U.S. patent application number 12/118409 was filed with the patent office on 2008-09-11 for method of treating and preventing infectious diseases via creation of a modified viral particle with immunogenic properties.
This patent application is currently assigned to Lipid Sciences, Inc.. Invention is credited to Bill E. Cham, Jo-Ann B. Maltais.
Application Number | 20080220017 12/118409 |
Document ID | / |
Family ID | 56290443 |
Filed Date | 2008-09-11 |
United States Patent
Application |
20080220017 |
Kind Code |
A1 |
Cham; Bill E. ; et
al. |
September 11, 2008 |
Method of Treating and Preventing Infectious Diseases via Creation
of a Modified Viral Particle with Immunogenic Properties
Abstract
The present invention relates to a method for reducing the
occurrence and severity of infectious diseases, especially
infectious diseases in which lipid-containing infectious organisms
are found in biological fluids, such as blood. The present
invention employs solvents useful for extracting lipids from the
lipid-containing infectious organism, thereby reducing the
infectivity of the infectious organism. The present invention uses
optimal solvent systems such that the lipid envelope around the
viral particle is dissolved while the viral particle remains
intact, resulting in a modified viral particle. The present
invention also provides an autologous vaccine composition,
comprising a lipid-containing infectious organism, treated with
solvents to reduce the lipid content of the infectious organism,
combined with a pharmaceutically acceptable carrier. The vaccine
composition is administered to an animal or a human to provide
protection against the lipid-containing infectious organism. The
present invention further provides a simple, inexpensive and easy
to use kit for delipidating fluids and for delipidation of
lipid-containing organisms in a fluid.
Inventors: |
Cham; Bill E.; (Port Vila,
VU) ; Maltais; Jo-Ann B.; (San Ramon, CA) |
Correspondence
Address: |
JOHN S. PRATT, ESQ;KILPATRICK STOCKTON, LLP
1100 PEACHTREE STREET
ATLANTA
GA
30309
US
|
Assignee: |
Lipid Sciences, Inc.
Pleasanton
CA
|
Family ID: |
56290443 |
Appl. No.: |
12/118409 |
Filed: |
May 9, 2008 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
10601656 |
Jun 20, 2003 |
|
|
|
12118409 |
|
|
|
|
10311679 |
Dec 18, 2002 |
|
|
|
PCT/IB01/01099 |
Jun 21, 2001 |
|
|
|
10601656 |
|
|
|
|
PCT/AU00/01603 |
Dec 28, 2000 |
|
|
|
10311679 |
|
|
|
|
60390066 |
Jun 20, 2002 |
|
|
|
Current U.S.
Class: |
424/204.1 |
Current CPC
Class: |
A61K 2039/55566
20130101; A61P 31/00 20180101; A61K 39/292 20130101; A61P 31/14
20180101; A61P 31/18 20180101; A61K 2039/5252 20130101; A61K 39/12
20130101; A61L 2/0011 20130101; A61P 43/00 20180101; A61P 1/16
20180101; C12N 7/00 20130101; C12N 2770/24363 20130101; A61L 2/0088
20130101; A61P 33/02 20180101; A61P 31/10 20180101; C12N 2770/24334
20130101; A61P 7/08 20180101; A61M 1/3486 20140204; A61P 31/12
20180101; A61K 2039/57 20130101; A61K 39/29 20130101; C12N
2730/10134 20130101; C12N 2730/10163 20130101; A61P 31/04
20180101 |
Class at
Publication: |
424/204.1 |
International
Class: |
A61K 39/12 20060101
A61K039/12; A61P 31/12 20060101 A61P031/12 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 29, 2000 |
AU |
PQ 8469 |
Claims
1-10. (canceled)
11. A method for provoking a positive immune response in an animal
or human having a plurality of lipid-containing viral particles
comprising the steps of: obtaining a fluid containing the
lipid-containing viral particles from the animal or the human;
contacting the fluid containing the lipid-containing viral
particles with a first organic solvent capable of extracting lipid
from the lipid-containing viral particles; mixing the fluid and the
first organic solvent; permitting organic and aqueous phases to
separate; collecting the aqueous phase containing modified viral
particles with reduced lipid content; and introducing the aqueous
phase containing the modified viral particles with reduced lipid
content into the animal or the human wherein the modified viral
particles with reduced lipid content provoke a positive immune
response in the animal or the human.
12. The method of claim 11, further comprising: contacting the
aqueous phase with a de-emulsifying agent capable of removing the
first organic solvent; and, separating the de-emulsifying agent
containing the removed first organic solvent from the contacted
aqueous phase.
13. The method of claim 11, wherein after the aqueous phase is
collected, the aqueous phase is contacted with a de-emulsifying
agent capable of removing the first organic solvent, and the
de-emulsifying agent containing the removed first organic solvent
is removed from the aqueous phase before introducing the aqueous
phase containing the modified viral particles with reduced lipid
content into the animal or the human.
14. A method for treating a viral infection in an animal or human
patient comprising: removing blood containing a plurality of
lipid-containing infectious viral particles from the animal or the
human; obtaining plasma from the blood, the plasma containing the
lipid-containing infectious viral particles; contacting the plasma
containing the lipid-containing infectious viral particles with a
first organic solvent capable of extracting lipid from the
lipid-containing infectious viral particles to produce modified
viral particles having reduced lipid content; mixing the plasma and
the first organic solvent; permitting organic and aqueous phases to
separate; collecting the aqueous phase containing the modified
viral particles; and introducing the aqueous phase containing the
modified viral particles into the animal or the human wherein the
modified viral particles have at least one exposed patient-specific
antigen that was not exposed in the plurality of lipid-containing
infectious viral particles.
15. The method of claim 14, wherein after the aqueous phase is
collected, the aqueous phase is contacted with a de-emulsifying
agent capable of removing the first organic solvent, and the
de-emulsifying agent containing the removed first organic solvent
from the contacted aqueous phase is separated and removed before
introducing the aqueous phase containing the modified viral
particles into the animal or the human.
16. The method of claim 14, further comprising adding cells to the
aqueous phase containing the modified viral particles before
introduction into the animal or the human.
17. The method of claim 15, further comprising adding cells to the
aqueous phase containing the modified viral particles before
introduction into the animal or the human.
18-19. (canceled)
20. A method of providing protection in an animal or a human
against a viral infection comprising the step of administering to
the animal or the human of an effective amount of a composition
comprising modified viral particles with reduced lipid content and
a pharmaceutically acceptable carrier, wherein the amount is
effective to provide a protective effect against infection by the
lipid-containing viral particle in the animal or the human.
21. The method of claim 20 further comprising administration of an
immunostimulant.
22. The method of claim 11, wherein the first organic solvent is an
alcohol, an ether, an amine, a hydrocarbon, or a combination
thereof.
23. The method of 11, wherein the first organic solvent is an
alcohol, an ether, or a combination thereof.
24. The method of claim 23 wherein the ether is C4 to C8 ether and
the alcohol is a C1 to C8 alcohol.
25. The method of 11, wherein the de-emulsifying agent is an
ether.
26. The method of claim 11, wherein the fluid is plasma, serum,
peritoneal fluid, lymphatic fluid, pleural fluid, pericardial
fluid, cerebrospinal fluid, or a fluid of the reproductive
system.
27. The method of claim 11, wherein the first organic solvent is an
alcohol, an ether, an amine, a hydrocarbon, an ester, a surfactant
or a combination thereof.
Description
RELATED APPLICATIONS
[0001] This application is a continuation-in-part of U.S.
non-provisional patent application Ser. No. 10/311,679 filed Dec.
18, 2002, which is a US national phase from PCT patent application
number PCT/IB01/01099 filed Jun. 21, 2001, which claims the benefit
of Australian patent application PQ8469 filed Jun. 29, 2000 and PCT
patent application number PCT/AU00/01603 filed Dec. 28, 2000. The
present application also claims the benefit of U.S. provisional
patent application Ser. No. 60/390,066 filed Jun. 20, 2002.
FIELD OF THE INVENTION
[0002] The present invention relates to a delipidation method
employing a solvent system useful for extracting lipids from a
virus, thereby creating a modified viral particle. The solvent
system of the present invention is optimally designed such that
upon delipidation of the virus, the viral particle remains
substantially intact. By dissolving the lipid envelope surrounding
the viral particle using the method of the present invention, the
resultant modified viral particle has exposed antigens (or
epitopes), which foster and promote antibody production. The
resulting modified viral particle of the present invention
initiates a positive immunogenic response in the species into which
it is re-introduced. The present invention can be applied to
delipidating viruses from a specific patient for future
reintroduction into the patient, to delipidating stock viruses, or
non-patient specific viruses, for use as a vaccine, or to
delipidating and combining both non-patient specific viruses and
patient specific viruses to create a therapeutic cocktail.
BACKGROUND OF THE INVENTION
Introduction
[0003] Viruses, of varied etiology, affect billions of animals and
humans each year and inflict an enormous economic burden on
society. Many viruses contain lipid as a major component of the
membrane that surrounds them. Viruses affect animals and humans
causing extreme suffering, morbidity, and mortality. These viruses
travel throughout the body in biological fluids such as blood,
peritoneal fluid, lymphatic fluid, pleural fluid, pericardial
fluid, cerebrospinal fluid, and in various fluids of the
reproductive system. Fluid contact at any site promotes
transmission of disease. Other viruses reside primarily in
different organ systems and in specific tissues, proliferate and
then enter the circulatory system to gain access to other tissues
and organs at remote sites. If the body does not exhibit a positive
immune response against these pathogens, they infect many cell
types within the body, inhibiting these cells from performing their
normal functions.
[0004] The human immune system is composed of various cell types
that collectively protect the body from different viruses. The
immune system provides multiple means for targeting and eliminating
foreign elements, including humoral and cellular immune responses,
participating primarily in antigen recognition and elimination. An
immune response to foreign elements requires the presence of
B-lymphocytes (B cells) or T-lymphocytes (T cells) in combination
with antigen-presenting cells (APC), which are usually macrophage
or dendrite cells. The APCs are specialized immune cells that
capture antigens. Once inside an APC, antigens are broken down into
smaller fragments called epitopes--the unique markers carried by
the antigen surface. These epitopes are subsequently displayed on
the surface of the APCs and are responsible for triggering an
antibody response in defense of the infection.
[0005] In a humoral immune response, when an APC displaying
antigens (in the form of unique epitope markers) foreign to the
body are recognized, B cells are activated, proliferating and
producing antibodies. These antibodies specifically bind to the
antigens present on the APC and block their ability to further
infect cells. After the antibody attaches, the APC engulfs the
entire antigen and kills it. This type of antibody immune response
is primarily involved in the prevention of viral infection.
[0006] In a cellular immune response, on recognizing the APC
displaying a foreign antigen, the T cells are activated. There are
two steps in the cellular immune response. The first step involves
activation of cytotoxic T cells (CTL) or CD8+ T killer cells that
proliferate and kill target cells that specifically present
antigens presented by APC. The second involves helper T cells (HTL)
or CD4+ T cells that regulate the production of antibodies and the
activity of CD8+ cells. The CD4+ T cells provide growth factors to
CD8+ T cells that allow them to proliferate and function
efficiently.
[0007] Certain infective pathogens are deemed "chronic" due to
their structure. For example, some viruses are able to evade an
immune response because of their ability to hide some of their
antigens from the immune system. Viruses contain an outer envelope
made up of lipids and fats derived from the host cell membrane
during the budding process. Viruses are comprised of virions,
non-cellular infectious agents consisting of a single type of
nucleic acid (either RNA or DNA), surrounded by a protein coat. The
outer protein covering of viruses is called a capsid, made up of
repeating subunits called capsomeres.
[0008] Since viruses are non-metabolic, they only reproduce within
living host cells. The virus codes the proteins of the viral
envelope while the host cell codes the lipids and carbohydrates.
Therefore, the lipid and carbohydrate content within a given viral
envelope is dependent on the particular host. The enveloped viral
particles therefore partially adopt the identity of the host cell,
via lipid and carbohydrate content, and are able to conceal
antigens associated with them, which would normally have initiated
an immune response. Instead, the viral particle confuses the host
immune system by presenting it with an antigenic complex that
contains components of host tissues, and is perceived by the host
immune system as partly "self" and partly "foreign". An immune
response that destroys the antigenic complex containing host tissue
elements can end up destroying host cells leading to severe
autoimmune disease. The immune system is forced to produce the
"compromise", ineffective antibodies which do not destroy the viral
particles, allowing them to proliferate and slowly cause severe
damage to the body, while destroying host cells.
[0009] Recent epidemics affecting the immune system include
acquired immune deficiency syndrome (AIDS), believed to be caused
by the human immunodeficiency virus (HIV). Related viruses affect
animal species, for example, simians and felines (SIV and FIV,
respectively). Other major viral infections include, but are not
limited to, meningitis, cytomegalovirus, and hepatitis in its
various forms.
Current Methods of Treatment
[0010] One prior art method of treating viruses of varied etiology
is via drug therapy. Most anti-viral drug therapies are directed to
preventing or inhibiting viral replication and appear to focus on
the initial attachment of the virus to the T4 lymphocyte or
macrophage, the transcription of viral RNA to viral DNA and the
assembly of new virus during reproduction. The high mutation rate
of the virus, especially in the case of HIV, is a major difficulty
with existing treatments because the various strains become
resistant to anti-viral drug therapy. Furthermore, anti-viral drug
therapy treatment may cause the evolution of resistant strains of
the virus. Other drawbacks to drug therapies are the undesirable
side effects and patient compliance requirements. In addition, many
individuals are afflicted with multiple viral infections such as a
combination of HIV and hepatitis. Such individuals require even
more aggressive and expensive drug regimens to counteract disease
progression, which in turn cause greater side effects and a greater
likelihood of multiple drug resistance.
[0011] Also known in the prior art is prevention of disease via the
use of vaccinations. Vaccines have been singularly responsible for
conferring immune response against several human pathogens. They
are designed to stimulate the immune system to protect against
various viral infections. In general, a vaccine is produced from an
antigen, isolated or produced from the disease-causing
microorganism, which can elicit an immune response. When a vaccine
is injected into the blood stream as a preventive measure to create
an effective immune response, the B cells in the blood stream
perceive the antigens contained by the vaccine as foreign or
`non-self` and respond by producing antibodies, which bind to the
antigens and inactivate them. Memory cells are thereby produced and
remain ready to mount a quick protective immune response against
subsequent infection with the same disease-causing agent. Thus when
an infective pathogen containing similar antigens as the vaccine
enters the body, the immune system will recognize the protein and
instigate an effective defense against infection.
[0012] The current methods of vaccination do have drawbacks, making
them less than optimally desirable for immunizing individuals
against particular pathogens, especially HIV. The existing vaccine
strategies aim to expose the body to the antigens associated with
infective pathogens so that the body builds an immune response
against these pathogens. For example, hepatitis B and HIV pathogens
are able to survive and proliferate in the human body despite
having an effective immune response. One explanation offered in the
prior art is that the antigens of these microorganisms change
constantly so the antibodies produced in response to a particular
antigen are no longer effective when the antigen mutates. The AIDS
virus is believed to undergo this antigenic variation. Although
antigenic variation has been addressed via the attempted use of
combination drugs or antigens, no prior art vaccine has succeeded
in addressing chronic infections such as HIV.
[0013] Another approach to treating viruses of varied etiology is
to inactivate the virus. Prior art methods of inactivating viruses
using chemical agents have relied on organic solvents such as
chloroform or glutaraldehyde. Although viral inactivation is
effective in reducing viral load of a patient and treating
contaminated blood to be used in blood transfusions, it does have
problems. For example, inactivation of a virus does not provide a
protective immune response against viral infection. In addition, it
is largely geared towards denaturing viral proteins, thereby
destroying the structure of the viral particle.
[0014] Drug therapy, as described above only provides a temporary
solution to viral infectivity and works only to decrease the viral
load of a patient. Chemical inactivation of the virus works to
temporarily decrease viral infectivity; however, once cells
replicate the level of infectivity will increase again. Moreover,
these destruction-type processes lead to total cell death and do
not initiate or promote a positive immunogenic response in the
patient. In sum, prior art methods have largely focused on
destroying, yet not suitably modifying, viral particles.
Current Methods of Manufacture of Viral Treatments and Medicaments
Viral Inactivation (or Chemical Kill)
[0015] Described in the prior art are methods of treating viral
particles with organic solvents and high temperatures thus
dissolving the lipid envelopes and subsequently inactivating the
virus. In those methods, blood is withdrawn from the patient and
separated into two phases--the first phase including red cells and
platelets and the second phase containing plasma, white cells, and
cell-free virus (virion). The second phase is treated with an
organic solvent, thereby killing the infected cells and virions,
and subsequently reintroduced into the patient. In addition to
dissolving the lipid envelope of the virus, the high organic
solvent concentrations cause cell death and damage to the antigens.
Essentially, this method results in a "chemical kill" of the
cell.
[0016] Glutaraldehyde is one such solvent whereby cell inactivation
is achieved as known by those of ordinary skill in the art by
fixation with a dilute solution of glutaraldehyde at about 1:250.
Although treating the virus with glutaraldehyde effectively
delipidates the virus, it also destroys the core. Destruction of
the core is not desirable for producing a modified viral particle
useful for inducing an immune response in a recipient.
[0017] Chloroform is another such solvent. Chloroform, however,
denatures many plasma proteins and is not suitable for use with
biological fluids, which will be reintroduced into the animal or
human. These plasma proteins deleteriously affected by chloroform
serve important biological functions including coagulation,
hormonal response, and immune response. These functions are
essential to life and thus damage to these proteins may have an
adverse effect on a patient's health, possibly leading to
death.
[0018] Other solvents or detergents such as B-propiolactone,
TWEEN-80, and dialkyl or trialkyl phosphates have been used, either
alone or in combination. Many of these methods, especially those
involving detergents, require tedious procedures to ensure removal
of the detergent before reintroduction of the treated plasma sample
into the animal or human. Further, many of the methods described in
the prior art involve extensive exposure to elevated temperature in
order to kill free virus and infected cells. Elevated temperatures
have deleterious effects on the proteins contained in biological
fluids, such as plasma.
Current Methods of Manufacturing Vaccines
[0019] To date, several manufacturing methods have been employed in
search for safe and effective vaccines for immunizing individuals
against infective pathogenic agents. To protect an individual from
a specific pathogenic infection, a target protein or antigen
associated with the infective pathogen is administered to the
individual. This includes presenting the protein as part of a
non-infective (inactivated) or less infective (attenuated) agent or
as a discreet protein composition. Known to one of ordinary skill
in the art are the following different types of vaccines: live
attenuated vaccines, whole inactivated vaccines, DNA vaccines,
combination vaccines, recombinant vaccines, live recombinant vector
vaccines, virus like particles and synthetic peptide vaccines.
[0020] In live attenuated vaccines, the viruses are rendered less
pathogenic to the host, either by specific genetic manipulation of
the virus genome or by passage in some type of tissue culture
system. In order to achieve genetic manipulation, an inessential
gene is deleted or one or more essential genes in the virus are
partially damaged. Upon genetic manipulation, the viral particles
become less virulent yet retain antigenic features. Live attenuated
vaccines can also be used as "vaccine vectors" for other genes,
wherein they act as carriers of genes from a second virus (or other
pathogen) against which protection is required. Attenuated vaccines
(less infective and not inactivated), however, pose several
problems. First, it is difficult to ascertain when the attenuated
vaccine is no longer pathogenic. The risk of viral infection from
the vaccine is too great to properly test for effective
attenuation. In addition, attenuated vaccines carry the risk of
reverting into a virulent form of the pathogen.
[0021] Whole inactivated vaccines are known in the art for
immunizing against infection by introducing killed or inactivated
viruses to introduce pathogen proteins to an individual's immune
system. The administration of killed or inactivated pathogen, via
heat or chemical means, into an individual introduces the pathogen
to the individual's immune system in a non-infective form thereby
instigating an immune response defense. Wholly inactivated vaccines
provide protection by directly generating cellular and humoral
immune responses against the pathogenic immunogens. There is little
threat of infection, because the viral pathogen is killed or
otherwise inactivated.
[0022] Subunit vaccines are yet another form of vaccination well
known to one of ordinary skill in the art. These consist of one or
more isolated proteins derived from the pathogen. These proteins
act as target antigens against which an immune response is
exhibited. The proteins selected for the subunit vaccine are
displayed by the pathogen so that upon infection of an individual
by the pathogen, the individual's immune system recognizes the
pathogen and instigates an immune response. Subunit vaccines are
not whole infective agents and are therefore incapable of becoming
infective. Subunit vaccines are the basic of AIDSVAX, the first
vaccine for HIV being tested for effectiveness in humans and which
contains a portion of HIV's outer surface (envelope) protein,
called gp120.
[0023] DNA vaccine is another type known in the art and uses actual
genetic material of pathogens. In addition, synthetic peptide
vaccines are made up of parts of synthetic, chemically engineered
HIV proteins called peptides. They comprise portions of HIV
proteins chosen specifically to achieve an anti-HIV immune
response. Also mentioned in the prior art are combination vaccines
that, when used in conjunction with one another, generate a broad
spectrum of immune responses. One example of a combination vaccine
is SHIV, which is a synthetic vaccine made from the HIV envelope
and SIV core.
