said recombinant
carrier is bacteria.
10. A composition according to claim 9, wherein said bacteria is
Bacillus Calmette-Guerin.
11. A composition according to claim 9, wherein said bacteria is
listeria monocytogenes.
12. A composition according to claim 9, wherein said composition
has been treated with neuraminidase, trypsin, or other appropriate
enzyme to remove sialic acid.
13. A composition according to claim 3, wherein said recombinant
carrier is yeast.
14. A composition according to claim 13, wherein said yeast is
Saccharomyces cerevisiae.
15. A composition according to claim 2, in which said binding site
is expressed by messenger RNA.
16. Use of the composition according to claim 2 for preparation of
a medication for eliciting an immune response to HIV.
17. Use of the composition according to claim 16 wherein the
medication inhibits the cleavage by Factor H of C3b into inactive
cell fragments.
18. A method of eliciting an immune response in an animal, which
comprises administering a composition comprising, in a
pharmaceutically acceptable support, an effective quantity of at
least one binding site of at least one HIV glycoprotein, wherein
said at least one HIV glycoprotein is selected from the group gp120
and gp41.
19. A method according to claim 18, wherein the composition is
administered, orally, transbucally, transmucosally, sublingually,
nasally, rectally, vaginally, intraocularly, intramuscularly,
intralymphatically, intravenously, subcutaneously, transdermally,
intradermally, intra tumor, topically, transpulmonarily, by
inhalation, by injection, or by implantation.
20. A method according to claim 18, wherein the composition is
administered, by capsule, gelcap, tablet, enteric capsule,
encapsulated particle, powder, suppository, injection, ointment,
cream, implant, patch, liquid, inhalant, or spray.
21. A composition according to claim 2, wherein said composition is
combined with an immune stimulant.
22. A composition according to claim 21, wherein said immune
stimulant is an adjuvant.
23. A composition according to claim 21, wherein said immune
stimulant comprises polysaccharides composed of at least one
mannose in a form capable of binding to said composition.
24. A composition according to claim 21, wherein said immune
stimulant comprises teichoic acid in a form capable of binding to
said composition.
25. A composition according to claim 21, wherein said immune
stimulant comprises zymosan in a form capable of binding to said
composition.
26. A composition according to claim 21, wherein said immune
stimulant comprises the polysaccharide capsule of cryptococcus
neoformans serotype C in a form capable of binding to said
composition.
27. A composition according to claim 21, wherein said immune
stimulant comprises protamine in a form capable of binding to
heparin.
28. A composition according to claim 21, wherein said immune
stimulant comprises a heparinase.
29. A composition according to claim 21, wherein said immune
stimulant comprises cobra venom factor in a form adapted to enhance
production of C3b.
30. A composition according to claim 29, wherein said cobra venom
factor is dCVF.
31. A composition according to claim 21, wherein said immune
stimulant comprises Nickel in a form adapted to enhance C3
convertase activity.
32. A composition according to claim 21, wherein said immune
stimulant comprises sulfated polyanions capable of absorbing Factor
H.
33. A composition according to claim 2, wherein polyanions within
the composition capable of potentiating Factor H are substantially
removed from the composition.
34. A composition able to provide an immune response to Factor H
binding on at least one HIV glycoprotein, comprising, in a
pharmaceutically acceptable support, a therapeutically effective
quantity of at least one Factor H binding site on at least one HIV
glycoprotein, wherein said at least one HIV glycoprotein is
selected from the group gp120 and gp41, and wherein said
composition inhibits the cleavage by Factor H of C3b into inactive
cell fragments.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority from U.S.
Provisional Application Ser. No. 60/513,827 filed 10/23/2003.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] This invention relates to the field of virology and
immunology. Particularly, but not exclusively, it relates to a
method of inducing an immune response, and a substance based on
certain glycoprotein factor H binding sites for achieving the
same.
[0004] 2. Description of the Related Art
[0005] Immunosuppressive diseases are medically challenging
because, among other things, they attack the ability of the host to
defend against viral invasion. The immune system can be considered
as having a non-specific aspect and a specific aspect. The
nonspecific aspect includes components, such as macrophages and
neutrophils, which simply engulf foreign organisms and kill them
without a need for antibodies. The specific aspect involves the
production of soluble proteins or antibodies that bind to foreign
antigens. Certain other cells then recognize the foreign organisms
and destroy them. If the immune response is suppressed, then the
individual becomes susceptible to opportunistic infections.
[0006] Some retroviruses that attack the immune system, such as
HIV-1, are variable and mutate readily, creating many strains of
varying genetic composition that hamper efforts to develop
effective treatment. (Cohen P. T., The AIDS Knowledge Base, 15-18,
21 (3rd ed. 1999)) These strains, which may be categorized into
groups or subtypes, have individual biological characteristics.
Sequences within a subtype may have genetic clustering or
similarities that sometimes reveal their common lineage. However,
variations in evolutionary rate can produce differences among
mutations even within a subtype. Further, the tendency of
retroviruses to recombine with related retroviruses complicate the
viral genome.
[0007] To date, research has not found a vaccine that would foster
an effective immune response against the immunosuppressive
retrovirus HIV-1. Most HIV vaccines use portions of the envelopes
of surface glycoproteins (gp160, gp120, and gp41) of the virus in
an attempt to induce production of neutralizing antibodies against
the envelope spikes of the virus. (Johnston et al., 2001) Some have
been successful in producing high titers of neutralizing
antibodies. The thought behind this approach is that the antibodies
that bind to these glycoproteins would neutralize the virus and
prevent infection. A functioning immune system could then activate
the complement system, which would cascade to lysis and destroy the
virus. The complement system is a series of circulating proteins
that "complements" the role of antibodies. The components of the
complement system are activated in sequence or turn, which is the
complement cascade. The conclusion of complement is a protein
complex, the Membrane Attack Complex (MAC) that seeks to attach to
an invading organism's surface and to destroy it by puncturing its
cell membrane. (Hoffman, Ronald et al., Hematology Basic Principles
and Practice, Ch. 37. (3d ed. 2000)) In addition, HIV components
can bear surface sialic acid, which can remain on preserved
structure of inactivated HIV. Sialic acids are typically found on
host proteins and cellular structures; high sialic acid content on
a virus, even if the virus is inactivated, would limit the host's
ability to recognize the virus and respond properly. (Michalek,
Michael T., et al., "Inhibition of the Alternative Pathway of Human
Complement by Structural Analogues of Sialic Acid," J. Immunology,
Vol 140, pp 1588-1594 (1988))
[0008] The high rate of mutation of HIV is believed to complicate
further the selection of appropriate neutralizing antibodies. In
addition, a substance known as Human Complement Factor H impedes or
disrupts the immune response complement cascade. Factor H does this
by inhibiting the activity of C3b, a molecule that is central for
the cascade sequences. (Jokiranta, T., FEBS Lett., 393(2-3):
297-302 (Sep. 16, 1996)) (Jokiranta, T., J of Biological Chemistry,
Vol 275 #36, 27657-27662 (Sep. 8, 2000)) (Hellwage, J., FEBS Lett.,
462(3): 345-352 (Dec. 3, 1999)) (Pangburn, M., Immun., Vol 164, pp
4742-4751 (2000)) Prior HIV vaccines have been limited in their
effectiveness, due at least in part to Factor H. A description of
the role of Factor H requires a summary of HIV and specific immune
response to it.
[0009] In 1965, Factor H became the first protein of its type to be
isolated in human serum. (Nilsson, U. R., et al., J. Exp. Med. 122,
277-298 (1965)) Since then Factor H-like protein 1
(FHL-1/reconectin) has also been isolated. FHL-1 is encoded from
the same genomic sequence as Factor H, but is a much smaller
molecule and is the result of alternate gene splicing by the human
cell. FHL-1 has similar immunological activity as Factor H.
(Friese, M. A., Molecular Immunology, Vol 36, pp 809-818 (1999))
(Zipfel, P. F., Immunology Today, (3):121-126 (1994)) (Skerka,
Kuhn, Immun., 155(12): 5663-5670 (1995))
[0010] Additionally, a group of proteins known as Factor H related
proteins (FHR1, FHR2, FHR3, FHR4 and FHR5) have been isolated and
characterized in human plasma. (Zipfel, P. F., Immunopharmacology,
May 41(1-3): 53-60 (1999)) (McRae, Jennifer, Biological Chemistry,
Vol 276, #9, Iss March 2, pp 6747-6754 (2001)) The FHR proteins are
coded by separate genetic sequences but are linked to the Factor H
protein on chromosome 1, specifically 1q31-32.1. Factor H, FHL-1,
FHR1, FHR2, FHR3, FHR4 and FHR5 are structurally similar. Each is
composed of short consensus/complement repeat (SCR) domains also
known as complement control proteins (CCP) or "sushi" units. Each
SCR is composed of approximately of 60 amino acids. Two disulfide
bonds exist in each SCR linking the first and third and second and
fourth amino acids. Although FHR behavior is currently unknown, it
is believed that some of the FHR proteins have immunological
activity similar to that of Factor H.
[0011] The Factor H family of proteins is primarily produced in the
liver. Other sites of synthesis include the lining of the
gastrointestinal tract, the lining of the genital urinary tract,
and various white blood cells.
[0012] The humoral arm of the immune system consists of B cells
that, upon stimulation, differentiate into antibody producing
plasma cells. The first antibodies to appear are IgM, followed by
IgG in blood, or IgA in secretory tissues. A major function of
these antibodies is to protect against infectious disease and their
toxins. Antibodies neutralize viruses and toxins, but also opsonize
microorganisms. Opsonization is a process by which antibodies make
viruses or bacteria more easily ingested and destroyed by
phagocytic cells. Phagocytic cells include both polymorphonuclear
neutrophils (PMNs) and tissue macrophages. PMNs comprise about 60%
of the leukocytes in the blood of an uninfected patient. The number
of PMNs and tissue macrophages may increase or decrease with
certain infectious disorders. For example, typhoid fever is
characterized by a decrease in the number of leukocytes (i.e.,
leukopenia). Both PMNs and macrophages phagocytose or consume
bacteria and viruses. PMNs do not present antigen to helper T
cells, whereas macrophages and dendritic cells do.
[0013] Phagocytosis includes (1) migration, (2) ingestion, and (3)
killing. Tissue cells in the infected area produce small
polypeptides known as chemokines. The chemokines attract PMNs and
macrophages to the site of an infection. Then the bacteria are
ingested by the invagination of the PMN cell membrane around the
bacteria to form a vacuole or phagosome. This engulfment is
enhanced by the binding of IgG antibodies (opsonins) to the surface
of the bacteria. The C3b component of the complement system also
enhances opsonization. (Hoffman, 2000) (William, Paul, Fundamental
Immunology, pp 967-995 (4th ed. 1999)) (Speth, C., et al., The
Middle European J. of Medicine, 111/10: 378-391 (1999)) (Lachmann,
P. J., Clin. Exp. Immul., Vol 21, pp 109-114 (1975)) The cell
membranes of PMNs and macrophage have receptors for the Fc portion
of IgG and the C3b molecule.
[0014] With engulfment, a metabolic pathway known as the
respiratory burst is triggered. As a result two microbicidal
agents, the superoxide radical and hydrogen peroxide are produced
within the phagosomes. These highly reactive compounds often called
reactive oxygen intermediates are synthesized by the following
reactions:
O.sub.2+e-->O.sub.2-
2O.sub.2-+2H+->H.sub.2O.sub.2 (Hydrogen peroxide)+O.sub.2
[0015] The first reaction reduces molecular oxygen to form the
superoxide radical, which is a weak microbicide. The second
reaction which is catalyzed by the enzyme superoxide disumtase
within the phagosome produces hydrogen peroxide radical. In
general, hydrogen peroxide is a more effective microbicide than the
superoxide. The respiratory burst also produces nitrous oxide (NO),
another microbicide. NO contains a free radical that participates
in the oxidative killing of ingested viruses and bacteria
phagocytosed by neutrophils and macrophages. The NO synthesis
within the phagosome is catalyzed by the enzyme NO Synthase, which
is induced by the process of phagocytosis.
[0016] The killing of the organism within the phagosome is a two
step process that consists of degranulation followed by the
production of hypochlorite ions, which is the most effective of the
microbicidal agents. Two types of granules are found within the
cytoplasm of the neutrophils or macrophages. These granules fuse
with the phagosome to form a phagolysosome. The contents of the
granules are then emptied. These granules are lysosomes that
contain a variety of enzymes essential to the killing and
degradation. Two types of lysosomal granules, which are
differentiated by their size, have been identified. The larger
lysosomal granule, which constitutes about 15% of the total,
contains several enzymes including myeloperoxidase, lysozyme, and
other degradative enzymes. The remaining 85% are smaller granules,
which contain lactoferrin and other degradative enzymes, such as
proteases, nucleases, and lipases. The actual killing or
destruction of microorganisms occurs by variety of mechanisms, some
oxygen-dependent and some oxygen-independent. The most important
oxygen-dependent mechanism is the production of the hypochlorite
ion catalyzed by myeloperoxidase:
Cl.sup.-+H.sub.2O.sub.2->ClO+H.sub.2O
[0017] Antibodies are glycoproteins, composed of light (L) and
heavy (H) polypeptide chains. The simplest antibody has a "Y" shape
and consists of four polypeptides: 2 H-chains and 2 L-chains.
Disulfide bonds link the four chains. An individual antibody
molecule will have identical H- and identical L-chains. L- and
H-chains are subdivided into two regions: variable and constant.
The regions have segments or domains, which are three-dimensionally
folded and repeating. An L-chain consists of one variable (V1) and
one constant (C1) domain. Most H chains consist of one variable
(VH) and three constant (CH) domains. The variable regions are
responsible for antigen (virus, bacteria or toxin) binding. The
constant regions encode several necessary biologic functions
including complement fixation and binding to cell surface
receptors. The complement binding site is located in the CH2
domain.
[0018] The variable regions of both L- and H-chains have three
highly variable (or hyper-variable) amino acids sequences at the
amino-terminal portion that makes up the antigen binding site. Only
5-10 amino acids in each hyper-variable region form this site.
Antigen-antibody binding involves electrostatic forces and van der
Waals' forces. In addition, hydrogen and hydrophobic bonds are
formed between the antigen and hyper-variable regions of the
antibody. The specificity or "uniqueness" of each antibody is in
the hyper-variable region; the hyper-variable region is the
thumbprint of the antibody.
[0019] The amino-terminal portion of each L-chain participates in
antigen binding. The carboxy-terminal portion contributes to the Fc
fragment. The Fc fragment (produced by proteolytic cleavage of the
hinge region of the antibody molecule separating the antigen
binding sites from the rest of the molecule or the Fc fragment)
expresses the biologic activities of the constant region,
specifically complement fixation. The H-chains are distinct for
each of the five immunoglobulin classes. The heavy chains of IgG,
IgA, IgM, IgE and IgD are designated .gamma., .alpha., .mu.,
.epsilon. and .delta. respectively. The IgG immunoglobulin class
opsonizes microorganisms; thus, this class of Ig (immunoglobulin)
enhances phagocytosis. (Hoffman, Ronald, et al., Hematology Basic
Principles & Practice, ch. 36 & 39 (3rd ed. 2000))
(Levinson, Warren, Medical Microbiology & Immunology, Ch. 59
& 63 (7th ed. 2002)). Receptors for the .gamma. H-chain of IgG
are found on the surface of PMNs and macrophages. IgM does not
opsonize microorganisms directly because there are no receptors on
the phagocyte surface for the .mu. H-chain. IgM does, however,
activate complement, and the C3b protein can opsonize because there
are binding sites for C3b on the surface of phagocytes. (Levinson,
2002) IgG and IgM, are able to initiate complement cascade. In
fact, a single molecule of IgM can activate complement. Activation
of complement by IgG requires two cross-linked IgG molecules (IgG1,
IgG2, or IgG3 subclasses, IgG4 has no complement activity). FIG. 1
shows the IgG antibody. A variety of non-immunologic molecules,
such as bacterial endotoxin, can also activate the complement
system directly.
[0020] The complement system consists of approximately twenty
proteins that are normally in serum. The term "complement"
indicates how these proteins complement or augment other components
in the immune system, such as antibodies and immunoglobulin.
Complement cascade has three important immune effects: (1) lysis of
microorganisms; (2) generation of mediators that participate in
inflammation and attract PMNs; and (3) opsonization.