[0024] What is needed is a therapeutic method and system for
providing patients with patient-specific antigens capable of
initiating a protective immune response. Accordingly, what is
needed is a simple, effective method that does not appreciably
denature or extract plasma proteins from the biological sample
being treated. What is also needed is an effective delipidation
process via which a viral particle is modified, rather than
destroyed, thereby both reducing and/or eliminating infectivity of
the viral particle and invoking a patient specific, autologous
immune response to reduce viral infection and prevent further
infection.
[0025] What is also needed is an effective means to immunize
individuals against viral pathogen infection that is unique to the
individual due to viral mutations. Preferably the means would
elicit a broad, biologically active protective immune response with
minimized risk of infecting the individual.
SUMMARY OF THE INVENTION
[0026] The present invention solves the problems described above by
providing a simple, effective and efficient method for treating and
preventing viral infection. The method of the present invention
affects the lipid envelope of a virus by utilizing an efficient
solvent system, which does not denature or destroy the virus. The
present invention employs an optimal solvent and energy system to
create, via delipidation, a non-synthetic, host-derived modified
viral particle that has its lipid envelope at least partially
removed, generating a positive immunologic response in a patient,
thereby providing that patient with some degree of protection
against the virus.
[0027] The present invention is also effective in producing an
autologous, patient-specific therapeutic vaccine against the virus,
by treating a biological fluid containing the virus such that the
virus is present in a modified form, but no longer infectious and
such that an immune response is initiated upon reintroduction of
the fluid into the patient. To create the vaccine, a biological
fluid (for example, blood) is removed from the patient, the plasma
is separated from the blood and treated to isolate the virus, and
the virus is delipidated using an optimal solvent system. A
lipid-containing virus, treated in this manner in order to reduce
its infectivity and create a modified viral particle, is
administered to a recipient, such as an animal or a human, together
with a pharmaceutically acceptable carrier in order to initiate an
immune response in the animal or human and create antibodies that
bind the exposed epitopes of the modified viral particle. Adjuvants
may also be administered with the modified viral particle in the
pharmaceutically acceptable carrier.
[0028] Thus an effective method is presented, by which new vaccines
can be developed out of lipid containing viruses by removing lipid
from the lipid envelope and exposing antigens hidden within the
lipid envelope or beneath the surface of the lipid envelope, in
turn generating a positive immune response when re-introduced into
the patient.
[0029] The present invention provides a modified viral particle
comprising at least a partially delipidated viral particle, wherein
the partially delipidated viral particle initiates a positive
immune response in an animal or human patient and incites
protection against an infectious organism in the animal or the
human patient.
[0030] The present invention provides a method for creating a
modified viral particle comprising the steps of: receiving a
plurality of viral particles, each having a viral envelope, in a
fluid; exposing the viral particles to a delipidation process; and,
partially delipidating the viral particles wherein the delipidation
process at least partially removes the viral envelopes to create
the modified viral particle and wherein the modified viral particle
is capable of provoking a positive immune response in a
patient.
[0031] The present invention also provides an antigen delivery
vehicle and a method for creating an antigen delivery vehicle
comprising the steps of: receiving a plurality of viral particles,
each having a viral envelope, in a fluid; exposing the viral
particles to a delipidation process; and, partially delipidating
the viral particles to create modified viral particles that act as
antigen delivery vehicles, wherein the delipidation process at
least partially removes the viral envelopes to expose at least one
antigen and wherein the at least one antigen is capable of
provoking a positive immune response in a patient.
[0032] The modified viral particles of the present invention
comprise at least a partially delipidated viral particle, wherein
the partially delipidated viral particle is produced by exposing a
non-delipidated viral particle to a delipidation process and
wherein the partially delipidated viral particle comprises at least
one exposed patient specific antigen that was not exposed in the
non-delipidated viral particle.
[0033] The present invention also provides a vaccine composition,
comprising at least a partially delipidated viral particle having
patient-specific antigens and a pharmaceutically acceptable
carrier, wherein the partially delipidated viral particle is
capable of provoking a positive immune response when the
composition is administered to a patient.
[0034] The present invention also provides a method for making a
vaccine comprising: contacting a lipid-containing viral particle in
a fluid with a first organic solvent capable of extracting lipid
from the lipid-containing viral particle; mixing the fluid and the
first organic solvent for a time sufficient to extract lipid from
the lipid-containing viral particle; permitting organic and aqueous
phases to separate; and collecting the aqueous phase containing a
modified viral particle with reduced lipid content wherein the
modified viral particle is capable of provoking a positive immune
response when administered to a patient.
[0035] The present invention also provides a method to protect an
animal or a human against an infectious viral particle comprising
administering to the animal or the human an effective amount of a
composition comprising a modified viral particle, wherein the
modification comprises at least partial removal of a lipid envelope
of the infectious viral particle, and a pharmaceutically acceptable
carrier, wherein the amount is effective to provide a protective
effect against infection by the infectious viral particle in the
animal or the human.
[0036] The present invention also provides a method for provoking a
positive immune response in an animal or human having a plurality
of lipid-containing viral particles, comprising the steps of:
obtaining a fluid containing the lipid-containing viral particles
from the animal or the human; contacting the fluid containing the
lipid-containing viral particles with a first organic solvent
capable of extracting lipid from the lipid-containing viral
particles; mixing the fluid and the first organic solvent:
permitting organic and aqueous phases to separate; collecting the
aqueous phase containing modified viral particles with reduced
lipid content; and introducing the aqueous phase containing the
modified viral particles with reduced lipid content into the animal
or the human wherein the modified viral particles with reduced
lipid content provoke a positive immune response in the animal or
the human.
[0037] The present invention also provides a method for treating a
viral infection in an animal or human patient comprising: removing
blood containing a plurality of lipid-containing infectious viral
particles from the animal or the human; obtaining plasma from the
blood, the plasma containing the lipid-containing infectious viral
particles; contacting the plasma containing the lipid-containing
infectious viral particles with a first organic solvent capable of
extracting lipid from the lipid-containing infectious viral
particles to produce modified viral particles having reduced lipid
content; mixing the plasma and the first organic solvent;
permitting organic and aqueous phases to separate; collecting the
aqueous phase containing the modified viral particles; and
introducing the aqueous phase containing the modified viral
particles into the animal or the human wherein the modified viral
particles have at least one exposed patient-specific antigen that
was not exposed in the plurality of lipid-containing infectious
viral particles.
[0038] As shown below, the characteristics of the modified viral
particle are exhibited in experimental data, showing mice having a
positive immunogenic response when vaccinated as compared with a
wholly inactivated vaccine. In addition, data exhibiting protein
recovery indicate retention of the structural integrity of the
viral particle, removing only its lipid-containing envelope.
[0039] Fluids which may be treated with the method of the present
invention include but are not limited to the following: plasma;
serum; lymphatic fluid; cerebrospinal fluid; peritoneal fluid;
pleural fluid; pericardial fluid; various fluids of the
reproductive system including but not limited to semen, ejaculatory
fluids, follicular fluid and amniotic fluid; cell culture reagents
such as normal sera, fetal calf serum or serum derived from any
other animal or human; and immunological reagents such as various
preparations of antibodies and cytokines.
[0040] The method of the present invention may be used to treat
viruses containing lipid in the viral envelope. Preferred viruses
to be treated with the method of the present invention include the
various immunodeficiency viruses including but not limited to human
(HIV) and subtypes such as HIV-1 and HIV-2, simian (SIV), feline
(FIV), as well as any other form of immunodeficiency virus. Other
preferred viruses to be treated with the method of the present
invention include but are not limited to hepatitis in its various
forms. Another preferred virus treated with the method of the
present invention is the bovine pestivirus. It is to be understood
that the present invention is not limited to the viruses provided
in the list above. Additional specific viruses are described in the
detailed description of this application. All viruses containing
lipid, especially in their viral envelope, are included within the
scope of the present invention.
[0041] The present invention also provides a simple, inexpensive
and easy to use kit for delipidating fluids and lipid-containing
viruses within fluids and to create modified viral particles. This
kit may be used in various situations, such as in the field, in a
clinic, by a physician in an emergency situation, in a laboratory
or elsewhere. Preferred fluids include biological fluids and
culture medium. A preferred biological fluid is plasma.
[0042] The kits of the present invention may be used to process
plasma from a patient. In a preferred embodiment, the plasma
contains lipid-containing virus. This delipidated plasma containing
modified viral particles may be stored in a blood bank for
subsequent autologous or heterologous administration. The kits of
the present invention may be used to process plasma from a patient
and then administer the delipidated plasma, containing modified
viral particles, to the patient. The kits of the present invention
may be used to process other fluids containing lipids or
lipid-containing viruses, such as culture media and cells cultured
in media. The modified viral particles produced with these kits may
be combined with a pharmaceutically acceptable carrier, and
optionally an adjuvant, and used as vaccines by administration to
an animal or human to cause an immune response to epitopes on or in
the modified viral particles.
[0043] The kits of the present invention generally comprise
containers used for different purposes. A first container generally
contains one or more first extraction solvents. This first
container contains means for removing the first extraction solvent
from the container. Such means may be a component of the first
container or a separate component adapted to function with the
first container. Such means include, but are not limited to, any
type of cap, spout, funnel, penetrable seal, penetrable diaphragm,
tube, pipette, or other means known to one skilled in the art for
removing the one or more first extraction solvents or for
introducing a fluid containing lipid or lipid-containing virus into
the first container. A second container contains the fluid
containing lipids or lipid-containing virus to be delipidated.
[0044] In one embodiment, a third container is used for contacting
or mixing the fluid containing lipids or lipid-containing virus to
be delipidated and the first extraction solvent. Mixing can occur
through agitation, inversion, shaking, or other means to agitate
the third container to a degree sufficient to mix the fluid and the
first extraction solvent. After the mixing step, the first
extraction solvent containing the dissolved lipids from the fluid
or from the lipid-containing organisms separates from the fluid. At
this point, the delipidated fluid may be removed through any means
such as pouring, decanting, pipetting, applying a vacuum connected
to a tube or pipette, or any other means known to one of ordinary
skill in the art of removing separated fluids.
[0045] A fourth container optionally receives the delipidated fluid
and modified viral particles from the third container.
Alternatively, the delipidated fluid and modified viral particles
are administered to the patient through a tube, catheter, an
intravenous line, an intraarterial line or other means without
introduction into a fourth container.
[0046] In one embodiment the first container contains sufficient
volume within it to receive the fluid containing lipids or
lipid-containing virus to be delipidated. In this embodiment,
mixing of the first extraction solvent and the fluid containing
lipids or lipid-containing virus to be delipidated occurs within
the first container. In this embodiment, a separate container for
mixing the fluid and the first extraction solvent, referred to as
the third container above, is not required. After mixing occurs,
the first extraction solvent containing the dissolved lipids
separates from the delipidated fluid. At this point, the
delipidated fluid may be introduced into another container,
analogous to the fourth container described above for subsequent
introduction into a patient or for optional additional extraction
of the first extraction solvent with a second extraction
solvent.
[0047] In another embodiment, when a second extraction solvent is
optionally employed to assist in removal of the first extraction
solvent, a fifth container is included which contains the second
extraction solvent. This second extraction solvent may be added to
the mixture described above in the third container, mixed and then
permitted to separate from the delipidated fluid. Alternatively,
the second extraction solvent may be added to the fourth container
described above, mixed and then permitted to separate from the
delipidated fluid if additional removal of residual first
extraction solvent is desired. In yet another alternative
embodiment, the second extraction solvent may be added to the first
container described above containing the mixture of the fluid and
the first extraction solvent, mixed and then permitted to separate
from the delipidated fluid, if mixing of the fluid and the first
extraction solvent, separation and additional extraction of the
first extraction solvent using a second extraction solvent are all
performed in the first container. The containers described above
may be graduated for easy determination of volume within a
container.
[0048] Accordingly, it is an object of the present invention to
provide a method for treating lipid containing virus in order to
create modified viral particles.
[0049] It is another object of the present invention to provide a
method for treating or preventing viral disease by administering
modified viral particles to a patient.
[0050] Another object of the present invention is to provide a
method for treating a biological fluid in order to reduce or
eliminate the infectivity of infectious viral organisms contained
therein.
[0051] It is further an object of the present invention to provide
a method for treatment of lipid-containing viruses within a fluid,
which minimizes deleterious effects on proteins contained within
the fluid, thereby creating a modified viral particle with
properties that are capable of initiating a positive immune
response in a patient.
[0052] It is a further object of the present invention to provide a
method for treatment of lipid-containing viruses within a fluid,
which minimizes deleterious effects on proteins contained within
the fluid, thereby creating a modified viral particle with
patient-specific antigens.
[0053] It is another object of the present invention to provide a
method for reducing the infectivity of viruses, wherein the method
does not employ elevated temperatures, chloroform, detergents, or
trialkyl phosphates.
[0054] It is another object of the present invention to provide a
method for reducing the infectivity of viruses, wherein the method
exposes antigenic determinants on the modified viral particle.
[0055] It is a farther object of the present invention to
completely or partially delipidate the viral particle, wherein the
viral particles comprise immunodeficiency virus, hepatitis in its
various forms, or any other lipid-containing virus, thereby
creating a modified viral particle.
[0056] It is a further object of the present invention to
completely or partially delipidate the viral particle, wherein the
viral particles comprise immunodeficiency virus, hepatitis in its
various forms, or any other lipid-containing virus, while retaining
the structural protein core of the virus.
[0057] It is another object of the present invention to provide a
method for reducing the infectivity of viruses, wherein the newly
formed viral particle can be used as an antigen delivery
vehicle.
[0058] Yet another object of the present invention is to treat
infectious organisms with the method of the present invention in
order to reduce their infectivity and provide a vaccine comprising
a delipidated, modified viral particle which may be administered to
an animal or a human together with a pharmaceutically acceptable
carrier and optionally an immunostimulant compound, to prevent or
minimize clinical manifestation of disease in a patient following
exposure to the virus.
[0059] Yet another object of the present invention is to treat
infectious organisms with the method of the present invention in
order to reduce their infectivity and provide a vaccine comprising
a delipidated, modified viral particle which may be administered to
an animal or a human together with a pharmaceutically acceptable
carrier and optionally an immunostimulant compound, to initiate a
positive immunogenic response in the animal or human.
[0060] It is another specific object of the present invention to
provide an anti-viral vaccine.
[0061] It is a further specific object of the present invention to
lessen the severity of a disease caused by a lipid-containing virus
in an animal or human receiving a vaccine comprising a composition
comprising a virus treated with the method of the present invention
in a pharmaceutically acceptable carrier.
[0062] It is another object of the present invention to combine
delipidated viral particles having patient specific antigens with
delipidated stock viral particles to create a therapeutic cocktail
for the treatment of diseases.
[0063] Accordingly it is an object of the present invention to
provide an inexpensive and easy to use kit for removal of lipids
from fluids and infectious organisms, preferably biological fluids,
plasma, or culture medium.
[0064] Another object of the present invention is to provide an
inexpensive and easy to use kit for removal of lipids from
lipid-containing viruses to create modified viral particles.
[0065] These and other features and advantages of the present
invention will become apparent after review of the following
drawings and detailed description of the disclosed embodiments.
Various modifications to the stated embodiments will be readily
apparent to those of ordinary skill in the art, and the disclosure
set forth herein may be applicable to other embodiments and
applications without departing from the spirit and scope of the
present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0066] The accompanying drawings, which are incorporated in and
form a part of the specification, illustrate preferred embodiments
of the present invention.
[0067] FIG. 1 is a schematic diagram of an embodiment of a kit of
the present invention containing a first container 10 with a screw
cap 15, containing first extraction solvent 20, and plasma 30, and
a second container 50 with a tube 60 leading from an opening 70,
the tube 60 being connected to a needle 62.
[0068] FIG. 2 is a schematic diagram of an embodiment of a kit of
the present invention containing a first container 10 with a screw
cap 15, containing first extraction solvent 20, a second container
50 with a screw cap 55 and containing plasma 30, a third container
70, with a screw cap 75 for mixing the first extraction solvent 20
and plasma 30 to form mixture 72, and a fourth container 80 with a
screw cap 85 for storing delipidated plasma 82.
[0069] FIG. 3 incorporates the elements of FIG. 2 and further
provides a fifth container 90 with a screw cap 95 containing a
second extraction solvent 92 and a sixth container 100 for storing
delipidated plasma 102 with reduced levels of residual first
extraction solvent, with a tube 110 leading from an opening 105,
the tube 110 being connected to a needle 112.
[0070] FIG. 4 is a schematic representation of an HIV viral
particle showing the lipid containing envelope (LE) or bilayer
derived from a host cell, the capsid (C), nuclear material (NM)
such as RNA, surface glycoproteins (GP) such as gp120 and gp41,
transmembrane proteins (TMP), p17 matrix protein, and capsid
proteins (CP) such as p24.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0071] By the term "fluid" is meant any fluid containing an
infectious organism, including but not limited to, a biological
fluid obtained from an organism such as an animal or human.
Preferred infectious organisms treated with the method of the
present invention are viruses. Such biological fluids obtained from
an organism include but are not limited to plasma, serum,
cerebrospinal fluid, lymphatic fluid, peritoneal fluid, follicular
fluid, amniotic fluid, pleural fluid, pericardial fluid,
reproductive fluids and any other fluid contained within the
organism. Other fluids may include laboratory samples containing
infectious organisms suspended in any chosen fluid. Other fluids
include cell culture reagents, many of which include biological
compounds such as fluids obtained from living organisms, including
but not limited to "normal serum" obtained from various animals and
used as growth medium in cell and tissue culture applications.
[0072] By the terms "first solvent" or "first organic solvent" "or
first extraction solvent" are meant a solvent, comprising one or
more solvents, used to facilitate extraction of lipid from a fluid
or from a lipid-containing biological organism. This solvent will
enter the fluid and remain in the fluid until being removed.
Suitable first extraction solvents include solvents that extract or
dissolve lipid, including but not limited to alcohols,
hydrocarbons, amines, ethers, and combinations thereof. First
extraction solvents may be combinations of alcohols and ethers.
First extraction solvents include, but are not limited to
n-butanol, di-isopropyl ether (DIPE), diethyl ether, and
combinations thereof
[0073] The term "second extraction solvent" is defined as one or
more solvents that facilitate the removal of a portion of the first
extraction solvent. Suitable second extraction solvents include any
solvent that facilitates removal of the first extraction solvent
from the fluid. Second extraction solvents include any solvent that
facilitates removal of the first extraction solvent including but
not limited to ethers, alcohols, hydrocarbons, amines, and
combinations thereof. Preferred second extraction solvents include
diethyl ether and di-isopropyl ether, which facilitate the removal
of alcohols, such as n-butanol, from the fluid. The term
"de-emulsifying agent" is a second extraction solvent that assists
in the removal of the first solvent which may be present in an
emulsion in an aqueous layer.
[0074] The term "delipidation" refers to the process of removing at
least a portion of a total concentration of lipids in a fluid or in
a lipid-containing organism. Lipid-containing organisms may be
found within fluids which may or may not contain additional
lipids.
[0075] The terms "pharmaceutically acceptable carrier" or
"pharmaceutically acceptable vehicle" are used herein to mean any
liquid including but not limited to water or saline, a gel, salve,
solvent, diluent, fluid ointment base, liposome, micelle, giant
micelle, and the like, which is suitable for use in contact with
living animal or human tissue without causing adverse physiological
responses, and which does not interact with the other components of
the composition in a deleterious manner.
[0076] The term "patient" refers to animals and humans in this
application.
A Modified Viral Particle
[0077] Practice of the method of the present invention to reduce
the lipid content of a virus creates a modified viral particle.
These modified viral particles are immunogenic. The present methods
expose epitopes that are not usually presented to the immune system
by untreated virus. A structural change occurs in the modified
viral particles, and proteins on, in, or near the surface of the
virus are modified such that a conformational change occurs. Some
of these proteins may also separate from the modified viral
particle. FIG. 4 is a schematic representation of HIV viral
particle showing the lipid containing envelope or bilayer derived
from a host cell, surface glycoproteins, transmembrane proteins,
the capsid, capsid proteins and nuclear material. The delipidation
process of the present invention modifies the viral particle. The
modified viral particle has a lower lipid content in the envelope,
displays modified proteins, loses infectivity and is
immunogenic.
Modified Viral Particle Resulting from Removal of Lipid from
Lipid-Containing Organisms
[0078] As described above, methods of treating the viral particles
with organic solvents and use of high temperatures, thus dissolving
the lipid envelopes and subsequently inactivating the virus are
well known in the prior art. In this method, blood is withdrawn
from the patient and separated into two phases--the first phase
including red cells and platelets and the second phase containing
plasma, white cells, and cell-free virus (virion). The second phase
is treated with an organic solvent, thereby killing the infected
cells and virions, and subsequently reintroduced into the patient.
In addition to dissolving the lipid envelope of the virus, however,
the high organic solvent concentrations cause cell death and damage
to the antigens. This method results in a "chemical kill" of the
cell. Another drawback is that elevated temperatures have
deleterious effects on the proteins contained in biological fluids
such as plasma. Glutaraldehyde is one such solvent whereby cell
inactivation is achieved as known by those of ordinary skill in the
art by fixation with a dilute solution of glutaraldehyde at about
1:250.
[0079] When a viral particle is sent through certain solvent
systems, lipids will be removed in the solvent because, when
treated appropriately, lipids are soluble in certain solvent
systems. Viruses are comprised of virions with the outer covering
comprised of a protein coat, or capsid, as described above. Since
viruses are non-metabolic, they only reproduce within living host
cells. The virus codes the proteins of the viral envelope while the
host cell codes the lipids and carbohydrates. Therefore, the lipid
and carbohydrate within a given viral envelope is dependent on the
particular host. The enveloped viral particles therefore partially
adopt the identity of the host cell and are able to conceal some
antigens associated with the virus, which normally would have
initiated an immune response.
[0080] Instead, the viral particle confuses the host immune system
by presenting it with an antigenic complex that contains components
of host tissues, and is perceived by the host immune system as
partly "self" and partly "foreign". An immune response that
destroys the antigenic complex containing host tissue elements can
destroy host cells leading to severe autoimmune disease. The immune
system is forced to produce the "compromise", ineffective
antibodies which do not destroy the viral particles, allowing them
to proliferate and slowly cause severe damage to the body, while
the host cells are destroyed.
[0081] Methods of the present invention can be used to solve this
problem because, by removing the lipid envelope of the virus, and
keeping the viral particle intact, the method of the present
invention exposes additional antigens. The host immune system is
forced to recognize the viral particle as wholly "foreign". Using
the method of the present invention, what is created is a modified
viral particle in which the antigenic core remains intact, thereby
using the epitopes of the actual viral particle to initiate a
positive immunogenic response in the patient into which it is
reintroduced. In addition, the method of the present invention
reduces the deleterious effect on the other plasma proteins,
measured by protein recovery, such that the plasma can be
reintroduced into the patient.
[0082] In creating this modified viral particle what is also
created is a patient-specific antigen that induces protection
against the viral particle in the species in which it is
introduced. The method of the present invention creates an
effective means to immunize individuals against viral pathogen
infection and elicit a broad, biologically active protective immune
response without risk of infecting the individual. New vaccines may
be developed from certain lipid containing viruses by removing the
lipid envelope and exposing antigens hidden beneath the envelope,
in turn generating a positive immune response. These "autologous
vaccines" can be created by the at least partial removal of the
lipid envelope using suitable solvent systems (one which would not
damage the antigens contained in the particle) exposing antigens
and/or forcing a structural modification in the viral protein
structures, which when introduced into the body, would provoke an
effective immune response.
Infectious Organisms Treated with the Present Invention p Viruses
are the preferred infectious organism treated with the method of
the present invention. Viral infectious organisms which may be
delipidated by the present invention to form modified viral
particles include, but are not limited to the lipid-containing
viruses of the following genuses: Alphavirus (alphaviruses),
Rubivurus (rubella virus), Flavivirus (Flaviviruses), Pestivirus
(mucosal disease viruses), (unnamed, hepatitis C virus),
Coronavirus, (Coronaviruses) severe acute respiratory syndrome
(SARS), Torovirus, (toroviruses), Arteivirus, (arteriviruses),
Paramyxovirus, (Paramyxoviruses), Rubulavirus (rubulavriuses),
Morbillivirus (morbillivuruses), Pneumovirinae (the pneumoviruses),
Pneumovirus (pneumoviruses), Vesiculovirus (vesiculoviruses),
Lyssavirus (lyssaviruses), Ephemerovirus (ephemeroviruses),
Cytorhabdovirus (plant rhabdovirus group A), Nucleorhabdovirus
(plant rhabdovirus group B), Filovirus (filoviruses),
Influenzavirus A, B (influenza A and B viruses), Influenza virus C
(influenza C virus), (unnamed, Thogoto-like viruses), Bunyavirus
(bunyaviruses), Phlebovirus (phleboviruses), Nairovirus
(nairoviruses), Hantavirus (hantaviruses), Tospovirus
(tospoviruses), Arenavirus (arenaviruses), unnamed mammalian type B
retroviruses, unnamed, mammalian and reptilian type C retroviruses,
unnamed, type D retroviruses, Lentivirus (lentiviruses), Spumavirus
(spumaviruses), Orthohepadnavirus (hepadnaviruses of mammals),
Avihepadnavirus (hepadnaviruses of birds), Simplexvirus
(simplexviruses), Varicellovirus (varicelloviruses),
Betaherpesvirinae (the cytomegaloviruses), Cytomegalovirus
(cytomegaloviruses), Muromegalovirus (murine cytomegaloviruses),
Roseolovirus (human herpes virus 6, 7, 8), Gammaherpesvirinae (the
lymphocyte-associated herpes viruses), Lymphocryptovirus
(Epstein-Bar-like viruses), Rhadinovirus (saimiri-ateles-like
herpes viruses), Orthopoxvirus (orthopoxviruses), Parapoxvirus
(parapoxviruses), Avipoxvirus (fowlpox viruses), Capripoxvirus
(sheeppoxlike viruses), Leporipoxvirus (myxomaviruses), Suipoxvirus
(swine-pox viruses), Molluscipoxvirus (molluscum contagiosum
viruses), Yatapoxvirus (yabapox and tanapox viruses), Unnamed,
African swine fever-like viruses, Iridovirus (small iridescent
insect viruses), Ranavirus (front iridoviruses), Lymphocystivirus
(lymphocystis viruses of fish), Togaviridae, Flaviviridae,
Coronaviridae, Enabdoviridae, Filoviridae, Paramyxoviridae,
Orthomyxoviridae, Bunyaviridae, Arenaviridae, Retroviridae,
Hepadnaviridae, Herpesviridae, Poxviridae, and any other
lipid-containing virus.
[0083] These viruses include the following human and animal
pathogens: Ross River virus, fever virus, dengue viruses, Murray
Valley encephalitis virus, tick-borne encephalitis viruses
(including European and far eastern tick-borne encephalitis
viruses, California encephalitis virus, St. Louis encephalitis
virus, sandfly fever virus, human coronaviruses 229-E and OC43 and
others causing the common cold, upper respiratory tract infection,
probably pneumonia and possibly gastroenteritis), human
parainfluenza viruses 1 and 3, mumps virus, human parainfluenza
viruses 2, 4a and 4b, measles virus, human respiratory syncytial
virus, rabies virus, Marburg virus, Ebola virus, influenza A
viruses and influenza B viruses, Arenaviruss: lymphocytic
choriomeningitis (LCM) virus; Lassa virus, human immunodeficiency
viruses 1 and 2, or any other immunodeficiency virus, hepatitis B
virus, hepatitis C virus, hepatitis G virus, Subfamily: human
herpes viruses 1 and 2, herpes virus B, Epstein-Barr virus),
(smallpox) virus, cowpox virus, monkeypox virus, molluscum
contagiosum virus, yellow fever virus, poliovirus, Norwalk virus,
orf virus, and any other lipid-containing virus.
Methods of Manufacture of the Modified Viral Particle
[0084] One of ordinary skill in the art would appreciate that there
may be multiple delipidation processes employed under the scope of
this invention. In a preferred embodiment, a solvent system
together applied energy, for example a mechanical mixing system, is
used to substantially delipidate the viral particle. The
delipidation process is dependent upon the total amount of solvent
and energy input into a system. Various solvent levels and mixing
methods, as described below, may be used depending upon the overall
framework of the process. Although a single solvent or multiple
solvents may be used for delipidation of virus, it is to be
understood that a single solvent is preferred since there is less
probability of destroying and denaturing the viral particle.
Exemplary Solvent Systems for Use in Removal of Lipid from Viruses
and Effective in Maintaining Integrity of the Viral Particle
[0085] The solvent or combinations of solvents to be employed in
the process of partially or completely delipidating
lipid-containing organisms may be any solvent or combination
thereof effective in solubilizing lipids in the viral envelope
while retaining the structural integrity of the modified viral
particle, which can be measured, in one embodiment, via protein
recovery. A delipidation process falling within the scope of the
present invention uses an optimal combination of energy input and
solvent to delipidate the viral particle, while still keeping it
intact. Suitable solvents comprise hydrocarbons, ethers, alcohols,
phenols, esters, halohydrocarbons, halocarbons, amines, and
mixtures thereof. Aromatic, aliphatic, or alicyclic hydrocarbons
may also be used. Other suitable solvents, which may be used with
the present invention, include amines and mixtures of amines. One
solvent system is DIPE, either concentrated or diluted in water or
a buffer such as a physiologically acceptable buffer. One solvent
combination comprises alcohols and ethers. Another solvent
comprises ether or combinations of ethers, either in the form of
symmetrical ethers, asymmetrical ethers or halogenated ethers.
[0086] The optimal solvent systems are those that accomplish two
objectives: first, at least partially delipidating the infectious
organism or viral particle and second, employing a set of
conditions such that there are few or no deleterious effects on the
other plasma proteins. In addition, the solvent system should
maintain the integrity of the viral particle such that it can be
used to initiate an immune response in the patient. It should
therefore be noted that certain solvents, solvent combinations, and
solvent concentrations may be too harsh to use in the present
invention because they result in a chemical kill.
[0087] It is preferred that the solvent or combination of solvents
has a relatively low boiling point to facilitate removal through a
vacuum and possibly heat without destroying the antigenic core of
the viral particle. It is also preferred that the solvent or
combination of solvents be employed at a low temperature because
heat has deleterious effects on the proteins contained in
biological fluids such as plasma. It is also preferred that the
solvent or combination of solvents at least partially delipidate
the viral particle.
[0088] Liquid hydrocarbons dissolve compounds of low polarity such
as the lipids found in the viral envelopes of the infectious
organisms. Particularly effective in disrupting the lipid membrane
of a viral particle are hydrocarbons which are substantially water
immiscible and liquid at about 37.degree. C. Suitable hydrocarbons
include, but are not limited to the following: C.sub.5 to C.sub.20
aliphatic hydrocarbons such as petroleum ether, hexane, heptane,
octane; haloaliphatic hydrocarbons such as chloroform,
1,1,2-trichloro-1,2,2-trifluoroethane, 1,1,1-trichloroethane,
trichloroethylene, tetrachloroethylene, dichloromethane and carbon
tetrachloride; thioaliphatic hydrocarbons each of which may be
linear, branched or cyclic, saturated or unsaturated; aromatic
hydrocarbons such as benzene; alkylarenes such as toluene;
haloarenes; haloalkylarenes; and thioarenes. Other suitable
solvents may also include saturated or unsaturated heterocyclic
compounds such as pyridine and aliphatic, thio- or halo-derivatives
thereof.
[0089] Suitable esters for use in the present invention include,
but are not limited to, ethyl acetate, propylacetate, butylacetate
and ethylpropionate. Suitable detergents/surfactants that may be
used include but are not limited to the following: sulfates,
sulfonates, phosphates (including phospholipids), carboxylates, and
sulfosuccinates. Some anionic amphiphilic materials useful with the
present invention include but are not limited to the following:
sodium dodecyl sulfate (SDS), sodium decyl sulfate,
bis-(2-ethylhexyl) sodium sulfosuccinate (AOT), cholesterol sulfate
and sodium laurate.
[0090] Solvents may be removed from delipidated viral mixtures
through the use of additional solvents. For example, demulsifying
agents such as ethers may be used to remove a first solvent such as
an alcohol from an emulsion. Removal of solvents may also be
accomplished through other methods, which do not employ additional
solvents, including but not limited to the use of charcoal.
Charcoal may be used in a slurry or alternatively, in a column to
which a mixture is applied. Pervaporation may also be employed to
remove one or more solvents from delipidated viral mixtures.
[0091] Examples of suitable amines for use in removal of lipid from
lipid-containing organisms in the present invention are those which
are substantially immiscible in water. Typical amines are aliphatic
amines--those having a carbon chain of at least 6 carbon atoms. A
non-limiting example of such an amine is
C.sub.6H.sub.13NH.sub.2.
[0092] Ether is a preferred solvent for use in the method of the
present invention. Particularly preferred are the C.sub.4-C.sub.8
containing-ethers, including but not limited to ethyl ether,
diethyl ether, and propyl ethers (including but not limited to
di-isopropyl ether). Asymmetrical ethers may also be employed.
Halogenated symmetrical and asymmetrical ethers may also be
employed.
[0093] Low concentrations of ethers may be employed to remove
lipids when used alone and not in combination with other solvents.
For example, a low concentration range of ethers include 0.5% to
30%. Such concentrations of ethers that may be employed include,
but are not limited to the following: 0.625%, 1.0% 1.25%, 2.5%,
5.0% and 10% or higher. It has been observed that dilute solutions
of ethers are effective. Such solutions may be aqueous solutions or
solutions in aqueous buffers, such as phosphate buffered saline
(PBS). Other physiological buffers may be used, including but not
limited to bicarbonate, citrate, Tris, Tris/EDTA, and Trizma.
Preferred ethers are di-isopropyl ether (DIPE) and diethyl ether
(DEE).
[0094] When used in the present invention, appropriate alcohols are
those which are not appreciably miscible with plasma or other
biological fluids. Such alcohols include, but are not limited to,
straight chain and branched chain alcohols, including pentanols,
hexanols, heptanols, octanols and those alcohols containing higher
numbers of carbons.
[0095] When alcohols are used in combination with another solvent,
for example, an ether, a hydrocarbon, an amine, or a combination
thereof, C.sub.1-C.sub.8 containing alcohols may be used. Alcohols
for use in combination with another solvent include C.sub.4-C.sub.8
containing alcohols. Accordingly, alcohols that fall within the
scope of the present invention are butanols, pentanols, hexanols,
heptanols and octanols, and iso forms thereof, in particular,
C.sub.4 alcohols or butanols (1-butanol and 2-butanol). The
specific alcohol choice is dependent on the second solvent
employed.
[0096] Ethers and alcohols can be used in combination as a first
solvent for treating the fluid containing the lipid-containing
virus, or viral particle. Any combination of alcohol and ether may
be used provided the combination is effective to at least partially
remove lipid from the infectious organism, without having
deleterious effects on the plasma proteins. In one embodiment,
lipid is removed from the viral envelope of the infectious
organism. When alcohols and ether are combined as a first solvent
for treating the infectious organism contained in a fluid, ratios
of alcohol to ether in this solvent are about 0.01%-60% alcohol to
about 40%-99.99% of ether, with a specific ratio of about 10%-50%
of alcohol with about 50%-90% of ether, with a more specific ratio
of about 20%-45% alcohol and about 55%-80% ether.
[0097] One combination of alcohol and ether is the combination of
butanol and di-isopropyl ether (DIPE). When butanol and DIPE are
combined as a first solvent for treating the infectious organism
contained in a fluid, ratios of butanol to DIPE in this solvent are
about 0.01%-60% butanol to about 40%-99.99% of DIPE, with a
specific ratio of about 10%-50% of butanol with about 50%-90% of
DIPE, with a more specific ratio of about 20%-45% butanol and about
55%-80% DIPE.
[0098] Another combination of alcohol and ether is the combination
of butanol with diethyl ether (DEE). When butanol is used in
combination with DEE as a first solvent, ratios of butanol to DEE
are about 0.01%-60% butanol to about 40%-99.99% of DEE, with a more
specific ratio of about 10%-50% of butanol with about 50%-90% of
DEE, with a most specific ratio of about 20%-45% butanol and about
55%-80% DEE. One specific ratio of butanol and DEE in a first
solvent is about 40% butanol and about 60% DEE. This combination of
about 40% butanol and about 60% DEE (vol:vol) has been shown to
have no significant effect on a variety of biochemical and
hematological blood parameters, as shown for example in U.S. Pat.
No. 4,895,558.
Biological Fluids and Treatment thereof for Reducing Infectivity of
Infectious, Lipid-Containing Organisms
[0099] As stated above, various biological fluids may be treated
with the method of the present invention in order to reduce the
levels of infectivity of the lipid-containing organism in the
biological fluid and to create modified viral particles. In a
preferred embodiment, plasma obtained from an animal or human is
treated with the method of the present invention in order to reduce
the concentration and/or infectivity of lipid-containing infectious
organisms within the plasma and to create modified viral particles.
In this embodiment, plasma may be obtained from an animal or human
patient by withdrawing blood from the patient using well-known
methods and treating the blood in order to separate the cellular
components of the blood (red and white cells) from the plasma. Such
methods for treating the blood are known to one of ordinary skill
in the art and include but are not limited to centrifugation and
filtration. One of ordinary skill in the art understands the proper
centrifugation conditions for separating such lipid-containing
organisms from the red and white cells. Filtration may include
diafiltration or filtration through membranes with pore sizes that
separate the lipid-containing organism, such as the cell-free
virus, from the red and white cells. Use of the present invention
permits treatment of lipid-containing organisms, for example those
found within plasma, without having deleterious effects on other
plasma proteins and maintaining the integrity of the viral
core.
[0100] Viruses are typically retained in the plasma and are
affected by the treatment of the plasma with the method of the
present invention. In cases where the lipid-containing organism to
be treated is substantially larger, and may pellet with red and
white blood cells under typical centrifugation conditions for
separating cells from plasma, the lipid-containing organism may be
separated from the red and white cells using techniques known to
one of ordinary skill in the art.
[0101] Treatment of lipid-containing organisms in biological fluids
other than blood and plasma does not generally involve separation
of the cells from the fluid prior to initiation of the delipidation
procedure. For example, follicular fluid and peritoneal fluid may
be treated with the present invention to affect the levels and
infectivity of lipid-containing organisms without deleterious
effects on protein components. The treated fluid may subsequently
be reintroduced into the animal or human from which it was
obtained. Treatment of these non-blood types of fluids affects the
lipid-containing organisms in the fluid, such as the virus.
[0102] Once a biological fluid, such as plasma, is obtained either
in this manner, or for example, from a storage facility housing
bags of plasma, the plasma is contacted with a first organic
solvent, as described above, capable of solubilizing lipid in the
lipid-containing infectious organism. The first organic solvent is
combined with the plasma in a ratio wherein the first solvent is
present in an amount effective to substantially solubilize the
lipid in the infectious organism, for example, dissolve the lipid
envelope that surrounds the virus. Exemplary ratios of first
solvent to plasma (expressed as a ratio of first organic solvent to
plasma) are described in the following ranges: 0.5-4.0:0.5-4.0;
0.8-3.0:0.8-3.0; and 1-2:0.8-1.5. Various other ratios may be
applied, depending on the nature of the biological fluid. For
example, in the case of cell culture fluid, the following ranges
may be employed of first organic solvent to cell culture fluid:
0.5-4.0:0.5-4.0; 0.8-3.0:0.8-3.0; and 1-2:0.8-1.5.
[0103] After contacting the fluid containing the infectious
organism with the first solvent as described above, the first
solvent and fluid are mixed, using methods including but not
limited to one of the following suitable mixing methods: gentle
stirring; vigorous stirring; vortexing; swirling; homogenization;
and end-over-end rotation.
[0104] The amount of time required for adequate mixing of the first
solvent with the fluid is related to the mixing method employed.
Fluids are mixed for a period of time sufficient to permit intimate
contact between the organic and aqueous phases, and for the first
solvent to at least partially or completely solubilize the lipid
contained in the infectious organism. Typically, mixing will occur
for a period of about 10 seconds to about 24 hours, possibly about
10 seconds to about 2 hours, possibly approximately 10 seconds to
approximately 10 minutes, or possibly about 30 seconds to about 1
hour, depending on the mixing method employed. Non-limiting
examples of mixing durations associated with different methods
include 1) gentle stirring and end-over-end rotation for a period
of about 10 seconds to about 24 hours, 2) vigorous stirring and
vortexing for a period of about 10 seconds to about 30 minutes, 3)
swirling for a period of about 10 seconds to about 2 hours, or 4)
homogenization for a period of about 10 seconds to about 10
minutes.
Separation of Solvents
[0105] After mixing of the first solvent with the fluid, the
solvent is separated from the fluid being treated. The organic and
aqueous phases may be separated by any suitable manner known to one
of ordinary skill in the art. Since the first solvent is typically
immiscible in the aqueous fluid, the two layers are permitted to
separate and the undesired layer is removed. The undesired layer is
the solvent layer containing dissolved lipids and its
identification, as known to one of ordinary skill in the art,
depends on whether the solvent is more or less dense than the
aqueous phase. An advantage of separation in this manner is that
dissolved lipids in the solvent layer may be removed.
[0106] In addition, separation may be achieved through means,
including but not limited to the following: removing the undesired
layer via pipetting; centrifugation followed by removal of the
layer to be separated; creating a path or hole in the bottom of the
tube containing the layers and permitting the lower layer to pass
through; utilization of a container with valves or ports located at
specific lengths along the long axis of the container to facilitate
access to and removal of specific layers; and any other means known
to one of ordinary skill in the art. Another method of separating
the layers, especially when the solvent layer is volatile, is
through distillation under reduced pressure or evaporation at room
temperature, optionally combined with mild heating. In one
embodiment employing centrifugation, relatively low g forces are
employed, such as 900.times.g for about 5 to 15 minutes to separate
the phases.
[0107] Another method of separating solvent is through the used of
charcoal, preferably activated charcoal. This charcoal is
optionally contained in a column. Alternatively the charcoal may be
used in slurry form. Various biocompatible forms of charcoal may be
used in these columns. Pervaporation methods and use of charcoal to
remove solvents are preferred methods for removing solvent.
[0108] Following separation of the first solvent from the treated
fluid, some of the first solvent may remain entrapped in the
aqueous layer as an emulsion. Optionally, a de-emulsifying agent is
employed to facilitate removal of the trapped first solvent. The
de-emulsifying agent may be any agent effective to facilitate
removal of the first solvent. A preferred de-emulsifying agent is
ether and a more preferred de-emulsifying agent is diethyl ether.