[0021] Complement cascade occurs via one of three paths: (1)
classic; (2) lectin; and (3) alternative. (Hoffman, 2000). These
pathways are diagrammed in FIG. 2. The dashed line shows that
proteolytic cleavage of the molecule at the tip of the arrow has
occurred. A line over a complex indicates that it is enzymatically
active. Although the large fragment for C2 is sometimes
interchangeably labeled C2a or C2b, for convention, here small
fragments are designated as "a," and all large fragments as "b."
Hence, the C3 convertase is C4b,2b. Note that proteases associated
with the mannose-binding lectin cleave C4 as well as C2. Each of
these pathways leads to the creation of the Membrane Attack Complex
(MAC).
[0022] With the antibody attached to a specific component of a
virus or bacteria, the MAC is able to perforate the microorganism's
protective cover and allow blood plasma and electrolytes to enter
the microorganism, and at the same time provide a means for egress
of the microorganism's internal structural components.
[0023] In the classic pathway, antigen-antibody complexes activate
C1 to form a protease, which cleaves C2 and C4 to form the C4b,2b
complex. C1 is composed of three proteins: C1q, C1r, and C1s. C1q
is composed of 18 polypeptides which assemble to form six identical
subunits each composed of three homologous chains. Each subunit
consists of a globular head, neck portion and stalk. C1q is
multivalent and can cross-link several immunoglobulin molecules.
C1s is a proenzyme that is cleaved to form an active protease and
calcium is required as a cofactor in the activation of C1. Further,
activation of C1 requires multi-point attachment of at least two
globular heads of C1q to the Fc domains of IgG and/or IgM. The
changes induced in C1q on binding multiple Fc immunoglobulins is
transmitted to the C1rs subunits, resulting in proteolytic
autoactivation of the C1r dimer, which then proteolytically
activates or cleaves C1s. As seen above, activated C1s possesses
the catalytic site for proteolytic splicing of C4 and C2. An enzyme
complex, C4b2b, is produced. This functions as a C3 convertase,
which cleaves C3 molecules into two fragments, C3a and C3b. C3b
forms a complex with C4b and C2b, producing a new enzyme,
(C4b,2b,3b) which is a C5 convertase.
[0024] In the lectin pathway, mannan-binding lectin (MBL, or
mannose-binding protein) binds to the surface of microbes
expressing mannan. MBP is a C-type lectin in plasma that has a
structure similar to that of C1q, and binds to C1q receptors
enhancing phagocytosis. Mannose is an aldohexose found on the
surface of a variety of microorganisms. The first component of the
lectin pathway is designated mannose (or mannan) binding protein
(MBP). A C-terminal carbohydrate recognition domain has affinity
for N-acetylglucosamine and confers the capacity for MBP to
directly opsonize microorganisms expressing mannose-rich surface
coats. In the blood, MBP circulates as a stable complex with a
C1r-like proenzyme and a C1s-like proenzyme (designated
MBP-associated serine protease, or MASP-1 and MASP-2 respectively).
The MBP-MASP-1, MASP-2 complex binds to the appropriate
carbohydrate surface. This results in conformational change in the
MBP protein which leads to auto-activation of MASP-1 by internal
peptide cleavage converting MASP-1 to an active serine protease.
Like C1r, active MASP-1 cleaves MASP-2 activating it. Active MASP-2
exhibits the capacity to proteolytically activate both C4 and C2 to
initiate assembly of the C4b,2b (C3 convertase) enzyme complex. As
with the classic pathway, this leads to the production of C5
convertase.
[0025] In the alternative pathway many unrelated cell surface
structures, such as bacterial lipopolysaccharides (endotoxin),
fungal cell walls, and viral envelopes, can initiate the process by
binding to C3(H.sub.20) and factor B. This complex is cleaved by a
protease, factor D, to produce C3b,Bb, which acts as a C3
convertase to generate more C3b. In contrast to the sequential
enzyme cascade of the classical pathway, the alternative pathway
uses positive feedback; the principal activation product, C3b, acts
as a cofactor for C3b,Bb, which is also responsible for its own
production. Thus, the alternative pathway is continuously primed
for explosive C3 activation. The rate of C3 activation is governed
by the stability of the C3b,Bb enzyme complex. Proteolysis of
factor B by factor D produces a small fragment (Ba) and a large
fragment (Bb). The larger Bb fragment combines with either
C3(H.sub.20) or C3b. Through a catalytic site in Bb, the complex
C3(H.sub.20),Bb can proteolytically convert C3 to C3a and C3b.
Nascent C3b generated by this mechanism is capable of binding
additional factor B. Therefore the alternative complement pathway
has at least two positive feedback loops enhancing the production
of C3b. As shown in FIG. 2, this route also leads to the production
of C5 convertase.
[0026] For each pathway the C5 convertase (C4b,2b,3b or C3b,Bb,C3b)
cleaves C5 into C5a and C5b. C5b binds to C6 and C7, to form a
complex that interacts with C8 and C9, ultimately producing MAC
(C5b,6,7,8,9). (Hoffman, 2000)
[0027] Regardless of which complement pathway is activated, the C3b
complex is a central molecule for complement cascade.
Immunologically C3b fulfills three roles:
[0028] 1. sequential combination with other complement components
to generate the C5 convertases, the enzymes that leads to
production of MAC (C5b,6, 7,8,9)
[0029] 2. opsonization of microorganisms. Phagocytes have receptors
for C3b on their cell surface.
[0030] 3. binding to its receptors on the surface of activated B
cells, which greatly enhances antibody production.
[0031] (Parham, Peter, The Immune System, Ch. 7 (2nd ed. 2004))
[0032] In addition, other proteins of the complement system have
immunologic functions. C5a and the C5b67 complex attract
neutrophils. C5a also enhances adhesion of neutrophils to the
endothelium. C3a, C4a and C5a are anaphylatoxins that cause
degranulation of mast cells with release of mediators such as
histamine, which lead to increased vascular permeability and smooth
muscle contraction. (Hoffman, 2000)
[0033] The complement cascade has several inherent immunologic
functions. Some host proteins have been identified that regulate
these functions. (Hoffman, 2000) (William, 1999) (Speth, 1999)
Inhibitors or regulators of complement cascade normally found in
plasma. These include:
[0034] 1. C1-INH: A serine protease inhibitor synthesized by
hepatocytes and macrophages that binds irreversibly to C1r and C1s
which have been proteolytically cleaved by C1q and are active. This
blocks their intrinsic enzymatic activity and breaks their
attachment to C1q.
[0035] 2. Carboxypeptidase N: a plasma hydrolase that removes the
terminal amino acid (arginine) at the free carboxyl end of C3a,
C4a, and C5a. This eliminates or reduces significantly impairs the
intrinsic activity of these anaphylatoxins (C3a, C4a, and C5a).
However, C5a retains approximately 10% of its chemotactic and
neutrophil-stimulating activity after removal of the arginine
moiety.
[0036] 3. C3b/C4b-binding proteins: Plasma proteins that bind C3b
or C4b, that are not attached to a cellular surface i.e. dissolved
in human plasma. Therefore, they regulate the assembly of C3/C5
convertases. Additionally, several of these proteins are plasma
membrane receptors. These proteins include Factor H and C4b-binding
protein:
[0037] a. Factor H: A plasma protein that demonstrates two basic
inhibitory functions. (1) Binding of C3b and inhibiting association
of C3b or C3(H.sub.2O) with Factor B. By restricting the assembly
of C3,bB proenzyme a C3 convertase is not formed. This is known as
decay acceleration. (2) Factor H also promotes dissociation of
assembled C3b,Bb enzyme complexes and serves as a cofactor for
proteolytic degradation of C3b by Factor 1. Limited intra-chain
proteolysis by Factor 1 initially converts C3b to inactivated C3b
(iC3b), which remains covalently bound to its original acceptor
surface. This is the cofactor activity for factor H.
[0038] b. C4b-Binding Protein (C4b-bp): An acute phase reactant
protein in plasma like CRP, noted in acute inflammatory reactions
that regulates complement activity at three levels. (1) Binding to
C4b, blocking its association with C2. (2) Accelerates the
dissociation of the assembled C4b,2b enzyme complex. (3) Cofactor
to facilitate proteolytic degradation of C4b by the serine protease
Factor 1. C4b-bp circulates in plasma as a complex with protein S,
a vitamin K-dependent protein with regulatory function for
coagulation linking the complement system with the coagulation
system.
[0039] 4. CD59 (HRF-20). A protein found on human blood cells and
vascular endothelial cells that inhibits the final step in the
formation of the MAC complex, insertion of C9 into the plasma
membrane.
[0040] Inhibitors or regulators of complement activity that are
plasma membrane bound:
[0041] 1. Decay-Accelerating Factor (DAF, CD55): A single chain
glycoprotein (William, Paul, Fundamental Immunology, pp 967-995
(4th ed. 1999)). normally expressed on the surface of red cells,
platelets, leukocytes, endothelium, and other host cells. DAF
accelerates dissociation of the subunits of the membrane-assembled
C4b,2b and C3b,Bb enzyme complexes protecting host cells from
complement activation. DAF does not act as a cofactor for
inactivation of C4b or C3b by Factor 1.
[0042] 2. Membrane Cofactor Protein (MCP, CD46): A glycoprotein
found on the cell membrane of all human leukocytes and platelets,
but not red blood cells. MCP binds C3b, C4b, and iC3b and
facilitates cofactor activity for Factor 1 mediated proteolysis of
C3b and C4b. DAF and MCP complement each other: DAF has
decay-accelerating activity, but no cofactor activity for Factor 1,
while MCP acts as cofactor for factor 1, but does not promote decay
of C3 convertase enzymes.
[0043] 3. CR1 (CD35): A principal cellular receptor found on
erythrocytes, monocytes/macrophages, eosinophils, neutrophils,
follicular dendritic cells, T cells and B cells for C3b and C4b.
Four polymorphic forms containing up to 34 short consensus repeat
units have been identified. CR1 erythrocytes absorb immune
complexes and transport them to the liver and spleen for removal.
CR1 on macrophages serves as an opsonin receptor. CR1 accelerates
the decay of C3b,Bb enzyme. Finally CR1 is an important cofactor
for the proteolysis of C3b and C4b by Factor 1 and therefore
inhibits the alternative and classic complement pathways on host
cells. (William, 1999)
[0044] 4. Vitronectin (S-protein) and Clusterin. Scavenger proteins
in plasma that binds C5b,6,7 preventing its attachment to other
cells and forming a MAC complex.
[0045] Any one of the aforementioned can reduce the effectiveness
of the complement system. (William, 1999) (Hoffman, 2000) Notably,
the genes for Factor H, C4b-bp, DAF, MCP and CR1 are linked on the
long arm of chromosome 1, and compose a family of closely related
genes designated regulators of complement activation (RCAs). Any
microorganism with the capacity to limit the activity of complement
cascade could theoretically protect itself against the humoral arm
of the immune system. (William, 1999) (Joiner, K. A., "Complement
Evasion," Annu, Rev. Microbio. Vol 42, pp 201-230 (1988)) Thus, the
complement cascade is the Achilles heel of the humoral arm.
[0046] Retroviruses can activate the complement system in the
absence of antibodies. (Haurum, John, "Complement Activation upon
binding of mannan-binding protein to HIV envelope glycoproteins,"
AIDS, Vol 7 (10), pp 1307-13 (1993)) Complement activation by HIV
envelope glycoproteins has been found to be mediated by the binding
of MBP to carbohydrates on natural envelope protein produced in
virus-infected cells, as well as on glycosylated recombinant
envelope proteins. (Haurum, 1993) (Speth, 1997) Activation of the
classical complement pathway and lectin pathway by retrovirus
envelopes can be initiated by the binding of MBP to carbohydrate
side chains of envelope glycoproteins. The transmembrane protein of
HIV-1, gp41, has been shown to be non-covalently associated with
gp120. Complement component, C1q, also binds to gp41. In the
cell-external part (ectodomain) of gp41, three sites (aa 526-538;
aa 601-613 and aa 625-655) bind both gp120 and C1q. Thus, C1q and
gp120 are both structurally and functionally homologous. The
interaction between gp41 and C1q is calcium dependent unlike the
association of gp41 and gp120 which is calcium independent.
[0047] When HIV triggers the classical and lectin pathway in an
antibody-independent manner, it enhances the infection of
complement receptor-positive cells by HIV. The binding of C1q to
gp41 may facilitate infection in different ways. C1q binds directly
to HIV-infected cells that are also infected with HIV-1. C1q
retains its ability to bind to the C1q receptor, also known as the
collectin receptor. Further, gp41 interacts directly with C1q
anchored on the plasma membrane of macrophages. In both cases, HIV
has the opportunity for C1q-mediated CD4 independent contact with
cells.
[0048] The homology of gp120 and C1q raises the possibility that
gp120 may interact directly with the C1q receptor, and thereby
facilitate the entry of HIV into macrophages in a CD4-independent
manner. (Stoiber, Heribert, European Journal of Immunology, Vol 24,
pp. 294-300 (1994)) Antibodies to gp120 are able to cross react
with C1q and may be responsible, at least in part, for the
significantly low C1q concentration in HIV-1 patients. C1q is one
of the factors responsible for the clearance of insoluble immune
complexes, and its absence might contribute to the significantly
high concentrations of insoluble immune complexes noted in
individuals infected with HIV. (Procaccia, S., AIDS Vol 5, p. 1441
(1991)) Hypocomplementemia which characterizes HIV disease is
correlated with HIV associated opportunistic infections and viral
associated malignancies.
[0049] Regulators of complement activity can be found attached to
plasma membranes. Others circulate freely in human plasma and
lymph. Many regulators of complement activity (RCA) have been
characterized and virtually every step in all three pathways is
subject to positive and negative controls. Three enzymatic
complexes (C3 convertases, C5 convertases, MAC complex) are focal
within the complement cascade and are subjected to multiple
inhibitors or catalysts.
[0050] Several proteins that control the complement activation
pathways circulate in plasma as freely soluble molecules, and can
either control C3 activation in the fluid phase or inhibit
formation of MAC on cell surfaces. Regulators of complement, such
as Factor H and low-molecular-weight Factor H-like proteins, have
been shown to mediate this function. Factor H interacts with gp120,
enhancing syncytium formation and soluble CD4 (sCD4) induced
dissociation of the envelope glycoprotein (env) complex. Factor H
only binds activated gp120 after it has engaged CD4, suggesting
that the binding site is hidden within the env complex, and becomes
exposed only after interaction of gp120 with CD4. (Pinter, Claudia,
Vol 11, (Nov. 8, 1995)) The gp120 molecule binds to the CD4
receptor on helper T cells. The virus then fuses with the T cell.
The fusion domain is located on gp41. Upon fusion, the gp120
fragment is shed. The gp41 ectodomain becomes exposed after
shedding gp120. Binding sites for C1q and factor H on gp41 become
unmasked. FIG. 3 is a model of the trans-membrane protein of gp41
spanning aa 510-665, while FIG. 4 is a model of the gp120
glycoprotein.
[0051] HIV activates human complement systems even in the absence
of specific antibodies. (Stoiber, Heribert, Journal Ann. Rev.
Immunology, Vol 15, page 649-674 (1997)) This would result in viral
inactivation if complement were unimpeded. The complement process
if unimpeded would produce membrane attack complex (MAC),
triggering virolysis. However, HIV avoids virolysis by
incorporating into its structure various molecules of the host
(e.g., DAF/CD55) that regulate complement. HIV includes these
molecules in the virus membrane upon budding from infected cells,
or by attachment to the gp41 and gp120 structures. (Stoiber,
Heribert, Journal Ann. Rev. Immunology, Vol 15, page 649-674
(1997)) This interaction with complement components enables HIV to
take advantage of complement components to enhance infectivity,
follicular localization, and broaden its target cell range. At the
same time, HIV defends against the humoral arm.
[0052] Proteins such as Factor H and CR1 have both cofactor and
decay accelerating activities on the C3 convertases. (Stoiber,
Heribert, Journal Ann. Rev. Immunology, Vol 15, page 649-674
(1997)) C3b integrity is essential for the complement cascade to
culminate in cell lysis. C3b is rapidly cleaved by a serine
protease (complement Factor 1-CF1) after interaction with
appropriate complement receptors. Proteins that mediate this
reaction possess cofactor activity for CF1. Some proteins down
regulate complement activation by inhibiting the assembly and/or by
favoring the dissociation of C3b and C5b generating enzymes
(convertases). This activity is termed decay acceleration and is
characteristic of the CD55 (DAF) protein molecule.