The de-emulsifying agent may be added to the fluid or in the
alternative the fluid may be dispersed in the de-emulsifying agent.
In vaccine preparation, alkanes in a ratio of about 0.5 to 4.0 to
about 1 part of emulsion (vol:vol) may be employed as a
de-emulsifying agent, followed by washing to remove the residual
alkane from the remaining delipidated organism used for preparing
the vaccine. Preferred alkanes include, but are not limited to,
pentane, hexane and higher order straight and branched chain
alkanes.
[0109] The de-emulsifying agent, such as ether, may be removed
through means known to one of skill in the art, including such
means as described in the previous paragraph. One convenient method
to remove the de-emulsifying agent, such as ether, from the system,
is to permit the ether to evaporate from the system in a running
fume hood or other suitable device for collecting and removing the
de-emulsifying agent from the environment. In addition,
de-emulsifying agents may be removed through application of higher
temperatures, for example from about 24 to 37.degree. C. with or
without pressures of about 10 to 20 mbar. Another method to remove
the de-emulsifying agent involves separation by centrifugation,
followed by removal of organic solvent through aspiration, further
followed by evaporation under reduced pressure (for example 50
mbar) or further supply of an inert gas, such as nitrogen, over the
meniscus to aid in evaporation. Yet another method of removing a
first solvent or a demulsifying agent is through the use of
adsorbants, such as charcoal. The charcoal is preferably activated
charcoal. This charcoal is optionally contained in a column, as
described above. Still another method of removing solvent is the
use of hollow fiber contactors. Pervaporation methods and charcoal
adsorbant methods of removing solvents are preferred.
Methods of Treating Biological Fluids (Delipidation)
[0110] It is to be understood that the method of the present
invention may be employed in either a continuous or discontinuous
manner. That is, in a continuous manner, a fluid may be fed to a
system employing a first solvent which is then mixed with the
fluid, separated, and optionally further removed through
application of a de-emulsifying agent. The continuous method also
facilitates subsequent return of the fluid containing delipidated
infectious organism to a desired location. Such locations may be
containers for receipt and/or storage of such treated fluid, and
may also include the vascular system of a human or animal or some
other body compartment of a human or animal, such as the pleural,
pericardial, peritoneal, and abdominopelvic spaces.
[0111] In one embodiment of the continuous method of the present
invention, a biological fluid, for example, blood, is removed from
an animal or a human through means known to one of ordinary skill
in the art, such as a catheter. Appropriate anti-clotting factors
as known to one of ordinary skill in the art are employed, such as
heparin, ethylenediaminetetraacetic acid (EDTA) or citrate. This
blood is then separated into its cellular and plasma components
through the use of a centrifuge. The plasma is then contacted with
the first solvent and mixed with the first solvent to effectuate
lipid removal from the infectious organism contained within the
plasma. Following separation of the first solvent from the treated
plasma, a de-emulsifying agent is optionally employed to remove
entrapped first solvent. After ensuring that acceptable levels
(non-toxic) of first solvent or de-emulsifying agent, if employed,
are found within the plasma containing the delipidated infectious
organism, the plasma is then optionally combined with the cells
previously separated from the blood to form a new blood sample
containing at least partially delipidated viral particles, also
called modified viral particles herein.
[0112] Through the practice of this method, the infectivity of the
infectious organism is greatly reduced or eliminated. Following
recombination with the cells originally separated from the blood,
this sample may be reintroduced into either the vascular system or
some other system of the human or animal. The effect of such
treatment of plasma removed from the human or animal and return of
the sample containing the partially or completely delipidated
infectious organism, or modified viral particle, to the human or
animal causes a net decrease in the concentration and infectivity
of the infectious organism contained within the vascular system of
the human or animal. In addition to decreasing the concentration
and infectivity of the infectious organism contained within the
vascular system, the modified viral particle serves to initiate an
autologous immune response in the patient. In this manner,
infectious viral load is reduced. In this mode of operation, the
method of the present invention is employed to treat body fluids in
a continuous manner--while the human or animal is connected to an
extracorporeal device for such treatment.
[0113] In yet another embodiment, the discontinuous or batch mode,
the human or animal is not connected to an extracorporeal device
for processing bodily fluids with the method of the present
invention. In a discontinuous mode of operation, the present
invention employs a fluid previously obtained from a human or
animal, which may include, but is not limited to plasma, lymphatic
fluid, or follicular fluid. The sample may be contained within a
blood bank or in the alternative, drawn from a human or animal
prior to application of the method. The sample may be a
reproductive fluid or any fluid used in the process of artificial
insemination or in vitro fertilization. The sample may also be one
not directly obtained from a human or animal but rather any fluid
containing a potentially infectious organism, such as cell culture
fluid. In this mode of operation, this sample is treated with the
method of the present invention to produce a new sample which
contains at least partially or completely delipidated infectious
organisms, or modified viral particles. One embodiment of this mode
of the present invention is to treat plasma samples previously
obtained from animals or humans and stored in a blood bank for
subsequent transfusion. These samples may be treated with the
method of the present invention to minimize or eliminate
transmission of infectious disease, such as HIV, hepatitis,
cytomegalovirus, from the biological sample.
[0114] Delipidation of an infectious organism can be achieved by
various means. A batch method can be used for fresh or stored
biological fluids, for example, fresh frozen plasma. In this case a
variety of the described organic solvents or mixtures thereof can
be used for viral inactivation. Extraction time depends on the
solvent or mixture thereof and the mixing procedure employed.
Kits
[0115] The kits of the present invention generally comprise
containers used for different purposes and are depicted in FIGS.
1-3. A first container 10 generally contains one or more first
extraction solvents 20. This first container 10 contains means 15
for removing the first extraction solvent from the opening 70 of
the container 10. Such means may be a component of the first
container 10 or a separate component adapted to function with the
first container 10. Such means include, but are not limited to, any
type of cap 15, spout, funnel, penetrable seal, penetrable
diaphragm, tube 60, pipette, or other means for removing the one or
more first extraction solvents 20 or for introducing a fluid 30
containing lipid-containing virus into the first container 10.
[0116] A second container 50 contains the fluid 30 containing
lipid-containing virus to be delipidated.
[0117] In one embodiment, a third container 70 is used for mixing
the fluid 30 containing lipid-containing virus to be delipidated
and the first extraction solvent 20. Mixing can occur through
agitation, inversion, shaking, or other means to agitate the third
container 70 to a degree sufficient to mix the fluid 30 and the
first extraction solvent 20 to form a mixture 72. After the mixing
step, the first extraction solvent containing the dissolved lipids
from the fluid or from the viruses separates from the fluid. At
this point, the delipidated fluid may be removed through any means
75 such as pouring, decanting, pipetting, applying a vacuum
connected to a tube or pipette, or any other means known to one of
ordinary skill in the art of removing separated fluids.
[0118] A fourth container 80 optionally receives the delipidated
fluid and modified viral particles 82 originating from the third
container 70. Alternatively, the delipidated fluid containing the
modified viral particles is administered into the patient through a
tube, catheter, an intravenous line, an intraarterial line or other
means without introduction into a fourth container 80.
[0119] In one embodiment, the first container 10 containing the
first extraction solvent 20, has sufficient additional volume
within it to receive the fluid 30 containing lipid-containing virus
to be delipidated. In this embodiment, mixing of the first
extraction solvent 20 and the fluid containing lipid-containing
virus 30 to be delipidated occurs within the first container 10. In
this embodiment, a separate container for mixing the fluid 30 and
the first extraction solvent 20, referred to as the third container
70, is not required. After mixing occurs, the first extraction
solvent containing the dissolved lipids separates from the
delipidated fluid and the modified viral particles. At this point,
the delipidated fluid and the modified viral particles may be
introduced into another container, analogous to the fourth
container described above, for subsequent introduction into a
patient or for optional additional extraction of the first
extraction solvent with a second extraction solvent 92.
[0120] In another embodiment, when a second extraction solvent 92
is optionally employed to assist in removal of the first extraction
solvent 20, a fifth container 90 is included which contains the
second extraction solvent 92. This second extraction solvent 92 may
be added to the mixture 72 described above in the third container
70, mixed and then permitted to separate from the delipidated fluid
and modified viral particles. Alternatively, the second extraction
solvent 92 may be added to the fourth container 80 described above,
mixed and then permitted to separate from the delipidated fluid and
modified viral particles if additional removal of residual first
extraction solvent is desired. In yet another alternative
embodiment, the second extraction solvent 92 may be added to the
first container 10 described above containing the mixture of the
fluid 30 containing the lipid containing virus and the first
extraction solvent 20, mixed, and then permitted to separate from
the delipidated fluid and modified viral particles if mixing of the
fluid 30 containing the lipid containing virus and the first
extraction solvent 20, separation and additional extraction of the
first extraction solvent 20 using a second extraction solvent 92
are all performed in the first container 10.
[0121] The containers described above may be graduated for easy
determination of volume within a container.
[0122] Optionally, means for removing or introducing a biological
fluid from a patient comprising a venipuncture system may be
included in a kit. Such systems are well known to those skilled in
the art of removing or replacing fluids, for example vascular
fluids, including without limitation a vac tube, hypodermic syringe
connected to an intravascular needle 62, 112, tubing 60, 110 or an
intravascular needle 62, 112, connected through tubing 60, 110 to a
bag for collection of blood. Any of these devices may be optionally
incorporated into the kit. A sensor may be added to the kit for
determining the level of a first extraction solvent and optionally
a second extraction solvent in the delipidated fluid.
[0123] Suitable materials for use in any of the apparatus
components as described herein include materials that are
biocompatible, approved for medical applications that involve
contact with internal body fluids, and in compliance with U.S. PV1
or ISO 10993 standards. Further, the materials should not
substantially degrade during at least a single use, from for
instance, exposure to the solvents used in the present invention.
The materials should typically be sterilizable, preferably by
radiation or ethylene oxide (EtO) sterilization. Such suitable
materials should be capable of being formed into objects using
conventional processes, such as, but not limited to, extrusion,
injection molding and others. Materials meeting these requirements
include, but are not limited to, nylon, polypropylene,
polycarbonate, acrylic, polysulphone, polyvinylidene fluoride
(PVDF), fluoroelastomers such as VITON, available from DuPont Dow
Elastomers L.L.C., thermoplastic elastomers such as SANTOPRENE,
available from Monsanto, polyurethane, polyvinyl chloride (PVC),
polytetrafluoroethylene (PTFE), polyphenylene ether (PFE),
perfluoroalkoxy copolymer (PFA), which is available as TEFLON PFA
from E.I. du Pont de Nemours and Company, and combinations
thereof.
[0124] Through the use of the kits and methods of the present
invention, levels of lipid in lipid-containing viruses in a fluid
are reduced, and the fluid, for example, delipidated plasma
containing the modified viral particles may be administered to the
patient. Such fluid contains modified viral particles that are not
infective. These modified viral particles induce an immune response
in the recipient to epitopes on the modified viral particles.
Alternatively the modified viral particles may be further isolated
from the delipidated fluid and combined with a pharmaceutically
acceptable carrier, and optionally an adjuvant and administered as
a vaccine composition to a human or an animal to induce an immune
response in the recipient.
Vaccine Production
[0125] The modified viral particle, which is at least partially or
substantially delipidated and has immunogenic properties is
combined with a pharmaceutically acceptable carrier to make a
composition comprising a vaccine. This vaccine composition is
optionally combined with an adjuvant or an immunostimulant and
administered to an animal or a human. It is to be understood that
vaccine compositions may contain more than one type of modified
viral particle or component thereof, in order to provide protection
against more than one disease after vaccination. Such combinations
may be selected according to the desired immunity. For example,
preferred combinations may be, but are not limited to HIV and
hepatitis or influenza and hepatitis. More specifically, the
vaccine can comprise a plurality of modified viral particles having
patient-specific antigens and modified viral particles having
non-patient specific antigens or stock viral particles that have
undergone the delipidation process of the present invention.
[0126] The remaining particles of the organism are retained in the
delipidated biological fluid, and when reintroduced into the animal
or human, are presumably ingested by phagocytes. The number of
viral particles isolated and modified by the delipidation treatment
is determined by counting the particles before and after
treatment.
Administration of Vaccine Produced With the Method of the Present
Invention
[0127] When a delipidated infectious organism, for example one in
the form of a modified viral particle with exposed antigenic
determinants, is administered to an animal or a human, it is
typically combined with a pharmaceutically acceptable carrier to
produce a vaccine, and optionally combined with an adjuvant or an
immunostimulant as known to one of ordinary skill in the art. The
vaccine formulations may conveniently be presented in unit dosage
form and may be prepared by conventional pharmaceutical techniques
known to one of ordinary skill in the art. Such techniques include
uniformly and intimately bringing into association the active
ingredient and the liquid carriers (pharmaceutical carrier(s) or
excipient(s)). Formulations suitable for parenteral administration
include aqueous and non-aqueous sterile injection solutions which
may contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents.
[0128] The formulations may be presented in unit-dose or multi-dose
containers--for example, sealed ampules and vials--and may be
stored in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example, water for
injections, immediately prior to use. The vaccine may be stored at
temperatures of from about 4.degree. C. to -100.degree. C. The
vaccine may also be stored in a lyophilized state at different
temperatures including room temperature. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders,
granules and tablets commonly used by one of ordinary skill in the
art. The vaccine may be sterilized through conventional means known
to one of ordinary skill in the art. Such means include, but are
not limited to filtration, radiation and heat. The vaccine of the
present invention may also be combined with bacteriostatic agents,
such as thimerosal, to inhibit bacterial growth.
[0129] Preferred unit dosage formulations are those containing a
dose or unit, or an appropriate fraction thereof, of the
administered ingredient. It should be understood that in addition
to the ingredients, particularly mentioned above, the formulations
of the present invention may include other agents commonly used by
one of ordinary skill in the art.
[0130] The vaccine may be administered through different routes,
such as oral, including buccal and sublingual, rectal, parenteral,
aerosol, nasal, intramuscular, subcutaneous, intradermal,
intravenous, intraperitoneal, and topical.
[0131] The vaccine of the present invention may be administered in
different forms, including but not limited to solutions, emulsions
and suspensions, microspheres, particles, microparticles,
nanoparticles, and liposomes. It is expected that from about 1 to 5
dosages may be required per immunization regimen. One of ordinary
skill in the medical or veterinary arts of administering vaccines
will be familiar with the amount of vaccine to be administered in
an initial injection and in booster injections, if required, taking
into consideration, for example, the age and size of an a
patient.
Vaccination Schedule
[0132] The vaccines of the present invention may be administered
before, during or after an infection. The vaccine of the present
invention may be administered to either humans or animals. In one
embodiment, the viral load (one or more viruses) of a human or an
animal may be reduced by delipidation treatment of the plasma. The
same individual may receive a vaccine directed to the one or more
viruses, thereby stimulating the immune system to combat against
the virus that remains in the individual. The optimal time for
administration of the vaccine is about one to three months before
the initial infection. However, the vaccine may also be
administered after initial infection to ameliorate disease
progression or to treat the disease.
Adjuvants
[0133] A variety of adjuvants known to one of ordinary skill in the
art may be administered in conjunction with the modified viral
particles in the vaccine composition. Such adjuvants include, but
are not limited to the following: polymers, co-polymers such as
polyoxyethylene-polyoxypropylene co-polymers, including block
co-polymers; polymer P1005; monotide ISA72; Freund's complete
adjuvant (for animals); Freund's incomplete adjuvant; sorbitan
monooleate; squalene; CRL-8300 adjuvant; alum; QS 21, muramyl
dipeptide; trehalose; bacterial extracts, including mycobacterial
extracts; detoxified endotoxins; membrane lipids; water-in-oil
mixtures, water-in-oil-in-water mixtures or combinations
thereof.
Suspending Fluids and Carriers
[0134] A variety of suspending fluids or carriers known to one of
ordinary skill in the art may be employed to suspend the vaccine
composition. Such fluids include without limitation: sterile water,
saline, buffer, or complex fluids derived from growth medium or
other biological fluids. Preservatives, stabilizers and antibiotics
known to one of ordinary skill in the art may be employed in the
vaccine composition.
[0135] The following experimental examples are illustrative in
showing that a delipidation process of the viral particle occurred
and in particular, that the viral particle was modified and noted
to exhibit a positive immunogenic response in the species from
which it was derived. It will be appreciated that other embodiments
and uses will be apparent to those skilled in the art and that the
invention is not limited to these specific illustrative examples or
preferred embodiments.
EXAMPLE 1
[0136] A. Delipidation of Serum Produces Duck Hepatitis B Virus
(DHBV) having Reduced Infectivity
[0137] A standard duck serum pool (Camden) containing 10.sup.6
ID.sub.50 doses of DHBV was used. ID.sub.50 is known to one of
ordinary skill in the art as the infective dosage (ID) effective to
infect 50% of animals treated with the dose. Twenty-one ducklings
were obtained from a DHBV negative flock on day of hatch. These
ducklings were tested at purchase and shown to be DHBV DNA negative
by dot-blot hybridization.
[0138] The organic solvent system was mixed in the ratio of 40%
butanol to 60% diisopropyl ether. The mixed organic solvent system
(4 ml) was mixed with the standard serum pool (2 ml) and gently
rotated for 1 hour at room temperature. The mixture was centrifuged
at 400.times.g for 10 minutes and the lower aqueous phase
(containing the plasma) removed at room temperature. The aqueous
phase was then mixed with an equal volume of diethyl ether and
centrifuged as before to remove any remaining lipid/solvent
mixture. The aqueous phase was again removed and mixed with an
equal volume of diethyl ether and re-centrifuged. The aqueous phase
was removed and any residual diethyl ether was removed by airing in
a fume cabinet at room temperature for about 1 hour. The
delipidated plasma, with or without viral particles was stored at
-20.degree. C.
[0139] The positive and negative control duck sera were diluted in
phosphate buffered saline (PBS). Positive controls: 2 ml of pooled
serum containing 10.sup.6ID.sub.50 doses of DHBV was mixed with 4
ml of PBS. Negative controls: 2 ml of pooled DHBV negative serum
was mixed with 4 ml of PBS. Residual infectivity was tested by
inoculation of 100 .mu.l of either test sample (n=7), negative
(n=7) or positive (n=7) controls into the peritoneal cavities of
day-old ducks. Controls were run with DHBV negative serum treated
with organic solvents and subsequently mixed with PBS and injected
into recipient ducks.
[0140] One of the positive control ducks died between 4 and 6 days
of age and was excluded from further analysis. A further 3 positive
control ducks died between 9 and 10 days of age, and two treatment
and one negative control died on day 11. It was decided to
terminate the experiment. The remaining ducklings were euthanized
on day 12 with sodium pentibarbitone, i.v., and their livers
removed for DHBV DNA analysis as described by Deva et al (J.
Hospital Infection 33:119-130, 1996). All seven negative control
ducks remained DHBV negative. Livers of all six positive control
ducks were DHBV positive. All seven test ducks remained negative
for DHBV DNA in their liver.
[0141] Delipidation of serum using the above solvent system
resulted in DHBV having reduced infectivity. None of the ducklings
receiving treated serum became infected. Although the experiment
had to be terminated on day 12 instead of day 14, the remaining
positive control ducks were positive for DHBV (3/3 were DHBV
positive by day 10). This suggests that sufficient time had elapsed
for the treated ducks to become DHBV positive in the liver and that
the premature ending of the experiment had no bearing on the
results.
B. Delipidated DHBV Positive Serum as a Vaccine to Prevent DHBV
Infection
[0142] The efficacy of the delipidation procedure to provide a
patient specific "autologous" vaccine against Duck Hepatitis B
Virus (DHBV) was examined. Approximately 16 Pekin cross ducklings
were obtained from a DHBV negative flock of ducklings on the day of
hatch. The ducklings were tested and determined to be DHBV negative
by analysis of DHBV DNA using dot-blot hybridization. The ducks
were divided into the following three groups:
TABLE-US-00001 TABLE 1 # of Ducks Vaccine Administered Results
GROUP 1 6 Test Vaccine 5/6 ducks remained DHBV negative following
challenge GROUP 2 4 Sham Vaccine [Glutaraldehyde- 4/4 ducks became
DHBV inactivated DHBV (chemical kill)] positive following
challenge. GROUP 3 6 Mock Vaccine 6/6 ducks became DHBV (Control)
[Phosphate Buffered Saline (PBS)] positive following challenge.
[0143] 1. Glutaraldehyde Inactivation
[0144] Glutaraldehyde inactivation was achieved as known by those
of ordinary skill in the art by fixation with a dilute solution of
glutaraldehyde at about 1:250. Glutaraldehyde is a well known cross
linking agent.
[0145] 2. Delipidation Procedure
[0146] An organic solvent system was employed to perform
delipidation of serum. The solvent system consisted of a ratio of
40% butanol (analytical reagent grade) and 60% diisopropyl ether
and was mixed with the serum in a 2:1 ratio. Accordingly, 4 ml of
the organic solvent was mixed with 2 ml of the serum and rotated
for 1 hour. This mixture was centrifuged at approximately
400.times.g for 10 minutes followed by removal of the aqueous
phase. The aqueous phase was then mixed with an equal volume of
diethyl ether and centrifuged at 400.times.g for 10 minutes. Next,
the aqueous phase was removed and mixed with an equal volume of
diethyl ether and rotated end-over-end at 30 rpm for about 1 hour,
and centrifuged at 400.times.g for 10 minutes. The aqueous phase
was removed and the residual diethyl ether was removed through
evaporation in a fume cabinet for approximately 10 to 30 minutes.
The treated serum remained following removal of diethyl ether and
was used to produce the vaccine. The delipidation procedure control
involved subjecting the DHBV negative serum to the same
delipidation procedure as the DHBV positive serum.
[0147] 3. Vaccine Production
TABLE-US-00002 TABLE 2 First Dose Second Dose Third Dose (injected
with 200 .mu.l of (injected with 300 .mu.l (injected with 300 .mu.l
of respective vaccine into of respective vaccine respective vaccine
peritoneal cavity on Day 8 intramuscularly on intramuscularly on
Day 22 Vaccine Type post-hatch) Day 16 post-hatch) post-hatch) TEST
A 40 .mu.l aliquot of the A 40 .mu.l aliquot of the A 200 .mu.l
aliquot of the delipidated serum was mixed delipidated serum
delipidated serum was mixed with 1960 .mu.l of phosphate was mixed
with with 1800 .mu.l of PBS and then buffered saline (PBS) 1960
.mu.l of PBS and emulsified in 1000 .mu.l of Freund's then
emulsified in Incomplete Adjuvant. 1000 .mu.l of Freund's
Incomplete Adjuvant. SHAM A 200 .mu.l aliquot of DHBV A 200 .mu.l
aliquot of A 200 .mu.l aliquot of DHBV positiv$$ (DHBV positive
serum pool #4 DHBV positive serum serum pool #4 (20.4.99) was SERUM
(20.4.99) was mixed with pool #4 (20.4.99) was mixed with 300 .mu.l
of PBS and CONTROL) 300 .mu.l of PBS and 100 .mu.l of a mixed with
300 .mu.l of 100 .mu.l 2% glutaraldehyde solution PBS and 100 .mu.l
Aidal Plus (Whiteley Chemicals) (Aidal Plus from Whiteley Aidal
Plus (Whiteley and incubated for 10 minutes to Chemicals) and
incubated for Chemicals) and inactivate the DHBV. A 40 .mu.l 10
minutes to inactivate the incubated aliquot of the inactivated
DHBV. A 40 .mu.l aliquot of the for 10 minutes to serum/PBS mixture
was added to inactivated serum/PBS inactivate the DHBV. 1960 .mu.l
PBS and emulsified in mixture was added to 1960 .mu.l A 40 .mu.l
aliquot of the 1000 .mu.l Freunds Incomplete PBS. inactivated
serum/PBS Adjuvant. mixture was added to 1960 .mu.l PBS and
emulsified in 1000 .mu.l Freunds Incomplete Adjuvant. MOCK PBS A
2000 .mu.l aliquot of A 2000 .mu.l aliquot of PBS was (DHBV PBS was
emulsified emulsified in 1000 .mu.l Freunds NEGATIVE in 1000 .mu.l
Freunds Incomplete Adjuvant. CONTROL) Incomplete Adjuvant.
[0148] 4. Experimental Procedure
[0149] Ducks were challenged with 1000 .mu.l of DHBV positive serum
(serum pool 20.1.97) on day 29, post-hatch. Serum pool 20.1.97 was
shown to have 1.8.times.10.sup.10 genome equivalent (gev)/ml by
dot-blot hybridization. One genome equivalent (gev) is
approximately one viral particle. Ducks were bled prior to full
vaccination on days 1 and 10, prior to challenge on days 17 and 23,
and post challenge on days 37, 43 and 52. Their serum was tested
for DHBV DNA by dot-blot hybridization as described by Deva et al.
(1995). Ducks were euthanized on day 58 and their livers removed,
the DNA extracted and tested for the presence of DHBV by dot-blot
hybridization as described by Deva et al. (1995).
[0150] 5. Analysis of Results
[0151] a. Test Ducks [0152] i. Five of the 6 test ducks vaccinated
with the test vaccine remained negative for DHBV DNA in the serum
and liver following challenge. One test duck became positive for
DHBV following challenge.
[0153] b. Sham Vaccinated Ducks [0154] i. All 4 of the ducks
vaccinated with glutaraldehyde inactivated serum became DHBV
positive following challenge with DHBV.
[0155] c. Mock Vaccinated Ducks
[0156] i. Five of the 6 mock-vaccinated negative control ducks
became DHBV positive following challenge.
[0157] The Chi-square analysis was used to compare differences
between treatments. Significantly more control ducks (mock
vaccinated) became DHBV positive following challenge than the ducks
vaccinated with delipidated serum p<0.05).
[0158] Vaccination of ducklings with delipidated DHBV positive
serum using the above protocol resulted in prevention of DHBV
infection following challenge with DHBV positive serum in 5 of 6
ducklings. This suggests that the delipidated serum vaccine is
capable of inducing a positive immunogenic response in vaccinated
ducks. It is further believed that the delipidation process exposed
patient-specific antigens that were previously unexposed and/or
caused a structural change in the viral particle structure to
enable the positive immunogenic response. In comparison 5 of 6 mock
vaccinated and 4 of 4 sham-vaccinated ducks became DHBV positive
following vaccination suggesting no induction of immunity in these
ducks due to lack of immune response.
EXAMPLE 2
A. Delipidation of Cattle Pestivirus (Bovine Viral Diarrhea Virus,
BVDV), as a Modelfor Hepatitis C
[0159] A standard cattle pestivirus isolate (BVDV) was used in
these experiments. This isolate, "Numerella" BVD virus, was
isolated in 1987 from a diagnostic specimen submitted from a
typical case of `Mucosal Disease` on a farm in the Bega district of
New South Wales (NSW), Australia. This virus is non-cytopathogenic,
and reacts with all 12 of a panel of monoclonal antibodies raised
at the Elizabeth Macarthur Agricultural Institute (EMAI), NSW,
Australia, as typing reagents. Therefore, this virus represents a
`standard strain` of Australian BVD viruses.
[0160] The Numerella virus was grown in bovine MDBK cells tested
free of adventitious viral agents, including BVDV. The medium used
for viral growth contained 10% adult bovine serum derived from EMAI
cattle, all of which tested free of BVDV virus and BVDV antibodies.
This serum supplement has been employed for years to exclude the
possibility of adventitious BVDV contamination of test systems, a
common failing in laboratories worldwide that do not take
precautions to ensure the test virus is the only one in the culture
system. Using these tested culture systems ensured high-level
replication of the virus and a high yield of infectious virus.
Titration of the final viral yield after 5 days growth in MDBK
cells showed a titer of 10.sup.6.8 infectious viral particles per
ml of clarified (centrifuged) culture medium.
[0161] 1. Treating Infectious BVDV
[0162] 100 ml of tissue-culture supernatant, containing 10.sup.6.8
viral particles/ml, was harvested from a 150 cm.sup.2
tissue-culture flask. The supernatant was clarified by
centrifugation (cell debris pelleted at 3000 rpm, 10 min, 4.degree.
C.) and 10 ml set aside as a positive control for animal
inoculation (non-treated virus). The remaining 90 ml, containing
10.sup.7.75 infectious virus, was treated using the following
protocol: 180 ml of a solvent mixture butanol:diisopropyl ether
(DIPE) (2:1) was added to a 500 ml conical flask and mixed by
swirling. The mixture was then shaken for 60 min at 30 rpm at room
temperature on an orbital shaker. It was then centrifuged for 10
min at 400.times.g at 4.degree. C., after which the organic solvent
phase was removed and discarded. In subsequent steps, the bottom
layer (aqueous phase) was removed from beneath the organic phase,
improving yields considerably.
[0163] The aqueous phase, after the butanol:DIPE treatment, was
washed four times with an equal volume of fresh diethyl ether (DEE)
to remove all contaminating traces of butanol. After each washing,
the contents of the flask was swirled to ensure even mixing of both
aqueous and solvent phases before centrifugation as above
(400.times.g, 10 min, 4.degree. C.). After four washes, the aqueous
phase was placed in a sterile beaker covered with a sterile tissue
fixed to the top of the beaker with a rubber band to prevent
contamination and placed in a fume hood running continuously
overnight (16 hr) to remove all remaining volatile ether residue
from the inactivated viral preparation. Subsequent culture of the
treated material demonstrated no contamination. The treated viral
preparation was then stored at 4.degree. C. under sterile
conditions until inoculation into tissue culture or animals to test
for any remaining infectious virus.
[0164] 2. Testing of Treated BVDV Preparation
[0165] a. Tissue-Culture Inoculation
[0166] 2 ml of the solvent-treated virus preparation, expected to
contain about 10.sup.7.1 viral equivalents, was mixed with 8 ml
tissue-culture medium Minimal Eagles Medium (MEM) containing 10%
tested-free adult bovine serum and adsorbed for 60 min onto a
monolayer of MDBK cells in a 25 cm.sup.2 tissue-culture flask. As a
positive control, 2 ml of non-treated or substantially
lipid-containing infectious virus (also containing about 10.sup.7.1
viral equivalents) was similarly adsorbed on MDBK cells in a 25
cm.sup.2 tissue-culture flask. After 60 min, the supernatant was
removed from both flasks and replaced with normal growth medium
(+10% ABS). The cells were then grown for 5 days under standard
conditions before the MDBK cells were fixed and stained using a
standard immunoperoxidase protocol with a mixture of 6
BVDV-specific monoclonal antibodies (EMAI panel, reactive with 2
different BVD viral proteins).
[0167] There were no infected cells in the monolayer of MDBK cells
that was inoculated with the organic solvent treated virus. In
contrast, approximately 90% of the cells in the control flask (that
was inoculated with non-inactivated BVDV) were positive for virus
as shown by heavy, specific, immunoperoxidase staining. These
results showed that, under in vitro testing conditions, no
infectious virus remained in the treated, at least partially
delipidated BVDV preparation.
[0168] b. Animal Inoculation
[0169] An even more sensitive in vivo test is to inoculate naive
(antibody negative) cattle with the at least partially delipidated
virus preparation. As little as one infectious viral particle
injected subcutaneously in such animals is considered to be an
infectious cow dose, given that entry into cells and replication of
the virus is extremely efficient for BVDV. A group of 10
antibody-negative steers (10-12 months of age) were randomly
allocated to 3 groups.
[0170] The first group of 6 steers was used to test whether BVDV
had reduced infectivity. The same at least partially delipidated
preparation of BVDV described above was used in this example. Two
steers were inoculated with a vaccine having at least partially
delipidated viral particles to act as a positive control for the
vaccine group. These two positive control animals were run under
separate, quarantined conditions to prevent them from infecting
other animals when they developed a transient viraemia after
infection (normally at 4-7 days after receiving live BVDV virus).
The two remaining steers acted as negative "sentinel" animals to
ensure there was no naturally-occurring pestivirus transmission
within the vaccinated group of animals. Antibody levels were
measured in all 10 animals using a validated, competitive ELISA
developed at EMAI. This test has been independently validated by
CSL Ltd and is marketed by IDEXX Scandinavia in Europe.
[0171] The six animals in the first group each received a
subcutaneous injection of 4.5 ml of the at least partially
delipidated BVDV preparation, incorporated in a commercial
adjuvant. Since each ml of the at least partially delipidated
preparation contained 10.sup.6.8 viral equivalents, the total viral
load before the delipidation process was 10.sup.7.4 tissue culture
infectious doses (TCID).sub.50. The positive-control animals
received 5 ml each of the non-delipidated preparation, that is,
10.sup.7.5 TCID.sub.50 injected subcutaneously in the same way as
for the first group. The remaining two `sentinel` animals were not
given any viral antigens, having been grazed with the first group
of animals throughout the trial to ensure there was no natural
pestivirus activity occurring in the group while the trial took
place.
[0172] There was no antibody development in any of the vaccinated
steers receiving the at least partially delipidated BVD virus
preparation until a second dose of vaccine was given. Thus, at 2
and 4 weeks after a single dose, none of the 6 steers seroconverted
showing that there was no infectious virus left in a total volume
of 27 ml of the at least partially delipidated virus preparation.
This is the equivalent of a total inactivation of 10.sup.8.2
TCID.sub.50. In contrast, there were high levels of both anti-E2
antibodies (neutralizing antibodies) and anti-NS3 antibodies at
both 2 and 4 weeks after inoculation in the two steers receiving 5
ml each of the viral preparation prior to delipidation. This
confirmed the infectious nature of the virus prior to delipidation.
These in vivo results confirm the findings of the in vitro
tissue-culture test. The two `sentinel` animals remained
seronegative throughout, showing the herd remained free of natural
pestivirus infections.
[0173] The panel of monoclonal antibodies used detected host
antibodies directed against the major envelope glycoprotein (E2),
which is a glycoprotein incorporated in the lipid envelope of the
intact virus. The test systems also detected antibodies directed
against the non-structural protein, NS3 that is made within cells
infected by the virus. This protein has major regulatory roles in
viral replication and is not present within the infectious virus.
There was no evidence in the vaccinated cattle that infectious
virus was present, indicating all infectious viral particles had
been destroyed. All pestiviruses are RNA viruses. Therefore, there
was no viral DNA present in the delipidated preparation. These
results demonstrate the efficacy of the present method to at least
partially delipidate virus such that substantially no infectious
virus is found in animals receiving the delipidated virus.
B. Delipidated BVDV Preparation as a Vaccine in Steers
[0174] All six steers that had received an initial dose of 4.5 ml
of the at least partially delipidated BVDV preparation described in
above in Section A were again injected subcutaneously with a
similar dose at 4 weeks after the first priming dose. At this time
there were no antibody responses after the initial dose. It is
normal for an animal to react after the second dose. Strong
secondary immune responses for anti-E2 antibody levels (equivalent
to serum neutralizing antibodies SNT) were observed in 3 of the 6
steers at 2 weeks after the second dose of the at least partially
delipidated virus. This response was more than 70% inhibition in a
competitive ELISA. The remaining 3 animals showed weak antibody
responses (23-31% inhibition).
[0175] In contrast to the anti-E2 antibody responses, only one
animal developed a strong anti-NS3 antibody response (93%
inhibition) at 2 weeks after the second dose of at least partially
delipidated BVDV. A second animal had a weak anti-NS3 response (29%
inhibition) and four animals showed no antibody following
administration of 2 doses. This was not unexpected since similar
responses following administration of at least partially
delipidated BVDV vaccines have been observed previously. The
antibody levels in steers following 2 doses of the at least
partially delipidated BVDV preparation demonstrate its potential as
a vaccine since antiE2 antibody levels were measurable in all 6
vaccinated steers at 2 weeks after the second dose.
EXAMPLE 3
[0176] Use of Delipidated SIV to Induce or Augment SIV Specific
Humoral and CD4+ T Cell Memory Responses in Mice--a Modelfor a New
Auto-Vaccination Strategy against Lentiviral Infection
[0177] The following studies focused on the simian equivalent of
human HIV, termed SIV. The purpose was to utilize delipidated
SIVmac251 (an uncloned highly pathogenic isolate of SIV) to carry
out studies to determine the relative immunogenicity of the
delipidated virus in mice. The complete nucleotide sequence of an
infectious clone of simian immunodeficiency virus of macaques,
SIVmac239, has been determined. Virus produced from this molecular
clone causes AIDS in rhesus monkeys in a time frame suitable for
laboratory investigation. The proviral genome including both long
terminal repeats is 10,279 base pairs in length and contains open
reading frames for gag, pol, vif, vpr, vpx, tat, rev, and env. The
nef gene contains an in-frame premature stop after the 92nd codon.
At the nucleotide level, SIVmac239 is closely related to SIVmac251
(98%) and SIVmac142 (96%). (Regier DA, Desrosiers Annual Review
Immunology. 1990;8:55-78.)
[0178] Experiments were performed to determine the minimal dose of
delipidated simian immunodeficiency virus (SIV) that would produce
a readily recognizable boosting of the virus specific humoral
and/or cellular immune response in previously primed Balb/c mice.
All experiments were carried out in a BSL3 facility.
[0179] The immunogenicity of the delipidated virus preparation was
compared with an aliquot of the same virus in its native form. The
quality (titer of antibody, the conformational and linear epitope
specificity of the antibody, the isotype content of the antibody
and the function of the antibody) and quantity of antibody induced
by immunization of mice with equivalent protein amounts of the
non-delipidated and delipidated virus preparation were ascertained
as described below. Total protein from an aliquot of wild type
virus and total protein recovered following delipidation of the
same aliquot of virus were determined using standard quantitative
protein assay (Biorad, BCA kit assay, Rockford, Illinois). The
total protein profile was determined using SDS-PAGE analysis of the
wild type virus and the delipidated virus preparation and the
relative epitope preservation was ascertained by Western Blot
comparison of wild type with delipidated virus.
[0180] Equivalent protein amounts of the chemically treated wild
type and the delipidated virus were analyzed for their ability to
boost virus specific immune response in groups of mice. The sera
from these immunized mice were assayed by ELISA and Western Blot
analysis for reactivity against native wild type and for comparison
the delipidated virus preparation. Spleen cells were assayed for
their CD4 and CD8 SIV virus env and gag specific immune response
enhancing capacity as outlined below. Standard statistical analyses
were performed for the analysis of the data.
[0181] Four to six week old healthy female Balb/c mice from the
Jackson labs, Bar Harbor, Me were purchased and housed in the
BSL2/3 mouse housing facility at Emory University. Twenty Balb/c
mice were each immunized subcutaneously with 25 ug of protein of
2-2 dithiopyridine-inactivated SIVmac251 incorporated in an equal
volume of Freunds incomplete adjuvant.
[0182] A sufficient quantity of SIVmac251 was delipidated to
provide the amount needed for boosting these mice per schedule.
Delipidation consisted of incubating SIVmac251 with 10% DIPE in
phosphate buffered saline (PBS). 1.0 ml of a 10% DIPE solution in
PBS was prepared and mixed on a vortexer until it appeared
cloudy.
[0183] The virus preparation: A 1 ml tube from Advanced
Biotechnologies SIVmac251 was used as seed stock (Sucrose Gradient
Purified Virus 1 mg/ml). The supplier reported a titer of
10.sup.6.7 with total protein of 1.074 mg/mL (Pierce BCA protein
method) and virus particle count of 6.95.sup.10/ml (EM). It was
confirmed that the virus had a titer of 10.sup.7.0 using CEMx174,
the first time as a rapid assay, and the second time in
quadruplicate cultures/dilution. A measurement of p27 in this
preparation revealed a value of 106 ug/ml. Next, 25 .mu.l of the
undiluted viral stock was introduced into 0.6 ml clear snap-cap
polypropylene Eppendorf tube.1 Then, 2.5 .mu.l of 10% DIPE solution
was added into the Eppendorf tube containing virus and vortexed for
15 seconds. The tube was spun (using an Eppendorf 5810R centrifuge)
at room temp at 1000.times.g for 2 minutes. No bulk solvent was
removed. The solvent was removed by vacuum centrifugation (Speedvac
Concentrator Model SVC200H) at 2000 rpm with no heat for 30
minutes. The volume in the tube was adjusted to 25 .mu.l with PBS.
Total protein recovery was measured using a Pierce BCA protocol.
Gels (12% SDS-PAGE) were employed for specific protein recoveries
(env protein, pol protein, gp41, p27 and gag protein) and stained
with Coomasie Blue and provided semi-quantitative results using OD.
Western blots were run using serum from SIV-infected monkeys to
measure envelope protein, gp66, gp41, p27, gag, and p6 gag. The
viral infectivity of the preparation was determined using a
luciferase assay and CEM-174 cells. The virus titer was 10.sup.4.5,
a 2.5 log reduction from that measured in undelipidated stock. This
delipidated SIV preparation appears to retain greater than 90% of
the major protein constituents of SIVmac251 such as the gag and env
proteins.
[0184] Next, the immunogenicity of the modified viral preparation
was determined in the twenty adult female Balb/c mice described
above that were each immunized subcutaneously with 25 ug of protein
of 2-2 dithiopyridine-inactivated SIVmac251. On day 14, groups 3-6
were boosted with 10 ug to 0.01 ug (based on total protein of
stock) of delipidated virus in 0.5 ml normal saline. The estimated
actual virus protein content was equal to 1/10 that of total
protein based on the ratio of total protein/p27 protein in stock.
The mice were injected with the delipidated vaccine composition as
follows:
TABLE-US-00003 TABLE 3 Initial Immunization s.c. 2-2
dithiopyridine- Groups (containing 4 inactivated Day 14 - Booster
mice each) SIVmac251 Injections i.v. GROUP 1 - Control
Non-immunized Administered-saline without delipidated virus GROUP 2
Immunized Not administered GROUP 3 Immunized 0.5 ml saline + 10 ug
of delipidated virus GROUP 4 Immunized 0.5 ml saline + 1.0 ug of
delipidated virus GROUP 5 Immunized 0.5 ml saline + 0.1 ug of
delipidated virus GROUP 6 Immunized 0.5 ml saline + 0.01 ug of
delipidated virus
Four days after the booster injection, the mice were anesthetized
and blood was collected via retro-orbital puncture and
intra-cardiac puncture. About 0.5 ml of blood was collected from
each mouse, primarily from intra-cardiac puncture. The blood was
permitted to clot at room temperature. The spleen of each mouse was
aseptically removed and transported to the lab under double bag
containment. The clotted blood from each mouse was centrifuged at
about 450.times.g at room temperature, and serum was collected from
tube, transferred to a sterile tube, and stored at -70.degree. C.
until use. ELISA was performed to determine antibody titers against
SIV for each serum sample.