[0053] Serum lacking Factor H will lyse HIV and infected cells, but
not cells that are uninfected. (Stoiber, Heribert, J. Exp. Med.,
Vol 183, pp 307-310 (1996)) In the presence of Factor H, lysis of
HIV has been shown to occur when the binding of Factor H was
inhibited by a monoclonal antibody directed to a Factor H binding
site in gp41. As demonstrated in FIG. 5, human serum that is devoid
of Factor H effectively lyses HIV virions. But to date, there has
been no indication of how to implement this growing knowledge of
the relationship of HIV and Factor H of the human complement
system.
[0054] Many microorganisms thwart the immune system by assimilating
Factor H, and/or FHL-1 into their structure, such as Streptococcus
pyogenes, Borrelia burgdorferi, Neisseria gonorrhea, Neisseria
meningitides, Yersinia enterocolitica, Echinococcus granulosus and
Onchocerca volvulus. (William, Paul, Fundamental Immunology pp.
967-995 (4th ed. 1999)) (Joiner, K..A., "Complement Evasion," Annu,
Rev. Microbio. Vol 42, pp 201-230 (1988)). (Speth C., et al., The
Middle European Journal of Medicine, 111/10: 378-391 (1999)).
Factor H inhibits complement cascade with HIV by inhibiting the
activity of 3Cb in the blood. Factor H directly binds to both gp120
and gp41 and inactivates C3b virtually as soon as it is deposited.
(Pinter, Claudia, et al., AIDS Research and Human Retroviruses, Vol
11, #5, pages 577-588 (1995)) (Pinter Claudia, et. al., AIDS
Research and Human Retroviruses, Vol 11, #8, pages 971-980 (1995))
(Stoiber Heribert, et al., "Human Complement Proteins C3b, C4b,
factor H and Properdin react with specific sites in gp120 and gp41,
The Envelope Proteins of HIV 1," Immunobiology, Vol 193, pp 98-113
(1995)) Both gp120 and gp41 contain sequences that are similar to
one of the Factor H binding sites on C3b. When Factor H is
deposited on gp120 or gp41, it interacts with covalently bound C3b
and promotes its cleavage into the inactive fragments C3dg and
iC3b. To different degrees, immunosuppressive viruses may reduce
complement destruction by incorporating cell-derived complement
controlling proteins such as CD55 (DAF) and CD59 into their
membranes as they bud from the membranes of host cells.
[0055] As described above, HIV has multiple means to escape the
host immune system. One is the incorporation of sialic acid into
its external glycoproteins which interferes with the complement
system. HIV has multiple mechanisms to evade the host complement
system. HIV can neutralize the antimicrobial activity of complement
by incorporating the host plasma protein Factor H into surface
glycoproteins, which protects the virus against destruction by the
humoral arm of the immune system. Yet the HIV virus can be
destroyed in human serum in vitro by inhibiting the activity of DAF
and Factor H using monoclonal antibodies. (Stoiber Heribert, et
al., J. Exp. Med., Vol 183, pp 307-310 (1996)) Resistance of HIV
and HIV-infected cells against complement-mediated lysis in vivo is
dependent on DAF and Factor H, and said resistance can be weakened
by inhibiting the function of DAF and Factor H. The present
invention stimulates the immune system by enhancing the cytotoxic
T-lymphocyte and antibody humoral response to HIV.
[0056] HIV and HIV-infected cells are resistant to the potentially
destructive effects of human complement, but are readily destroyed
by other animal sera. Indeed, HIV activates complement in most
animal models rendering them unsuitable models for some forms of
study. (Spear G. T., et al., Immunology, Vol 73, pp 377-382 (1991))
In humans the complement pathway is interrupted at the level of C3b
deposition, but in animals complement proceeds to the production of
the MAC and effective virion lysis. (Spear, 1991)
[0057] The structure of Factor H has been defined. (Ripoche, Jean,
et al., Biochem. J., Vol 249, pp 593-602 (1988)) (Aslam, Mohammed,
et al., Molecular Biology, Vol 309, Issue 5, pp 1117-1138 (2002))
It consists entirely of 20 short complement/consensus repeat (SCR)
domains, each 61 amino acids long. The SCR domains constitute the
most abundant type domain in the complement proteins and are
cysteine rich. The cofactor and the decay accelerating activity are
located within the four N-terminal domains, SCR-1 to SCR-4, which
bind to intact C3b. A second C3 site is located within SCR-6 to
SCR-10 which binds to the C3c region of C3b, and a third site is
located within SCR-16 and SCR-20 which binds to the C3d region of
C3b. The discovery of the binding of C3 to Factor H reveals that
the primary binding activity of gp41 and gp120 is with Factor H,
and is not with some combination of C3 and Factor H. It has also
been revealed that sialic acid residue facilitate Factor H binding
to gp41 and gp120. (Meri, Seppo, et al, Proc. Natl. Acad. Sci.,
USA, Vol 87, pp 3982-3986 (1990)) These revelations, in view of the
role of Factor H in reducing cytotoxicity, indicate that a subunit
vaccine that inhibited Factor H from binding to gp41 and gp120
would be immunogenic.
[0058] Factor H also binds to heparin and other polyanionic
substrates enhancing its activity and therefore modulating the
complement regulatory functions of Factor H. (Aslam, 2002)
(Hellwage 1999) (Blackmore, T. K., et al., Immunology, Vol. 157,
Iss 12, pp 5422-5427 (1996)) Heparin binding sites have been
located in SCR-7 and SCR-20 and a third heparin binding site maybe
located at or near SCR-13. The SCR domains act synergistically to
enable Factor H to achieve differential control of complement
activation. 19 flexible peptide links join the 20 SCR domains. This
flexibility allows Factor H to assume a bent back structure
enabling the multiple Factor H binding sites for C3 and heparin to
come into close proximity.
[0059] Heparin induces a conformational change in the reactive site
of antithrombin III (ATIII), and allows a target protease to
interact more efficiently with this site. Heparin induces a similar
conformational change in Factor H, increasing its ability to
enhance the proteolytic cleavage of C3b by Factor 1. (Giannakis,
Eleni, et al., Int'l. Immunopharmacology, pp 433443 (2001)) (Meri,
1990) Both heparin and Factor 1 are serine proteases. Heparin
sulfate binds HIV with high affinity increasing viral infectivity.
Factor H binding to heparin, a substance found in normal human
serum, also increases viral infectivity by enhancing factor H
activity. Acting as a cofactor for Factor 1 in the plasma, C3b is
inactivated by Factor 1, which cleaves a fragment from the C3b
.alpha.--chain to generate iC3b. Proteolysis by Factor 1 requires
binding of C3b either to plasma component Factor H or to the
membrane receptors CR1 or MCP. Further proteolysis by Factor 1
liberates the larger fragment C3c, leaving C3dg covalently bound to
the target surface via the carbonyl of the internal thioester.
[0060] In addition, heparin is characterized with both
anti-complement and anti-coagulant activity. (Weiler, John M., et
al., J. Exp. Med., Vol. 147, pp 409-421 (1978)) Heparin inhibits
C1q binding to immune complexes as well as the interaction of C1s
with C4 and C2. The anti-complement activity of the heparin
molecule is structurally distinct from its anti-coagulant
activity.
[0061] The inactive fragments of C3b produced by Factor H include
C3dg and iC3b. Both of these fragments are covalently bound to
gp120 and gp41, as well as the complement receptors CR1, CR2 and
CR3. These receptors are found on dendritic cells (APCs),
monocytes, macrophages, B cells, and T cells. C3dg and iC3b are
usually only bound to viral fragments that have been previously
destroyed by complement. When localized to lymph nodes, these
fragments bind dendritic cells and initiate the activation of naive
T and B cells. In HIV infections, live virus concentrates in lymph
nodes and the thymus because the virus is able to avoid complement
cascade. (Stoiber Heribert, et al., Ann. Rev. Immunology, Vol. 15,
pp 649-674 (1997)) Under normal circumstances with viral pathogens
other than HIV disease, live virus does not become concentrated in
the lymph nodes or the thymus gland. (Thieblemont, et. al,
Immunology, Vol 155, p. 4861 (1995))
[0062] Within the lymph nodes, the live virus bound to C3dg and
iC3b is targeted by APCs, T cells, B cells, and macrophages..
(Doepper, Susi, et al, Current Molecular Medicine, Vol, 2, Iss 8,
pp 703-711 (2002)) (Stoiber H, et. al., Ann. Rev. Immunology, Vol.
15, pp 649-674 (1997)) (Dierich, M. P., et al., Immunology Today,
Vol 14, Iss 9, pp 435-440 (1993)) When inactive C3dg and iC3b
triggers the macrophage CR1 and CR3 receptors, the cellular
transcription factor NF-kB becomes activated, which results in
enhanced viral replication in latently infected T cells. Finally,
the activation of CR3 receptor by C3dg and iC3b has been shown in
vivo to result in the suppression of two cytokines, IL-12 and gamma
interferon.
[0063] Activated dendritic cells produce IL-12, a cytokine that
amplifies the immune response, promoting the differentiation of the
T helper 1 lymphocyte subset. This, in turn, sustains the natural
killer (NK) cell activity of the innate immune system. Initially in
HIV infection a type 1 pattern predominates. As the disease
progresses a decline in IL-12 shifts the cytokine milieu from type
1 to type 2. (Cohen, P. T., 1999, The AIDS Knowledge Base, Ch. 18
(3rd ed. 1999))
[0064] CR1 and CR3 also mediate HIV infection of monocytes,
macrophages, and thymic T cells with complement-opsonized HIV.
Therefore, CD4 is not the only cell surface molecule that HIV uses
to bind to and enter its target cells. (Stoiber Heribert, et al.,
Ann. Rev. Immunology, Vol. 15, pp 649-674 (1997)) (Doepper, 2002)
Virus coated with complement fragments interacts efficiently with
cells bearing complement receptors of various types. This mechanism
has shown to be most relevant at low concentrations of the virus.
HIV-1 is characterized by a unique function: an ability to infect
cells not infected with HIV disease that is enhanced by low viral
titers. Low viral titers are one goal of antiretroviral therapy.
Achievement of this goal comes at a price: increased HIV targeting,
binding, and entry into non-infected cells. (Legendre C., et al.,
Febs. Lett. 381:227-232 (1996)) (Reisenger, E. C., et al., AIDS,
Vol 4, pp 961-965 (1990)) Interestingly HIV-specific treatment
decreases viral titers, but increases the virulence of the virus.
Dendritic cells can bind HIV through receptors for complement
fragments, and these cells can transmit HIV to other cells and
promote extensive viral replication when they interact with CD4+ T
cells.
[0065] Because of the homology with human C3, direct interaction of
viral envelope glycoproteins with CR3 or indirect interaction
(i.e., via fixed C3 fragments or immune complexes--specific
antibody plus complement fragments) on the HIV surface are
possible. This can facilitate the infection of CD4+ cells and/or
broaden the host range to include cells that have no CD4, or on
which the use of CD4 is blocked by antibodies against CD4.
(Reisenger, 1990)
[0066] HIV induces various cytokine (TNF-a,IL-1, IL-6) production
in the brain of HIV patients. (Gasque, P., Imunol., 149:1381-87
(1992)) These cytokines then induce the production and release of
complement proteins within the central nervous system. In the
brain, HIV is opsonized with complement fragments. In accordance
with the enhancement of HIV infection of peripheral blood cells
increased infectivity for CR-positive CNS cells, like microglial
cells, is possible. These cells are one of the main targets of HIV
in the CNS. Because CR3 is found on microglial cells and can
directly interact with gp41, it can facilitate entry of virus into
the cells after attachment to receptors via gp120. (Levy, J. A.,
Microbiol., Rev. 57:183-289 (1993))
[0067] HIV mimics a variety of complement proteins, including C1q,
C3, Properdin, and C4bp. (Morrow, W. J., Clin. Immunol.
Immunopathol. 40:515-24 (1996)) The antibodies induced by HIV, or
by vaccination with recombinant proteins, not only recognize viral
proteins, but also cross react with different members of the
complement system. C1q, which is involved in the clearance of
immune complexes, may be blocked by gp120 induced auto-antibodies.
This occurs because C1q binds directly to the gp120 molecule. This
contributes to the high levels of immune complexes found in
HIV-infected individuals. (Morrow, 1996) Four sites in gp41 share
homology to C3. Because of the central role of C3 in the complement
cascade, auto-antibodies against C3 which are detectable in
HIV-positive sera, adversely affect complement-mediated immune
responses.
[0068] Among the first targets for infection with HIV are
epithelial cells and Langerhans cells in the skin, rectal and
vaginal mucosa; this is due to complement directed follicular/APC
virus localization. (Stoiber, Heribert, et al., Ann. Rev.
Immunology, Vol. 15, pp 649-674 (1997)) Their susceptibility to
infection seems to be clade dependent; clade E-type viruses are the
most efficient. (Stoiber Heribert, et al., Ann. Rev. Immunology,
Vol. 15, pp 649-674 (1997)).
[0069] An efficient cellular response in the early stages of HIV
infection correlates with a rapid decrease in viremia. (Stoiber H,
et al., Ann. Rev. Immunology, Vol. 15, p 649-674 (1997)) This
advantageous aspect, however, may be detrimental since HIV infected
CD4+ cells may be killed through a conventional, virus-specific CD8
cell-mediate mechanism in which one part of the immune system
eliminates the other. Moreover, the cytotoxic T lymphocyte (CTL)
response may efficiently eliminate most virus-infected cells and
those presenting peptides of viral proteins. (Stoiber, Heribert, et
al, Ann. Rev. Immunology, Vol. 15, pages 649-674 (1997)) This will
select cells bearing HIV in a latent form that escapes
recognition.
[0070] HIV triggers a domino effect by its inactivation of
complement cascade. The pivotal immune effect of HIV is its
capability to assimilate Factor H into its surface glycoproteins.
(Pinter, Claudia, AIDS Research in Human Retroviruses, Vol 11,
November 8 (1995)) (Pinter Claudia, et al., AIDS Research and Human
Retroviruses, Vol 11, #5, pp 577-588 (1995)) (Stoiber, Heribert, et
al., Immunobiology, Vol 193, pp 98-113 (1995)) HIV escapes
complement cascade, enhances its own replication, and sequesters
itself within the lymph nodes of the human body. This distorts or
destroys basic lymph node architecture and function. Additionally,
the inactive C3b fragments enhance viral replication.
[0071] Because Factor H is a host or cellular protein, it is not
subject to the viral mutation common in HIV. (Ripoche, 1988)
(Aslam, 2002) (Kirkitadze, Marina, et al., Immun. Rev., Vol 180, pp
146-151 (2001)) (Feifel, Elisabeth, et al., Immunogenetics, Vol 36,
pp 104-109 (1992)) (Discipio, R. G., J. Immunology, Vol 149, Iss 8,
pp 2592-2599 (1992)). (Ault, Bettina, et al., Biological Chemistry,
Vol 272, #40, pp 25,168-25,175 (1997)) The amino acid sequences
that comprise the primary structure of Factor H are constant and
immutable. Accordingly, the binding sites for Factor H and related
proteins on gp120 and gp41 are also conserved. (Pinter, 1995) If
there were mutations at the binding site, then the humoral arm of
the immune system would then be able to proceed normally to
inactivate the virus. Thus, HIV cannot tolerate mutations at the
Factor H binding sites, providing another advantage for use in a
subunit vaccine.
[0072] The present invention is a composition and subunit vaccine
composed of the Factor H binding epitope of glycoprotein gp120,
gp41, or preferably, a combination of gp120 and gp41. The
immunogenic composition may include the binding sites for Factor H,
FHL-1, and similarly behaving FHR 1-5. Factor H in circulation
assumes two separate conformational states or two separate tertiary
structures, .phi..sub.1 and .phi..sub.2. The composition may
include binding sites for both of these conformational states on
both of the gp120 and gp41 glycoproteins. By keeping Factor H away
from the viral surface, HIV resistance against human complement is
weakened, enabling human complement to function.
SUMMARY OF THE INVENTION
[0073] The present invention is an immunogenic composition and
vaccine composed of a viral epitope of the gp120 and/or gp41
glycoproteins or portion thereof that will bind with Human
Complement Component Factor H, FHL-1, and similarly behaving FHR
(1-5) of Factor H (herein referred to generally as Factor H.)
Sialic acid is removed to reveal to the host that the composition
(the binding sites for factor H on HIV) is foreign and to impede
Factor H binding.