SIV ELISA Protocol
[0185] Stocks of positive and negative serum and fluids to be
tested were frozen in aliquots to be used on every plate to
standardize each run.
[0186] Coated Corning Easy-Plates were washed with 100 ul per well
of poly-1-lysine at a concentration of 10 ug per ml of PBS, pH
7.2-7.4. Plates were covered and incubated overnight at 4.degree.
C. Several plates were coated at one time and stored for subsequent
use. Next, excess polylysine was removed and the plate dried for a
few minutes. About 100 ul of 2% Triton-X was added to 100 ul of the
stock ABI SiVmac251 the samples sat for 5 minutes. Next, 50 ul of
coating buffer of pH 9.6 was added. Next, 100 ul of the viral
antigen was added to each well of 5 plates, which were covered and
incubated at 4.degree. C. overnight.
[0187] After the overnight incubation, wells were washed 3 times
with PBS-T. The wells then received 200 ul per well of 2% nonfat
dry milk in PBS for one hour at room temperature to block
non-specific binding. Excess fluid was removed. About 100 ul of
test or control serum diluted at 1/100 in 10% RPMI 1640 or PBS with
10% calf serum was added to duplicate wells and incubated for 2
hours at 37.degree. C. Wells were washed 4 times with PBS-T. Next
100 ul of Southern Biotech (from Fisher) alkaline phosphatase anti
Mouse IgG ( diluted 1/800 in media or PBS with 10% calf serum) was
added and incubated 1 hour at 37.degree. C. Wells were washed 4
times with PBS-T.
[0188] The BIORAD Alkaline Phosphatase Substrate kit was used to
develop a reaction product. One substrate tablet was added for each
5 ml of 1.times. buffer and mixed. Next 100 ul was added per well
and evaluated at about 5, 10, 15, 30 and then at 1 hour intervals
for color development.
[0189] Blank readings were obtained from the media controls when
the positive control was above 1.500 and the negative control was
0.100 to 0.200 for the serum. The results were then recorded and
the means and the standard deviations of the negative control,
positive control and the experimental samples were calculated. The
negative cutoff value was the mean of the negative control plus
0.150.
Immunogenicity Results
[0190] The immunogenicity of the delipidated SIV virus preparation
in mice was examined with an ELISA assay. The mean optical density
(O.D.) was examined at 405 nm at various dilutions of serum. Table
4 provides the results of the ELISA test on serum samples.
TABLE-US-00004 TABLE 4 Serum No 10 dil. boost ug boost 1 ug boost
0.1 ug boost 0.01 ug boost 1/100 2.541 3.663 3.289 2.846 2.627
1/500 1.035 2.86 2.055 1.458 1.257 1/2500 0.449 1.239 0.855 0.601
0.445 1/12500 0.194 0.463 0.304 0.229 0.181 1/62500 0.127 0.151
0.153 0.129 0.123 1/312500 0.11 0.116 0.108 0.108 0.107
Analysis of Responses of Dissociated Spleen Cells Obtained from
Immunized Mice
[0191] A single cell suspension of spleen cells was prepared from
each individual mouse by gently teasing the splenic capsule and
passing the cells through a 25 gauge needle. Spleen cells were
dissociated into a single cell suspension in medium (RPMI 1640
supplemented with 100 ug/ml penicillin, 100 ug/ml streptomycin, 2
mM L-glutamine), washed twice in medium and subsequently adjusted
to 10 million cells/ml. 0.1 ml of this cell suspension from each
mouse was dispensed into each well of a 96 well round bottom
microtiter plate containing medium. Remaining cells were
cryopreserved. These spleen cell cultures were then assessed for
the ability of CD4+ and CD8+ T cells to synthesize IFN-gamma by
standard intracellular cytokine staining (ICC) and flow
cytometry.
[0192] Two individual wells containing the duplicate cell cultures
from an individual mouse received either a) 0.1 ml of medium
containing 2 ug/ml of each of a pool of 9 SIV envelope (SE)
peptides (n=14 pools), or b) 0.1 ml of medium containing a pool of
7 SIV gag (SG) peptides (n=17 pools). Each pool contained 2 ug/ml
of 7 peptides each for SIV env and SIV gag. Controls consisted of
spleen cell cultures that received media alone (background control)
or a previously determined optimum concentration of phorbol
myristic acetate (PMA 1 ug/ml)+ionomycin (0.25 ug/ml) for maximal
IFN-gamma staining (positive control). The SIVenv peptides (n=49
individual peptides) were mixed in a grid fashion of a 7.times.7
matrix and the SIV gag peptides (n=72 peptides) were mixed in a
grid fashion of a 9.times.8 matrix which permitted identification
of individual peptide specific immune responses. The SIV env and
gag peptides were synthetic 20 mer peptides that overlapped each
other by 12 amino acids and encompassed the entire SIV env and gag
sequence. Peptide pools were made to contain 2.0 ug/ml of each
peptide. For each spleen cell preparation there were 36 wells of
culture. The components of the 9 pools and 7 pools of env and gag
overlapping peptides are described below. Shown are the peptides
that compose the pools with their respective position within
SiVmac239gag (SG) and env (SE).
TABLE-US-00005 TABLE 5 Pool arrangement of individual SIVmac239 env
peptides. 7 SG Peptide pools SG18 1 2 3 4 5 6 7 SG19 8 9 10 11 12
13 14 SG20 15 16 17 G-6* G-5* 20 21 SG21 22 23 24 25 26 27 28 SG22
29 30 31 32 33 34 35 SG23 36 37 38 39 40 41 42 SG24 43 44 45 46 47
48 49
TABLE-US-00006 TABLE 6 Pool arrangement of individual SIVmac239 env
peptides. 9 SE Peptide pools SE9 1 2 3 4 5 6 7 8 SE10 9 10 11 12 13
14 15 16 SE11 17 18 19 20 21 22 23 24 SE12 25 26 27 28 29 30 31 32
SE13 33 34 35 36 37 38 39 40 SE14 41 42 43 44 45 46 47 48 SE15 49
50 51 52 53 54 55 56 SE16 57 58 59 60 61 62 63 64 SE17 65 66 67 68
69 70 71 72
TABLE-US-00007 TABLE 7 SIVmac239 gag overlapping peptides for
epitope mapping SEQ ID NO: 1 MGVRNSVLSGKKADELEKIRLR SG1 1-22 SEQ ID
NO: 2 KKADELEKIRLRPNGKKKYMLK SG2 11-32 SEQ ID NO: 3
LRPNGKKKYMLKHVVWAANELD SG3 21-42 SEQ ID NO: 4
LKHVVWAANELDRFGLAESLLE SG4 31-52 SEQ ID NO: 5
LDRFGLAESLLENKEGCQKILS SG5 41-62 SEQ ID NO: 6
LENKEGCQKILSVLAPLVPTGS SG6 51-72 SEQ ID NO: 7
LSVLAPLVPTGSENLKSLYNTV SG7 61-82 SEQ ID NO: 8
GSENLKSLYNTVCVIWCIHAEE SG8 71-92 SEQ ID NO: 9
TVCVIWCIHAEEKVKHTEEAKQ SG9 81- 102 SEQ ID NO: 10
EEKVKHTEEAKQIVQRHLVVET SG10 91- 112 SEQ ID NO: 11
KQIVQRHLVVETGTTETMPKTS SG11 101- 122 SEQ ID NO: 12
ETGTTETMPKTSRPTAPSSGRG SG12 111- 132 SEQ ID NO: 13
TSRPTAPSSGRGGNYPVQQIGG SG13 121- 142 SEQ ID NO: 14
RGGNYPVQQIGGNYVHLPLSPR SG14 131- 152 SEQ ID NO: 15
GGNYVHLPLSPRTLNAWVKLIE SG15 141- 162 SEQ ID NO: 16
PRTLNAWVKLIEEKKFGAEVVP SG16 151- 172 SEQ ID NO: 17
IEEKKFGAEVVPGFQALSEGCT SG17 161- 182 SEQ ID NO: 18
VPGFQALSEGCTPYDINQMLNCVGD G-6 171- 195* SEQ ID NO: 19
GCTPYDINQMLNCVGDHQAA G-5 180- 199* SEQ ID NO: 20
NCVGDHQAAMQIIRDIINEEAAD SG20 191- 213 SEQ ID NO: 21
IIRDIINEEAADWDLQHPQPAP SG21 202- 223 SEQ ID NO: 22
ADWDLQHPQPAPQQGQLREPSG SG22 212- 233 SEQ ID NO: 23
APQQGQLREPSGSDIAGTTSSV SG23 222- 243 SEQ ID NO: 24
SGSDIAGTTSSVDEQIQWMYRQ SG24 232- 253 SEQ ID NO: 25
SVDEQIQWMYRQQNPIPVGNIY SG25 242- 263* (*) SEQ ID NO: 26
RQQNPIPVGNIYRRWIQLGLQK SG26 252- 273(*) SEQ ID NO: 27
IYRRWIQLGLQKCVRMYNPTNIL SG27 262- 284(*) SEQ ID NO: 28
KCVRMYNPTNILDVKQGPKEPF SG28 273- 294 SEQ ID NO: 29
ILDVKQGPKEPFQSYVDRFYKS SG29 283- 304 SEQ ID NO: 30
PFQSYVDRFYKSLRAEQTDAAV SG30 293- 314 SEQ ID NO: 31
KSLRAEQTDAAVKNWMTQTLLI SG31 303- 324 SEQ ID NO: 32
AVKNWMTQTLLIQNANPDCKLV SG32 313- 334 SEQ ID NO: 33
LIQNANPDCKLVLKGLGVNPTL SG33 323- 344 SEQ ID NO: 34
LVLKGLGVNPTLEEMLTACQGV SG34 333- 354 SEQ ID NO: 35
TLEEMLTACQGVGGPGQKARLM SG35 343- 364 SEQ ID NO: 36
GVGGPGQKARLMAEALKEALAP SG36 353- 374 SEQ ID NO: 37
LMAEALKEALAPVPIPFAAAQQ SG37 363- 384 SEQ ID NO: 38
APVPIPFAAAQQRGPRKPIKCW SG38 373- 394 SEQ ID NO: 39
AQQRGPRKPIKCWNCGKEGHSA SG39 382- 403 SEQ ID NO: 40
KCWNCGKEGHSARQCRAPRRQG SG40 392- 413 SEQ ID NO: 41
SARQCRAPRRQGCWKCGKMDHV SG41 402- 423 SEQ ID NO: 42
RQGCWKCGKMDHVMAKCPDRQAG SG42 411- 433 SEQ ID NO: 43
HVMAKCPDRQAGFLGLGPWGKK SG43 422- 443 SEQ ID NO: 44
AGFLGLGPWGKKPRNFPMAQVH SG44 432- 453 SEQ ID NO: 45
KKPRNFPMAQVHQGLMPTAPPE SG45 442- 463 SEQ ID NO: 46
VHQGLMPTAPPEDPAVDLLKNY SG46 452- 473 SEQ ID NO: 47
PEDPAVDLLKNYMQLGKQQREK SG47 462- 483 SEQ ID NO: 48
NYMQLGKQQREKQRESREKPYK SG48 472- 493 SEQ ID NO: 49
EKQRESREKPYKEVTEDLLHLN SG49 482- 503 SEQ ID NO: 50
YKEVTEDLLHLNSLFGGDQ SG50 492- 510 *denotes peptides containing
defined or (*)semi defined gag epitopes (156-158)
TABLE-US-00008 TABLE 8 Overlapping peptides in Env of SIVmac239
(25-mer with 13-mer overlapping) SEQ ID MGCLGNQLLIAILLLSVYGIYCTLY
SE1 1-25 NO: 51 SEQ ID LLLSVYGIYCTLYVTVFYGVPAWRN SE2 13-37 NO: 52
SEQ ID YVTVFYGVPAWRNATIPLFCATKNR SE3 25-49 NO: 53 SEQ ID
NATIPLFCATKNRDTWGTTQCLPDN SE4 37-61 NO: 54 SEQ ID
RDTWGTTQCLPDNGDYSEVALNVTE SE5 49-73 NO: 55 SEQ ID
NGDYSEVALNVTESFDAWNNTVTEQ SE6 61-85 NO: 56 SEQ ID
ESFDAWNNTVTEQAIEDVWQLFETS SE7 73-97 NO: 57 SEQ ID
QAIEDVWQLFETSIKPCVKLSPLCI SE8 85-109 NO: 58 SEQ ID
SIKPCVKLSPLCITMRCNKSETDRW SE9 97-121 NO: 59 SEQ ID
TMRCNKSETDRWGLTKSITTTAST SE10 109-133 NO: 60 SEQ ID
WGLTKSITTTASTTSTTASAKVDMV SE11 121-145 NO: 61 SEQ ID
TTSTTASAKVDMVNETSSCIAQDNC SE12 133-157 NO: 62 SEQ ID
VNETSSCIAQDNCTGLEQEQMISCK SE13 145-169 NO: 63 SEQ ID
CTGLEQEQMISCKFNMTGLKRDKKK SE14 157-181 NO: 64 SEQ ID
KFNMTGLKRDKKKEYNETWYSADLV SE15 169-193 NO: 65 SEQ ID
KEYNETWYSADLVCEQGNNTGNESR SE16 181-205 NO: 66 SEQ ID
VCEQGNNTGNESRCYMNHCNTSVIQ SE17 193-217 NO: 67 SEQ ID
RCYMNHCNTSVIQESCDKHYWDAIR SE18 205-229 NO: 68 SEQ ID
QESCDKHYWDAIRFRYCAPPGYALL SE19 217-241 NO: 69 SEQ ID
RFRYCAPPGYALLRCNDTNYSGFMP SE20 229-253 NO: 70 SEQ ID
LRCNDTNYSGFMPKCSKVVVSSCTR SE21 241-265 NO: 71 SEQ ID
PKCSKVVVSSCTRMMETQTSTWFGF SE22 253-277 NO: 72 SEQ ID
RMMETQTSTWFGFNGTRAENRTYIY SE23 265-289 NO: 73 SEQ ID
FNGTRAENRTYIYWHGRDNRTIISL SE24 277-301 NO: 74 SEQ ID
YWHGRDNRTIISLNKYYNLTMKCRR SE25 289-313 NO: 75 SEQ ID
LNKYYNLTMKCRRPGNKTVLPVTIM SE26 301-325 NO: 76 SEQ ID
RPGNKTVLPVTIMSGLVFHSQPIND SE27 313-337 NO: 77 SEQ ID
MSGLVFHSQPINDRPKQAWCWFGGK SE28 325-349 NO: 78 SEQ ID
DRPKQAWCWFGGKWKDAIKEVKQTI SE29 337-361 NO: 79 SEQ ID
KWKDAIKEVKQTIVKHPRYTGTNNT SE30 349-373 NO: 80 SEQ ID
IVKHPRYTGTNNTDKINLTAPGGGD SE31 361-385 NO: 81 SEQ ID
TDKINLTAPGGGDPEVTFMWTNCRG SE32 373-397 NO: 82 SEQ ID
DPEVTFMWTNCRGEFLYCKMNWFLN SE33 385-409 NO: 83 SEQ ID
GEFLYCKMNWFLNWVEDRNTANQKP SE34 397-421 NO: 84 SEQ ID
NWVEDRNTANQKPKEQHKRNYVPCH SE35 409-433 NO: 85 SEQ ID
PKEQHKRNYVPCHIRQIINTWHKVG SE36 421-445 NO: 86 SEQ ID
HIRQIINTWHKVGKNVYLPPREGDL SE37 433-457 NO: 87 SEQ ID
GKNVYLPPREGDLTCNSTVTSLIAN SE38 445-469 NO: 88 SEQ ID
LTCNSTVTSLIANIDWIDGNQTNIT SE39 457-481 NO: 89 SEQ ID
NIDWIDGNQTNITMSAEVAELYRLE SE40 469-493 NO: 90 SEQ ID
TMSAEVAELYRLELGDYKLVEITPI SE41 481-505 NO: 91 SEQ ID
ELGDYKLVEITPIGLAPTDVKRYTT SE42 493-517 NO: 92 SEQ ID
IGLAPTDVKRYTTGGTSRNKRGVFV SE43 505-529 NO: 93 SEQ ID
TGGTSRNKRGVFVLGFLGFLATAGS SE44 517-541 NO: 94 SEQ ID
VLGFLGFLATAGSAMGAASLTLTAQ SE45 529-553 NO: 95 SEQ ID
SAMGAASLTLTAQSRTLLAGIVQQQ SE46 541-565 NO: 96 SEQ ID
QSRTLLAGIVQQQQQLLDVVKRQQE SE47 553-577 NO: 97 SEQ ID
QQQLLDVVKRQQELLRLTVWGTKNL SE48 565-589 NO: 98 SEQ ID
ELLRLTVWGTKNLQTRVTAIEKYLK SE49 577-601 NO: 99 SEQ ID
LQTRVTAIEKYLKDQAQLNAWGCAF SE50 589-613 NO: 100 SEQ ID
KDQAQLNAWGCAFRQVCHTTVPWPN SE51 601-625 NO: 101 SEQ ID
FRQVCHTTVPWPNASLTPKWNNETW SE52 613-637 NO: 102 SEQ ID
NASLTPKWNNETWQEWERKVDFLEE SE53 625-649 NO: 103 SEQ ID
WQEWERKVDFLEENITALLEEAQIQ SE54 637-661 NO: 104 SEQ ID
ENITALLEEAQIQQEKNMYELQKLN SE55 649-673 NO: 105 SEQ ID
QQEKNMYELQKLNSWDVFGNWFDLA SE56 661-685 NO: 106 SEQ ID
NSWDVFGNWFDLASWIKYIQYGVYI SE57 673-697 NO: 107 SEQ ID
ASWIKYIQYGVYIVVGVILLRIVIY SE58 685-709 NO: 108 SEQ ID
IVVGVILLRIVIYIVQMLAKLRQGY SE59 697-721 NO: 109 SEQ ID
YIVQMLAKLRQGYRPVFSSPPSYFQ SE60 709-733 NO: 110 SEQ ID
YRPVFSSPPSYFQQTHIQQDPALPT SE61 721-745 NO: 111 SEQ ID
QQTHIQQDPALPTREGKERDGGEGG SE62 733-757 NO: 112 SEQ ID
TREGKERDGGEGGGNSSWPWQIEYI SE63 745-769 NO: 113 SEQ ID
GGNSSWPWQIEYIHFLIRQLIRLLT SE64 757-781 NO: 114 SEQ ID
IHFLIRQLIRLLTWLFSNCRTLLSR SE65 769-793 NO: 115 SEQ ID
TWLFSNCRTLLSRVYQILQPILQRL SE66 781-805 NO: 116 SEQ ID
RVYQILQPILQRLSATLQRIREVLR SE67 793-817 NO: 117 SEQ ID
LSATLQRIREVLRTELTYLQYGWSY SE68 805-829 NO: 118 SEQ ID
RTELTYLQYGWSYFHEAVQAVWRSA SE69 817-841 NO: 119 SEQ ID
YFHEAVQAVWRSATETLAGAWGDLW SE70 829-853 NO: 120 SEQ ID
ATETLAGAWGDLWETLRRGGRWILA SE71 841-865 NO: 121 SEQ ID
WETLRRGGRWILAIPRRIRQGLELTLL SE72 853-877 NO: 122
The cultures were incubated overnight at 37.degree. C. in a 7%
CO.sub.2 humidified atmosphere. Cells from each well were gently
removed, transferred to 5.0 ml FACS test tubes and washed. One set
of cells was stained with anti-CD3+ anti-CD4+. The other duplicate
set was stained with anti-CD3+ anti-CD8+ (see below). These cell
surface stained cells were then permeabilized and stained for
intracellular content of IFN-gamma using an anti-IFN-gamma staining
antibody using standard intracellular staining protocols. Each
stained cell population (about 10,000 cells from each tube) was
then analyzed using a FACS flow cytometer and the frequency of CD3+
CD4+ and CD3+ CD8+ T cells synthesizing IFN-gamma was determined.
The negative and positive controls were utilized for background
control and for positive control references. About 1000 analyses
were performed in this manner during this experiment.
[0193] The frequency of CD4+ T cells (y axis) that expressed
IFN-gamma by spleen cells from the six groups of mice in response
to pools of SIV env peptide (9 pools) and SIV gag peptides (7
pools) were determined. Also determined was the frequency of CD8+ T
cells (y axis) that express IFN-gamma by spleen cells from the same
six groups of mice in response to pools of SIV env peptide (9
pools) and SIV gag peptides (7 pools). Data were the mean value
from 4 mice/group. Results of these initial studies indicated that
delipidated SIVmac251 at a dose of 10 ug or 1.0 ug led to marked
augmentation of the SIV specific humoral responses in previously
primed BALB/c mice. Even a dose of 0.1 ug (5.times.10.sup.6 viral
particles) led to detectable enhancement of the SIV specific
humoral responses in these mice. A dose of 1.0 ug, but not 10 ug,
led to markedly broad breadth of SIV env and SIV gag peptide
specific CD4+ T cell responses as measured by IFN-g synthesis in
previously primed BALB/c mice.
EXAMPLE 4
[0194] Charcoal Removal of Solvents after Plasma Delipidation
[0195] A charcoal column was generated by loading 2 ml of
PBS-washed Hemasorba charcoal into 3-ml BD LuerLock syringe
containing a Whatman filter frit. The column was washed with 5%
glucose/PBS (5 to 10 column volumes). The column was incubated in
5% glucose/PBS for 30 min. This column was used to remove solvents
from treated plasma.
[0196] About 2 ml of freshly isolated human plasma (ACD) was mixed
with 1 ml of one the following solvents: 1% DIPE; 10% DIPE; or
butanol/DIPE (25:75). The mixture was vortexed for 15 seconds and
then centrifuged 5 min at 3000 rpm (.about.1000.times.g). The
solvent layer was aspirated. The plasma was passed through the
charcoal column described in the preceding paragraph. About 0.5 ml
of PBS was used to wash the column. Washing may occur several times
as needed. The results are shown in Table 9. Total cholesterol
(TC), triglycerides (TG), phospholipid (PL), apolipoprotein Al
(ApoAl), apolipoprotein B (ApoB) and HDL were measured. The results
show good recoveries of ApoAl, ApoB and HDL compared to
controls
TABLE-US-00009 TABLE 9 Analysis of Plasma Delipidated and Passed
Through Charcoal Syringe Columns Sample TC TG PL ApoA1 ApoB HDL
Assay CSI 132.8 83.5 154.2 106.0 60.5 17.3 Assay CSII 197.4 164.7
224.0 87.2 107.7 21.3 Control 1 66.2 76.0 57.4 47.8 31.5 10.5
Control 2 103.6 113.2 98.7 79.6 48.8 15.6 1% DIPE (1 pass) 48.1
59.2 38.6 39.4 15.6 10.1 1% DIPE (2 pass) 40.1 46.5 29.3 26.0 15.7
6.8 10% DIPE (1 pass) 56.9 60.3 45.0 37.0 20.1 8.9 10% DIPE (2
pass) 58.9 61.7 52.4 42.2 25.5 8.5 But/DIPE (1 pass) 57.4 65.0 54.1
47.7 24.8 9.1 But/DIPE (2 pass) 81.7 84.4 73.8 53.8 34.2 9.2
Plasma Virus Recovery After Passage through Charcoal Column
[0197] Freshly isolated human plasma (ACD) was combined with HIV-1
to 1 ug/ml p24. HIV was added to the plasma such that the final
concentration or particle content was 1 ug/ml of virus p24 antigen.
Next, 1 ml of this plasma was passed through the column followed by
1 ml of PBS wash. The flow through and wash were combined. This
procedure was repeated twice on fresh columns using 1 ml of the
plasma. The flow through and wash from each of these three runs
were analyzed separately. The results showed excellent recovery of
p24 from the columns. P24 was measured by a standard capture ELISA
protocol with a monoclonal antibody coated plate (for capture) and
a polyclonal antibody for detection. Standard curves with known
amounts of p24 are used to determined the p24 content of
unknowns.
Direct Delipidation of HIV-1 and Removal of Solvents with Charcoal
Column and Retention of HIV Proteins
[0198] About 25 ul of 1000.times. HIV-1 IIIB was mixed with 1)
nothing; 2) 12.5 ul butanol/DIPE (25:75); 3) 2.5 ul 100% DIPE; or
4) 12.5 ul 1% DIPE in PBS and the samples were vortexed for 15
seconds. Charcoal columns (0.5-ml) were prepared as described
above. The virus-solvent mixtures were loaded individually onto
separate columns. The columns were eluted with 1 ml of PBS. The
elution volumes were measured and samples assayed for p24 by ELISA,
protein, and subjected to Western blotting.
[0199] The samples treated with 1% DIPE showed excellent p24
recovery compared to controls. The samples treated with 10% DIPE or
butanol/DIPE showed slightly less p24 recovery. The total protein
recovery was similar in terms of percentage relative to control, to
the p24 results obtained 1% DIPE, 10% DIPE or butanol/DIPE.
[0200] Western blot analysis, performed in a similar manner to the
protocol provided below in this example, revealed numerous
immunoreactive bands when probed with human anti-HIV IgG with
butanol/DIPE, 10% DIPE or 1% DIPE solvent treatments. Western blot
analysis also revealed positive immunoreactive bands corresponding
to p24 with butanol/DIPE, 10% DIPE or 1% DIPE. Positive
immunoreactive bands were observed for gp41 using 10% DIPE or 1%
DIPE. Additional positive immunoreactive bands were observed for
gp120 with butanol/DIPE, 10% DIPE or 1%DIPE, although the intensity
of staining was higher with 10% DIPE or 1% DIPE.
SIV and HIV Western Blot Analysis
[0201] Reagents for comparison included delipidated SIVmac251, heat
inactivated SIVmac251 and a rabbit polyclonal antibody against
whole SIV (available through the AIDS reagent repository,
Rockville, Md.). About 1 ug of protein was required to visualize
most of the SIV bands in the Western blot. SDS-polyacrylamide gel
electrophoresis (SDS-PAGE) was performed on the viral lysates
(lysate buffer:50 mM Tris-HCI, pH 7.4; 1% NP-40; 0.25% sodium
deoxycholate; 150 mM NaCl; 1 mM EGTA; 1 mM PMSF; 1 ug/ml each of
aprotinin, leupeptin and pepstatin; 1 mM sodium vanadate; 1 mM
NaF).
[0202] A silver stain was used to visualize the bands which reveal
the various viral proteins present following delipidation with
respect to molecular weight standards. The heat inactivated
SIVmac251 proteins were compared with the delipidated SIVmac251
proteins on the gels. A similar SDS-PAGE was run and the proteins
are transferred to nitrocellulose. The blotted nitrocellulose was
washed twice with water. A minimum of three blots each for the
delipidated SIVmac251 and the heat inactivated SIVmac251 were
run.
[0203] The blotted nitrocellulose was blocked in freshly prepared
PBS containing 3% nonfat dry milk (MLK) for 20 min at 20-25.degree.
C. with constant agitation. The nitrocellulose strips were
incubated with a freshly prepared pre-determined optimum
concentration of the rabbit polyclonal anti-SIV antiserum (about 5
ml of a 1:1000 dilution of the antiserum in PBS-MLK) overnight with
agitation. The nitrocellulose strips were washed twice with water.
The strips were incubated with horseradish peroxidases
(HRP)-conjugated goat anti-rabbit IgG 1:3000 dilution in PBS-MLK
for 90 min at room temperature with agitation. The nitrocellulose
was washed with water twice and then with PBS-0.05% Tween 20 for
3-5 min. The nitrocellulose strips were washed with 4-5 changes of
water. Detection of the developed bands was achieved via detection
of the developed bands. The bands developed using the heat
inactivated SIV with the delipidated SIV were compared.
[0204] A similar approach was used for Western blot analysis of
solvent treated HIV-1 passed through charcoal columns and probed
for p24, gp41, gp120, and also for HIV antigens using an human
anti-HIV IgG. Western blotting was performed on SDS-PAGE separated
virus samples transferred onto nitrocellulose membranes. The
membranes are probed with polyclonal and monoclonal antibodies to
viral proteins and developed with secondary antibodies conjugated
with peroxidase and enhanced chemiluminescence reagents.
EXAMPLE 5
Use of a Kit for Delipidation of a Plasma Sample Containing HIV and
Production of Delipidated HIV Viral Particles
[0205] A 200 ml plasma sample, stored in a plasma bag with a tube
connected to an opening in the bag, is obtained from blood drawn
from a 22 year old patient afflicted with the human
immunodeficiency virus (HIV) and showing symptoms of acquired
immunodeficiency syndrome (AIDS). The patient requires a reduction
in the viral load in the blood. The plasma sample is exposed to a
first extraction solvent to remove lipid from the viral envelope of
the HIV virus.
[0206] A first container with a 500 ml capacity is removed from the
kit. The first container, which is graduated, contains a known
volume (about 200 ml) of a first extraction solvent. The entire
plasma sample is added to the first container through a removable
screw cap. The first container is agitated through repeated
inversion, thereby mixing the first extraction solvent and the
plasma sample sufficiently to create a mixture. The first container
is placed on a counter and the mixture settles into two phases.
[0207] The delipidated plasma phase is removed with a manual
pipette or a pipette connected to a vacuum, and placed in a second
container from the kit. The volatile components of the first
extraction solvent evaporate. Mild heating may be employed at this
step. A tube, obtained from the kit, is inserted into the second
container. The tube serves as, or is connected to, an intravascular
line leading to a needle introduced into the antecubital vein of
the patient. The delipidated fluid containing delipidated plasma
and delipidated HIV viral particles with reduced infectivity is
introduced into the vascular system through the force of gravity by
elevating the second container above the patient. The needle is
optionally obtained from the kit. Administration of the delipidated
HIV viral particles into the vascular system induces an immune
response in the patient to epitopes on the delipidated HIV viral
particles.
EXAMPLE 6
Use of a Kit for Delipidation of a Plasma Sample Containing HIV and
Production of Delipidated HIV Viral Particles
[0208] A 200 ml plasma sample, stored in a plasma bag with a tube
connected to an opening in the bag, is obtained from blood drawn
from a 22 year old patient afflicted with the human
immunodeficiency virus (HIV) and showing symptoms of AIDS. The
patient requires a reduction in the viral load in the blood. The
plasma sample is exposed to a first extraction solvent to remove
lipid from the viral envelope of the HIV virus.
[0209] A first container with a 500 ml capacity is removed from the
kit. The first container, which is graduated, contains a known
volume (about 200 ml) of a first extraction solvent. The entire
plasma sample is added to a second container through a removable
screw cap. The contents of the first container and the second
container are added to a third container obtained from the kit. The
third container is agitated through repeated inversion, thereby
mixing the first extraction solvent and the plasma sample
sufficiently to create a mixture. The third container is placed on
a counter and the mixture settles into two phases.
[0210] The delipidated plasma phase is removed with a manual
pipette or a pipette connected to a vacuum, and placed in a fourth
container from the kit. The volatile components of the first
extraction solvent evaporate. Mild heating may be employed at this
step. A tube, obtained from the kit, is inserted into the fourth
container. The tube serves as, or is connected to, an intravascular
line leading to a needle introduced into the antecubital vein of
the patient. The delipidated fluid containing delipidated plasma
and delipidated HIV viral particles with reduced infectivity is
introduced into the vascular system through the force of gravity by
elevating the fourth container above the patient. The needle is
optionally obtained from the kit. Administration of the delipidated
HIV viral particles into the vascular system induces an immune
response in the patient to epitopes on the delipidated HIV viral
particles.
EXAMPLE 7
Use of a Kit for Delipidation of a Plasma Sample Containing HIV and
Production of Delipidated HIV Viral Particles
[0211] A 200 ml plasma sample, stored in a plasma bag with a tube
connected to an opening in the bag, is obtained from blood drawn
from a 22 year old patient afflicted with the human
immunodeficiency virus (HIV) and showing symptoms of AIDS. The
patient requires a reduction in the viral load in the blood. The
plasma sample is exposed to a first extraction solvent to remove
lipid from the viral envelope of the HIV virus.
[0212] A first container with a 500 ml capacity is removed from the
kit. The first container, which is graduated, contains a known
volume (about 200 ml) of a first extraction solvent. The entire
plasma sample is added to a second container through a removable
screw cap. The contents of the first container and the second
container are added to a third container obtained from the kit. The
third container is agitated through repeated inversion, thereby
mixing the first extraction solvent and the plasma sample
sufficiently to create a mixture. The third container is placed on
a counter and the mixture settles into two phases.
[0213] The delipidated plasma phase is removed with a manual
pipette or a pipette connected to a vacuum, and placed in a fourth
container from the kit. The volatile components of the first
extraction solvent evaporate. Mild heating may be employed at this
step. A second extraction solvent, contained in a graduated fifth
container, is poured into the fourth container in order to remove
residual first extraction solvent. The fourth container is agitated
through repeated inversion, thereby mixing residual first
extraction solvent, the partially delipidated plasma sample and the
second extraction solvent sufficiently to create a mixture. The
fourth container is allowed to sit and the mixture separates into a
delipidated plasma layer and a solvent layer containing the second
extraction solvent and residual first extraction solvent. The
delipidated plasma layer is removed and placed in a sixth container
obtained from the kit. A tube, obtained from the kit, is inserted
into the sixth container. The tube serves as or is connected to an
intravascular line leading to a needle introduced into the
antecubital vein of the patient. The delipidated fluid containing
delipidated plasma and delipidated HIV viral particles with reduced
infectivity is introduced into the vascular system through the
force of gravity by elevating the sixth container above the
patient. The needle is optionally obtained from the kit.
Administration of the delipidated HIV viral particles into the
vascular system induces an immune response in the patient to
epitopes on the delipidated HIV viral particles.
[0214] All patents, publications and abstracts cited above are
incorporated herein by reference in their entirety. It should be
understood, of course, that the foregoing relates only to preferred
embodiments of the present invention and that numerous
modifications or alterations may be made therein without departing
from the spirit and the scope of the invention as set forth in the
appended claims.
Sequence CWU 1
1
122122PRTArtificial SequenceSynthetic 1Met Gly Val Arg Asn Ser Val
Leu Ser Gly Lys Lys Ala Asp Glu Leu1 5 10 15Glu Lys Ile Arg Leu
Arg20222PRTArtificial SequenceSynthetic 2Lys Lys Ala Asp Glu Leu
Glu Lys Ile Arg Leu Arg Pro Asn Gly Lys1 5 10 15Lys Lys Tyr Met Leu
Lys20322PRTArtificial SequenceSynthetic 3Leu Arg Pro Asn Gly Lys
Lys Lys Tyr Met Leu Lys His Val Val Trp1 5 10 15Ala Ala Asn Glu Leu
Asp20422PRTArtificial SequenceSynthetic 4Leu Lys His Val Val Trp
Ala Ala Asn Glu Leu Asp Arg Phe Gly Leu1 5 10 15Ala Glu Ser Leu Leu
Glu20522PRTArtificial SequenceSynthetic 5Leu Asp Arg Phe Gly Leu
Ala Glu Ser Leu Leu Glu Asn Lys Glu Gly1 5 10 15Cys Gln Lys Ile Leu
Ser20622PRTArtificial SequenceSynthetic 6Leu Glu Asn Lys Glu Gly
Cys Gln Lys Ile Leu Ser Val Leu Ala Pro1 5 10 15Leu Val Pro Thr Gly
Ser20722PRTArtificial SequenceSynthetic 7Leu Ser Val Leu Ala Pro
Leu Val Pro Thr Gly Ser Glu Asn Leu Lys1 5 10 15Ser Leu Tyr Asn Thr
Val20822PRTArtificial SequenceSynthetic 8Gly Ser Glu Asn Leu Lys
Ser Leu Tyr Asn Thr Val Cys Val Ile Trp1 5 10 15Cys Ile His Ala Glu
Glu20922PRTArtificial SequenceSynthetic 9Thr Val Cys Val Ile Trp
Cys Ile His Ala Glu Glu Lys Val Lys His1 5 10 15Thr Glu Glu Ala Lys
Gln201022PRTArtificial SequenceSynthetic 10Glu Glu Lys Val Lys His
Thr Glu Glu Ala Lys Gln Ile Val Gln Arg1 5 10 15His Leu Val Val Glu
Thr201122PRTArtificial SequenceSynthetic 11Lys Gln Ile Val Gln Arg
His Leu Val Val Glu Thr Gly Thr Thr Glu1 5 10 15Thr Met Pro Lys Thr
Ser201222PRTArtificial SequenceSynthetic 12Glu Thr Gly Thr Thr Glu
Thr Met Pro Lys Thr Ser Arg Pro Thr Ala1 5 10 15Pro Ser Ser Gly Arg
Gly201322PRTArtificial SequenceSynthetic 13Thr Ser Arg Pro Thr Ala
Pro Ser Ser Gly Arg Gly Gly Asn Tyr Pro1 5 10 15Val Gln Gln Ile Gly
Gly201422PRTArtificial SequenceSynthetic 14Arg Gly Gly Asn Tyr Pro