[0074] Alternatively, the nucleic acid (RNA and/or DNA) encoding
the factor H binding sites can be used as an immunogen or a
vaccine. The nucleic acid may be administered as a naked RNA or DNA
molecule, plus the appropriate adjuvants to enhance immunogenicity
if needed. Furthermore, the nucleic acid may be recombined into a
bacterial or viral vector which would then be used as the immunogen
or the vaccine. The nucleic acid may be either native viral DNA or
RNA or complementary DNA (cDNA).
[0075] In an alternate embodiment the messenger RNA (mRNA) encoding
the factor H binding site(s) on gp120 and/or gp41 can be used as an
immunogen or vaccine. Finally, the above methodologies can be used
independently of each other, as a multivalent immunogen or used
sequentially.
BRIEF DESCRIPTION OF THE DRAWINGS
[0076] FIG. 1 shows the IgG antibody.
[0077] FIG. 2 shows the Classic, Lectin, and Alternative pathways
of the complement system.
[0078] FIG. 3 is a model of the trans-membrane protein of gp41
spanning aa 510-665. The fusion domain (aa 510-526), the
immunosuppressive (aa 570-585) and the immunodominant (aa 587-605)
domains are shown. Furthermore the binding sites for Factor H and
Properdin are illustrated.
[0079] FIG. 4 is a model of the gp120 glycoprotein. Complement
binding sites for the C3b protein, C4b protein and Factor H are
shown
[0080] FIG. 5 shows the effect of the addition of Factor H to
Factor H depleted serum, as shown in Stoiber, H. et al., J. of
Experimental Medicine, Vol 183, 307-310.
[0081] FIG. 6 depicts the categories of embodiments for the present
immunogenic composition.
[0082] FIG. 7 is a graph of the exemplary carriers available for
recombinant DNA.
[0083] FIG. 8 is a chart demonstrating splicing of genetic material
encoding the Factor H binding site genetic material into
recombinant bacterial compositions or vaccines.
[0084] FIG. 9 is a chart demonstrating splicing of genetic material
encoding the Factor H binding site genetic material into
recombinant viral compositions or vaccines.
[0085] FIG. 10 is a list of immune stimulants for use with naked
DNA compositions
[0086] FIG. 11 describes customary routes of administration for
DNA.
[0087] FIG. 12 demonstrates how increasing polysaccharide length
enhances immunogenicity up to a maximum of 16 monosaccharides.
[0088] FIG. 13 is a schematic drawing showing the chain structures
of C3 and CVF and their relationship.
DESCRIPTION OF THE INVENTION
[0089] The following detailed description is not to be taken in a
limiting sense, but is made merely for the purpose of illustrating
general principles of the invention. In particular, the subunit
immunogenic composition described is based on a viral epitope of
the gp120 and/or gp41 glycoproteins that will bind with Complement
Factor H, FHL-1, and similarly behaving FHR (1-5) (herein referred
to generally as Factor H.) Factor H binds to only one site on the
gp120 glycoprotein, which can be mapped specifically to the site
Env 105-119 (HEDIISLWDQSLKPC). Four sites on the gp41 glycoprotein
have been shown to bind to Factor H, including the amino acids
561-585, 587-607, 615-635 and 651-675. The portion of the gp120
glycoprotein that binds to Factor H can be synthesized in vitro
using a commercially available amino acid synthesizer. The four
areas of gp41 glycoprotein to which Factor H maps have also been
sequenced:
[0090] Amino Acids
1 Amino Acids 561-585 (LRAIEAQQHLLQLTVWPIKQLQARI) 587-605
(AVERYLKDQQLLGIWGCSG) 651-675 (IEESQNQQEKNEQELLELDKWASLW) 615-635
(WNASWSNKSLEQIVNNMTWME).
[0091] A commercially available amino acid synthesizer can be used
to synthesize all four areas of gp41 glycoprotein that bind to
Factor H.
[0092] B. Subunit Compositions
[0093] The present subunit immunogen is comprised of a protein or
portions thereof or the genetic sequences encoding for the protein
or protein segments in order to create an immune response and
immune memory. In the present invention, the desired immune
response is directed to the critical surface factor H binding site
of HIV, or portions thereof. Importantly, the composition should be
presented properly to the immune system. Isolation and use of
nucleic acids, peptides, and proteins are familiar to those of
ordinary skill in the art, and as described herein.
[0094] One of the advantages of a subunit composition is a lack of
infectivity in therapeutic applications. Therefore subunit
compositions may serve when a virus is extremely virulent, as with
HIV. Some viruses such as HIV undergo profound mutation and
therefore an attenuated strain used in a vaccine or therapy can
undergo spontaneous reversion to a more virulent strain. Therefore
with HIV the use of live viral vectors would be risky. Also subunit
compositions or vaccines can be used when the virus cannot be grown
conveniently in culture. Subunit compositions may be produced
quickly and relatively inexpensively.
[0095] For example, a subunit vaccine is currently available using
the hepatitis B virus surface antigen obtained by expression of a
cloned gene in yeast cells. This vaccine has been successfully used
in Taiwan and it appears to have reduced the incidence of primary
liver cancer in young children. (Wagner, 1999)
[0096] Direct administration of a protein would not induce a
cell-mediated response in the same way that a live virus vaccine
would. Yet the advantages of a subunit vaccine include a lack of
potential infectivity, either mild in the case of an attenuated
strain or severe in the case of the virulent strains. Further, the
present invention is contemplated for use in conjunction with
immune stimulants and other immunogenic compositions.
[0097] A strong stimulation of B cells and an antibody response are
evident against all of the major HIV proteins soon after infection.
(Goudsmit, 1988) For unknown reasons, this does not lead to the
production of effective neutralizing antibodies. On the contrary
these antibodies may enhance uptake of HIV by cells other than CD4
lymphocytes, and thereby promote a more efficient localization on
the antigen presentation cells (APC), due to deposition of
complement fragments on the virus surface. (Stoiber, 1997) In the
conversion of neutralizing antibodies into enhancing antibodies,
follicular dendritic cells may play an important role. So far,
efforts to generate neutralizing antibodies by vaccination have
been unsuccessful. (Cohen P. T., The AIDS Knowledge Base, Ch. 22
(3rd ed. 1999)
[0098] Thus, the composition of the present invention includes a
method for inducing an immune response in a human or other animal.
General references to "animal" include humans. The method comprises
preparing the composition and administering to an animal capable of
mounting a humoral or a cellular immune response. An immune
response may be detected using common methods of measurement known
in the art. The present invention may be used to develop laboratory
tools and research immune response. Furthermore this invention will
aid the development of a vaccine for administration to an HIV
infected subject or for producing an immune response in a subject
that is not infected, but for whom an immune response is
desired.
[0099] C. Method of Preparation
[0100] A variety of methods of development, preparation, and
administration are contemplated by the present invention. It is
expected that such methods shall be selected based on efficacy for
the particular strain and response of the subject animal. As shown
in FIG. 6 this subunit composition may be categorized for
preparation purposes as protein isolation, messenger RNA
expression, or nucleic acid DNA/RNA expression.
[0101] Thus, the present invention may be prepared using any one or
more of a variety of methods available to those in the field,
including but not limited to:
[0102] 1. Purification and isolation of the factor H binding site
from HIV
[0103] 2. Messenger RNA cloning and expression of the factor H
binding site into a suitable bacteria such as escherichia coli,
yeast, or virus; alternatively the mRNA can be used as a naked
nucleic acid vaccine with the appropriate adjuvants if
necessary
[0104] 3. Naked or recombinant DNA/RNA cloning and expression of
the factor H binding sites into a suitable bacterium such as
escherichia coli or yeast or virus. (Aroeti, 1993)
[0105] Antigen presenting cells take up exogenous proteins by
phagocytosis, leading to presentation of the immunogen and immune
response. With reference to the list above, embodiment 1 relies on
protein fragments, while embodiments 2-3 rely on nucleic acids and
recombinant technology. Embodiments 2-3 may also include the
synthetic in vitro manufacturing of the nucleic acid. (Aroeti,
1993)
[0106] C.1. Protein Based Compositions
[0107] The factor H binding sites may be isolated from a single
viral particle or a viral culture. In the case of a single
particle, enzymatic (proteolytic) degradation may be used. For
example, a mature F protein may be isolated from viral particles by
degrading and enzymatically digesting the mature viral particles
into individual protein components. "Purification" means merely
that the protein is sufficiently free of other cell subunits or
contaminants so as to enable therapeutic use. The composition need
not be totally pure. The factor H binding sites may also be
isolated from a viral culture. In vivo each protein of the viral
structure is produced in quantities exceeding that necessary for
viral replication. Therefore individual viral proteins may be
isolated and separated from viral isolates based on that protein's
characteristic size, shape, solvency characteristics, electrostatic
potential, density and/or buoyancy and sedimentation rate in a
variety of media. Therefore, this approach involves the use of
specific protein fragments or peptides to elicit an immune
response.
[0108] C.2. Nucleic Acid Based Compositions
[0109] In general, nucleic acid based compositions may comprise
naked DNA/RNA, recombinant DNA/RNA, or messenger RNA. A composition
based on naked DNA would use the DNA of the viral antigen encoding
the factor H binding sites that has been stripped of histones
(small unfolded chromosomal proteins) and protein, usually by
exposure to detergents or phenols. Recombinant DNA is genetically
engineered DNA made by recombining fragments of DNA from different
organisms, as discussed in detail below. DNA/RNA or mRNA for both
embodiments may be isolated, purified, and amplified using
procedures that are known in the art, and are partially described
herein.
[0110] In general, immunogenic compositions or vaccines based on
DNA/RNA employ the genes of the viral antigens instead of an
isolation of the antigen itself. Nucleic acid based compositions
may employ a genetic sequence for the factor H binding sites of HIV
that will yield proteins that are incapable of the final steps of
maturation, which occur only upon and after integration into the
whole viral particle. This embodiment would not produce a complete
HIV virion, so a fully mature factor H binding site would not be
realized. However, host cellular enzymes would glycosylate and
modify the tertiary structure of the protein in the same manner
that protein modification would occur in an HIV infected
individual. Accordingly, in one embodiment, the factor H binding
sites or immunogenic fragment thereof may be chosen by selecting
from the group of genome segments that encode the factor H binding
sites sequences. Preferably, the strain of HIV would match that of
strain of highest concern, whether of infection or probability of
exposure.
[0111] In addition, and as described below, mRNA based immunogenic
compositions and vaccines may be an alternative concept to using
naked DNA/DNA or rDNA sequence coding for protein. Messenger RNA
(mRNA) is an intermediary between the two (DNA and protein), and
can be used to transfect cells and undergo translation within a
host cell to produce the viral proteins in question.
[0112] C.2.1. Isolation of DNA and RNA
[0113] Procurement of nucleic acid(s) requires three basic steps
(1) lysing of the cells to expose the nucleic acids preferred for
processing; (2) separation of the nucleic acids from other cell
components; and (3) recovering of the nucleic acid in purified
form. (Nicholls Desmond, An Introduction to Genetic Engineering,
Ch. 3 (2nd ed. 2002)) "Purification" means merely that the nucleic
acid is sufficiently free of other cell subunits or contaminants so
as to enable therapeutic use.
[0114] A plethora of modalities may be used to recover nucleic
acids. Many are quite simple requiring only a few steps. More
complex purification procedures involving several different stages
are standard in the industry. Commercially available kits readily
enable purification of nucleic acids from a range of sources.
[0115] The first step in any isolation protocol is disrupting the
starting material. The method used to open cell walls should be as
gentle as possible, preferably utilizing enzymatic degradation of
cell wall material and detergent lysis of cell membranes. If more
vigorous methods of cell disruption are required, there is the
danger of sheering large DNA molecules and this can hamper the
production of representative recombinant molecules during
subsequent processing.
[0116] Following cell disruption, cell proteins are removed. Phenol
or phenol/chloroform mixture is often used in the extraction
procedure(s). Upon centrifugation to separate the phases, protein
molecules partition into the phenol phase and accumulate at the
interface. Due to their inherent hydrophilicity nucleic acids
remain mostly in the upper aqueous space and may be precipitated
from solution using isopropanol or ethanol.
[0117] If a DNA preparation is required, the enzyme ribonuclease
(RNase) can be used to digest the RNA in preparation. If mRNA is
needed for the cDNA synthesis, a further purification can be
performed using oligo(dT)-cellulose to bind to the poly (A) tails
of eukaryotic mRNAs. This gives substantial enrichment for mRNA and
enables most contaminating DNA, rRNA and tRNA to be removed.
[0118] Gradient centrifugation is frequently used to isolate DNA,
particularly plasmid (pDNA). DNA is dissolved into a caesium
chloride (CsCl) solution and spun at high speed in an
ultracentrifuge. Over time (in some cases up to 48 hours) a density
gradient is formed. The pDNA forms an easily identifiable band or
line at one position in the centrifuge tube. This band is devoid of
cellular contaminants and may be removed. Using dialysis, the CsCl
is removed to give a pure preparation of pDNA. Size exclusion
chromatography can be used as an alternative to
ultracentrifugation. Many plasmid DNAs however, are commercially
available. (Nicholls, 2002)
[0119] Amplification of a preferred DNA sequence can be
accomplished by the polymerase chain reaction (PCR). (Nicholl,
2002) Simplicity, elegance and high specificity characterize PCR,
which has replaced traditional cloning methodology. In the PCR
process the DNA duplex is heated denaturing and unwinding the
double helix and separating the strands. Each single strand is
copied by a DNA polymerase. The process is repeated many times
resulting in an exponential increase in the number of copies.
[0120] C.2.2. Recombinant Technologies
[0121] The methods used in producing recombinant DNA are
conceptually straightforward and known in the art. Genes of the HIV
factor H binding sites may be engineered into the DNA of a carrier,
such as Escherichia coli; a list of suggested carriers is in FIG.
7. As shown in FIG. 8 bacterial carriers may include rDNA by
plasmid, chromosome integration, or a combination. As shown in FIG.
9, viral carriers may be used for recombinant technology, by
chromosome integration, insertion of proteins into the viral coat,
or a combination. When the carrier reproduces, the immunogen is
propagated if the immunogen is inserted into the host chromosome.
Plasmid DNA can undergo replication within a non replicating cell.
The cutting or isolation of the genes with restriction enzymes is
as described herein and known.
[0122] Preparation of rDNA
[0123] Electrophoresis enables the separation, identification, and
purification of DNA fragments. The porosity of the matrix
determines the degree of separation achieved. Two gel types are
commonly used in the field, agarose and polyacrylamide. Agarose,
extracted from seaweed, is available commercially as a dry powder,
which is melted in buffer at an appropriate concentration. On
cooling, agarose sets or gels. Polyacrylamide gels are used to
separate small nucleic acid molecules because the pore size of
polyacrylamide is smaller than agarose. Polyacrylamide can separate
DNA molecules that differ in length by only one nucleotide.
Electrophoresis may be carried out by placing nucleic acid samples
in a gel and applying an electrical potential across it. DNA
contains negative charged phosphate groups and will therefore
migrate towards the positive electrode. When a marker dye, usually
bromophenol blue (added to the sample prior to loading), reaches
the end of the gel the electrical potential is removed. The nucleic
acids in the gel may be visualized by staining with the
intercalating dye ethidium bromide and examined under UV light.
(Nicholls, 2002) Large DNA fragments containing as many as 100,000
base pairs can be separated by another process known as pulsed gel
electrophoresis.
[0124] Pulsed field gel electrophoresis (PFGE) and simple gel
electrophoresis permit DNA fragments to be separated on the basis
of size: the smaller the fragment, the more rapid the migration.
Overall rate of migration and optimal range of size for separation
are determined by the chemical nature of the gel and by the degree
of its cross-linking. Highly crossed linked gels optimize the
separation of small DNA fragments. The dye ethidium bromide forms a
brightly fluorescent adduct as it binds to DNA. Small amounts of
separated DNA fragments can be isolated on gels. This dye binds
between the DNA bases (intercalates) and fluoresces orange when
illuminated with ultraviolet light. (Nicholls, 2002) The
identification of a specific DNA fragment can be accomplished by
probes containing complementary sequences.