Val Gln Gln Ile Gly Gly Asn Tyr Val His1 5 10 15Leu Pro Leu Ser Pro
Arg201522PRTArtificial SequenceSynthetic 15Gly Gly Asn Tyr Val His
Leu Pro Leu Ser Pro Arg Thr Leu Asn Ala1 5 10 15Trp Val Lys Leu Ile
Glu201622PRTArtificial SequenceSynthetic 16Pro Arg Thr Leu Asn Ala
Trp Val Lys Leu Ile Glu Glu Lys Lys Phe1 5 10 15Gly Ala Glu Val Val
Pro201722PRTArtificial SequenceSynthetic 17Ile Glu Glu Lys Lys Phe
Gly Ala Glu Val Val Pro Gly Phe Gln Ala1 5 10 15Leu Ser Glu Gly Cys
Thr201825PRTArtificial SequenceSynthetic 18Val Pro Gly Phe Gln Ala
Leu Ser Glu Gly Cys Thr Pro Tyr Asp Ile1 5 10 15Asn Gln Met Leu Asn
Cys Val Gly Asp20 251920PRTArtificial SequenceSynthetic 19Gly Cys
Thr Pro Tyr Asp Ile Asn Gln Met Leu Asn Cys Val Gly Asp1 5 10 15His
Gln Ala Ala202023PRTArtificial SequenceSynthetic 20Asn Cys Val Gly
Asp His Gln Ala Ala Met Gln Ile Ile Arg Asp Ile1 5 10 15Ile Asn Glu
Glu Ala Ala Asp202122PRTArtificial SequenceSynthetic 21Ile Ile Arg
Asp Ile Ile Asn Glu Glu Ala Ala Asp Trp Asp Leu Gln1 5 10 15His Pro
Gln Pro Ala Pro202222PRTArtificial SequenceSynthetic 22Ala Asp Trp
Asp Leu Gln His Pro Gln Pro Ala Pro Gln Gln Gly Gln1 5 10 15Leu Arg
Glu Pro Ser Gly202322PRTArtificial SequenceSynthetic 23Ala Pro Gln
Gln Gly Gln Leu Arg Glu Pro Ser Gly Ser Asp Ile Ala1 5 10 15Gly Thr
Thr Ser Ser Val202422PRTArtificial SequenceSynthetic 24Ser Gly Ser
Asp Ile Ala Gly Thr Thr Ser Ser Val Asp Glu Gln Ile1 5 10 15Gln Trp
Met Tyr Arg Gln202522PRTArtificial SequenceSynthetic 25Ser Val Asp
Glu Gln Ile Gln Trp Met Tyr Arg Gln Gln Asn Pro Ile1 5 10 15Pro Val
Gly Asn Ile Tyr202622PRTArtificial SequenceSynthetic 26Arg Gln Gln
Asn Pro Ile Pro Val Gly Asn Ile Tyr Arg Arg Trp Ile1 5 10 15Gln Leu
Gly Leu Gln Lys202723PRTArtificial SequenceSynthetic 27Ile Tyr Arg
Arg Trp Ile Gln Leu Gly Leu Gln Lys Cys Val Arg Met1 5 10 15Tyr Asn
Pro Thr Asn Ile Leu202822PRTArtificial SequenceSynthetic 28Lys Cys
Val Arg Met Tyr Asn Pro Thr Asn Ile Leu Asp Val Lys Gln1 5 10 15Gly
Pro Lys Glu Pro Phe202922PRTArtificial SequenceSynthetic 29Ile Leu
Asp Val Lys Gln Gly Pro Lys Glu Pro Phe Gln Ser Tyr Val1 5 10 15Asp
Arg Phe Tyr Lys Ser203022PRTArtificial SequenceSynthetic 30Pro Phe
Gln Ser Tyr Val Asp Arg Phe Tyr Lys Ser Leu Arg Ala Glu1 5 10 15Gln
Thr Asp Ala Ala Val203122PRTArtificial SequenceSynthetic 31Lys Ser
Leu Arg Ala Glu Gln Thr Asp Ala Ala Val Lys Asn Trp Met1 5 10 15Thr
Gln Thr Leu Leu Ile203222PRTArtificial SequenceSynthetic 32Ala Val
Lys Asn Trp Met Thr Gln Thr Leu Leu Ile Gln Asn Ala Asn1 5 10 15Pro
Asp Cys Lys Leu Val203322PRTArtificial SequenceSynthetic 33Leu Ile
Gln Asn Ala Asn Pro Asp Cys Lys Leu Val Leu Lys Gly Leu1 5 10 15Gly
Val Asn Pro Thr Leu203422PRTArtificial SequenceSynthetic 34Leu Val
Leu Lys Gly Leu Gly Val Asn Pro Thr Leu Glu Glu Met Leu1 5 10 15Thr
Ala Cys Gln Gly Val203522PRTArtificial SequenceSynthetic 35Thr Leu
Glu Glu Met Leu Thr Ala Cys Gln Gly Val Gly Gly Pro Gly1 5 10 15Gln
Lys Ala Arg Leu Met203622PRTArtificial SequenceSynthetic 36Gly Val
Gly Gly Pro Gly Gln Lys Ala Arg Leu Met Ala Glu Ala Leu1 5 10 15Lys
Glu Ala Leu Ala Pro203722PRTArtificial SequenceSynthetic 37Leu Met
Ala Glu Ala Leu Lys Glu Ala Leu Ala Pro Val Pro Ile Pro1 5 10 15Phe
Ala Ala Ala Gln Gln203822PRTArtificial SequenceSynthetic 38Ala Pro
Val Pro Ile Pro Phe Ala Ala Ala Gln Gln Arg Gly Pro Arg1 5 10 15Lys
Pro Ile Lys Cys Trp203922PRTArtificial SequenceSynthetic 39Ala Gln
Gln Arg Gly Pro Arg Lys Pro Ile Lys Cys Trp Asn Cys Gly1 5 10 15Lys
Glu Gly His Ser Ala204022PRTArtificial SequenceSynthetic 40Lys Cys
Trp Asn Cys Gly Lys Glu Gly His Ser Ala Arg Gln Cys Arg1 5 10 15Ala
Pro Arg Arg Gln Gly204122PRTArtificial SequenceSynthetic 41Ser Ala
Arg Gln Cys Arg Ala Pro Arg Arg Gln Gly Cys Trp Lys Cys1 5 10 15Gly
Lys Met Asp His Val204223PRTArtificial SequenceSynthetic 42Arg Gln
Gly Cys Trp Lys Cys Gly Lys Met Asp His Val Met Ala Lys1 5 10 15Cys
Pro Asp Arg Gln Ala Gly204322PRTArtificial SequenceSynthetic 43His
Val Met Ala Lys Cys Pro Asp Arg Gln Ala Gly Phe Leu Gly Leu1 5 10
15Gly Pro Trp Gly Lys Lys204422PRTArtificial SequenceSynthetic
44Ala Gly Phe Leu Gly Leu Gly Pro Trp Gly Lys Lys Pro Arg Asn Phe1
5 10 15Pro Met Ala Gln Val His204522PRTArtificial SequenceSynthetic
45Lys Lys Pro Arg Asn Phe Pro Met Ala Gln Val His Gln Gly Leu Met1
5 10 15Pro Thr Ala Pro Pro Glu204622PRTArtificial SequenceSynthetic
46Val His Gln Gly Leu Met Pro Thr Ala Pro Pro Glu Asp Pro Ala Val1
5 10 15Asp Leu Leu Lys Asn Tyr204722PRTArtificial SequenceSynthetic
47Pro Glu Asp Pro Ala Val Asp Leu Leu Lys Asn Tyr Met Gln Leu Gly1
5 10 15Lys Gln Gln Arg Glu Lys204822PRTArtificial SequenceSynthetic
48Asn Tyr Met Gln Leu Gly Lys Gln Gln Arg Glu Lys Gln Arg Glu Ser1
5 10 15Arg Glu Lys Pro Tyr Lys204922PRTArtificial SequenceSynthetic
49Glu Lys Gln Arg Glu Ser Arg Glu Lys Pro Tyr Lys Glu Val Thr Glu1
5 10 15Asp Leu Leu His Leu Asn205019PRTArtificial SequenceSynthetic
50Tyr Lys Glu Val Thr Glu Asp Leu Leu His Leu Asn Ser Leu Phe Gly1
5 10 15Gly Asp Gln5125PRTArtificial SequenceSynthetic 51Met Gly Cys
Leu Gly Asn Gln Leu Leu Ile Ala Ile Leu Leu Leu Ser1 5 10 15Val Tyr
Gly Ile Tyr Cys Thr Leu Tyr20 255225PRTArtificial SequenceSynthetic
52Leu Leu Leu Ser Val Tyr Gly Ile Tyr Cys Thr Leu Tyr Val Thr Val1
5 10 15Phe Tyr Gly Val Pro Ala Trp Arg Asn20 255325PRTArtificial
SequenceSynthetic 53Tyr Val Thr Val Phe Tyr Gly Val Pro Ala Trp Arg
Asn Ala Thr Ile1 5 10 15Pro Leu Phe Cys Ala Thr Lys Asn Arg20
255425PRTArtificial SequenceSynthetic 54Asn Ala Thr Ile Pro Leu Phe
Cys Ala Thr Lys Asn Arg Asp Thr Trp1 5 10 15Gly Thr Thr Gln Cys Leu
Pro Asp Asn20 255525PRTArtificial SequenceSynthetic 55Arg Asp Thr
Trp Gly Thr Thr Gln Cys Leu Pro Asp Asn Gly Asp Tyr1 5 10 15Ser Glu
Val Ala Leu Asn Val Thr Glu20 255625PRTArtificial SequenceSynthetic
56Asn Gly Asp Tyr Ser Glu Val Ala Leu Asn Val Thr Glu Ser Phe Asp1
5 10 15Ala Trp Asn Asn Thr Val Thr Glu Gln20 255725PRTArtificial
SequenceSynthetic 57Glu Ser Phe Asp Ala Trp Asn Asn Thr Val Thr Glu
Gln Ala Ile Glu1 5 10 15Asp Val Trp Gln Leu Phe Glu Thr Ser20
255825PRTArtificial SequenceSynthetic 58Gln Ala Ile Glu Asp Val Trp
Gln Leu Phe Glu Thr Ser Ile Lys Pro1 5 10 15Cys Val Lys Leu Ser Pro
Leu Cys Ile20 255925PRTArtificial SequenceSynthetic 59Ser Ile Lys
Pro Cys Val Lys Leu Ser Pro Leu Cys Ile Thr Met Arg1 5 10 15Cys Asn
Lys Ser Glu Thr Asp Arg Trp20 256024PRTArtificial SequenceSynthetic
60Thr Met Arg Cys Asn Lys Ser Glu Thr Asp Arg Trp Gly Leu Thr Lys1
5 10 15Ser Ile Thr Thr Thr Ala Ser Thr206125PRTArtificial
SequenceSynthetic 61Trp Gly Leu Thr Lys Ser Ile Thr Thr Thr Ala Ser
Thr Thr Ser Thr1 5 10 15Thr Ala Ser Ala Lys Val Asp Met Val20
256225PRTArtificial SequenceSynthetic 62Thr Thr Ser Thr Thr Ala Ser
Ala Lys Val Asp Met Val Asn Glu Thr1 5 10 15Ser Ser Cys Ile Ala Gln
Asp Asn Cys20 256325PRTArtificial SequenceSynthetic 63Val Asn Glu
Thr Ser Ser Cys Ile Ala Gln Asp Asn Cys Thr Gly Leu1 5 10 15Glu Gln
Glu Gln Met Ile Ser Cys Lys20 256425PRTArtificial SequenceSynthetic
64Cys Thr Gly Leu Glu Gln Glu Gln Met Ile Ser Cys Lys Phe Asn Met1
5 10 15Thr Gly Leu Lys Arg Asp Lys Lys Lys20 256525PRTArtificial
SequenceSynthetic 65Lys Phe Asn Met Thr Gly Leu Lys Arg Asp Lys Lys
Lys Glu Tyr Asn1 5 10 15Glu Thr Trp Tyr Ser Ala Asp Leu Val20
256625PRTArtificial SequenceSynthetic 66Lys Glu Tyr Asn Glu Thr Trp
Tyr Ser Ala Asp Leu Val Cys Glu Gln1 5 10 15Gly Asn Asn Thr Gly Asn
Glu Ser Arg20 256725PRTArtificial SequenceSynthetic 67Val Cys Glu
Gln Gly Asn Asn Thr Gly Asn Glu Ser Arg Cys Tyr Met1 5 10 15Asn His
Cys Asn Thr Ser Val Ile Gln20 256825PRTArtificial SequenceSynthetic
68Arg Cys Tyr Met Asn His Cys Asn Thr Ser Val Ile Gln Glu Ser Cys1
5 10 15Asp Lys His Tyr Trp Asp Ala Ile Arg20 256925PRTArtificial
SequenceSynthetic 69Gln Glu Ser Cys Asp Lys His Tyr Trp Asp Ala Ile
Arg Phe Arg Tyr1 5 10 15Cys Ala Pro Pro Gly Tyr Ala Leu Leu20
257025PRTArtificial SequenceSynthetic 70Arg Phe Arg Tyr Cys Ala Pro
Pro Gly Tyr Ala Leu Leu Arg Cys Asn1 5 10 15Asp Thr Asn Tyr Ser Gly
Phe Met Pro20 257125PRTArtificial SequenceSynthetic 71Leu Arg Cys
Asn Asp Thr Asn Tyr Ser Gly Phe Met Pro Lys Cys Ser1 5 10 15Lys Val
Val Val Ser Ser Cys Thr Arg20 257225PRTArtificial SequenceSynthetic
72Pro Lys Cys Ser Lys Val Val Val Ser Ser Cys Thr Arg Met Met Glu1
5 10 15Thr Gln Thr Ser Thr Trp Phe Gly Phe20 257325PRTArtificial
SequenceSynthetic 73Arg Met Met Glu Thr Gln Thr Ser Thr Trp Phe Gly
Phe Asn Gly Thr1 5 10 15Arg Ala Glu Asn Arg Thr Tyr Ile Tyr20
257425PRTArtificial SequenceSynthetic 74Phe Asn Gly Thr Arg Ala Glu
Asn Arg Thr Tyr Ile Tyr Trp His Gly1 5 10 15Arg Asp Asn Arg Thr Ile
Ile Ser Leu20 257525PRTArtificial SequenceSynthetic 75Tyr Trp His
Gly Arg Asp Asn Arg Thr Ile Ile Ser Leu Asn Lys Tyr1 5 10 15Tyr Asn
Leu Thr Met Lys Cys Arg Arg20 257625PRTArtificial SequenceSynthetic
76Leu Asn Lys Tyr Tyr Asn Leu Thr Met Lys Cys Arg Arg Pro Gly Asn1
5 10 15Lys Thr Val Leu Pro Val Thr Ile Met20 257725PRTArtificial
SequenceSynthetic 77Arg Pro Gly Asn Lys Thr Val Leu Pro Val Thr Ile
Met Ser Gly Leu1 5 10 15Val Phe His Ser Gln Pro Ile Asn Asp20
257825PRTArtificial SequenceSynthetic 78Met Ser Gly Leu Val Phe His
Ser Gln Pro Ile Asn Asp Arg Pro Lys1 5 10 15Gln Ala Trp Cys Trp Phe
Gly Gly Lys20 257925PRTArtificial SequenceSynthetic 79Asp Arg Pro
Lys Gln Ala Trp Cys Trp Phe Gly Gly Lys Trp Lys Asp1 5 10 15Ala Ile
Lys Glu Val Lys Gln Thr Ile20 258025PRTArtificial SequenceSynthetic
80Lys Trp Lys Asp Ala Ile Lys Glu Val Lys Gln Thr Ile Val Lys His1
5 10 15Pro Arg Tyr Thr Gly Thr Asn Asn Thr20 258125PRTArtificial
SequenceSynthetic 81Ile Val Lys His Pro Arg Tyr Thr Gly Thr Asn Asn
Thr Asp Lys Ile1 5 10 15Asn Leu Thr Ala Pro Gly Gly Gly Asp20
258225PRTArtificial SequenceSynthetic 82Thr Asp Lys Ile Asn Leu Thr
Ala Pro Gly Gly Gly Asp Pro Glu Val1 5 10 15Thr Phe Met Trp Thr Asn
Cys Arg Gly20 258325PRTArtificial SequenceSynthetic 83Asp Pro Glu
Val Thr Phe Met Trp Thr Asn Cys Arg Gly Glu Phe Leu1 5 10 15Tyr Cys
Lys Met Asn Trp Phe Leu Asn20 258425PRTArtificial SequenceSynthetic
84Gly Glu Phe Leu Tyr Cys Lys Met Asn Trp Phe Leu Asn Trp Val Glu1
5 10 15Asp Arg Asn Thr Ala Asn Gln Lys Pro20 258525PRTArtificial
SequenceSynthetic 85Asn Trp Val Glu Asp Arg Asn Thr Ala Asn Gln Lys
Pro Lys Glu Gln1 5 10 15His Lys Arg Asn Tyr Val Pro Cys His20
258625PRTArtificial SequenceSynthetic 86Pro Lys Glu Gln His Lys Arg
Asn Tyr Val Pro Cys His Ile Arg Gln1 5 10 15Ile Ile Asn Thr Trp His
Lys Val Gly20 258725PRTArtificial SequenceSynthetic 87His Ile Arg
Gln Ile Ile Asn Thr Trp His Lys Val Gly Lys Asn Val1 5 10 15Tyr Leu
Pro Pro Arg Glu Gly Asp Leu20 258825PRTArtificial SequenceSynthetic
88Gly Lys Asn Val Tyr Leu Pro Pro Arg Glu Gly Asp Leu Thr Cys Asn1
5 10 15Ser Thr Val Thr Ser Leu Ile Ala Asn20 258925PRTArtificial
SequenceSynthetic 89Leu Thr Cys Asn Ser Thr Val Thr Ser Leu Ile Ala
Asn Ile Asp Trp1 5 10 15Ile Asp Gly Asn Gln Thr Asn Ile Thr20
259025PRTArtificial SequenceSynthetic 90Asn Ile Asp Trp Ile Asp Gly
Asn Gln Thr Asn Ile Thr Met Ser Ala1 5 10 15Glu Val Ala Glu Leu Tyr
Arg
Leu Glu20 259125PRTArtificial SequenceSynthetic 91Thr Met Ser Ala
Glu Val Ala Glu Leu Tyr Arg Leu Glu Leu Gly Asp1 5 10 15Tyr Lys Leu
Val Glu Ile Thr Pro Ile20 259225PRTArtificial SequenceSynthetic
92Glu Leu Gly Asp Tyr Lys Leu Val Glu Ile Thr Pro Ile Gly Leu Ala1
5 10 15Pro Thr Asp Val Lys Arg Tyr Thr Thr20 259325PRTArtificial
SequenceSynthetic 93Ile Gly Leu Ala Pro Thr Asp Val Lys Arg Tyr Thr
Thr Gly Gly Thr1 5 10 15Ser Arg Asn Lys Arg Gly Val Phe Val20
259425PRTArtificial SequenceSynthetic 94Thr Gly Gly Thr Ser Arg Asn
Lys Arg Gly Val Phe Val Leu Gly Phe1 5 10 15Leu Gly Phe Leu Ala Thr
Ala Gly Ser20 259525PRTArtificial SequenceSynthetic 95Val Leu Gly
Phe Leu Gly Phe Leu Ala Thr Ala Gly Ser Ala Met Gly1 5 10 15Ala Ala
Ser Leu Thr Leu Thr Ala Gln20 259625PRTArtificial SequenceSynthetic
96Ser Ala Met Gly Ala Ala Ser Leu Thr Leu Thr Ala Gln Ser Arg Thr1
5 10 15Leu Leu Ala Gly Ile Val Gln Gln Gln20 259725PRTArtificial
SequenceSynthetic 97Gln Ser Arg Thr Leu Leu Ala Gly Ile Val Gln Gln
Gln Gln Gln Leu1 5 10 15Leu Asp Val Val Lys Arg Gln Gln Glu20
259825PRTArtificial SequenceSynthetic 98Gln Gln Gln Leu Leu Asp Val
Val Lys Arg Gln Gln Glu Leu Leu Arg1 5 10 15Leu Thr Val Trp Gly Thr
Lys Asn Leu20 259925PRTArtificial SequenceSynthetic 99Glu Leu Leu
Arg Leu Thr Val Trp Gly Thr Lys Asn Leu Gln Thr Arg1 5 10 15Val Thr
Ala Ile Glu Lys Tyr Leu Lys20 2510025PRTArtificial
SequenceSynthetic 100Leu Gln Thr Arg Val Thr Ala Ile Glu Lys Tyr
Leu Lys Asp Gln Ala1 5 10 15Gln Leu Asn Ala Trp Gly Cys Ala Phe20
2510125PRTArtificial SequenceSynthetic 101Lys Asp Gln Ala Gln Leu
Asn Ala Trp Gly Cys Ala Phe Arg Gln Val1 5 10 15Cys His Thr Thr Val
Pro Trp Pro Asn20 2510225PRTArtificial SequenceSynthetic 102Phe Arg
Gln Val Cys His Thr Thr Val Pro Trp Pro Asn Ala Ser Leu1 5 10 15Thr
Pro Lys Trp Asn Asn Glu Thr Trp20 2510325PRTArtificial
SequenceSynthetic 103Asn Ala Ser Leu Thr Pro Lys Trp Asn Asn Glu
Thr Trp Gln Glu Trp1 5 10 15Glu Arg Lys Val Asp Phe Leu Glu Glu20
2510425PRTArtificial SequenceSynthetic 104Trp Gln Glu Trp Glu Arg
Lys Val Asp Phe Leu Glu Glu Asn Ile Thr1 5 10 15Ala Leu Leu Glu Glu
Ala Gln Ile Gln20 2510525PRTArtificial SequenceSynthetic 105Glu Asn
Ile Thr Ala Leu Leu Glu Glu Ala Gln Ile Gln Gln Glu Lys1 5 10 15Asn
Met Tyr Glu Leu Gln Lys Leu Asn20 2510625PRTArtificial
SequenceSynthetic 106Gln Gln Glu Lys Asn Met Tyr Glu Leu Gln Lys
Leu Asn Ser Trp Asp1 5 10 15Val Phe Gly Asn Trp Phe Asp Leu Ala20
2510725PRTArtificial SequenceSynthetic 107Asn Ser Trp Asp Val Phe
Gly Asn Trp Phe Asp Leu Ala Ser Trp Ile1 5 10 15Lys Tyr Ile Gln Tyr
Gly Val Tyr Ile20 2510825PRTArtificial SequenceSynthetic 108Ala Ser
Trp Ile Lys Tyr Ile Gln Tyr Gly Val Tyr Ile Val Val Gly1 5 10 15Val
Ile Leu Leu Arg Ile Val Ile Tyr20 2510925PRTArtificial
SequenceSynthetic 109Ile Val Val Gly Val Ile Leu Leu Arg Ile Val
Ile Tyr Ile Val Gln1 5 10 15Met Leu Ala Lys Leu Arg Gln Gly Tyr20
2511025PRTArtificial SequenceSynthetic 110Tyr Ile Val Gln Met Leu
Ala Lys Leu Arg Gln Gly Tyr Arg Pro Val1 5 10 15Phe Ser Ser Pro Pro
Ser Tyr Phe Gln20 2511125PRTArtificial SequenceSynthetic 111Tyr Arg
Pro Val Phe Ser Ser Pro Pro Ser Tyr Phe Gln Gln Thr His1 5 10 15Ile
Gln Gln Asp Pro Ala Leu Pro Thr20 2511225PRTArtificial
SequenceSynthetic 112Gln Gln Thr His Ile Gln Gln Asp Pro Ala Leu
Pro Thr Arg Glu Gly1 5 10 15Lys Glu Arg Asp Gly Gly Glu Gly Gly20
2511325PRTArtificial SequenceSynthetic 113Thr Arg Glu Gly Lys Glu
Arg Asp Gly Gly Glu Gly Gly Gly Asn Ser1 5 10 15Ser Trp Pro Trp Gln
Ile Glu Tyr Ile20 2511425PRTArtificial SequenceSynthetic 114Gly Gly
Asn Ser Ser Trp Pro Trp Gln Ile Glu Tyr Ile His Phe Leu1 5 10 15Ile
Arg Gln Leu Ile Arg Leu Leu Thr20 2511525PRTArtificial
SequenceSynthetic 115Ile His Phe Leu Ile Arg Gln Leu Ile Arg Leu
Leu Thr Trp Leu Phe1 5 10 15Ser Asn Cys Arg Thr Leu Leu Ser Arg20
2511625PRTArtificial SequenceSynthetic 116Thr Trp Leu Phe Ser Asn
Cys Arg Thr Leu Leu Ser Arg Val Tyr Gln1 5 10 15Ile Leu Gln Pro Ile
Leu Gln Arg Leu20 2511725PRTArtificial SequenceSynthetic 117Arg Val
Tyr Gln Ile Leu Gln Pro Ile Leu Gln Arg Leu Ser Ala Thr1 5 10 15Leu
Gln Arg Ile Arg Glu Val Leu Arg20 2511825PRTArtificial
SequenceSynthetic 118Leu Ser Ala Thr Leu Gln Arg Ile Arg Glu Val
Leu Arg Thr Glu Leu1 5 10 15Thr Tyr Leu Gln Tyr Gly Trp Ser Tyr20
2511925PRTArtificial SequenceSynthetic 119Arg Thr Glu Leu Thr Tyr
Leu Gln Tyr Gly Trp Ser Tyr Phe His Glu1 5 10 15Ala Val Gln Ala Val
Trp Arg Ser Ala20 2512025PRTArtificial SequenceSynthetic 120Tyr Phe
His Glu Ala Val Gln Ala Val Trp Arg Ser Ala Thr Glu Thr1 5 10 15Leu
Ala Gly Ala Trp Gly Asp Leu Trp20 2512125PRTArtificial
SequenceSynthetic 121Ala Thr Glu Thr Leu Ala Gly Ala Trp Gly Asp
Leu Trp Glu Thr Leu1 5 10 15Arg Arg Gly Gly Arg Trp Ile Leu Ala20
2512227PRTArtificial SequenceSynthetic 122Trp Glu Thr Leu Arg Arg
Gly Gly Arg Trp Ile Leu Ala Ile Pro Arg1 5 10 15Arg Ile Arg Gln Gly
Leu Glu Leu Thr Leu Leu20 25
* * * * *