[0125] All methods of electrophoresis rely on the polyanionic
nature of nucleic acids (RNA & DNA, single stranded and double
stranded) at neutral pH, i.e., nucleic acids carry multiple
negative charges on the phosphate groups. This means that the
molecules will migrate towards the positive electrode when placed
in an electric field. As negative charges are distributed evenly
along the DNA molecule, the charge/mass ratio is constant, thus
mobility depends on fragment length. The technique is preferably
executed on a gel matrix which separates the nucleic acid molecules
according to size. (Nicholls, 2002)
[0126] Restriction enzymes or endonucleases allow bacteria to
distinguish between homologous and heterologous DNA. These enzymes
hydrolyze and cleave DNA at specific sites known as restriction
sites. This specificity of sequence recognition allows the precise
selectivity of DNA fragment preparation, which is the foundation
for DNA vaccines. Bacteria that possess a restriction enzyme system
disguise recognition sites in its own DNA by modifying them. The
addition of a methyl group to an adenine or cytosine residue near
or at the site of cleavage protects its own nucleic acid. (Brooks,
Geo., Medical Microbiology 102 (23rd ed. 2004))
[0127] Restriction modification systems of bacteria fall into two
broad classes: Type 1 systems in which the restriction and
modification activities are combined in a single multi-subunit
protein, and Type 2 systems which consist of separate endonucleases
and methylases. (Brooks, 2004)
[0128] An analogy between restriction endonucleases that have
become standard laboratory devices and a surgeon's knife is
evident. Restriction endonucleases are usually named by a three or
four letter abbreviation of the named organism from which the
enzyme has been isolated. (Brooks, 2004) The generic and specific
names of the organism in which the enzyme is formed are used to
provide the first part of the designation which comprise the first
letter of the generic name and is the first two letters of the
specific name. Thus an enzyme from the strain of Escherichia coli
is termed Eco and one from Bacillus amyloliquefaciens is Bam.
(Nicholls, 2002)
[0129] Restriction endonucleases generally cleave phosphodiester
bonds in both DNA strands in a mirror like fashion. A restriction
enzyme recognizes and cleaves at the same DNA sequence and only
cleaves at that particular sequence. Most of the DNA sequences
recognized by restriction enzymes are palindromes; that is, both
strands of DNA have the same basic sequence running in opposite
directions on either side of the axis of symmetry when read in a 5'
to 3' direction (self-complementary). The cuts made by these
enzymes are usually "sticky" (i.e., the products are
single-stranded at the ends with one strand overhanging the other.)
However, sometimes the products are blunt with double stranded
ends. Over five hundred restriction enzymes with different
specificities have been isolated and characterized. Most are
readily available as laboratory tools.
[0130] Restriction fragments of DNA may be used to identify
variations in base sequence in a gene. However they can also be
used to synthesize a recombinant DNA also called chimeric DNA,
which is composed of molecules of DNA from different sources that
have been recombined in vitro. The sticky ends of two unrelated DNA
fragments may be joined to each other if they have complementary
sticky ends. Complementary ends may be obtained by cleaving
unrelated DNAs strands with the same restriction enzyme if the
restriction enzyme recognizes palindromic strands. After the sticky
ends of the fragments base pair with each other, the fragments can
then be covalently attached by the action of a DNA ligase. (Smith,
Coleen, Basic Medical Biochemistry: A Clinical Approach, Ch. 17 (2d
ed. 1996)) DNA ligase is a cellular enzyme that repairs broken
phosphodiester bonds that may occur at random or as a consequence
of DNA replication or recombination. (Nicholls, 2002) The DNA
ligase most often used is T4 DNA ligase, which may be purified from
E. coli cells infected with bacteriophage T4. Although the enzyme
is most efficient when sealing gaps in fragments that are held
together by cohesive ends, it will also join blunt-ended DNA
molecules together under appropriate conditions. DNA ligase
produces a phosphodiester bond between a 5' phosphate and a 3'
hydroxyl (OH) group. The enzyme is most effective at 37.degree. C.,
but may be used at lower temperatures. Thermodenaturation of the
single strand ends however, occurs at higher temperatures
(37.degree. C.). Therefore this enzymatic process if often
accomplished at lower temperatures to affect a higher purity
although the overall process is somewhat slower. (Nicholls,
2002)
[0131] The length of DNA fragments produced by restriction enzymes
varies tremendously because of the individuality of DNA sequences.
Most restriction enzymes recognize palindromic sequences which
occur somewhat randomly. Furthermore the average length of a DNA
fragment is determined, in large part, by the number of specific
base pairs recognized by the restriction enzyme. Restriction
enzymes recognizing up to 15 base sequences have been
characterized, however most recognize four, six, or eight base
sequences. Recognition of four bases yields fragments with an
average length of 250 base pairs, and therefore is generally useful
for analysis or manipulation of gene fragments. As the number of
base pairs recognized by the restriction enzyme increases the
average length of the nucleotide sequence increases
logarithmically. For instance restriction enzymes that recognize
six bases produce fragments with an average size of about 4,000
base pairs. Restriction enzymes that recognize eight bases produce
fragments with a typical size of 64,000 base pairs and are
therefore useful for analysis of larger genetic regions. (Brooks,
2004)
[0132] In the production of DNA vaccines, plasmid DNA derived from
eukaryotic cells such as bacteria and yeast is often used as the
donor vehicle. A plasmid is a genetic particle physically separate
from the nucleus of the host cell. The nuclei of prokaryotes are
not enveloped. Plasmid can independently function and replicate,
that is independent of the nucleus of the cell. Plasmids usually
confer some survival or growth advantage to the host cell, but are
not essential to the cell's basic function. For example, a
resistance plasmid carries genes responsible for antibiotic or
antibacterial drug resistance. Plasmids are small circles of DNA;
however the three dimensional structure is often that of a figure
eight or more complex structure. Nonetheless, the small size of
plasmids renders them amenable to genetic manipulation in vitro.
Furthermore, after genetic manipulation their small size permits
introduction into other cells. Therefore, plasmids are frequently
used in genetic engineering and are the basis of most DNA vaccines.
(Brooks, 2004)
[0133] Because many restriction enzymes cleave asymmetrically and
produce DNA fragments with cohesive (sticky) ends, hybridization of
DNA is possible. This DNA can be used as a donor with plasmid
recipients to form genetically engineered recombinant plasmids.
Cleavage of a plasmid with the same restriction enzyme produces a
linear fragment with cohesive ends that are identical to each
other. To prevent the two ends of the plasmid from reannealling
enzymatic removal of the free phosphate groups from these ends is
performed. This ensures that the original circular plasmid is
structurally incompetent and cannot function. Ligation in the
presence of other DNA fragments from other sources containing free
phosphate groups produces recombinant plasmids or chimeric plasmids
which contain DNA fragments as inserts in covalently now circular
DNA. Plasmids must be in a circular form in order to replicate in
the bacterial host. (Brooks, 2004)
[0134] The amino acid sequences of the present subunit, the factor
H binding sites, have been deduced. Each amino acid is coded by a
separate codon. A codon is a set of three consecutive nucleotides
in a strand of DNA or RNA that provides the genetic information to
code for a specific amino acid which will be incorporated into a
protein chain or serve as a termination signal. Therefore,
knowledge of the present subunit permits deduction of the
nucleotide sequences of the DNA and/or RNA for the factor H binding
sites. The origin for elongation of a DNA sequence is determined by
a DNA primer that can be synthesized by known nucleotide
synthesizing devices for chemical oligonucleotide synthesis. Such
devices can produce DNA strands containing 75 or more
oligonucleotides. (Brooks, 2004)
[0135] Chemically synthesized oligonucleotides can serve as primers
for polymerase chain reaction (PCR) which is a procedure that
allows amplification and sequencing of DNA between the primers.
Thus, in many instances, DNA need not be cloned in order to be
sequenced or to be made available for engineering.
[0136] DNA sequencing can also be performed using the Maxam-Gilbert
technique, which relies on the relative chemical liability of
different nucleotide bonds and the Sanger (dideoxytermination)
method, which disrupts the elongation of DNA sequences by
incorporating dideoxynucleotides into the sequences. Furthermore a
procedure known as shotgunning allows the sequencing and analysis
of entire genomes in viruses. In this procedure, DNA is randomly
fragmented to create a random fragment library. These unordered
fragments are sequenced by automated DNA sequencers and may be
reassembled in correct order using computer software available in
the field. (Brooks, 2004)
[0137] The essential components of a plasmid DNA designed for
vaccination include a start signal (promoter-enhancer) and stop
signal (polyadenylation signal/transcript termination sequence).
The start and stop signals can be selected from a variety of
sources viral, bacterial or mammalian. A marker of activity of the
plasmid such as antibiotic resistance or specific enzymatic
activity can be included and may be advantageous if only to
demonstrate that a fully functional plasmid has been developed. It
is also advantageous to include intron-containing sequences that
have been shown to greatly improve expression within transfected
cell lines for many constructs even through introns contain
sequences that are ultimately not translated into protein. The
promoters/enhancers that have been mostly used for DNA vaccines are
the CMV immediate early promoter (pCMVIE) enhancer and the Rous
sarcoma virus (RSV) LTR. Hundreds of plasmids are available
commercially from different suppliers. A basic plasmid vaccine
vector is known as V1J. This is comprised of pCMVIE, intron A
derived from CMV, bovine growth hormone (BGH)
polyadenylation/transcript termination sequence and a gene (ampr)
coding for ampicillin resistance. A pUC plasmid DNA sequence from
which the lac operon and multicloning site have been deleted,
serves as the basic construct for this recombinant plasmid
structure. Two separate restriction enzyme sites have been mapped
for insertion of donor DNA. V1J does not replicate in mammalian
cells and does not contain any sequences known to promote plasmid
integration into host genomic DNA ensuring a wide safety margin.
Furthermore it can be produced in large quantities by growth in E.
coli. These properties help ensure the safety of the recombinant
DNA process by minimizing the probability for cell-transforming
integration events.
[0138] Best results for vaccination in animals have been obtained
by using normal saline solutions of plasmid. Other vehicles
including solutions of bupivicaine and sucrose have been used, but
there has been no enhanced immunogenicity with these methodologies
in animals. (Kaufman, Stefen, Concepts in Vaccine Development, ch
3.7.3, (1996)) A small percentage of myotubules take up and express
DNA following intramuscular injection of a plasmid saline
formulation. This however, has been sufficient for obtaining
significant immune responses. (Kaufmann, Stefan, Concepts in
Vaccine Development Ch. 3.7 (1996))
[0139] Both humoral and cytotoxic T cell responses are noted to
occur with naked DNA vaccines. Strong proliferation of T cells was
observed at low DNA doses in animal models down to one microgram
even in the absence of measurable antigen-specific serum antibody
responses, indicating that less antigen may be required to elicit T
cell responses by DNA vaccines than for antibody generation.
Therefore, since the most likely correlate of immunity to HIV
disease would be a robust cytotoxic T cell response directed toward
HIV disease, less (antigen) with HIV vaccine technology means more.
The development of a strong humoral response to HIV disease has
been associated with a poorer prognosis. Low dose DNA vaccines
stimulate the production of Type 1 helper T cells (T.sub.H1).
T.sub.H1 cells generate cytokines II-2 and gamma-interferon which
have been shown to promote cellular immune responses by stimulating
CD8.sup.+ activity. (Kaufmann, 1996)
[0140] For HIV infections, strong T.sub.H1-like responses have been
important in maintaining high CD4 cell counts and low viral titers
as well as prevention of secondary opportunistic infections.
(Kaufmann, 1996)
[0141] The advantages of expressing antigens in the host rather
than administering antigens such as inactivated viruses,
recombinant proteins or peptides, include the following: (1)
circumventing potential loss of antigenicity by an inactivation
process (e.g., chemical cross linking) inherent in the host cell;
(2) synthesis of proteins with conformation and post translational
modifications including carbohydrate and lipid linkages encoded by
the host cell; (3) intracellular antigen processing and
presentation by MHC class I molecules leading to the induction of
cytotoxic T lymphocyte (CTL) responses; and (4) allowing for MHC
determinant selection. (Kiyono, Hiroshi, Mucosal Vaccines Ch. 8
(1996))
[0142] Antigen presentation after IM DNA vaccination results in a
robust cytotoxic T cell response. Three models for inducing the CTL
response with IM DNA vaccines have been proposed:
[0143] 1. Uptake of DNA and expression of antigens by antigen
presenting cells including dendritic cells, macrophages and
langerhans cells;
[0144] 2. Antigen presentation by transfected myocytes acting as or
assuming the role of antigen presenting cells; and
[0145] 3. Transfer of antigens from transfected myocytes to antigen
presenting cells which in turn present the antigen to the
appropriate T cell. (Kiyono,1996)
[0146] DNA vaccines have been used to elicit specific immune
responses, antibody, CD8 cell and CD4 cell, against a variety of
antigens in animal species, including but not limited to the
following:
[0147] 1. Hepatitis B surface antigen in mice (Davis, et. al.,
1993, 1994)
[0148] 2. Herpes simplex virus 1 glycoprotein B in mice (Manickan
et. al., 1995)
[0149] 3. Bovine herpesvirus 1 glycoprotein IV in cattle (Cox et.
al., 1993)
[0150] 4. Rabies virus glycoprotein in mice (Xiang, et. al., 1994,.
1995)
[0151] 5. Malaria circumsporozoite protein in mice (Sedegah, et.
al., 1994; Hoffman et. al., 1994)
[0152] 6. Leishmania gp63 in mice (Xu and Liew 1995)
[0153] 7. Lymphocytic choriomeningitis virus (LCMV) NP in mice
(Pedroz Martins, et al. 1995; Yokoyama et. al., 1995)
[0154] 8. Carcinoembryonic antigen in mice (Conry, et. al.,
1994)
[0155] 9. MHC class I antigen in rats (Geissler, et. al., 1994)
[0156] 10. Cottontail rabbit papillomavirus (CRPV) L1 in rabbits
(Donnelly et. al., 1996)
[0157] 11. M tuberculosis antigen 85 complex proteins in mice
(Huygen et. al., 1996) (Kaufmann, 1996)
[0158] More specifically, the ability of DNA vaccines to induce CTL
responses has also been demonstrated several times. It was first
demonstrated using influenza NP (nucleoprotein). NP is a conserved
internal protein of the virus and a target for cross reactive CTL.
The NP DNA induced a CTL response in mice which demonstrated an
element of longevity implying the potential for vaccination.
Interestingly cell mediated immunity induced by DNA encoding
influenza NP or matrix protein also played a role in protection of
ferrets as measured by reduction of virus shedding in nasal
secretions. DNA vaccine induced CTL response has been demonstrated
for the following as well:
[0159] 1. Rabies virus glycoprotein (Xiang, et al., 1994)
[0160] 2. Malaria circumsporozoite protein (Sedegah, et al.,
1994)
[0161] 3. Lymphocytic choriomeningitis virus NP (Pedroz Martins, et
al., 1995; Yokoyama, et. al., 1995; Zarozinski et al., 1995)
[0162] 4. HIV envelope protein (Wang, et al., 1994; Shiver et al.,
1995)
[0163] 5. Human Factor IX (Katsumi, et al., 1994)
[0164] 6. MHC class I (Geissler, et al., 1994; Plautz, et al.,
1994; Hui et al., 1994)
[0165] Detection of CTL responses for one to two years after
immunization has been noted in some of the above models. Dosing of
the DNA vaccine should start at 1 mcg. CTL assays should be
performed and the lowest dose at which an adequate CTL response is
noted is a preferable dose for administration.
[0166] As discussed below, cationic lipids formulated with IM DNA
vaccine actually resulted in a lower level of gene expression.
However, the use of cationic lipids to facilitate DNA uptake has
been noted with mucosal delivery systems. Cationic lipids
facilitate DNA uptake on mucosal surfaces via a non-specific
mechanism or a specific plasma membrane transport mechanism yet to
be characterized. Mucosal delivery of DNA can potentially transfect
many cell types lining the GI and GU tract as well as the cells
beneath their respective basement membranes including Peyer's
patches which are preferred sites of HIV replication. In addition
to potential facilitation of cellular uptake on mucosal surfaces,
cationic lipids also protect DNA from degradation. In vitro studies
have shown that DNA/cationic lipids have a longer half life than
uncomplexed DNA. (Puyal, et al., 1995) Therefore the preferred
embodiment for mucosal DNA vaccines will include cationic
lipids.
[0167] Parenteral administration of DNA vaccines induces strong
systemic humoral and cell mediated immune responses (dose
dependent), but does not result in the generation of significant
mucosal immune responses. Therefore in certain instances it may be
desirable to design a vaccine that could induce both mucosal and
systemic immune responses. (Kiyono, 1996) This can be achieved by
DNA vaccines delivered by different routes (parenteral and
mucosal). This approach has been tested in several systems using
parenteral priming followed by mucosal boosting (Keren, et al.,
Infect. Immun., 56: 910-915 (1988)) and vice versa (Forrest, et
al., Infect. Immun. 60: 465-471 (1992)). With some vectors mucosal
administration of DNA/cationic lipids resulted in both local and
systemic immune responses. A recombinant BCG vaccine induced local
IgA and serum IgG antibodies against heterologous antigen
(Langerman, et al., 1994) and a recombinant Salmonella vector given
orally induced cell mediated immunity (Aggarwal, et al., 1990).
[0168] A preferred embodiment utilizing DNA vaccine technology
would be a combination of a naked DNA vaccine administered
parenterally (preferably intramuscularly) and a cationic lipid/DNA
vaccine applied mucosally.
[0169] Therefore in summary, to produce a recombinant bacteria DNA
vaccine, the following steps will be followed:
[0170] 1. Selecting a suitable plasmid vector from commercially
available sources
[0171] 2. Isolating the subject HIV DNA
[0172] 3. Effecting restriction enzyme cleavage/modification of
plasmid DNA and HIV DNA
[0173] 4. Isolating the specified gene(s) from HIV
[0174] 5. PCR amplifying selected HIV DNA gene(s)
[0175] 6. Enzymatically removing free phosphate (PO.sub.4) groups
from plasmid DNA
[0176] 7. Transforming the plasmid DNA into a bacterial cell such
as E. coli.
[0177] 8. Administering ligase to seal the DNA strands together
[0178] To accomplish the process of transformation the recipient
cells need to be made competent. Competence relates to the ability
of a cell to assimilate foreign RNA or DNA. The steps to accomplish
this are:
[0179] 1. Soaking the recipient cells in an ice cold solution of
calcium chloride (this induces competency in a way that is still
not fully understood);
[0180] 2. Mixing the plasmid DNA with the cells and incubating them
on ice for 20 to 30 minutes;
[0181] 3. Heat shocking (two minutes at 42.degree. C.) to enable
the DNA to enter the cells;
[0182] 4. Incubating the transformed cells in a nutrient broth at
37.degree. C. for 60 to 90 minutes. This allows the plasmid to
become established and ultimately permit phenotypic expression of
the plasmid nucleic acid; and
[0183] 5. Placing the cells with the plasmid vector onto a selected
media suitable for replication.
[0184] As shown in FIG. 7, rDNA/RNA may be delivered by a bacterial
or viral carrier.
[0185] C.2.3 Recombinant Carriers
[0186] C.2.3.1 Bacterial Carriers
[0187] Live attenuated bacteria may serve as carriers for DNA/RNA.
Bacteria may carry and express genes that are encoded with the
factor H binding sites on the capsid protein or portions thereof.
The bacteria provide an environment in which the capsid protein
DNA/RNA may be amplified, purified and administered. Bacterial
carriers may include those customary in the art, exemplary types
being Salmonella, BCG, E. Coli, Streptococcus gordonii,
Lactococci/Lactobacilli, Vibrio Cholerae, Yersinia enterocolitica,
Shigella flexneri, and Listeria monocytogenes. Salmonella, BCG, and
E. coli are preferable.
[0188] Among the bacteria thus far explored for recombination,
attenuated Salmonella sp. has received the most intense scrutiny.
Other bacteria including Bacillus Calmette-Guerin (BCG) have also
been investigated. Attenuated enteric pathogens including E. coli,
Vibrio, Yersinia and Shigella have been used as platforms for
recombinant vaccine technology. Other organisms generally
considered as commensals including the gram positives Streptococcus
gordonii, Staphylococcus xylosus and the lactococci or lactobacilli
have been used in recombinant methodologies. Recently Listeria
monocytogenes has been introduced as a potential recombinant
vaccine vector. Most of these organisms by virtue of their ability
to colonize and/or infect mucosal surfaces lend themselves to
delivery to these surfaces. Therefore the gut associated lymphoid
tissue (GALT) is being stimulated directly through mucosal
immunization rather than antibody diffusion from the serum
subsequent to parenteral inoculation. GALT including Peyer's
Patches is the primary site of HIV infection and replication in
sexual transmission of the disease.
[0189] The preponderance of attention is focused on enteric
pathogens, particularly Salmonella. The bacteria undergo the
process of attenuation before recombination can occur. In doing so,
the bacteria become avirulent and are unable to cause typhoid fever
or other salmonella derived diseases. The first description of such
mutation appeared in 1951 in the metabolic pathway for
p-aminobenzoic acid (pab). Subsequently gal E mutants of S.
typhimurium and S. typhi (strain Ty21a) were isolated which
resulted in the cytoplasmic accumulation of galactose-1-phosphate
leading to the lysis of cells. Hoiseth and Stocker in S.
typhimurium introduced the widely used salmonella auxotrophic
mutant, aro A, which encodes the enzyme
5-enolpyruvyl-shikimate-3-phosphate synthetase, an essential
element in the aromatic pathway. Additional mutations made in this
pathway involving aro C and aro D genes in S. typhimurium result in
highly attenuated organisms. Mutations in the regulatory genes cya,
crp which encode for adenylate cyclase and the cyclic AMP receptor
protein respectively have also been proven highly successful.
Furthermore the cya and the crp mutations are often used in
conjunction with mutations in asd encoding aspartate
gamma-semialdehyde dehydrogenase which is essential for
peptidoglycan synthesis. In addition, mutations of other regulatory
genes such as phop (phosphatase) and ompR (outer membrane proteins)
have proved successful as attenuators of vaccine vector strains.
(Hughes, Huw, Bacterial Vectors for Vaccine Delivery, Designer
Vaccines Principles for Successful Prophylaxis, Ch. 8 (1998))
[0190] Three separate methods have been used for expression of
heterologous antigens in salmonella have been delineated: (1)
plasmids; (2) integration of the foreign gene into the salmonella
chromosome; and (3) transportation of foreign antigens to the cell
surface by various carrier proteins of the salmonella bacteria
including flagellin, Neisseria, IgA protease precursor, 1 anB,
phoE, ompA. Other carriers of epitopes which target alternative
cellular compartments include fusions with maltose-binding proteins
(malE), LTB, the C fragment of tetanus toxin (tetC),
-galactosidase, pagC and the core antigen (HBcAg) of hepatitis B.
(Hughes, 1998)
[0191] Recombinant salmonella has been used successfully to express
a number of viral antigens with induction of both humoral and cell
mediated responses to the heterologous antigen in animal studies.
Various proteins of influenza have been successfully expressed
using the salmonella bacterial vector in animals, including the
nucleoprotein (NP) and an epitope of the hemagglutinin protein
(HA). Other viral DNA sequences have been successfully integrated
into salmonella includes hepatitis B virus, HIV, and herpes
simplex.
[0192] Most studies have used the oral delivery system for foreign
antigens but others have used parenteral immunization protocols.
Both can be used concomitantly or sequentially with recombinant
vaccines. Other variables that need to be addressed with
recombinant bacterial vaccines with HIV disease include the
targeting of foreign antigens to the specific cell compartments.
Interestingly, BCG and listeria appear to be more advantageous for
eliciting a cellular response and therefore would be the preferable
routes for recombinant vaccine technology with HIV disease.
(Hughes, 1998)
[0193] Using attenuated salmonella bacteria does have an advantage
in that it initially replicates in the large intestines and immune
response occurs in Peyer's patches, which are the immunologic
vehicles lining the terminal colon and are the sites for initial
HIV replication in most cases where the virus is transmitted
sexually. Therefore salmonella bacteria would offer a preferred
methodology for recombinant vaccine technology with HIV
disease.
[0194] The techniques of transformation and transfection represent
the simplest methods available for getting recombinant DNA into
cells. In the context of cloning E. coli cells, transformation
refers to the uptake of plasmid DNA and transfection to the uptake
of bacteriophage DNA. A bacteriophage is a virus that infects
bacteria. Like other viruses they contain either (but never both)
RNA or DNA, and vary in structure from the seemingly simple
filamentous bacterial virus to a relatively complex form with
contractile tails. Their relationships to the host bacteria are
highly specific. Transformation is also used more generally to
describe uptake of any DNA by any cell. (Nicholls, 2003)
[0195] Only a small percentage of competent cells undergo
transformation. Thus, the process can become the rate limiting step
in a cloning experiment where a large number of individual
recombinants is required or when the starting material is limited.
Properly performed, 10.sup.9 transformed cells (transformants) per
microgram of input DNA can be realized, although transformation
frequencies of about 10.sup.6 or 10.sup.7 transformants per
microgram are more realistic. (Nicholls, 2003)
[0196] An alternative to transformation procedures is to introduce
DNA into the cell by a physical method. One exemplary technique is
microinjection, or using a very fine needle and injecting the DNA
directly into the nucleus. This technique has been used
successfully with both plant and animal cells. The cell is held on
a glass tube by mild suction, and the needle is used to pierce the
membrane. The technique requires a mechanical micromanipulator and
a microscope and is done by hand. (Nicholls, 2003) Microinjection
offers a preferred embodiment for DNA bacterial recombinant vaccine
production with HIV disease.
[0197] C.2.3.2 Viral Carriers
[0198] Recombinant viral vaccines may be engineered to express
genes from the pathogen against which the host is to be protected.
The vector serves as a vehicle to carry the foreign gene into the
host, and after transcription and translation of the nucleic acid
present the protein encoded by the nucleic acid to the immune
system of the host. As with any vaccine, of course, the major
criteria for acceptability are safety and efficacy. Safety may be
approached from two perspectives. The safety of the immunogen can
be assured by using viral vectors with good safety records due to
prior attenuation or prior vaccination of the host to the carrier
virus. Secondly, viruses may be engineered to improve safety in a
rational and reliable manner. (Hughes, 1998) The utilization of
viral vectors to which the host has already been immunized does
have a disadvantage in that the immunogen would be rapidly
destroyed by a memory immune response. Nonetheless some
transcription and translation of recombinant DNA or RNA would
occur. A preferred methodology would be use of an attenuated
nonvirulent virus (without prior immunization to the carrier virus)
as a carrier for the recombinant vaccine.
[0199] Thus, viruses like bacteria or yeast may also be used in
recombinant technology. As carriers, viruses easily infect cells
and stimulate cytotoxic T cell immune responses. Because the
carrier virus may be able to replicate, a full and complete immune
response may be generated. Both the humoral and cellular arms of
the immune system would then be activated. General viral carriers
may include Poliovirus, Adenovirus Strains 2, 4, 5, and 7, and
Poxviruses. Some of the poxviruses used in recombinant technology
include vaccinia, canarypox, ALVAC (derived from canarypox),
fowlpox, pigeonpox, swinepox, herpesvirus (HSV-1, VZV (herpes
zoster), EBV (Epstein Barr Virus)), Alphaviruses, Paramyxoviruses,
Influenza, and Hepatitis D. Of these, a preferred embodiment is
based on poliovirus due to extensive existing knowledge of the
virus structure and lifecycle. Prior immunization to Polio would be
a consideration in limiting the immune response. Chronic viral
infections such as HSV-1 offer an attractive alternative since the
host immune system would receive low dose background immunogen
stimulating cytotoxic activity.
[0200] The introduction of genes from one microorganism into the
genome of another microorganism may result in a virulent strain. To
avoid this, the carrier virus should be modified to ensure that any
use of the composition in treatment is, in fact, avirulent. This
would allow for a myriad of viral mosaic combinations to be
developed. The gene(s) introduced may replace genes not required
for replication of the carrier virus when it is used as a vaccine
or it could be added to the viral genome. (Wagner, 1999) Methods
for practicing recombinant technology in the production and use of
immunogenic compositions or vaccines for viral infections are known
and currently available to those in the field. (Porter, 1995)
(Stahl, 1997)
[0201] Among the viruses used for recombinant viral vaccine vectors
are pox viruses (vaccinia virus which includes fowlpox, canarypox,
pigeonpox, myxoma and swinepox), adenovirus (particularly types 2
and 5 which have been sequenced and adenovirus types 4 and 7 have
been widely used as vaccines, not commercially but for the U. S.
military without evidence of adverse effects), herpes virus, polio
virus and influenza virus. HIV genes have been spliced into
vaccinia virus vectors with some limited success in animals. With
adenovirus, genes can be inserted into the non-essential E3 region
(up to four kb) or in the essential E1 region. Interestingly,
construction of recombinant adenovirus expressing the glycoprotein
B of herpes simplex virus (HSV) from the E3 region has been
performed by McDermott et al. Inoculating mice with this
recombinant virus produced antibodies specific for gB which
neutralized HSV in vitro. In addition, mice were protected from a
lethal HSV challenge after a single inoculation with the adenovirus
recombinant. Jacobs, et al., have utilized the E1 region to express
and non structural gene, NS1, from the tick-borne encephalitis
(TBE) virus. They have demonstrated protection against lethal
challenge in a murine model using this replication defective
system. The E1 deleted adenoviruses have an extra safety factor
introduced by their replication defective nature. The E3 gene
confers immunoprotection to the virus. Therefore, recombinant
adenovirus vectors missing the E3 gene are attenuated and avirulent
and represent a preferred embodiment using adenoviral vectors with
recombinant viral technology. The gp19 protein encoded by the E3
region reduces expression of the major histocompatibility complex
(MHC) class I antigens in infected cells. The gp19 protein may act
at the level of transcription, translation, protein modification in
the endoplasmic reticulum or Golgi apparatus or combination
thereof. Adenovirus vectors deficient in this gene may be more
efficient in presenting the proteins encoded in their foreign genes
to the immune system in a more effective manner eliciting a more
robust CD8 cytotoxic response. Furthermore, hepatitis B surface
antigen has been expressed from adenovirus strains 4 and 7, both
with and without deletions of E3, and in animal models a good
antibody response was noted in those vectors lacking the E3
sequences. Vectors containing a functional E3 sequence generated
only weak or negligible responses. (Hughes, 1998)
[0202] Herpes viruses have a large genome and several genes have
been identified as non essential in vitro and more importantly in
vivo. The deletion of non-essential genes would allow recombination
at several sites and allow more than one recombination event per
virion. A limited number of examples of herpes virus vaccine
vectors have been tested in a natural host with some success. For
example, Dan Ziji, et al. has reported the protection of pigs
against pseudo-rabies virus as well as hog cholera virus.
[0203] Influenza has been recently added to the list of potential
viral vaccine vectors in recombinant vaccine technology Influenza
in an uncompromised host is relatively nonvirulent. Manipulation of
the influenza nucleic acid can be accomplished with reverse
genetics. Castrucci, et al. have constructed a recombinant
influenza virus expressing a CTL epitope from the LCMV
nucleoprotein in the stalk of the influenza neuraminidase enzyme
which cleaves sialic acid. A single dose of this recombinant
vaccine protected mice against future challenge by virulent non
attenuated LCMV. Many influenza strains have been characterized,
and many of those vary only in the hemagglutinin and neuraminidase
proteins they express. Therefore, different influenza strains can
be used sequentially to vaccinate a host to a specific viral
protein without the problem of developing immunity to the viral
vector itself which would limit the effectiveness of repeated
inoculations. Cold-adapted attenuated influenza viruses have been
used extensively for years as vaccines. Stocks of these vaccines
could be used for recombinant virus vaccines, particularly if
several inoculations were required.
[0204] Rodriguez, et al. tested the efficacy of recombinant
influenza vectors The CD8.sup.+ T cell epitope of the
circumsporozoite protein of Plasmodium yoelii, a rodent malaria
parasite, was expressed in two distinct influenza proteins,
hemagglutinin and neuraminidase in the same virion. In addition a
vaccinia virus recombinant expressing only one copy of the same
epitope was constructed. Both vectors systems were found to induce
comparative levels of epitope-specific T cells. The most
efficacious protocol consisted of priming with the influenza
recombinant followed by boosting with a vaccinia recombinant.
(Hughes, 1998)
[0205] Two separate recombinant viral vectors may be used
sequentially or concomitantly for optimum immune response with HIV
disease.
[0206] Live vaccines against polio (Sabin) are attenuated strains
of the virus itself. Although these vaccines proved to be extremely
safe and effective (introduced in 1961), occasional reversion to
virulence complicated the methodology. The American Academy of
Pediatrics endorsed the older Salk vaccine (introduced in 1955),
which is not capable of active replication. However, despite its
safety, the Salk vaccine produces a less competent immunologic
response. Due to the tight compartmentalization of the poliovirus
virion, only small DNA sequences coding for a few amino acids can
be cleaved into the viral genome for recombinant technology.
[0207] Polio virus is classified as an enterovirus because of its
fecal/oral transmission route. Polio is a plus stranded RNA virus
as is HIV disease. To differentiate between the two, although both
are positive sense RNA, the retroviruses require RNA to be
converted to DNA by a virion-associated enzyme (reverse
transcriptase). Polio however does not require a reverse
transcription enzyme. The polio RNA functions like a cellular
messenger RNA. Both viruses are encased in icosahedral structures.
Polio is non-enveloped; HIV however is an enveloped virus.
[0208] Polio-specific cellular immune responses have recently been
studied. The generation of a cell mediated response to the polio
virus has been demonstrated in orally vaccinated volunteers.
(Simmons, et al., 1993; Graham et al., 1993) This is important
because as mentioned before, T cell immunity will be the best
correlate with immune protection to HIV disease. (Kiyono, 1996)
[0209] Interestingly, the polio virus can be delivered not only
orally but nasally to stimulate both the systemic and mucosal
antibodies. The development of a recombinant vaccine vector based
on polio virus has been facilitated because of the immense
knowledge available about the virus. The complete viral RNA genome
has been sequenced and the viral proteins identified. (Kitamura,
1981) (Racaniello, 1981) An infectious cDNA of the viral genome has
been generated, making it possible to manipulate the virus
genetically. (Racaniello, 1981) (Semler, 1984) The three
dimensional structure of the complete virus is known and the major
antigenic epitopes have been identified on the molecular level.
(Hogle, 1985) The receptor (PVR) that polio virus utilizes to gain
entry into the cells has been cloned and the nucleic acid sequence
has been determined. (Mendelsohn, 1989; Ren, 1992) Furthermore,
transgenic mice have been bred with expressed polio virus receptor
and are therefore susceptible to polio virus infection. Therefore,
an animal model exists to study recombinant polio virus vectors
with all diseases, especially HIV disease.
[0210] The vast information available on the polio virus makes it
an ideal target for the development of recombinant poliovirus/HIV
vectors. Since poliovirus vaccines can be administered to mucosal
sites and since polio replicates in Peyer's patches after initially
inoculating tonsillar tissue, recombinant polio vaccines are a
preferred embodiment for recombinant viral vaccines for HIV
disease.
[0211] The availability of an infectious polio virus cDNA has
prompted further investigation into the regions of the polio virus
genome that can be deleted without compromising the replication
capacity of the RNA. (Racaniello, 1981) (Semler, 1984) These RNA
molecules or replicons retain the property for self-replication
when introduced into cells. Early studies by Kaplan and Racaniello
describe polio virus replicons with deletions encompassing the
majority of the P1 region. (Kaplan, 1988) Polio virus replicons
containing fragments of up to 1.5 kb of the HIV-1 gag, pol or env
genes have been the subject of laboratory investigations. (Choi,
1991) The foreign genes were inserted so the translational reading
frame was maintained between the remaining capsid sequences
encoding the P2- and the P3- proteins. Transfection of these RNAs
into cells resulted in the replication of these genomes as well as
the expression of foreign proteins as a fusion protein with the
flanking capsid proteins. (Kiyono, 1996)
[0212] The polio virus cDNA has been modified to accommodate larger
genes for expression of recombinant proteins. In these vectors the
complete P1 region of the polio virus was deleted, and a replicon
was constructed which contained the complete gene for HIV-1 gag
(approximately 1.5 kb). Transfection of this replicon into cells
resulted in the production of the HIV-1 Gag precursor protein,
Pr.sub.55.sup.gag which was eluted from the supernatant of the
cells after centrifugation and visualized with electron microscopy.
(Porter, 1996) (Kiyono, 1996)
[0213] In conclusion, it is possible to express a wide variety of
foreign genes including genes encoding glycosylated proteins using
the polio virus replicon system. (Kiyono, 1996)
[0214] C.2.4 mRNA Expression
[0215] The activation of a host cell results in HIV transcription
of viral DNA into messenger RNA (mRNA). In HIV, viral RNA acts as
both a messenger and genomic RNA. The viral DNA is transcribed into
mRNA. The viral mRNA migrates into the cytoplasm where it becomes
associated with cellular ribosomes and cellular transfer RNA to
produce viral protein. Messenger RNA is a stable strand of genetic
material that communicates the genetic information of the virus.
Messenger RNA is attractive for use in an immunogenic composition
for its stability and efficiency. Messenger RNA is more efficient
than DNA in coding for protein.
[0216] RNA or DNA encodes for various proteins. An intermediate
step is the production of mRNA. The mRNA for a protein or group of
proteins is identical to the DNA strand (or RNA strand) encoding
for it, with the exception that thymidine in DNA is substituted for
uracil in RNA. Also in DNA the sugar moiety is deoxyribose in RNA
the sugar moiety is ribose. The mRNA undergoes the process of
capping where at the 5' end a 7-methylguanosine triphosphate is
added and at the 3' end a poly(A)tail of about 100 bases is added
to the untranslated segment of the 3' end. The cap is necessary for
the proper binding of the ribosome and the tail signals an end to
the ribosomal translation. Transcription is the process where DNA
"transcribes" into mRNA. Translation is the process where mRNA is
"translated" into proteins.
[0217] There are many theoretical advantages to mRNA within an
immunogenic composition. These include but are not limited to: (1)
mRNA does not need to cross through the nuclear membrane; (2) mRNA
does not need to enter nucleoplasm; (3) mRNA does not need to
integrate into host DNA; (4) mRNA does not need to undergo the
process of transcription; (5) the host translational enzymes and
ribosomes are available to the mRNA within the cell cytoplasm to
allow for translation of the mRNA into protein; (6) a quicker
immune response should be noted with mRNA in comparison to
intracellular DNA because many steps in the production of viral
protein are circumvented; (7) mRNA can be re-used several times so
that many protein sequences can be translated from one mRNA
template; therefore only minute quantities of mRNA need enter into
the cell cytoplasm; and (8) because the intracellular production of
proteins will be accomplished with mRNA, these proteins will be
associated with MHC class I proteins on the cell surface and will
elicit a CD8.sup.+ cytotoxic T cell response.
[0218] The production of mRNA is straightforward. With the
knowledge of a specific amino acid sequence of a specified HIV
protein the RNA sequence complementary to this can be deduced. Then
the RNA sequence can be capped and tailed at the 5' and 3' ends
respectively. Furthermore mRNA can be produced by automated nucleic
acid sequencing synthesis, as is known in the art.
[0219] C.2.5 Enhancing CD8+ T Cell Response for Naked DNA/RNA based
Compositions
[0220] DNA-based compositions may offer a number of potential
advantages over conventional vaccines. Single dosing, long-lasting
immunity, cell-mediated immunity as well as humoral responses can
be realized with intracellular production of viral particles
introduced by recombinant DNA technology. In contrast subunit
vaccines based on proteins internalized by endocytosis generally do
not sensitize cells for CD8.sup.+ T cell recognition.
[0221] One evasion strategy of HIV and other viral pathogens is to
penetrate and replicate in non immunologic cells For example,
epithelial cells are invaded by Chlamydia sp. and Rickettsia sp.,
while hepatocytes are targets for Plasmodium sp. and L.
monocytogenes. As described above, although HIV targets primarily
CD4 cells, other non immunologic tissues are invaded, such as the
central nervous system. In stimulating an enhanced CD8 cytotoxic
response, a broader scope of target cells may be recognized by the
immune system. As described above, CD8.sup.+ T cells recognize
antigens in the context of MHC class I molecules that are present
on all nucleated cells and enables the CD8.sup.+ T cells to detect
infected host cells of any type. In contrast, CD4.sup.+ T cells are
restricted to MHC class 2 expressing host cells and are thus much
more limited in scope. Macrophages, dendritic cells and B cells
bear MHC class I as well as MHC class II molecules. Furthermore,
Langerhans cells of the skin possess both class I and class 11 MHC
proteins. (Kaufmann, 1996) Accordingly, constituents enhancing CD8+
T cell response are contemplated for the present invention. As
shown in FIG. 10, a variety of constituents may be combined to
naked DNA/RNA embodiments to enhance CD8+ T cell response, some of
which are described here.
[0222] For example, it has been demonstrated that specific
hypomethylated CpG motifs within bacterially derived DNA can
exhibit a potent adjuvant effect that is, in part, responsible for
induction of Th1-type response that is a characteristic feature of
DNA based vaccines. A significant feature of DNA based vaccines,
unlike most conventional vaccines, is the unique ability to
stimulate humoral and cell mediated responses in immunized animals.
The ability to induce a potent Th1-type immune response is of
considerable importance because with many pathogens (viral,
bacterial, and parasitic), cell-mediated immunity and not the
presence of antibodies is correlated with protection. (Lewis,
1999)
[0223] An additional method of enhancing cytotoxic T cell activity
is to link the mycobacterium tuberculosis heat shock protein 70
(HSP70) to actual naked DNA/RNA that encodes the subunit. HSP70 is
a cytosolic HSP that functions in protein folding, transfer, and
degradation. (Chen, 2000) HSP reactive T cells can exert a strong
helper effect by reacting to conjugated peptides; HSP can induce a
T-helper pro-inflammatory response and induce the secretion of
TNF-.alpha. and IFN. (Chen, 2000) Related to HSPs is calreticulin
(CRT), which is a Ca.sup.2+ binding protein, located in the
endoplasmic reticulum. It associates with peptides delivered to the
endoplasmic reticulum by transporters associated with antigen
processing and presentation. (Wen-fang Cheng, 2002) CRT enhances
CD8 activity.
[0224] Proteasomal degradation of antigen can enhance MHC class I
presentation. (Chien-fu-hung, 2003) Thus, an additional method of
enhancing cytotoxic T cell activity is to link gamma-tubulin to the
DNA/RNA sequence. A centrosome is a sub-cellular compartment rich
in proteasomes. Centrosomes are important in mitosis and the
production of tubules. Centrosomes are also an important locus for
MHC Class I antigen processing. Linking gamma-tubulin to DNA/RNA
will result in cellular localization of the protein to the
centrosomes, enhancing CD8+ T cell immune response. (Chan, 2000)
Similarly, the present composition may use a DNA/RNA sequence
encoding for the lysosome associated membrane protein (LAMP-1)
linked to a DNA/RNA sequence for the capsid protein to enhance
B-Cell response. (Chen, 2000) (Chien-fu-hung, 2003)
[0225] C.2.6. Enhancing CD8+ T Cell Response for Subunit Based
Compositions
[0226] As noted above, subunit protein vaccines may not sensitize
cells for CD8.sup.+ T cell recognition. However priming of CTL
responses with intact proteins has been achieved by incorporation
of the antigen into immunostimulating complexes such as ISCOMs (a
matrix of lipid micelles containing viral proteins that deliver
antigens to the cytosol and allows induction of cytotoxic T cells)
or liposomes. Furthermore cationic lipids have been used to enhance
class I MHC pathways of antigen presenting cells in animals. One
cationic lipid used is DOTAP (N-[1-(2,3-dioleoyloxy)
propyl]-N,N,N-trimethylammonium methyl sulfate) which is a
commercially available cationic lipid used for DNA transfection.
Other cationic lipids which can sensitize target cells are
available commercially. These lipids are similar in structure to
DOTAP with two long hydrophobic alkyl chains coupled to one or more
positively charged ammonium groups. The proposed mechanism of
action for the cationic lipids involves an interaction between the
macromolecule-lipid complex carrying an overall positive charge and
the negatively charged cell surface followed by fusion with the
cell membrane. In contrast, pH sensitive liposomes are thought to
destabilize upon contact with the acidic environment of the
endosome and rupture and/or fuse with the endosomal membrane to
release their contents into the cytoplasm. (Walker, 1992)
[0227] ISCOMs contain Saponin which is a complex glycoside found in
plants. Saponin possesses an adjuvant quality. Saponin has a
hydrophilic oligosaccharide sequence of about 8 to 10
monosaccharides. The preparation of ISCOMs is know to those
familiar with the art. Since ISCOMs also possess a steroid or
triterpene their basic structure is amphiphatic. This allows ISCOMs
to form a lipid matrix associated with hydrophobic proteins. The
lipid quality of ISCOMs allows membrane fusion with a target cell.
The proteins suspended in lipid matrix of the ISCOMs become
internalized in the target cell and are subjected to immunologic
clearance. (Kiyono, 1996)
[0228] Formation of complexes between the soluble protein of a
subunit vaccine and DOTAP occurs by ionic interactions between the
negative charge of the protein and the cationic lipid. Thus the
maturation or modification of a subunit vaccine is not required.
Association therefore requires only mixing of the subunit protein
in the DOTAP solution or other cationic lipid prior to application
to cells or injection into experimental animals or humans. Thus
cationic lipids are readily available delivery vehicles for study
of intracellular events that lead to class I MHC presentation of
antigen and they can serve as an alternative to recombinant viruses
for enhancing CD8.sup.+ T cell response to viruses. (Walker,
1992)
[0229] The ISCOMs or lipid carriers act as adjuvants but with
minimal toxicity. They load proteins and peptides into the cell
cytoplasm allowing class I restricted T cell responses to peptides.
Therefore they can be used with subunit vaccines to enhance CD8
activity. To gain access to the cytoplasm of the cell, the lipid
micelles of the ISCOMs fuse with the cell membranes as noted above,
and the particles trapped within the ISCOMs can be transported to
the endoplasmic reticulum. Once inside the endoplasmic reticulum,
these particles are bound to newly synthesized MHC class I
molecules. For final protein modification the particles pass
through the Golgi apparatus. They are then transported to the cell
surface as peptide MHC class I complexes. (Parham, Peter, The
Immune System, Ch. 12 (2004))
[0230] Therefore, the present composition should preferably be
incorporated into ISCOMs, liposomes, and/or dissolved in cationic
lipids to enhance T cell activity or to prime the CTL responses
[0231] C.3. Conclusion--Method of Preparation
[0232] Thus, the present invention comprises both a protein based
composition and a nucleic acid based composition that could be used
to induce an immune response against the factor H binding sites on
the capsid proteins, and to create immune memory thereto. Nucleic
acid based compositions may be DNA, RNA, or mRNA. Recombinant
nucleic acid carriers may be bacterial or viral. Preferably, the
composition includes one or more constituents for enhancing CD8+ T
cell response.
[0233] Protein based compositions may be developed and administered
using methods that are known in the art. For the purposes of
compositions or vaccines that are based on nucleic acids and are
administered to animals, then commercially available gene guns are
a preferred method for delivery. This technique utilizes an
instrument designed to propel DNA-coated gold particles directly
into cells within the epidermis and dermis. DNA enters directly
into dendritic cells, which leads to direct priming of CD8+ T
cells. (Chen, 2000) In particular, gene gun delivery by DNA coated
gold beads may thus be preferable for use with composition
constituents enhancing CD8+ T cell immune response for nucleic acid
based subunit compositions. (Chien-Fu Hung, 2003) Routes of
administration for nucleic acid based compositions are summarized
in FIG. 11 and below.
[0234] D. Description of Additional Alternative Embodiments and
Immune Stimulants
[0235] The immune response contemplated by the present invention
may be enhanced by the use of non-specific or specific substances
stimulating immune response. The present invention may be mixed
with appropriate immune stimulant or adjuvant, including those
described as alternative embodiments below. Such compositions may
be used as appropriate for the application. Customary stimulants or
adjuvant known in the art include incomplete Freund's adjuvant,
liposomes, etc. A preferred embodiment includes one or more
stimulant taken from customary adjuvants and/or those compositions
described further herein. In addition, DNA enhances complement
activity and therefore, may be used concurrently as a DNA vaccine
and an adjuvant. (The DPT vaccine is composed of three separate
vaccine particles. The pertussis component acts as an adjuvant for
the other two. (Parham, 2004) An analogous situation exists here,
where a DNA vaccine (preferably encoding the sequences for the
factor H binding sites) for HIV disease would act as an adjuvant
for a factor H subunit vaccine.)
[0236] To enhance immunogenicity of a recombinant bacterial or
viral vector sialic acid needs to be removed from the plasma
membrane of the bacteria or the protein coat and or envelope (if
virus is enveloped) structure of the virus. Treatment with
neuraminidase would effectively remove sialic acid residues without
altering the protein structure of the bacteria or virus.
[0237] In an alternative embodiment, the composition may be bound
covalently or otherwise to polysaccharides composed of mannose or
mannan. Binding or coupling may be accomplished using methods known
to those in the field. Mannose is a sugar found only on
microorganisms and pathogens not ordinarily found within the human
body. Mannose binding protein (MBP) is a collectin, a C-type lectin
that contains regions of collagenous structure. It is present in
normal human serum and consists of subunits each composed of three
polypeptide chains, forming a collagen-like triple helix and three
C-terminal globular carbohydrate recognition domains (CRDs). Six
subunits together form an overall structure resembling the bouquet
of tulip-like structure of C1q of the classical complement pathway.
Binding of MBP to carbohydrate initiates the classical complement
pathway to the activation of C1r.sub.2 C1s.sub.2. This may result
in complement killing either directly through insertion of the
terminal membrane attack complex or through opsonization by
deposition of complement on the microbial surface. MBP may also
activate C2 and C4 via another newly described serine protease
called MASP (1 and 2) serine proteases. Thus, MBP also exhibits
complement independent opsonizing activity, probably mediated by
binding of the collagenous stalks to the collectin receptor of
phagocytic cells. (Presanis J. S., et al., Biochemistry and
Genetics of Mannan-binding Lectin (MBL), Biochemical Society
Transactions, Vol. 31, pp 748-752 (2003) Any organism with mannose
or mannan on its surface will stimulate the lectin pathway of
complement activation. A composition bound to such polysaccharides
will bind with mannose binding lectin in the serum, activating the
lectin pathway of the complement system. Thus, this alternative
embodiment would thereby enhance the overall immunologic response
to the vaccine.
[0238] In another alternate embodiment, the composition may be
combined with substances that stimulate or activate the alternative
complement pathway. For example, it is known that certain forms of
teichoic acid are potent activators of the alternative complement
pathway. (Winkelstein J. A., J. of Immun., Vol. 120, pp 174-178
(1978)) In addition, zymosan, which may be derived from yeast
cells, can induce cytokines and stimulate immune response in
conjunction with the alternative pathway of the complement system.
Zymosan is phagocytosed by macrophages with or without
opsonization, and therefore has a useful immunologic property of
activating the alternative pathway of complementation. The zymosan
macrophage interaction is believed to enhance the Th-1 response.
CD4 cells can be divided into Th-1 and Th-2 cells. Th-1 cells
activate cytotoxic T cells by producing IL-2; whereas Th-2 cells
activate B-cells by producing primarily IL-4 and IL-5. The level of
Th-1 response produced by zymbsan is regulated by C3 cleavage
fragments, C3b and iC3b. The amplified C3b deposits on the accepted
surface of zymosan and assembles macrophages, dendritic cells or
other antigen-presenting cells. Macrophages, dendritic cells, and
antigen-presenting cells make an antigen presentation to Th-1 cells
after opsonizing zymosan, and after antigen-specific macrophage
activation occurs. (Fearon D. T., et al., Proc. Natl. Acad. Sci,
Vol. 74, pp 1683-1687 (1977)) Zymosan can therefore be used as an
adjuvant; it enhances both humoral and cell-mediated immune
responses to HIV disease. Thus, the composition may be bound
covalently or otherwise to substances that stimulate the
alternative complement pathway, such as teichoic acid or
zymosan.
[0239] The adjuvant effect of zymosan on HIV specific DNA vaccine
was demonstrated recently using a plasma vector (pCMV160 IIIb). In
laboratory mice the plasmid vaccine was inoculated with and without
the zymosan. Higher levels of both humoral immune response and HIV
specific delayed type hypersensitivity (DTH) response were observed
when zymosan was co-inoculated with the plasmid vector as to that
using the plasmid vector alone. HIV specific cytotoxic T cell
lymphocyte activity was also enhanced. The effects are suggested to
be based on the consequences of its (zymosan) recruitment and
activation of macrophages, dendritic cells, or antigen-presenting
cells through complement activation and especially through the
alternative pathway. These results suggest zymosan as an effective
immunologic stimulant. (Ara, 2001)
[0240] Therefore, to enhance the immunogenicity of the composition,
mannose, teichoic acid, zymosan, or some combination thereof may be
bonded to the protein component of the subunit vaccine. Preferably,
the polysaccharides will consist of sixteen separate saccharide
units. (Pangburn, Michael K., Immun., Vol. 142, pp 2766-2770
(1989)) The preferred source for the carbohydrate/adjuvant
component of the subunit vaccine would be the capsular
polysaccharide of the yeast cell, Cryptococcus neoformans serotype
C. (Sahu Arvind, et al., Biochem. J., Vol 302, pp 429-436 (1994))
This yeast cell exhibits four branching xylose sugars from each
trimannose repeat unit. The thioester site of the C3 complement
component demonstrates a strong preference for this specific
carbohydrate sequence. This results in the cleavage of C3 into the
C3a fragment and C3b. The C3b molecule is a focal point in all
three complement pathways.
[0241] FIG. 12 demonstrates the loss of activity of Factors H and
Factor 1 on C3b with increasing polysaccharide length to a maximum
of 16.
[0242] Additionally, all glucose molecules and polysaccharides are
to be removed from the composition. The addition of insulin to a
cell culture will facilitate the transport of extracellular glucose
across the plasma membrane and into the cytoplasm of the cells.
Free soluble glucose molecules inhibit both the rate and the extent
of C3b deposition. (Sahu Arvind, 1994)
[0243] In an alternate embodiment, the effect of heparin may be
inhibited. Heparin is a cofactor necessary for effective Factor H
function. (Maillet, Francoise, et al., Mol. Immun., Vol. 25, pp
917-923 (1988)) (Maillet, Francoise, et al., Molecular Immun., Vol.
20, pp 1401-1404 (1983)) As noted above, Factor H is a major
limiting protein in the alternative complement pathway. The
alternative complement pathway is the first arm of the immune
system to respond to microorganisms or vaccines. Protamine binds
heparin and is used to reduce the effective heparin in patients
undergoing anticoagulation. (Furie, Bruce, Oral Anticoagulant
Therapy, Hematology Basic Principles & Practice, Ch. 121 (3rd
ed. 2000)) Recently, a less toxic heparin antagonist, low molecular
weight protamine (LMWP) has become available. Protamine, or
preferably LMWP for this embodiment, may be included as a component
of the composition in order to impair the activity of Factor H in
limiting the alternative complement pathway. (Liang J. F, et al.,
Biochemistry, Vol. 68, pp 116-120 (2002)) Alternatively, Heparinase
is known to degrade Heparin enzymatically.
[0244] Branched partially hydrolyzed polysaccharides of glucose
known as dextrans have been used for effective plasma expanders.
(Hoffman, Ronald, Hematology Basic Principles and Practice, 2177
(3rd ed. 2000)) Dextran sulfate is a sodium salt of sulfuric acid
esters of the polysaccharide dextran. Soluble dextran sulfate with
a molecular weight greater than 5.times.10.sup.3 is an inducer of
the alternative pathway of complement. The number of sulfate groups
per hundred glucose residues in the dextran determined the
activation potency of the dextran in the alternative pathway. The
optimal degree of sulphation was 50-60 SO.sub.4/100 glucose
molecules. (Burger, R., et al., Immunology, Vol. 29. pp 549-554
(1975))
[0245] Sulphated sephadex (SS) is a cross-linked insoluble form of
dextran. Like soluble dextran sulphate SS activate the alternative
pathway of complement and the classical pathway as well. Three
variables control the activity of SS with both pathways of
complement activity:
[0246] (1) Amount of sulphation; the higher the sulphated content
up to 15.6% by weight resulted in higher complement activation. No
complement activation was noted with sulphate content less than
2.43%;
[0247] (2) Concentration of SS; higher concentrations result in
complement activation with a maximum C3 turnover at 40-50 .mu.g/ml;
and
[0248] (3) Temperature; maximum C3 turnover was noted at 37.degree.
C. with a total loss of activity at 4.degree. C.
[0249] (Burger, R., et al., Immunology 33:827 (1977)) Both soluble
and insoluble forms of dextran (>5000 molecular weight) activate
the alternative pathway of complement. This is accomplished by
blocking the effect of factor H. (Burger, R., et al., European J.
Immunology, pp. 291-295 (1981)) Low molecular weight dextran
sulfate (<5000) enhances factor H binding therefore it limits
the activity of the alternative pathway of complement. (Seppo Meri,
et. al., Proc. Natl. Acad. Sci, Vol 87, pp 3982-3986 (1990) DNA
like heparin also increases factor H binding. (Gardner, William D.,
Biochemical and Biophysical Research Communications, Vol. 94, pp
61-67 (1980))
[0250] Therefore, to enhance immunogenicity dextran sulfate with a
molecular weight >5000 with 50-60 SO.sub.4/100 glucose molecules
may be included in the compound. Likewise SS with 15.6% SO.sub.4 by
weight at a concentration of 40-50 .mu.g/ml at a temperature of
37.degree. would enhance the immunogenicity of the compound. Low
molecular weight dextran would not be included in the formulation
since it would increase factor H binding and decrease complement
activation.
[0251] In a further alternate embodiment, substances that stabilize
C3 convertase may be used with the present invention. All three
complement pathways lead to the production of C3b, which bonds
covalently to the surface of microorganisms or components of the
microorganisms presented in such an immunogenic composition. C3b is
produced by enzymes known as C3 convertase. Cobra venom factor
(CVF), derived from the snake Naja kaouthia stabilizes this enzyme.
(Alper, C. A., et al., Science, Vol. 191, pp 1275-1276 (1976) The
half life of CVFC3b,Bb C3/C5 convertase is seven hours, in contrast
to that of endogenously produced alternative complement pathway C3
convertase (C3b,Bb), which is 1.5 minutes. C3b,Bb is disassembled
by Factor H and C3b is inactivated by the combined action of Factor
H and Factor I. In contrast Factor CVFC3,Bb is resistant to all
regulatory complement proteins. (Kock, Michael A., et al., J. of
Biol. Chemistry, Vol. 279 pp 30836-30843 (2004)) C3b,Bb requires
additional C3b to act on C5 whereas CVF,Bb can cleave C5 directly.
Therefore, the CVF,Bb enzyme continuously activates C3 and C5.
(Kock, 2004)
[0252] The biological function of CVF in cobra venom is believed to
facilitate the entry of the toxic venom components into the
bloodstream. This is achieved by complement activation causing
release of the anaphylatoxins C3a, C5a and Bb, which increase
vascular permeability. (Vogel, Carl W., Immunoconjugates, Ch. 9
(1987)) CVF, despite its derivation from cobra venom, is a
non-toxic protein; CVF can be isolated from the other enzymes,
polypeptides, etc., from cobra venom, which includes toxins.
[0253] Thus, administration of CVF results in an explosive
production of C3b. (Vogel, 1987) (Kock, 2004) FIG. 13 illustrates
the structural homology between C3 and CVF. C3b on the surface of
microorganisms is recognized by follicular dendritic cells within
the lymph nodes as well as T cells and B cells in the peripheral
circulation and within the germinal centers of the lymph nodes. C3b
is a powerful opsonin. Opsonins trigger several arms of the immune
system simultaneously. (Hoffman, Ronald, Hematology Basic
Principles and Practice, Ch. 27 (3rd ed. 2000)) Thus, in an
alternative embodiment, CVF may be used as a component of the
composition.
[0254] The preferred form of CVF is dCVF (De-.alpha.-galactosylated
CVF). (Gowda, D. C., et al., "Immunoreactivity and function of
Oligosaccharides in Cobra Venom Factor," J. of Immun., pp.
2977-2986, (Dec. 21, 1993)) Naturally occurring CVF is
characterized by an unusual polysaccharide which is a fucosylated
biantennary complex-type N-linked chain containing an
a-galactosylated Lex antigenic epitope, Gal.alpha.1-3Gal.beta.1-4
(Fucal-3) GlcNAc.beta.1. Removal of this polysaccharide can be
accomplished by incubating CVF with peptide-N-glycosidase F
(N-glycanase) at 37.degree. C. for 18 to 23 hours at a ph of 8.0.
Removal of this novel polysaccharide from CVF is necessary since 1%
of human IgG reacts with the terminal Gal.alpha.1-3Gal.beta.1
sequence of CVF. However removal of this polysaccharide does not
interfere with the complement fixation character of the molecule
nor does it result in a shorter half life of the molecule. dCVF
will be covalently bound to the polysaccharide unit(s) comprising
the immunogenic composition.
[0255] In another embodiment, nickel compounds may be added to the
composition. It has been shown that nickel is effective in
enhancing the C3 convertase activity of both the lectin and the
alternative complement pathways. (Fishelson, Z., et al., J. of
Immun., Vol. 129, pp 2603-2607 (1982)) Average nickel intake for
average adults is estimated to be 60 to 260 micrograms per day,
with an environmental health reference dose of 0.02 milligram per
kilogram body weight per day (mg/kg/d). (U.S. EPA, 1986) It is
contemplated that the present invention would include Nickel
preferable in the form of nickel chloride on the order of average
daily intake well below the reference dose. Therefore, the present
invention may be produced using nickel to enhance immune
response.
[0256] E. Summary
[0257] To prepare the composition that constitutes the vaccine
agent for the invention, it is possible to use known methods of
purification, synthesis, or genetic engineering. Practitioners
skilled in the art may isolate and purify a fragment, or prepare a
sequence encoding of such capsid protein binding site for factor H.
Protein fragments, naked DNA/RNA, recombinant DNA/RNA, or messenger
RNA may be incorporated into pharmaceutical compositions
appropriate for the anticipated method of administration, such as
carriers or excipients. An animal or subject for which an immune
response according to the present invention is desired may be
administered the composition; a therapeutically effective dose
would be that amount necessary to reverse specific immune
suppression, to the extent desired, and determined using standard
means, such as Chromium Release Assay, Intracellular Cytokine
Assay, Lympho-proliferative Assay (LPA), Interferon Gamma
(IFN-gamma) ELISpot Assays, and MHC Tetramer Binding Assays. The
MHC Tetramer Binding Assay is preferable. These same laboratory
tests would be applied to measure the immune response of an
uninfected subject.
[0258] The analysis and development of the immunogenic composition
should incorporate a wide range of doses of inactivated particulate
for evaluation. Animal trials should consider differences in size,
species, and immunological characteristics; it is anticipated that
immunological differences between humans and animals may relegate
animal trials to toxicity analysis. Clinical trials will involve at
least the standard three phase model, ranging from safety and
dosage in a small population, safety and immunogenicity in a second
phase of several hundred volunteers, to a large scale effectiveness
phase. The clinical trials should include appropriate exclusionary
criteria as is customary, such as exclusion for other immune
suppression conditions, pregnancy, active drug use, etc. A starting
dose for trials with subunit proteins may be 10 micrograms/strain
for juveniles and 20 micrograms/strain for adults. For naked DNA
vaccines a starting dose of 1 microgram/strain for all ages would
be appropriate.
[0259] Administration may be made in a variety of routes, for
example orally, transbucally, transmucosally, sublingually,
nasally, rectally, vaginally, intraocularly, intramuscularly,
intralymphatically, intravenously, subcutaneously, transdermally,
intradermally, intra tumor, topically, transpulmonarily, by
inhalation, by injection, or by implantation, etc. Various forms of
the composition may include, without limitation, capsule, gel cap,
tablet, enteric capsule, encapsulated particle, powder,
suppository, injection, ointment, cream, implant, patch, liquid,
inhalant, or spray, systemic, topical, or other oral media,
solutions, suspensions, infusion, etc. Because some of the first
targets for infection with HIV are epithelial cells and Langerhans
cells in the skin and rectal and vaginal mucosa, then a preferable
embodiment of delivery is dermal combined with rectal and/or
vaginal suppositories. HIV is contracted predominantly by rectal
and vaginal intercourse. Therefore rectal and/or vaginal
suppository administration of the vaccine would be the preferred
administration methodology. In addition, the present invention may
be combined with other therapeutic agents, such as cytokines,
including natural, recombinant and mutated forms, fragments, fusion
proteins, and other analogues and derivatives of the cytokines,
mixtures, other biologically active agents and formulation
additives, etc. Those skilled in the art will recognize that for
injection, formulation in aqueous solutions, such as Ringer's
solution or a saline buffer may be appropriate. Liposomes,
emulsions, and solvents are other examples of delivery vehicles.
Oral administration would require carriers suitable for capsules,
tablets, liquids, pills, etc, such as sucrose, cellulose, etc.
[0260] While the description above refers to particular embodiments
of the present invention, it will be understood that many
modifications may be made without departing from the spirit
thereof. The accompanying claims are intended to cover such
modifications as would fall within the true scope and spirit of the
present invention.
* * * * *